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OTAR3088
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Cellfinder-cleaned_CoNNL2

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[ "BackgroundMuch", "of", "our", "current", "knowledge", "of", "the", "molecular", "expression", "profile", "of", "human", "embryonic", "stem", "cells", "(hESCs)", "is", "based", "on", "transcriptional", "approaches.", "These", "analyses", "are", "only", "partly", "predictive", "of", "protein", "expression", "however,", "and", "do", "not", "shed", "light", "on", "post-translational", "regulation,", "leaving", "a", "large", "gap", "in", "our", "knowledge", "of", "the", "biology", "of", "pluripotent", "stem", "cells.ResultsHere", "we", "describe", "the", "use", "of", "two", "large-scale", "western", "blot", "assays", "to", "identify", "over", "600", "proteins", "expressed", "in", "undifferentiated", "hESCs,", "and", "highlight", "over", "40", "examples", "of", "multiple", "gel", "mobility", "variants,", "which", "are", "suspected", "protein", "isoforms", "and/or", "post-translational", "modifications.", "Twenty-two", "phosphorylation", "events", "in", "cell", "signaling", "molecules,", "as", "well", "as", "potential", "new", "markers", "of", "undifferentiated", "hESCs", "were", "also", "identified.", "We", "confirmed", "the", "expression", "of", "a", "subset", "of", "the", "identified", "proteins", "by", "immunofluorescence", "and", "correlated", "the", "expression", "of", "transcript", "and", "protein", "for", "key", "molecules", "in", "active", "signaling", "pathways", "in", "hESCs.", "These", "analyses", "also", "indicated", "that", "hESCs", "exhibit", "several", "features", "of", "polarized", "epithelia,", "including", "expression", "of", "tight", "junction", "proteins.ConclusionOur", "approach", "complements", "proteomic", "and", "transcriptional", "analysis", "to", "provide", "unique", "information", "on", "human", "pluripotent", "stem", "cells,", "and", "is", "a", "framework", "for", "the", "continued", "analyses", "of", "self-renewal.", "Human", "embryonic", "stem", "cells", "(hESCs)", "are", "pluripotent", "cells", "isolated", "from", "the", "inner", "cell", "mass", "of", "the", "blastocyst", "[1].", "They", "can", "be", "maintained", "for", "prolonged", "periods", "in", "culture", "and", "differentiate", "to", "representatives", "of", "the", "three", "germ", "layers", "as", "well", "as", "trophoblasts", "and", "germ", "cells.", "This", "differentiation", "potential", "may", "be", "used", "to", "model", "certain", "aspects", "of", "human", "embryogenesis,", "including", "the", "development", "and", "differentiation", "of", "pluripotent", "and", "other", "stem", "cell", "types", "during", "the", "processes", "of", "gastrulation,", "neurogenesis", "and", "organogenesis.", "Thus,", "hESCs", "provide", "a", "unique", "and", "powerful", "system", "to", "study", "otherwise", "intractable", "aspects", "of", "human", "development.", "Furthermore,", "these", "approaches", "have", "the", "potential", "to", "provide", "differentiated", "cell", "types", "for", "cell", "replacement", "therapies", "of", "degenerative", "disorders", "such", "as", "Parkinson's", "disease", "and", "Type", "I", "diabetes", "[2,3].", "Before", "these", "cell", "therapy", "applications", "are", "developed,", "an", "understanding", "of", "the", "molecular", "and", "cellular", "mechanisms", "that", "drive", "self-renewal", "and", "differentiation", "is", "required.", "Fundamental", "to", "this", "understanding", "is", "the", "elucidation", "of", "the", "transcriptome", "and", "proteome", "of", "hESCs,", "using", "approaches", "that", "lay", "a", "framework", "for", "functional", "analyses", "of", "the", "unique", "properties", "of", "these", "cells.", "Large-scale", "gene", "expression", "analyses", "such", "as", "microarray,", "massive", "parallel", "signature", "sequencing", "(MPSS),", "expressed", "sequenced", "tag", "(EST)", "enumeration,", "and", "serial", "analysis", "of", "gene", "expression", "(SAGE)", "have", "been", "used", "to", "compare", "multiple", "hESC", "lines", "[4-7];", "hESCs", "to", "germ", "cell", "tumors", "[8];", "or", "to", "differentiated", "derivatives", "in", "embryoid", "bodies", "[9-11]", "or", "neural", "populations", "[12].", "These", "approaches", "have", "highlighted", "an", "expanded", "set", "of", "transcripts", "that", "mark", "the", "pluripotent", "state", "[4,13,14],", "cross-species", "commonalities", "in", "the", "molecular", "profile", "of", "ESCs", "[6,12,15],", "prominent", "receptors", "expressed", "by", "hESCs", "[8]", "and", "pathways", "that", "may", "play", "a", "role", "in", "the", "regulation", "of", "pluripotency", "[16,17].", "Nevertheless,", "cataloguing", "the", "cellular", "transcriptome", "is", "only", "predictive", "of", "protein", "expression", "and", "typically", "does", "not", "shed", "light", "on", "post-transcriptional", "regulation.", "For", "example,", "while", "tens", "of", "thousands", "of", "transcripts", "can", "be", "followed", "simultaneously", "with", "SAGE,", "microarrays", "and", "MPSS,", "these", "methods", "do", "not", "routinely", "detect", "differences", "in", "transcript", "splice", "variants,", "or", "polyadenylation", "status.", "These", "differences", "may", "have", "profound", "effects", "on", "translation,", "as", "well", "as", "the", "isoform", "and", "function", "of", "the", "protein", "produced.", "Finally,", "numerous", "post-translational", "modifications", "are", "known", "to", "regulate", "protein", "function,", "including", "enzymatic", "cleavage,", "covalent", "coupling", "to", "other", "molecules,", "glycosylation,", "phosphorylation", "and", "ubiquitination.", "These", "issues", "all", "highlight", "potential", "shortfalls", "in", "our", "understanding", "of", "the", "hESC", "proteome.", "Several", "practical", "approaches", "for", "proteomic", "analyses", "are", "currently", "available,", "the", "most", "established", "of", "which", "is", "the", "2-dimensional", "(2D)", "separation", "of", "proteins", "by", "polyacrylamide", "gel", "electrophoresis", "(PAGE).", "HPLC-tandem", "mass", "spectrometry", "(HPLC-MS/MS)", "based", "technology", "is", "rapidly", "evolving", "and", "has", "recently", "been", "used", "to", "detect", "protein", "expression", "in", "multiple", "cell", "types.", "An", "alternate", "approach", "is", "the", "recent", "large-scale", "adaptation", "of", "standard", "western", "blotting", "[18].", "In", "this", "procedure,", "a", "large", "well", "is", "used", "to", "separate", "the", "sample", "by", "PAGE", "and", "lanes", "are", "created", "on", "the", "membrane", "containing", "immobilized", "protein", "with", "the", "use", "of", "a", "manifold.", "Compatible", "combinations", "of", "primary", "antibodies", "are", "predetermined,", "with", "the", "criterion", "of", "being", "able", "to", "identify", "proteins", "that", "do", "not", "co-migrate.", "Different", "combinations", "of", "primary", "antibodies", "are", "added", "to", "each", "well,", "with", "appropriate", "dilutions", "of", "each", "primary", "antibody", "so", "that", "expressed", "proteins", "are", "detected", "in", "a", "single", "condition.", "The", "scalability", "of", "the", "system", "depends", "on", "defining", "suitable", "combinations", "of", "primary", "antibodies,", "with", "up", "to", "1000", "antibodies", "in", "200", "lanes", "being", "used", "in", "the", "largest", "screens", "thus", "far.", "Detection", "software", "is", "used", "to", "identify", "proteins", "based", "on", "their", "expected", "and", "observed", "gel", "mobility.", "Unlike", "2D", "PAGE", "and", "HPLC-MS/MS,", "large-scale", "western", "blotting", "only", "identifies", "proteins", "for", "which", "antibodies", "are", "already", "available.", "While", "this", "is", "not", "an", "appropriate", "screen", "for", "identifying", "uncharacterized", "proteins,", "it", "greatly", "simplifies", "the", "verification", "and", "functional", "analyses", "of", "proteins", "that", "are", "detected.", "In", "addition,", "this", "approach", "is", "highly", "flexible,", "and", "if", "desired", "can", "be", "focused", "to", "particular", "sets", "of", "proteins", "or", "protein", "function,", "such", "as", "cell", "signaling", "molecules.", "Importantly,", "the", "foundation", "of", "this", "approach", "is", "the", "large", "amount", "of", "data", "on", "individual", "antibodies,", "which", "are", "already", "available", "and", "characterized", "in", "the", "literature.", "More", "recently,", "two", "research", "groups", "have", "conducted", "proteomic", "analyses", "of", "hESCs", "using", "MS", "[19-22].", "In", "the", "present", "study,", "we", "used", "two", "large-scale", "western", "blot", "systems", "to", "examine", "the", "expression", "of", ">", "1000", "proteins", "in", "hESCs", "and", "detected", ">", "600", "proteins", "that", "were", "grouped", "into", "18", "functional", "classes.", "In", "addition,", "we", "identified", "42", "examples", "of", "multiple", "bands", "for", "a", "single", "protein,", "likely", "to", "be", "protein", "isoforms", "and/or", "post-translational", "modifications,", "and", "22", "phosphorylation", "events", "in", "cell", "signaling", "molecules.", "We", "correlated", "the", "expression", "of", "members", "of", "key", "active", "pathways", "in", "our", "transcriptional", "and", "proteomic", "databases", "and", "confirmed", "the", "validity", "of", "this", "approach.", "Using", "these", "approaches", "we", "identified", "new", "markers", "for", "undifferentiated", "hESCs", "and", "highlighted", "unrecognized", "epithelial", "characteristics", "of", "hESCs.", "Our", "data", "confirm", "the", "importance", "of", "proteomic", "analyses", "in", "complementing", "transcriptional", "profiling", "and", "provide", "a", "framework", "for", "continued", "analyses", "of", "the", "molecular", "and", "cellular", "biology", "of", "pluirpotent", "hESCs.", "PowerBlot", "analysis", "of", "hESCsWe", "first", "employed", "a", "large-scale", "western", "blot", "screen,", "the", "PowerBlot", "system,", "to", "profile", "protein", "expression", "in", "undifferentiated", "hESCs.", "This", "system", "used", "934", "antibodies", "toward", "proteins", "representing", "22", "diverse", "classes", "of", "function,", "such", "as", "transcription", "factors,", "the", "MAP", "kinase", "(MAPK)", "pathway,", "and", "apoptosis,", "among", "others.", "To", "expand", "a", "large-scale", "culture", "of", "BG01", "cells", "for", "this", "assay,", "a", "collagenase-", "and", "trypsin-", "based", "passaging", "method", "was", "used", "[23].", "While", "these", "conditions", "have", "been", "associated", "with", "the", "accumulation", "of", "trisomies", "of", "chromosomes", "12,", "17", "and", "X", "[24],", "the", "ease", "of", "use", "of", "these", "cultures", "and", "similarity", "in", "gene", "expression", "and", "differentiation", "potential", "to", "karyotypically", "normal", "BG01", "hESCs", "[11,24,25]", "make", "them", "suitable", "for", "such", "large", "scale", "applications.", "For", "the", "PowerBlot", "screen,", "whole", "cell", "lysate", "from", "BG01", "hESCs", "was", "separated", "on", "five", "4–15%", "gradient", "gels.", "Each", "blot", "contained", "size", "markers", "and", "39", "lanes.", "Each", "lane", "was", "screened", "with", "1–8", "antibodies", "in", "combinations", "that", "had", "been", "predetermined", "to", "enable", "accurate", "identification", "of", "well-separated", "proteins", "(Fig.", "1A–E).", "The", "gels", "and", "blots", "were", "performed", "in", "duplicate", "and", "expressed", "proteins", "were", "identified", "by", "their", "predicted", "size", "and", "verified", "by", "visual", "inspection.Figure", "1PowerBlot", "analysis", "of", "undifferentiated", "BG01", "hESCs.", "This", "large-scale", "western", "blot", "consisted", "of", "five", "gels", "run", "in", "duplicate", "and", "probed", "with", "934", "antibodies.", "(A-E)", "One", "set", "of", "blots", "is", "shown", "at", "a", "contrast", "that", "highlights", "most", "bands.", "(F)", "A", "representative", "lane", "(gel", "C,", "lane", "24)", "aligned", "with", "protein", "markers", "used", "for", "band", "identification.", "(G)", "Scatter-plot", "of", "the", "normalized", "average", "intensity", "(i.u.)", "values", "for", "each", "protein", "indicating", "a", "linear", "relationship", "between", "duplicate", "blots.", "Datasets", "for", "this", "analysis", "are", "in", "Additional", "Tables", "1", "and", "2.A", "total", "of", "545", "antibodies", "detected", "bands", "of", "appropriate", "size,", "which", "could", "be", "compressed", "to", "529", "proteins", "with", "unique", "SwissProt", "identification", "numbers", "(Fig.", "1A–E", "and", "Additional", "File", "1).", "An", "enlargement", "of", "a", "representative", "lane", "(lane", "24", "of", "Blot", "C)", "alongside", "protein", "markers", "is", "shown", "in", "Fig.", "1F.", "Thirteen", "proteins", "including", "AKT,", "caveolin1", "and", "ERK1", "were", "detected", "in", "multiple", "lanes", "using", "the", "same", "or", "different", "antibodies.", "Information", "on", "the", "antibody", "catalogue", "number", "and", "dilution,", "band", "intensity", "for", "each", "repeat", "and", "the", "averaged", "value,", "description", "of", "protein", "function,", "and", "Entrez", "gene", "and", "SwissProt", "database", "identification", "numbers", "is", "shown", "in", "Additional", "File", "1.", "Three", "hundred", "and", "eighty", "three", "antibodies", "did", "not", "detect", "bands", "in", "this", "screen,", "indicating", "lack", "of", "expression,", "or", "possibly", "technical", "issues", "with", "detection", "under", "standard", "conditions", "(Additional", "File", "1).The", "size", "of", "the", "detected", "proteins", "ranged", "from", "15", "kD", "(GS15)", "to", "280", "kD", "(ABP-280).", "The", "average", "intensity", "of", "the", "detected", "proteins", "ranged", "from", "195", "to", "117926", "normalized", "intensity", "units", "(i.u.),", "with", "an", "average", "of", "5367", "i.u.", "The", "proteins", "with", "the", "highest", "band", "intensity", "were", "the", "B2", "Bradykinin", "Receptor", "(117926", "i.u.),", "Karyopherin", "α", "(80698", "i.u.),", "and", "BiP", "(74922", "i.u.),", "whilst", "the", "proteins", "with", "the", "lowest", "intensity", "that", "could", "be", "verified", "by", "visual", "inspection", "were", "Inhibitor", "2", "(247", "i.u.),", "Caspase", "8", "(201", "i.u.),", "and", "OXA1Hs", "(195", "i.u.).", "Finally,", "the", "consistency", "of", "this", "assay", "was", "demonstrated", "by", "plotting", "the", "normalized", "average", "intensity", "values", "for", "each", "protein,", "which", "revealed", "a", "linear", "relationship", "between", "the", "duplicate", "samples", "(Fig.", "1G).", "Kinexus", "analysis", "of", "hESCsA", "more", "focused", "screen", "was", "used", "to", "profile", "expression", "of", "protein", "kinases,", "phosphatases", "and", "phosphorylated", "sites", "in", "cell", "signaling", "molecules", "in", "hESCs.", "The", "Kinexus", "assays", "contained", "140", "antibodies", "to", "these", "related", "classes", "of", "proteins", "and", "phospho-sites.", "Karyotypically", "normal", "BG03", "hESCs", "grown", "on", "a", "fibronectin", "matrix", "in", "MEF-CM", "[26]", "were", "used", "for", "this", "analysis,", "and", "whole", "cell", "lysate", "was", "separated", "on", "four", "12.5%", "gels", "for", "western", "blotting.", "Eighty", "five", "immunoreactive", "bands", "were", "identified,", "representing", "38", "protein", "kinases", "and", "16", "phosphatases,", "their", "isoforms,", "and", "22", "phosphorylated", "sites", "in", "signaling", "molecules", "(Fig.", "2A–D,", "Additional", "File", "1).", "Sixty-four", "antibodies", "did", "not", "detect", "their", "corresponding", "antigen", "(Additional", "File", "1).Figure", "2Kinexus", "blots", "of", "undifferentiated", "BG03", "cells.", "Four", "blots", "were", "used", "to", "probe", "BG03", "lysate", "with", "(A,", "B)", "76", "antibodies", "for", "protein", "kinases,", "(C)", "27", "antibodies", "for", "phosphatases", "and", "(D)", "37", "antibodies", "for", "phosphoylated", "sites", "in", "cell", "signaling", "molecules.", "Identified", "bands", "are", "indicated", "(*).", "Datasets", "for", "this", "analysis", "are", "in", "Additional", "Tables", "1", "and", "2.", "Functional", "classification", "of", "proteins", "expressed", "in", "hESCsThe", "PowerBlot", "and", "Kinexus", "assays", "identified", "a", "diverse", "range", "of", "proteins", "expressed", "in", "hESCs.", "To", "further", "annotate", "these", "data,", "the", "detected", "proteins", "were", "ordered", "into", "18", "subgroups", "based", "on", "protein", "function", "(Additional", "File", "2).", "For", "example,", "16", "factors", "with", "known", "or", "implied", "roles", "in", "the", "regulation", "of", "self-renewal", "or", "pluripotency", "of", "mESCs", "or", "hESCs,", "such", "as", "Oct4", "[27],", "STAT3", "[28],", "members", "of", "the", "FGF", "[29],", "PI3", "kinase", "[30],", "Src", "[31]", "or", "MAPK", "pathways", "[32],", "and", "phosphorylated", "isoforms", "of", "GSK3,", "STAT3", "and", "p38", "MAPK,", "were", "grouped", "under", "\"Pluripotency\"", "(Fig.", "3A", "and", "Additional", "File", "2).", "Another", "functional", "group", "(Cell", "surface)", "consisted", "of", "20", "transmembrane", "or", "cell", "surface", "proteins", "(Additional", "File", "2).", "This", "included", "several", "receptors", "for", "peptides", "and", "growth", "factors,", "such", "as", "neurotensin", "receptor", "3,", "the", "B2", "bradykinin,", "endothelin", "1,", "and", "thrombin", "receptors,", "and", "the", "glial", "derived", "neurotrophic", "factor", "receptor", "α", "(Fig.", "3B).", "These", "molecules", "may", "be", "useful", "as", "targets", "for", "cell", "sorting", "experiments,", "and", "expression", "of", "these", "receptors", "could", "identify", "bioactive", "peptides", "or", "growth", "factors", "that", "may", "influence", "hESC", "self-renewal", "or", "differentiation.Figure", "3Functional", "classification", "and", "mobility", "variants", "of", "proteins", "detected", "in", "hESCs.", "(A)", "Proteins", "with", "known", "or", "suggested", "roles", "in", "self-renewal", "are", "shown,", "including", "Oct4,", "STAT3,", "Smad2/3", "and", "FGF2", "(Additional", "Table", "2,", "\"Pluripotency\").", "Isoforms", "of", "FGF2,", "and", "phospho-GSK3", "are", "indicated", "(*).", "(B)", "Cell", "surface", "proteins", "are", "shown,", "including", "Connexin", "43,", "E-Cad", "and", "GDNFRα", "(Additional", "Table", "2,", "\"Cell", "Surface\").", "Other", "functional", "classes", "of", "proteins", "are", "indicated", "in", "Additional", "Table", "2.", "(C)", "A", "total", "of", "42", "proteins,", "including", "FGF2,", "HSP70", "and", "ERK1,", "were", "found", "to", "have", "multiple", "bands", "in", "either", "the", "PowerBlot", "or", "Kinexus", "blots.", "These", "bands", "migrated", "closely", "but", "were", "sufficiently", "separated", "from", "other", "detected", "proteins.", "Bands", "predicted", "to", "be", "isoforms", "of", "the", "indicated", "protein", "are", "highlighted", "in", "some", "panels", "(*).Other", "functional", "classification", "of", "the", "proteins", "detected", "by", "the", "PowerBlot", "screen", "included:", "transcription", "factors", "(71", "proteins),", "nucleus", "and", "nuclear", "transport", "(144),", "cytoskeleton", "(75),", "cell", "adhesion", "(45),", "MAP", "kinase", "pathway", "(24),", "protein", "kinase", "A", "(13),", "protein", "kinase", "C", "(20),", "tyrosine", "kinases", "(15),", "adaptors", "and", "tyrosine", "kinase", "substrates", "(51),", "protein", "phosphatases", "(17),", "GTPases", "and", "regulators", "(42),", "calcium", "signaling", "(23),", "cell", "cycle", "(87),", "apoptosis", "(61),", "membrane", "research", "(62),", "and", "other", "functions", "(51)", "(Additional", "File", "1).", "Some", "proteins", "were", "included", "in", "multiple", "functional", "categories", "due", "to", "overlapping", "properties,", "such", "as", "AIM-1,", "which", "was", "included", "in", "the", "cell", "cycle", "as", "well", "as", "in", "the", "nucleus/nuclear", "transport", "categories.", "The", "Kinexus", "expression", "data", "was", "organized", "separately", "into", "cell", "signaling-related", "functional", "groups", "(Additional", "File", "1).", "In", "addition,", "35", "proteins", "were", "detected", "by", "both", "the", "PowerBlot", "and", "Kinexus", "systems", "(Table", "1).Table", "1Proteins", "detected", "by", "both", "PowerBlot", "and", "Kinexus", "systemsProtein", "nameSwiss", "NrProtein", "nameSwiss", "NrBMXP51813MEK2P36506CaM", "Kinase", "KinaseQ64572MKP2Q62767Casein", "Kinase", "I", "epsilonP49674p38", "alpha/SAPK2aQ16539Casein", "Kinase", "II", "alpha/CK2aP19139PaxillinP49024Cdk1/Cdc2P06493PKA", "CP17612Cdk5Q00535PKC", "betaP05771Cdk7P50613PKC", "deltaQ05655DAP", "KinaseP53355PP2A", "Catalytic", "alphaP05323DAP3P51398PP5/PPTP53042ERK1Q63538PTP1BP18031ERK2P27703PTP1C/SHP1P29350FAKQ00944PTP1D/SHP2Q06124GSK-3", "betaP18266RbP13405I", "kappa", "B", "alphaP25963RskQ15418IKK", "betaO14920Stat1A46159JAK1P23458Stat3P52631JNK1P45983VHRP51452MEK1Q02750", "Detection", "of", "protein", "isoforms", "or", "post-translational", "variantsUnlike", "many", "cDNA-based", "gene", "expression", "assays,", "western", "blotting", "has", "the", "capacity", "to", "detect", "multiple", "protein", "isoforms", "due", "to", "translation", "of", "different", "mRNA", "splice", "variants,", "as", "well", "as", "post-translational", "modifications", "such", "as", "enzymatic", "cleavage,", "glycosylation,", "or", "phosphorylation.", "Examination", "of", "the", "blots", "described", "here", "identified", "42", "examples", "of", "multiple", "banding", "for", "a", "single", "target", "antigen", "(Fig.", "3C).", "These", "candidates", "exhibited", "closely", "migrating", "multiple", "bands,", "which", "were", "close", "to", "their", "predicted", "size", "but", "were", "sufficiently", "separated", "from", "other", "proteins.", "For", "example,", "four", "closely", "migrating", "bands", "were", "observed", "for", "FGF2", "(Fig.", "3C,", "top", "panel),", "which", "may", "represent", "known", "glycosylation", "variants", "of", "this", "growth", "factor", "[33].", "Other", "known", "examples", "of", "post-translational", "modifications", "included", "those", "of", "HSP70,", "IKKgamma", "and", "ERK1.", "Verification", "of", "protein", "expression", "by", "immunocytochemistryThe", "PowerBlot", "and", "Kinexus", "assays", "identified", "proteins", "based", "on", "their", "expected", "and", "observed", "molecular", "weight,", "using", "combinations", "of", "antibodies", "that", "had", "been", "predetermined", "to", "detect", "proteins", "of", "sufficiently", "different", "sizes.", "Proteins", "known", "to", "be", "expressed", "by", "hESCs", "and", "also", "identified", "by", "these", "assays,", "included", "Oct4,", "E-CAD,", "Connexin", "43", "and", "Hsp70.", "To", "verify", "expression", "using", "a", "complementary", "approach,", "we", "performed", "immunoflurorescent", "staining", "for", "10", "proteins", "not", "previously", "reported", "to", "be", "expressed", "in", "hESCs", "by", "immunocytochemistry,", "using", "karyotypically", "normal", "BG01", "cultures", "(Fig.", "4A–K).", "These", "included", "ABP-280,", "a", "homodimeric", "actin-binding", "protein", "often", "associated", "with", "membrane", "glycoproteins;", "CtBP1", "and", "CtBP2,", "two", "C", "terminal", "binding", "proteins", "that", "are", "a", "class", "of", "transcription", "corepressors;", "GS-28,", "a", "golgi", "protein;", "HDJ-2,", "a", "member", "of", "the", "DnaJ-related", "Hsp40", "(heat", "shock", "protein", "40)", "subfamily;", "L-Caldesmon,", "a", "cytoplasmic", "actin-binding", "protein;", "Rabaptin,", "a", "GTP-binding", "protein;", "phosphorylated-p130", "Cas,", "a", "docking", "protein", "with", "an", "amino-terminal", "SH3", "domain", "that", "may", "function", "as", "a", "molecular", "switch", "that", "regulates", "CAS", "(Crk-associated", "substrate)", "tyrosine", "phosphorylation;", "Ras-GAP", "and", "phosphorylated", "Ras-GAP", "(p-Y460),", "a", "protein", "that", "down-regulates", "the", "signal", "transducer", "p21ras;", "and", "ShcC,", "a", "protein", "with", "an", "N-terminal", "phosphotyrosine-binding", "domain.", "These", "proteins", "were", "all", "expressed", "by", "hESCs,", "with", "the", "expected", "subcellular", "localization", "(Fig.", "4A–K).", "Oct4", "was", "used", "as", "a", "positive", "control", "(Fig.", "4L).", "These", "results", "suggested", "that", "most", "of", "the", "bands", "in", "the", "PowerBlot", "and", "Kinexus", "assays", "were", "likely", "to", "be", "correctly", "identified.Figure", "4Verification", "of", "protein", "expression", "using", "immunocytochemistry.", "(A-K)", "Ten", "proteins", "that", "were", "detected", "in", "undifferentiated", "hESCs", "by", "western", "blotting", "were", "also", "detected", "by", "immunofluorescence", "of", "BG01", "cells", "grown", "in", "MEF-CM.", "Ras-GAP", "(pY460)", "is", "a", "phosphorylated", "form", "of", "Ras-GAP.", "The", "same", "antibodies", "were", "used", "in", "this", "analysis", "as", "in", "the", "PowerBlot", "assay,", "except", "phospho-p130", "Cas", "(Tyr165).", "(L)", "Oct4", "was", "used", "as", "a", "positive", "control.", "(M-R)", "Oct4,", "TNIK", "and", "p130", "Cas", "as", "markers", "of", "undifferentiated", "hESCs.", "BG01", "cultures", "were", "partially", "differentiated", "by", "exposure", "to", "10%", "fetal", "bovine", "serum", "for", "3", "days.", "(M)", "Oct4", "was", "expressed", "uniformly", "in", "undifferentiated", "cells,", "(P)", "but", "was", "downregulated", "in", "morphologically", "differentiated", "areas", "after", "3", "days", "in", "serum", "(arrowhead).", "(N)", "TNIK", "expression", "was", "localized", "to", "the", "cytoplasm,", "and", "(N,", "Q)", "expression", "appeared", "to", "be", "restricted", "to", "morphologically", "undifferentiated", "cells", "(arrowhead).", "(O)", "p130", "Cas", "was", "detected", "in", "a", "membrane/peripheral-cytoplasmic", "pattern", "in", "undifferentiated", "cells,", "(R)", "but", "this", "distribution", "was", "substantially", "altered", "in", "differentiating", "cells", "with", "a", "flattened", "morphology,", "which", "exhibited", "a", "general", "cytoplasmic,", "or", "perinuclear", "profile.", "Scale", "bar", "for", "A-L:", "(A,", "L)", "200", "μm;", "(C,", "D,", "F,", "H,", "I,", "J,", "K)", "100", "μm;", "(B,", "E,", "G)", "50", "μm.", "Scale", "bar", "for", "M-R:", "(M,", "N,", "P,", "Q):", "100", "μm", ";", "(O,", "R):", "50", "μm.Preliminary", "analyses", "also", "indicated", "that", "expression", "of", "some", "of", "these", "proteins", "was", "downregulated", "in", "differentiated", "cells,", "including", "p130", "Cas", "and", "the", "Traf2-", "and", "Nck-interacting", "kinase", "(TNIK).", "TNIK", "is", "known", "to", "be", "involved", "in", "the", "inhibition", "of", "cell", "spreading", "via", "disruption", "of", "F-actin", "[34,35].", "Immunofluorescence", "was", "used", "to", "examine", "the", "expression", "of", "TNIK", "and", "p130", "Cas", "during", "early", "differentiation", "of", "hESCs.", "BG01", "cultures", "were", "partially", "differentiated", "by", "growth", "in", "serum", "containing", "media", "for", "3", "days.", "This", "condition", "generated", "heterogeneous", "populations", "containing", "Oct4+", "cells", "with", "characteristic", "hESC", "morphology", "and", "less", "tightly", "packed,", "and", "morphologically", "differentiated", "areas,", "lacking", "expression", "of", "Oct4", "(Fig", "4M,", "P).", "TNIK", "was", "expressed", "highly", "in", "undifferentiated", "hESCs,", "and", "in", "the", "undifferentiated", "areas", "at", "day", "3,", "but", "was", "downregulated", "in", "areas", "undergoing", "morphological", "differentiation", "(Fig", "4N,", "Q).", "This", "may", "indicate", "that", "TNIK", "is", "active", "in", "hESCs", "and", "degraded", "rapidly", "upon", "differentiation.", "p130", "Cas", "was", "detected", "in", "a", "membrane/peripheral-cytoplasmic", "pattern", "in", "hESCs", "(Fig", "4O).", "The", "distribution", "of", "p130Cas", "was", "substantially", "altered", "in", "differentiating", "cells", "with", "a", "flattened", "morphology,", "exhibiting", "a", "general", "cytoplasmic,", "or", "perinuclear", "profile", "(Fig", "4R).", "This", "could", "indicate", "an", "alteration", "in", "the", "function", "of", "p130", "Cas", "as", "pluripotent", "cells", "differentiate.", "These", "analyses", "suggested", "that", "the", "change", "in", "expression", "or", "distribution", "of", "these", "proteins", "could", "be", "used", "as", "markers", "for", "undifferentiated", "hESCs.", "Comparison", "of", "proteomic", "and", "transcriptional", "profiles", "of", "hESCsWe", "have", "previously", "employed", "the", "Illumina", "Bead", "Array", "system", "for", "the", "large-scale", "profiling", "of", "gene", "expression", "in", "hESCs", "using", "24,000", "transcript", "probes", "[11].", "To", "compare", "proteomic", "and", "transcriptional", "analyses", "of", "hESCs,", "the", "levels", "of", ">", "600", "proteins", "detected", "using", "large", "scale", "blotting", "were", "correlated", "with", "the", "levels", "of", "transcripts", "detected", "with", "the", "Illumina", "platform", "(Additional", "File", "3).", "In", "general,", "a", "close", "match", "between", "the", "expression", "level", "of", "transcript", "and", "protein", "was", "observed:", "transcripts", "for", "nearly", "all", "the", "detected", "proteins", "were", "also", "identified", "in", "the", "Illumina", "analysis,", "and", "most", "proteins", "expressed", "at", "high", "levels", "also", "exhibited", "high", "mRNA", "levels.We", "reasoned", "that", "a", "focused", "comparison", "of", "specific", "signaling", "pathways", "using", "a", "combination", "of", "proteomic", "and", "transcriptional", "data", "was", "likely", "to", "be", "much", "more", "informative", "than", "a", "global", "interrogation", "of", "hESCs.", "Several", "major", "signal", "pathways", "that", "have", "been", "suggested", "to", "be", "involved", "in", "self-renewal", "were", "examined", "to", "test", "this", "approach.", "These", "included", "the", "FGF,", "TGFβ,", "GSK3β/Wnt/β-catenin", "and", "Jak/Stat", "pathways", "[17,29,36-39],", "as", "well", "as", "the", "more", "recently", "suggested", "MAPK/ERK", "and", "Gap", "junction", "pathways", "[32,40].", "Correlating", "transcriptional", "and", "proteomic", "data", "provided", "direct", "confirmation", "that", "these", "pathways", "were", "present", "and", "likely", "functional", "in", "hESCs", "(Table", "2).", "For", "example,", "FGF2", "protein", "was", "expressed", "highly", "in", "hESCs", "and", "expression", "of", "key", "members", "of", "the", "TGFβ,", "Wnt,", "Jak/Stat", "and", "Gap", "junction", "pathways,", "namely", "Stat1,", "SMADs,", "GSK3β,", "β-catenin", "and", "Connexin", "43,", "were", "detected", "in", "both", "transcriptional", "and", "proteomic", "databases.Table", "2Signal", "pathways", "that", "may", "be", "active", "in", "hESCsNameProteinmRNATGF", "βStat1++++++PAI-1/SERPINE1+++-Smad2/3++++Jun+++Smad4/DPC4+++Endoglin+-WntCtBP2+++++++PP2A", "Catalytic", "alpha/PPP2CA+++++++EBP50/SLC9A3R1++++++beta-Catenin/Ctnnb1++++Cyclin", "D3/CCND3++++GSK-3", "beta++++Jun+++Casein", "Kinase", "II", "alpha/CSNK2A1++++Jak-StatStat1++++++Crk++++Stat3/2+++++Stat6+++++PTP1B+++++JAK1++-Glucocorticoid", "R/NR3C1++-Thrombin", "Receptor/PAR1/F2R+++SHPS-1/PTPNS1++++MCM5+++++Smad2/3++++Tyk2++++Jun+++Bcl-x/BCL2L1++++Smad4/DPC4+++Stat5A++GPCRB2", "Bradykinin", "Receptor/BDKRB2++++-Neurotensin", "Receptor", "3/SORT1+++-Endopeptidase", "3.4.24.16/NLN++++IP3R-3++++SHC++++Gap", "JunctionCdk1/Cdc2++++++GRB2++++++MEK1/MAP2K1++++++PKA", "C++-PKA", "RI", "alpha++-PKC", "alpha++-C-Raf/RAF1++++ZO-1/TJP1+++++Connexin-43/GJA1++++IGFPKC", "iota++++++MEK1/MAP2K1++++++Rsk/RPS6KA1+++++GRB2++++++MEK2/MAP2K2++++++PI3Kinase/PIK3R1+++++pan", "ERK/MAPK1+++++Crk++++eIF-4E+++++ShcC+++-PAI-1/SERPINE1+++-C-Raf++++SHC+++++PKC", "beta/PRKCB1++++NCK++++PKB", "alpha/Akt+++GSK-3", "beta++++Ercc-1++++Fatty", "Acid", "Synthase/FASN+++++Jun+++RAFT1/FRAP++++PTP1D/SHP2/PTPN11++++SCAMP1++++Bcl-x/BCL2L1++++p70s6k/RPS6KB1+-PI3-Kinase", "p170/PIK3C2A++PTP1B/PTPN1+++Dok1/p62dok+++PI3-Kinase", "p110", "alpha/PIK3CA+-ERBBEphA4/Sek++++-ShcC/SHC3+++-c-erb-B2/ERBB2++++C-Raf/RAF1++++SHC/SHC1+++++GDNFI", "kappa", "B", "epsilon/NFKBIE++++++GRB2++++++MEK2++++++NCK+++++C-Raf++++Ras-GAP/RASA1++++SHC+++++GDNFR-alpha/Gfra1++-Jun+++IKK", "beta++++pan-JNK/SAPK1/MAPK10+++NBS1/ARTN++Dok1/p62dok+++Tight", "JunctionPTEN++++++PP2A", "Catalytic", "alpha+++++++PKC", "iota++++++Sec8/SEC8L1+++++beta-Catenin/CTNNB1++++CDC42+++++AF6/MLLT4+++++PKC", "alpha++-Yes++++Rho/ARHA+++++ZO-1/TJP1+++++CASK+++Symplekin/SYMPK++-Ras/NRAS++++Casein", "Kinase", "II", "alpha/CSNK2A1++++VAP33/VAPA+++alpha-Catenin/Ctnna1+++MAPKpan", "ERK++++++MEK1++++++Rsk++++++ERK2++++++MEK2/Map2k2++++++MST3/STK25++++++ERK1+++++CDC42+++++C-Raf++++p38", "alpha/SAPK2a++-G3BP+++++TFII-I/GTF2IRD1++++MST1/STK4++++MKP2/Dusp4++++Ras++++Phospho-p38MAPK", "(T180/Y182)+++pan-JNK/SAPK1+++Inhibitor2/PPP1R2++++ABP-280++++++14-3-3", "epsilon/YWHAE++++MAPKAPK-5+++TAO1+*PBK+++MKK3b/Map2k3++Protein", "expression", "level:", ">", "10,000:", "++++;", "5,000–10,000:", "+++;", "1,000–5,000:++;", "100–1,000:", "+", "mRNA", "gene", "expression", "level:", ">", "5,000:++++;", "1,000–5,000:", "+++;", "100–1,000:", "++;", "30–100:", "+*:", "not", "included", "in", "the", "gene", "expression", "arrayThis", "independent", "confirmation", "of", "known", "networks", "led", "us", "to", "examine", "other", "pathways", "that", "showed", "a", "similar", "correlation", "but", "have", "not", "been", "identified", "as", "key", "regulators", "of", "either", "self-renewal", "or", "differentiation,", "or", "suggest", "unappreciated", "characteristics", "of", "hESCs.", "Four", "signaling", "pathways", "(IGF,", "ERBB2,", "GPCR,", "and", "GDNF)", "and", "the", "tight", "junction", "complex", "were", "highlighted", "by", "this", "analysis", "(Table", "2),", "and", "expression", "of", "key", "proteins", "in", "these", "pathways", "was", "confirmed.", "A", "detailed", "study", "demonstrating", "the", "importance", "of", "the", "IGF", "and", "ERBB2", "pathways", "in", "hESC", "self-renewal", "has", "been", "performed", "and", "enabled", "the", "development", "of", "a", "defined", "medium", "for", "hESC", "maintenance", "(TCS", "and", "AJR,", "submitted).", "Tight", "junctions", "are", "apical", "cell-cell", "junctions", "found", "in", "epithelia", "that", "establish", "a", "barrier", "to", "the", "extracellular", "environment", "and", "a", "border", "for", "apical-basolateral", "polarity.", "While", "hESCs", "grow", "in", "colonies", "that", "are", "highly", "reminiscent", "of", "epithelia,", "and", "have", "been", "shown", "to", "be", "coupled", "by", "gap", "junctions", "[40],", "the", "formation", "of", "tight", "junction", "complexes", "has", "not", "been", "described.", "hESCs", "expressed", "the", "ZO1", "and", "occludin", "tight", "junction", "proteins", "along", "cell", "borders", "as", "expected", "in", "polarized", "epithelia.", "The", "distribution", "of", "ZO1", "expression", "changed", "dramatically", "as", "hESCs", "proliferated", "in", "culture.", "When", "tight", "junction", "complexes", "were", "disrupted", "by", "disaggreagation", "to", "single", "cells,", "only", "a", "subset", "of", "cells", "showed", "ZO1", "staining", "4", "days", "after", "plating", "(Fig.", "5).", "Continued", "proliferation", "to", "a", "confluent", "monolayer", "on", "day", "7", "was", "accompanied", "by", "widespread", "expression", "of", "ZO1,", "suggesting", "the", "formation", "of", "a", "general", "tight", "junction", "barrier.", "These", "cultures", "were", "undifferentiated", "and", "retained", "uniform", "expression", "of", "Oct4", "protein", "(not", "shown).", "ERBB2", "and", "3", "are", "members", "of", "the", "epidermal", "growth", "factor", "(EGF)-receptor", "family,", "which", "regulate", "epithelial", "proliferation", "via", "EGF-family", "ligands.", "ERBB2", "and", "3", "transcripts", "are", "expressed", "by", "hESCs", "[8],", "are", "known", "to", "function", "as", "a", "heterodimer", "[41],", "and", "transmit", "a", "strong", "proliferative", "signal", "for", "hESCs", "by", "Heregulin", "1β", "(an", "EGF-family", "ligand)", "(TCS", "and", "AJR,", "submitted).", "Immunofluorescence", "revealed", "general", "cell", "surface", "expression", "of", "ERBB2", "on", "hESCs.", "Conversely,", "ERBB3", "was", "highly", "localized", "to", "a", "concentrated", "area,", "and", "observed", "in", "cells", "that", "also", "expressed", "ZO1.", "Epithelial", "cells", "are", "known", "to", "localize", "ERBB", "receptors", "to", "the", "basolateral", "side", "of", "tight", "junctions,", "which", "serves", "to", "functionally", "separate", "receptors", "from", "ligands", "[42,43].", "This", "is", "a", "basic", "epithelial", "wound", "healing", "mechanism,", "whereby", "disruption", "of", "the", "tight", "junction", "barrier", "by", "injury", "immediately", "exposes", "receptors", "to", "extracelluar", "ligands", "[43].", "These", "staining", "patterns", "are", "also", "suggestive", "of", "basolateral", "sorting", "of", "ERBB3", "in", "hESCs.", "The", "pathways", "and", "complexes", "identified", "by", "these", "analyses", "lay", "a", "framework", "for", "future", "functional", "analyses", "of", "signaling", "networks", "in", "hESCs.Figure", "5Tight", "junction", "proteins", "and", "ERBB2/3", "expression", "in", "hESCs.", "BG01", "hESCs", "were", "disaggregated", "to", "single", "cells", "using", "accutase", "[52]", "and", "cultured", "in", "defined", "conditions.", "(A)", "ZO1", "expression", "four", "and", "(B)", "seven", "days", "after", "plating,", "indicating", "progressive", "tight", "junction", "formation.", "(C)", "Occludin", "expression", "5", "days", "after", "plating.", "(D)", "General", "cell", "surface", "expression", "of", "ERBB2,", "in", "the", "same", "field", "of", "view", "as", "(A).", "(E)", "Localized", "expression", "of", "ERBB3,", "in", "the", "same", "field", "of", "view", "as", "(B).", "(F)", "Higher", "magnification", "of", "ERBB3", "localization", "in", "ZO1", "expressing", "BG01", "cells,", "5", "days", "after", "plating.", "Nuclei", "were", "stained", "with", "DAPI.", "Attempts", "to", "harness", "the", "potential", "of", "hESCs", "for", "models", "of", "human", "embryogenesis", "and", "cell", "therapy", "applications", "will", "be", "greatly", "enhanced", "by", "a", "detailed", "understanding", "of", "their", "molecular", "characteristics.", "This", "includes", "definition", "of", "the", "transcripts,", "splice", "variants,", "and", "protein", "isoforms", "expressed", "by", "these", "cells.", "Post-translational", "modifications", "such", "as", "phosphorylation", "and", "glycosylation,", "and", "the", "receptors", "and", "signaling", "pathways", "active", "in", "the", "pluripotent", "state,", "or", "during", "early", "differentiation,", "also", "need", "to", "be", "determined.", "This", "should", "also", "be", "complemented", "by", "an", "understanding", "of", "epigenetic", "characteristics", "of", "pluripotency,", "including", "methylation,", "imprinting", "and", "chromatin", "conformation.", "Such", "a", "comprehensive", "definition", "of", "the", "molecular", "state", "of", "hESCs", "will", "enable", "more", "accurate", "prediction", "and", "testing", "of", "the", "conditions", "used", "for", "growth", "and", "differentiation", "of", "hESCs,", "by", "precise", "genetic", "modification", "or", "application", "of", "specific", "growth", "factor", "cocktails", "and", "reagents.", "For", "example,", "a", "scalable,", "fully", "defined", "and", "GMP-certified", "culture", "system", "will", "need", "to", "be", "developed", "for", "the", "eventual", "development", "of", "hESC-based", "cellular", "therapies.", "Progress", "has", "been", "made", "in", "defining", "growth", "factor", "conditions", "that", "support", "self-renewal", "[44-46],", "and", "hESC", "lines", "have", "been", "isolated", "in", "the", "absence", "of", "mouse", "embryonic", "fibroblasts", "and", "in", "animal", "protein", "free", "culture", "conditions", "[47,48].", "A", "more", "refined", "understanding", "of", "the", "biology", "of", "hESCs", "has", "contributed", "the", "development", "of", "a", "defined", "medium", "utilizing", "ligands", "for", "IGF1R", "and", "ERBB2/3", "receptors", "to", "promote", "in", "self-renewal", "(TCS", "and", "AJR,", "submitted).", "We", "and", "others", "have", "performed", "transcriptional", "analyses", "of", "hESCs,", "using", "cDNA", "and", "oligonucleotide", "microarrays,", "SAGE,", "MPSS", "and", "EST", "enumeration.", "These", "techniques", "have", "enabled", "the", "collation", "and", "comparison", "of", "transcriptional", "profiles", "from", "multiple", "hESC", "lines", "and", "their", "differentiated", "derivatives", "and", "have", "highlighted", "an", "expanded", "set", "of", "hESC", "specific", "markers", "and", "signaling", "pathways", "that", "may", "regulate", "self-renewal", "or", "differentiation.", "Using", "pathway", "analysis", "we", "were", "also", "able", "to", "identify", "key", "pathways", "that", "are", "active", "in", "ESCs", "(reviewed", "in", "[16]).", "While", "these", "efforts", "have", "been", "highly", "valuable", "in", "defining", "the", "transcriptional", "profile", "of", "undifferentiated", "hESCs,", "they", "are", "only", "predictive", "of", "translation", "and", "do", "not", "shed", "light", "on", "post-translational", "events", "in", "this", "unique", "cell", "type.", "These", "processes", "may", "also", "be", "highly", "regulated,", "which", "could", "contribute", "significantly", "to", "the", "overall", "conversion", "of", "genetic", "information", "to", "actual", "protein", "function.", "We", "report", "here", "a", "proteomic", "analysis", "of", "pluripotent", "hESCs", "by", "using", "two", "large-scale", "western", "blotting", "systems", "and", "highlight", "post-translational", "events", "in", "undifferentiated", "hESCs.", "The", "expression", "of", "545", "bands", "was", "detected,", "potentially", "representing", "529", "proteins,", "or", "their", "migratory", "isoforms.", "In", "addition,", "one", "hundred", "and", "forty", "phospho-specific", "antibodies", "were", "used", "to", "identify", "85", "different", "phosphorylated", "sites,", "on", "76", "proteins", "in", "these", "cells.", "The", "detected", "proteins", "were", "annotated", "into", "functional", "classes", "representing", "diverse", "cellular", "processes.", "For", "example,", "multiple", "proteins", "were", "detected", "that", "have", "been", "suggested", "to", "regulate", "the", "pluirpotent", "state", "in", "mouse", "ESCs", "or", "hESCs.", "Defining", "the", "interplay", "of", "these", "multiple", "signaling", "pathways", "will", "be", "critical", "in", "understanding", "the", "self-renewal", "versus", "differentiation", "decisions", "of", "hESCs.", "Therefore,", "our", "data", "provide", "a", "powerful", "framework", "for", "the", "functional", "analysis", "of", "specific", "proteins,", "protein", "classes,", "or", "molecular", "pathways.", "In", "particular,", "the", "availability", "of", "antibodies", "for", "candidate", "proteins", "is", "a", "major", "benefit", "of", "this", "approach", "compared", "to", "2D-gel", "or", "HPLC-MS/MS", "based", "proteomics.", "Although", "these", "western", "blotting", "approaches", "are", "currently", "more", "limited", "in", "scope", "than", "most", "large-scale", "cDNA", "based", "assays,", "detecting", "up", "to", "1000", "proteins", "compared", "to", "tens", "of", "thousands", "of", "transcripts,", "they", "have", "the", "potential", "to", "highlight", "translational", "events", "and", "post-translational", "modifications.", "By", "comparison,", "SAGE", "and", "MPSS", "are", "limited", "to", "detecting", "short", "sequence", "\"tags\"", "adjacent", "to", "the", "poly-A", "tail", "of", "transcripts,", "and", "may", "not", "distinguish", "splice", "variants", "with", "the", "same", "3'", "exon.", "We", "detected", "42", "proteins", "with", "multiple", "closely", "migrating", "bands", "(Fig.", "3C),", "suggestive", "of", "closely", "related", "isoforms", "or", "post-translational", "modifications", "such", "as", "phosphorylation.", "These", "focused", "proteomic", "approaches", "are", "therefore", "likely", "to", "be", "highly", "complimentary", "to", "transcriptional", "analyses", "in", "investigating", "the", "functional", "expression", "of", "the", "genome", "in", "hESCs", "and", "during", "cellular", "differentiation.", "One", "potential", "issue", "with", "this", "approach", "is", "that", "multiple", "antibodies", "are", "included", "in", "each", "lane,", "which", "could", "possibly", "lead", "to", "misidentification", "of", "bands.", "To", "demonstrate", "that", "identified", "proteins", "were", "expressed", "in", "hESCs,", "the", "same", "antibodies", "used", "in", "the", "PowerBlot", "assay", "were", "used", "to", "confirm", "expression", "of", "10", "representative", "proteins", "by", "immunofluorescence", "(Fig.", "4).", "Furthermore,", "13", "proteins", "were", "detected", "with", "multiple", "different", "antibodies,", "and", "35", "proteins", "(Table", "1)", "were", "detected", "in", "both", "the", "PowerBlot", "and", "Kinexus", "assays.", "This", "provided", "internal,", "or", "independent,", "confirmation", "of", "expression", "of", "these", "proteins.", "Other", "studies", "have", "also", "demonstrated", "the", "expression", "of", "several", "of", "the", "proteins", "we", "detected", "in", "hESCs.", "These", "include", "Oct4,", "a", "key", "marker", "of", "the", "pluripotent", "state,", "Connexin", "43", "and", "GSK3β,", "confirming", "the", "reliability", "of", "large-scale", "western", "blotting.", "Finally,", "several", "proteins", "detected", "by", "our", "assays", "were", "also", "detected", "in", "hESCs", "by", "MS", "approaches", "including", "Karyopherin", "α", "[19].", "Additionally,", "the", "PowerBlot", "assay", "was", "performed", "in", "duplicate,", "and", "was", "shown", "to", "be", "highly", "reproducible.", "This", "suggested", "that", "this", "approach", "should", "be", "informative", "when", "comparing", "hESCs", "to", "their", "differentiated", "derivatives.", "Two", "candidate", "proteins,", "TNIK", "and", "p130", "Cas,", "were", "downregulated,", "or", "exhibited", "altered", "localization", "upon", "spontaneous", "differentiation", "of", "hESCs,", "respectively.", "This", "indicated", "that", "they", "were", "novel", "markers", "of", "undifferentiated", "cells", "and", "molecules", "that", "could", "be", "functionally", "involved", "with", "self-renewal.", "It", "is", "impossible", "in", "an", "initial", "manuscript", "to", "analyze", "and", "rigorously", "test", "all", "the", "predictions", "that", "could", "be", "made", "from", "comparing", "transcriptional", "and", "proteomic", "data", "sets.", "However,", "we", "did", "examine", "key", "features", "to", "illustrate", "the", "power", "of", "this", "methodology.", "Potential", "new", "markers", "for", "hESCs", "were", "identified,", "the", "expression", "and", "activation", "of", "proteins", "in", "key", "self-renewal", "pathways", "were", "confirmed,", "and", "a", "diverse", "range", "of", "proteins", "were", "detected", "and", "expression", "correlated", "with", "transcriptional", "analyses.", "In", "addition,", "we", "highlighted", "several", "candidate", "signaling", "pathways", "that", "may", "be", "relevant", "to", "self-renewal.", "Examination", "of", "tight", "junction", "protein", "expression", "indicated", "that", "undifferentiated", "hESCs", "could", "form", "polarized", "epithelia,", "which", "has", "also", "been", "recently", "suggested", "by", "ultrastructural", "analyses", "[49].", "Discrete", "localization", "of", "ERBB3", "may", "also", "suggest", "basolateral", "separation", "of", "this", "receptor", "from", "soluble", "ligand.", "These", "analyses", "highlight", "that", "predictions", "from", "a", "combination", "of", "transcriptional", "and", "proteomic", "approaches", "will", "serve", "to", "focus", "the", "investigation", "of", "hESCs", "in", "the", "future.", "In", "summary,", "we", "generated", "a", "focused", "proteome", "of", "hESCs", "using", "large-scale", "western", "blotting", "and", "sorted", "the", "detected", "proteins", "according", "to", "function", "and", "signaling", "pathways.", "This", "characterization", "provides", "important", "basic", "information", "on", "expressed", "proteins,", "their", "isoforms", "and", "post-translational", "modifications,", "and", "tools", "for", "the", "continued", "investigation", "of", "the", "underlying", "molecular", "characteristics", "of", "hESCs.", "Importantly,", "we", "provide", "a", "list", "of", "tools,", "in", "the", "form", "of", "commercially", "available", "antibodies,", "which", "can", "be", "used", "to", "interrogate", "the", "function", "of", "these", "molecules", "in", "self-renewal", "or", "differentiation.", "Culture", "of", "human", "embryonic", "stem", "cellsFor", "the", "PowerBlot", "analysis,", "enzymatically", "passaged", "BG01", "hESCs", "were", "grown", "as", "described", "previously", "[23].", "These", "conditions", "were", "necessary", "to", "scale", "up", "the", "culture", "to", "generate", "the", "milligram", "amounts", "of", "protein", "lysate", "required", "for", "this", "analysis.", "These", "conditions", "maintain", "cell", "populations", "that", "express", "the", "appropriate", "markers", "of", "pluripotency", "and", "can", "differentiate", "to", "representatives", "of", "all", "three", "germ", "layers,", "but", "may", "lead", "to", "eventual", "accumulation", "of", "trisomies", "for", "chromosomes", "12,", "17", "or", "X", "[26].", "For", "the", "Kinexus", "assays,", "BG03", "hESCs", "were", "maintained", "in", "MEF-conditioned", "medium", "(MEF-CM)", "without", "the", "accumulation", "of", "karyotypic", "abnormalities", "as", "described", "previously", "[14,26].hESCs", "were", "also", "maintained", "in", "a", "defined", "medium", "as", "indicated.", "These", "conditions", "are", "described", "in", "detail", "elsewhere", "(TCS,", "AJR,", "submitted).", "Briefly,", "the", "media", "consisted", "of", "DMEM/F12", "(Invitrogen),", "2%", "fatty", "acid-free", "Cohn's", "fraction", "V", "BSA", "(Serologicals),", "1×", "nonessential", "amino", "acids,", "50", "U/ml", "penicillin/streptomycin,", "50", "μg/ml", "ascorbic", "acid,", "10", "μg/ml", "bovine", "transferrin,", "0.1", "mM", "β-mecaptoethanol", "(all", "from", "Invitrogen),", "1×", "Trace", "Elements", "A,", "B", "&", "C", "(Mediatech),", "10", "ng/ml", "hergulin1β", "(Peprotech),", "10", "ng/ml", "activinA", "(R&D", "Systems),", "200", "ng/ml", "LR3-IGF1", "(JRH", "Biosciences),", "and", "8", "ng/ml", "FGF2", "(R&D", "Systems).", "Cultures", "were", "passaged", "using", "Collagenase", "IV", "and", "plated", "on", "growth", "factor", "depleted", "Matrigel", "(BD", "Biosciences)", "diluted", "1:200.", "These", "cultures", "were", "karyotypically", "normal.To", "partially", "differentiate", "hESC", "cultures", "for", "immunostaining", "analysis,", "karyotypically", "normal", "BG01", "cells", "were", "plated", "on", "matrigel", "and", "grown", "for", "three", "days", "in", "DMEM/F12", "containing", "10%", "fetal", "calf", "serum", "(HyClone),", "1×", "nonessential", "amino", "acids,", "20", "mM", "L-glutamine,", "50", "U/ml", "penicillin/streptomycin,", "and", "0.1", "mM", "β-mecaptoethanol.", "PowerBlot", "assaysBG01", "hESC", "lysate", "was", "prepared", "in", "10", "mM", "Tris-HCl", "pH", "7.4,", "1", "mM", "sodium", "orthovanadate", "and", "1%", "SDS,", "and", "the", "PowerBlot", "assays", "were", "performed", "by", "BD", "Biosciences", "(BD", "Biosciences).", "Briefly,", "200", "μg", "of", "protein", "lysate", "was", "loaded", "in", "a", "single,", "gel-wide", "well,", "on", "a", "SDS-4–15%", "gradient", "polyacrylamide", "gel.", "The", "full", "PowerBlot", "screen", "consisted", "of", "five", "gels,", "which", "were", "blotted", "and", "probed", "with", "934", "antibodies,", "and", "was", "performed", "in", "duplicate", "with", "the", "same", "cell", "lysate.", "The", "gel", "dimensions", "were", "130", "×", "100", "×", "0.5", "mm,", "and", "proteins", "were", "separated", "at", "150", "volts", "for", "1.5", "hours,", "and", "transferred", "to", "an", "Immobilon-P", "membrane", "(Millipore).", "The", "membranes", "were", "blocked", "and", "clamped", "in", "a", "manifold", "that", "created", "40", "lanes", "across", "each", "membrane.", "A", "mix", "of", "1", "to", "8", "mouse", "monoclonal", "primary", "antibodies", "was", "added", "to", "each", "lane,", "in", "dilutions", "and", "combinations", "that", "had", "been", "predetermined", "to", "enable", "accurate", "identification", "of", "well-separated", "proteins.", "The", "predicted", "sizes", "of", "detectable", "proteins", "in", "the", "blots", "ranged", "from", "10–540", "kD,", "and", "the", "dilutions", "of", "the", "primary", "antibodies", "ranged", "from", "1:250", "to", "1:15,000.The", "blots", "were", "removed", "from", "the", "manifolds,", "washed", "and", "incubated", "with", "goat", "anti-mouse", "secondary", "antibody", "conjugated", "to", "the", "Alexa680", "fluorophore", "(Molecular", "Probes).", "The", "membranes", "were", "scanned", "using", "the", "Odyssey", "Imaging", "System", "(LI-COR).", "Molecular", "weight", "standards", "were", "generated", "by", "adding", "a", "cocktail", "of", "antibodies", "to", "P190", "(190", "kD),", "Adaptin", "beta", "(106", "kD),", "STAT-3", "(92", "kD),", "PTP1D", "(72", "kD),", "Mek-2", "(46", "kD),", "RACK-1", "(36", "kD),", "GRB-2", "(24", "kD)", "and", "Rap2", "(21", "kD)", "to", "lane", "40", "of", "gels", "A-D.", "Molecular", "standards", "for", "gel", "E", "were", "generated", "by", "adding", "a", "cocktail", "of", "antibodies", "to", "Exportin-1/CRM1", "(112", "kD),", "MCM", "(83", "kD),", "Nucleoporin", "p62", "(62", "kD),", "α-tubulin", "(55", "kD),", "Actin", "(42", "kD),", "KNP-1/HES1", "(28", "kD)", "and", "NTF2", "(15", "kD)", "to", "lane", "16,", "and", "antibodies", "to", "p190", "(190", "kD),", "Hip1R", "(120", "kD),", "Transportin", "(101", "kD),", "Calreticulin", "(60", "kD),", "Arp3", "(50", "kD),", "eIF-6", "(27", "kD)", "and", "Rap2", "(21", "kD)", "to", "lane", "17.Bands", "were", "detected", "and", "raw", "signal", "intensity", "captured", "automatically", "using", "the", "PDQuest", "software", "(Bio-Rad).", "To", "normalize", "the", "signal", "intensities,", "the", "total", "raw", "quantity", "of", "each", "band", "was", "divided", "by", "the", "average", "intensity", "value", "of", "the", "molecular", "standards", "in", "that", "image", "and", "the", "normalized", "values", "for", "the", "duplicate", "samples", "were", "averaged", "and", "expressed", "as", "normalized", "intensity", "units", "(i.u.).", "These", "values", "represent", "the", "relative", "signal", "intensity", "observed", "for", "each", "identified", "protein", "band,", "rather", "than", "relative", "expression", "levels", "of", "different", "proteins,", "due", "to", "differences", "in", "the", "efficiencies", "of", "antibody", "binding", "and", "dilution", "of", "the", "primary", "antibodies", "used.", "Proteins", "were", "identified", "based", "on", "the", "similarity", "of", "expected", "and", "observed", "band", "migration", "profiles", "and", "bands", "that", "could", "not", "be", "identified", "were", "excluded", "from", "the", "analysis.", "All", "identified", "proteins", "were", "verified", "by", "visual", "inspection,", "and", "proteins", "exhibiting", "a", "low", "signal", "intensity,", "with", "an", "averaged", "signal", "of", "<", "1000", "i.u.,", "were", "verified", "by", "visual", "inspection", "using", "contrast", "enhancement", "in", "Adobe", "Photoshop.", "Bands", "with", ">", "800", "i.u.", "could", "typically", "be", "observed", "without", "additional", "image", "enhancement.", "Microsoft", "Excel", "files", "were", "generated", "that", "contained", "information", "on:", "gel", "number,", "lane", "number,", "antibody", "catalogue", "number", "(BD", "Biosciences),", "protein", "name,", "expected", "size,", "observed", "size,", "repeat", "1", "i.u.", "value,", "repeat", "2", "i.u.", "value,", "averaged", "i.u.", "value,", "antibody", "dilution,", "outline", "of", "protein", "function,", "Entrez", "gene", "and", "SwissProt", "identification", "numbers.", "These", "tables", "were", "used", "to", "list", "expressed", "proteins", "(Additional", "File", "1).", "Kinexus", "assaysPreparation", "of", "the", "BG03", "cell", "lysate", "and", "western", "blotting", "was", "performed", "according", "to", "published", "protocols", "[50].", "Briefly,", "cell", "lysate", "was", "prepared", "in", "20", "mM", "MOPS", "pH", "7.0,", "2", "mM", "EGTA,", "5", "mM", "EDTA,", "30", "mM", "sodium", "fluoride,", "40", "mM", "β-glycerolphosphate", "pH", "7.2,", "20", "mM", "sodium", "pyrophosphate,", "1", "mM", "sodium", "orthovanadate,", "1", "mM", "PMSF,", "3", "mM", "benzamidine,", "5", "μM", "pepstatin,", "10", "μM", "leupeptin,", "0.5%", "nonidet", "P-40,", "with", "the", "final", "pH", "adjusted", "to", "7.2.", "The", "Kinexus", "assays", "for", "protein", "kinases", "(KPKS-1.2A", "and", "B", "[76", "antibodies]),", "phosphatases", "(KPPS-1.2", "[27", "antibodies])", "and", "phosporylated", "sites", "in", "cell", "signaling", "molecules", "(KPSS-3.1", "[37", "antibodies])", "were", "performed", "by", "Kinexus.", "The", "Bio-Rad", "Mini-PROTEAN", "3", "electrophoresis", "system", "was", "used", "to", "separate", "proteins", "by", "SDS-PAGE.", "For", "each", "assay,", "250", "μg", "of", "cell", "lysate", "was", "loaded", "in", "a", "single", "well", "spanning", "the", "width", "of", "the", "stacking", "gel,", "then", "separated", "through", "a", "12.5%", "SDS-Polyacrylamide", "gel", "and", "transferred", "to", "a", "PVDF", "membrane.", "A", "20-lane", "manifold", "was", "placed", "over", "the", "membrane", "and", "a", "different", "mixture", "of", "up", "to", "3", "primary", "antibodies", "was", "added", "to", "each", "well.", "The", "combinations", "of", "primary", "antibodies", "had", "been", "predetermined", "to", "detect", "well-separated", "proteins,", "avoiding", "crossreaction", "to", "different", "proteins", "that", "co-migrate.", "The", "primary", "antibodies", "were", "rabbit", "and", "goat", "polyclonal,", "and", "mouse", "monoclonal", "antibodies,", "diluted", "1:1000.", "After", "incubation", "with", "the", "primary", "antibodies,", "the", "membranes", "were", "removed", "from", "the", "manifolds,", "washed", "and", "incubated", "with", "a", "mix", "of", "the", "appropriate", "secondary", "antibodies.", "The", "secondary", "antibodies", "were", "donkey", "anti-rabbit", "(at", "1:5000),", "sheep", "anti-mouse", "(at", "1:10,000)", "and", "bovine", "anti-goat", "(at", "1:10,000),", "all", "conjugated", "with", "horse", "radish", "peroxidase.", "The", "membranes", "were", "washed", "and", "immunoreactive", "bands", "detected", "by", "enhanced", "chemiluminescence", "(Amersham-Pharmacia)", "using", "a", "FluorS", "Max", "Multi-imager", "(Bio-Rad).", "Prestained", "size", "markers", "(201.5,", "156.8,", "106,", "79.7,", "48.4,", "37.8,", "23.3,", "and", "18.2", "kD)", "and", "predetermined", "human-specific", "protein", "migration", "profiles", "were", "used", "to", "accurately", "identify", "proteins", "using", "the", "Kinexus", "immuno-reactivity", "identification", "system", "(IRIS)", "software.", "Detected", "proteins", "were", "verified", "by", "visual", "inspection.", "ImmunocytochemistryImmunocytochemistry", "and", "staining", "procedures", "were", "as", "described", "previously", "[51].", "Briefly,", "cells", "were", "fixed", "with", "4%", "paraformaldehyde", "for", "half", "an", "hour,", "blocked", "in", "blocking", "buffer", "(5%", "goat", "serum,", "1%", "BSA,", "0.1%", "Triton", "X-100)", "for", "1", "hour", "followed", "by", "incubation", "with", "the", "primary", "antibody", "at", "4°C", "overnight.", "Appropriately", "coupled", "secondary", "antibodies", "(Molecular", "Probes)", "were", "used", "for", "single", "and", "double", "labeling.", "All", "secondary", "antibodies", "were", "tested", "for", "cross", "reactivity", "and", "non-specific", "immunoreactivity.", "The", "following", "antibodies", "were", "used:", "ABP-280", "(1:250,", "BD", 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9, 9, 5, 9, 9, 9, 9, 5, 9, 9, 9, 9, 9, 5, 9, 9, 9, 9, 5, 15, 9, 9, 9, 9, 5, 9, 9, 9, 9, 5, 9, 9, 9, 9, 5, 9, 9, 9, 9, 5, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 7, 9, 7, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9 ]
[ "Human", "embryonic", "stem", "cells", "(hESCs)", "and", "neural", "progenitor", "(NP)", "cells", "are", "excellent", "models", "for", "recapitulating", "early", "neuronal", "development", "in", "vitro,", "and", "are", "key", "to", "establishing", "strategies", "for", "the", "treatment", "of", "degenerative", "disorders.", "While", "much", "effort", "had", "been", "undertaken", "to", "analyze", "transcriptional", "and", "epigenetic", "differences", "during", "the", "transition", "of", "hESC", "to", "NP,", "very", "little", "work", "has", "been", "performed", "to", "understand", "post-transcriptional", "changes", "during", "neuronal", "differentiation.", "Alternative", "RNA", "splicing", "(AS),", "a", "major", "form", "of", "post-transcriptional", "gene", "regulation,", "is", "important", "in", "mammalian", "development", "and", "neuronal", "function.", "Human", "ESC,", "hESC-derived", "NP,", "and", "human", "central", "nervous", "system", "stem", "cells", "were", "compared", "using", "Affymetrix", "exon", "arrays.", "We", "introduced", "an", "outlier", "detection", "approach,", "REAP", "(Regression-based", "Exon", "Array", "Protocol),", "to", "identify", "1,737", "internal", "exons", "that", "are", "predicted", "to", "undergo", "AS", "in", "NP", "compared", "to", "hESC.", "Experimental", "validation", "of", "REAP-predicted", "AS", "events", "indicated", "a", "threshold-dependent", "sensitivity", "ranging", "from", "56%", "to", "69%,", "at", "a", "specificity", "of", "77%", "to", "96%.", "REAP", "predictions", "significantly", "overlapped", "sets", "of", "alternative", "events", "identified", "using", "expressed", "sequence", "tags", "and", "evolutionarily", "conserved", "AS", "events.", "Our", "results", "also", "reveal", "that", "focusing", "on", "differentially", "expressed", "genes", "between", "hESC", "and", "NP", "will", "overlook", "14%", "of", "potential", "AS", "genes.", "In", "addition,", "we", "found", "that", "REAP", "predictions", "are", "enriched", "in", "genes", "encoding", "serine/threonine", "kinase", "and", "helicase", "activities.", "An", "example", "is", "a", "REAP-predicted", "alternative", "exon", "in", "the", "SLK", "(serine/threonine", "kinase", "2)", "gene", "that", "is", "differentially", "included", "in", "hESC,", "but", "skipped", "in", "NP", "as", "well", "as", "in", "other", "differentiated", "tissues.", "Lastly,", "comparative", "sequence", "analysis", "revealed", "conserved", "intronic", "cis-regulatory", "elements", "such", "as", "the", "FOX1/2", "binding", "site", "GCAUG", "as", "being", "proximal", "to", "candidate", "AS", "exons,", "suggesting", "that", "FOX1/2", "may", "participate", "in", "the", "regulation", "of", "AS", "in", "NP", "and", "hESC.", "In", "summary,", "a", "new", "methodology", "for", "exon", "array", "analysis", "was", "introduced,", "leading", "to", "new", "insights", "into", "the", "complexity", "of", "AS", "in", "human", "embryonic", "stem", "cells", "and", "their", "transition", "to", "neural", "stem", "cells.Author", "SummaryDeriving", "neural", "progenitors", "(NP)", "from", "human", "embryonic", "stem", "cells", "(hESC)", "is", "the", "first", "step", "in", "creating", "homogeneous", "populations", "of", "cells", "that", "will", "differentiate", "into", "myriad", "neuronal", "subtypes", "necessary", "to", "form", "a", "human", "brain.", "During", "alternative", "RNA", "splicing", "(AS),", "noncoding", "sequences", "(introns)", "in", "a", "pre-mRNA", "are", "differentially", "removed", "in", "different", "cell", "types", "and", "tissues,", "and", "the", "remaining", "sequences", "(exons)", "are", "joined", "to", "form", "multiple", "forms", "of", "mature", "RNA,", "playing", "an", "important", "role", "in", "cellular", "diversity.", "The", "authors", "utilized", "Affymetrix", "exon", "arrays", "with", "probes", "targeting", "hundreds", "of", "thousands", "of", "exons", "to", "study", "AS", "comparing", "human", "ES", "to", "NP.", "To", "accomplish", "this,", "a", "novel", "computational", "method,", "REAP", "(Regression-based", "Exon", "Array", "Protocol),", "is", "introduced", "to", "analyze", "the", "exon", "array", "data.", "The", "authors", "showed", "that", "REAP", "candidates", "are", "consistent", "with", "other", "types", "of", "methods", "for", "discovering", "alternative", "exons.", "In", "addition,", "REAP", "candidate", "alternative", "exons", "are", "enriched", "in", "genes", "encoding", "serine/theronine", "kinases", "and", "helicase", "activities.", "An", "example", "is", "the", "alternative", "exon", "in", "the", "SLK", "(serine/threonine", "kinase", "2)", "gene", "that", "is", "included", "in", "hESC,", "but", "excluded", "in", "NP", "as", "well", "as", "in", "other", "differentiated", "tissues.", "Finally,", "by", "comparing", "genomic", "sequences", "across", "multiple", "mammals,", "the", "authors", "identified", "dozens", "of", "conserved", "candidate", "binding", "sites", "that", "were", "enriched", "proximal", "to", "REAP", "candidate", "exons.", "The", "human", "central", "nervous", "system", "is", "composed", "of", "thousands", "of", "neuronal", "subtypes", "originating", "from", "neural", "stem", "cells", "(NSCs)", "that", "migrate", "from", "the", "developing", "neural", "tube.", "Such", "neuronal", "complexity", "is", "generated", "by", "a", "vast", "repertoire", "of", "molecular,", "genetic,", "and", "epigenetic", "mechanisms,", "such", "as", "the", "active", "retrotransposition", "of", "transposable", "elements", "[1],", "alternative", "promoter", "usage,", "alternative", "RNA", "splicing", "(AS),", "alternative", "polyadenylation,", "RNA", "editing,", "post-translational", "modifications,", "and", "epigenetic", "modulation", "[2].", "Understanding", "the", "processes", "that", "generate", "neuronal", "diversity", "is", "key", "to", "gaining", "insights", "into", "neuronal", "development", "and", "paving", "new", "avenues", "for", "biomedical", "research.", "Human", "embryonic", "stem", "cells", "(hESCs)", "are", "pluripotent", "cells", "that", "propagate", "perpetually", "in", "culture", "as", "undifferentiated", "cells", "and", "can", "be", "induced", "to", "differentiate", "into", "a", "multitude", "of", "cell", "types", "both", "in", "vitro", "and", "in", "vivo", "[3].", "As", "hESCs", "can", "theoretically", "generate", "all", "cell", "types", "that", "make", "up", "an", "organism,", "they", "serve", "as", "an", "important", "model", "for", "understanding", "early", "human", "embryonic", "development.", "In", "addition,", "the", "hESCs", "are", "a", "nearly", "infinite", "source", "for", "generating", "specialized", "cells", "such", "as", "neurons", "and", "glia", "for", "potential", "therapeutic", "purposes", "[4,5].", "In", "recent", "years,", "methods", "have", "been", "introduced", "to", "induce", "hESCs", "to", "differentiate", "into", "neural", "progenitors", "(NPs)", "[6,7]", "and", "neuronal", "and", "glial", "subtypes", "[8–12].", "The", "therapeutic", "interest", "in", "understanding", "the", "molecular", "basis", "of", "pluripotency", "and", "differentiation", "has", "led", "to", "many", "studies", "comparing", "transcriptional", "profiles", "in", "different", "hESC", "lines", "and", "the", "study", "of", "expression", "changes", "during", "the", "differentiation", "of", "hESCs", "to", "various", "lineages", "[13–17].", "NSCs", "and", "progenitor", "cells", "(NPs)", "are", "present", "throughout", "development", "and", "persist", "into", "adulthood", "[18–20].", "They", "are", "critical", "for", "both", "basic", "research", "and", "developing", "approaches", "to", "treat", "neurological", "disorders,", "such", "as", "Parkinson", "disease", "and", "amyotrophic", "lateral", "sclerosis", "(ALS),", "and", "stroke", "or", "head", "injuries", "[21,22].", "NSCs", "and", "NPCs", "can", "be", "isolated", "from", "human", "fetal", "brain", "tissue", "[23–26],", "as", "well", "as", "from", "several", "regions", "of", "the", "adult", "human", "brain,", "such", "as", "the", "cortex,", "hippocampus,", "and", "the", "subventricular", "zone", "(SVZ)", "of", "the", "lateral", "ventricles", "[26–35].", "Several", "studies", "have", "explored", "expression", "patterns", "of", "NPCs.", "For", "example,", "Wright", "et", "al.", "identified", "“expressed”", "and", "“not", "expressed”", "genes", "in", "NPCs", "isolated", "from", "the", "human", "embryonic", "cortex", "[24];", "Cai", "et", "al.", "used", "the", "massively", "parallel", "signature", "sequencing", "profiling", "(MPSS)", "technique", "to", "analyze", "expression", "of", "fetal", "NPCs", "in", "comparison", "to", "hESCs", "and", "astrocyte", "precursors", "[27];", "Maisel", "et", "al.", "used", "Affymetrix", "Gene", "Chip", "arrays", "to", "compare", "adult", "and", "fetal", "NPCs", "propagated", "in", "neurospheres", "[35].", "However,", "as", "with", "hESCs,", "the", "focus", "thus", "far", "has", "been", "primarily", "on", "transcriptional", "differences,", "which", "ignores", "differential", "RNA", "processing", "such", "as", "AS,", "polyadenylation,", "degradation,", "or", "promoter", "usage.", "AS", "is", "frequently", "used", "to", "regulate", "gene", "expression", "and", "to", "generate", "tissue-specific", "mRNA", "and", "protein", "isoforms", "[36–39].", "Recent", "studies", "using", "splicing-sensitive", "microarrays", "suggested", "that", "up", "to", "75%", "of", "human", "genes", "undergo", "AS,", "where", "multiple", "isoforms", "are", "derived", "from", "the", "same", "genetic", "loci", "[40].", "This", "functional", "complexity", "underscores", "the", "challenge", "and", "importance", "of", "elucidating", "AS", "regulation.", "AS", "appears", "to", "play", "a", "dominant", "role", "in", "regulating", "neuronal", "gene", "expression", "and", "function", "[41,42].", "Examples", "of", "splicing", "regulators", "that", "are", "enriched", "and", "function", "specifically", "in", "neuronal", "cells", "include", "the", "brain-specific", "splicing", "factor", "Nova", "[43,44]", "and", "neural-specific", "polypyrimidine", "tract", "binding", "protein", "(nPTB),", "which", "antagonizes", "its", "paralogous", "PTB", "to", "regulate", "exon", "exclusion", "in", "neuronal", "cells", "[45–47].", "Finally,", "an", "early", "report", "estimating", "that", "15%", "of", "point", "mutations", "disrupt", "splicing", "underscores", "the", "importance", "of", "splicing", "in", "human", "disease", "[48].", "Indeed,", "the", "disruption", "of", "specific", "AS", "events", "has", "been", "implicated", "in", "several", "human", "genetic", "diseases,", "such", "as", "frontotemporal", "dementia", "and", "parkinsonism,", "Frasier", "syndrome,", "and", "atypical", "cystic", "fibrosis", "[49].", "While", "insights", "into", "the", "regulation", "of", "AS", "have", "come", "predominantly", "from", "the", "molecular", "dissection", "of", "individual", "genes", "[36,49],", "it", "is", "becoming", "clear", "that", "molecular", "rules", "can", "be", "identified", "from", "large-scale", "studies", "of", "both", "constitutive", "splicing", "and", "AS", "[40].", "Most", "systematic", "global", "analyses", "on", "AS", "have", "focused", "on", "comparisons", "across", "differentiated", "human", "tissues", "[50–52].", "Only", "one", "study,", "utilizing", "expressed", "sequence", "tag", "(EST)", "collections", "from", "stem", "cells,", "has", "attempted", "to", "find", "AS", "differences", "between", "embryonic", "and", "hematopoietic", "stem", "cells", "[53].", "However,", "utilizing", "ESTs", "to", "identify", "AS", "has", "intrinsic", "problems,", "as", "ESTs", "tend", "to", "be", "biased", "for", "the", "3′", "ends", "of", "genes,", "and", "full", "coverage", "of", "the", "genome", "by", "ESTs", "is", "severely", "limited", "by", "sequencing", "costs.", "The", "commercial", "availability", "of", "Affymetrix", "exon", "arrays", "provides", "an", "alternative", "approach", "to", "interrogate", "the", "expression", "of", "every", "known", "and", "predicted", "exon", "in", "the", "human", "genome.", "The", "Affymetrix", "GeneChip", "Human", "Exon", "1.0", "ST", "array", "contains", "∼5.4", "million", "features", "used", "to", "interrogate", "∼1", "million", "exon", "clusters", "(collections", "of", "overlapping)", "of", "known", "and", "predicted", "exons", "with", "more", "than", "1.4", "million", "probesets,", "with", "an", "average", "of", "four", "probes", "per", "exon.", "Our", "goal", "was", "to", "identify", "and", "characterize", "AS", "events", "that", "distinguish", "pluripotent", "hESCs", "from", "multipotent", "NPs,", "paving", "the", "way", "for", "future", "candidate", "gene", "approaches", "to", "study", "the", "impact", "of", "AS", "in", "hESCs", "and", "NPs.", "However,", "as", "different", "hESC", "lines", "were", "established", "under", "different", "culture", "conditions", "from", "embryos", "with", "unique", "genetic", "backgrounds,", "we", "expected", "that", "hESCs", "and", "their", "derived", "NPs", "might", "have", "distinct", "epigenetic", "and", "molecular", "signatures", "[54].", "As", "both", "common", "and", "cell-line", "specific", "alternatively", "spliced", "exons", "are", "likely", "to", "be", "important", "in", "regenerative", "research,", "in", "our", "study", "two", "separate", "hESC", "lines", "were", "used,", "with", "independent", "protocols", "for", "differentiating", "the", "hESCs", "into", "NPs", "positive", "for", "Sox1,", "an", "early", "neuroectodermal", "marker.", "As", "an", "endogenously", "occurring", "population", "of", "NPs,", "human", "central", "nervous", "system", "stem", "cells", "grown", "as", "neurospheres", "(hCNS-SCns)", "were", "utilized", "as", "a", "natural", "benchmark", "for", "derived", "NPs.", "We", "developed", "an", "approach", "called", "REAP", "(Regression-based", "Exon", "Array", "Protocol),", "which", "is", "based", "on", "robust", "regression", "that", "analyzed", "signal", "estimates", "from", "Affymetrix", "exon", "array", "data", "to", "identify", "AS", "exons.", "Experimental", "validation", "revealed", "alternative", "exons", "that", "distinguish", "hESCs", "from", "NPs;", "some", "of", "them", "also", "distinguish", "hESCs", "from", "a", "variety", "of", "differentiated", "tissues.", "A", "comparison", "of", "REAP-predicted", "alternative", "events", "with", "independent", "methods,", "such", "as", "using", "publicly", "available", "transcripts", "(ESTs", "and", "mRNAs)", "and", "computational", "predictions", "based", "on", "genomic", "sequence", "information", "alone", "[55],", "showed", "a", "strong", "concordance", "of", "REAP-identified", "AS", "exons", "with", "AS", "events", "identified", "from", "these", "orthogonal", "methods.", "Finally,", "using", "analysis", "of", "the", "sequences", "flanking", "REAP-identified", "alternative", "exons,", "we", "were", "able", "to", "discover", "known", "and", "novel", "cis-regulatory", "elements", "that", "potentially", "regulate", "these", "AS", "events.", "Derivation", "of", "Neural", "Progenitors", "from", "Embryonic", "Stem", "CellsNPs", "were", "independently", "derived", "from", "two", "hESC", "lines,", "and", "RNA", "extracted", "from", "the", "cell", "lines", "was", "processed", "and", "hybridized", "onto", "Affymetrix", "Human", "1.0", "ST", "exon", "arrays.", "Immunohistochemical", "and", "reverse-transcriptase", "polymerase", "chain", "reaction", "(RT-PCR)", "analyses", "demonstrated", "that", "the", "hESCs", "expressed", "pluripotent", "marker", "genes,", "and", "the", "derived", "NPs", "expressed", "multipotent", "and", "neurogenic", "markers", "similar", "to", "hCNS-SCns.", "Undifferentiated", "Cythera", "(Cyt-ES)", "and", "HUES6", "(HUES6-ES)", "hESC", "lines", "were", "maintained", "in", "culture", "as", "previously", "described", "[12,23,56].", "Utilizing", "specific", "antibodies,", "we", "observed", "that", "undifferentiated", "Cyt-ES", "and", "HUES6-ES", "cells", "were", "positive", "for", "the", "pluripotent", "markers", "Oct4,", "SSEA-4,", "and", "Tra-1–80", "(unpublished", "data).", "NPs", "were", "derived", "from", "the", "hESC", "cell", "lines", "using", "protocols", "optimized", "for", "each", "line", "(see", "Materials", "and", "Methods).", "Greater", "than", "90%", "of", "derived", "NP", "cells", "(Cyt-NP", "from", "Cyt-ES", "and", "HUES6-NP", "from", "HUES6-ES)", "were", "positive", "for", "Sox1,", "an", "early", "neuroectodermal", "marker,", "and", "Nestin", "(Figure", "1A),", "and", "negative", "for", "Oct4", "(unpublished", "data).", "As", "a", "natural", "benchmark", "for", "the", "derived", "NPs,", "we", "utilized", "hCNS-SCns,", "which", "were", "previously", "isolated", "from", "fresh", "human", "fetal", "brain", "tissues", "using", "antibodies", "to", "cell-surface", "markers", "and", "fluorescence-activated", "cell", "sorting", "[12,23].", "The", "hCNS-SCns", "form", "neurospheres", "in", "culture", "which", "are", "greater", "than", "90%", "Nestin", "and", "Sox1", "positive,", "and", "differentiate", "into", "both", "neurons", "and", "glial", "cells", "in", "vitro", "[12,23].", "Immunohistochemical", "analysis", "confirmed", "that", "hCNS-SCns", "were", "negative", "for", "Oct4", "(unpublished", "data)", "and", "positive", "for", "Sox1", "and", "Nestin", "(Figure", "1A).Figure", "1Molecular", "Characterization", "of", "Human", "Embryonic", "Stem", "Cell", "Lines", "and", "Neuronal", "Progenitors(A)", "Immunohistochemical", "analysis", "of", "markers", "in", "NPs", "derived", "from", "the", "hESC", "lines", "(Cyt-NP", "from", "Cyt-ES;", "and", "HUES6-NP", "from", "HUES6-ES)", "and", "in", "hCNS-SCns.", "Cyt-NP,", "HUES6-NP,", "and", "hCNS-SCns", "cells", "were", "Nestin", "and", "Sox1", "positive.", "Nuclei", "stained", "positive", "for", "Dapi.", "White", "horizontal", "bar", "indicated", "15", "μm.(B)", "Gene-level", "signal", "estimates", "of", "marker", "genes", "(GAPDH,", "Oct4,", "Nanog,", "Nestin,", "Notch1,", "DNER,", "and", "Sox1)", "from", "Affymetrix", "exon", "array", "analysis.", "Vertical", "bars", "indicated", "average", "log2", "normalized", "signal", "estimates,", "and", "error", "bars", "represented", "standard", "deviations", "from", "three", "independent", "replicate", "experiments", "per", "cell", "type.(C)", "RT-PCR", "of", "marker", "genes", "(GAPDH,", "Oct4,", "Nanog,", "Nestin,", "Notch1,", "DNER,", "and", "Sox1).Here,", "known", "molecular", "markers", "were", "subjected", "to", "RT-PCR", "measurements,", "which", "were", "compared", "to", "gene-level", "signal", "estimates", "generated", "from", "the", "exon", "array", "data.", "Total", "RNA", "was", "extracted,", "and", "labeled", "cDNA", "targets", "were", "generated", "from", "three", "independent", "preparations", "of", "each", "cell", "type,", "namely", "Cyt-ES,", "HUES6-ES,", "Cyt-NP,", "HUES6-NP,", "and", "hCNS-SCns.", "To", "facilitate", "downstream", "analyses,", "instead", "of", "utilizing", "the", "meta-gene", "sets", "available", "from", "the", "manufacturers,", "we", "generated", "our", "own", "gene", "models", "by", "clustering", "alignments", "of", "ESTs", "and", "mRNAs", "to", "annotated", "known", "genes", "from", "the", "University", "of", "California", "Santa", "Cruz", "(UCSC)", "Genome", "Browser", "Database.", "After", "hybridization,", "scanning,", "and", "extraction", "of", "signal", "estimates", "for", "each", "probeset", "on", "the", "exon", "arrays,", "gene-level", "estimates", "were", "computed", "based", "on", "our", "gene", "models", "using", "available", "normalization", "and", "signal", "estimation", "software", "from", "Affymetrix.", "For", "every", "gene,", "a", "t-statistic", "and", "corresponding", "p-value", "were", "computed", "representing", "the", "relative", "enrichment", "of", "the", "expression", "of", "the", "gene", "in", "hESC", "versus", "NP,", "such", "as", "in", "Cyt-ES", "versus", "Cyt-NP.", "After", "correcting", "for", "multiple", "hypothesis", "testing", "using", "the", "Benjamini-Hochberg", "method,", "a", "p-value", "cutoff", "of", "0.01", "was", "used", "to", "identify", "enriched", "genes.", "Close", "inspection", "of", "all", "pairs", "of", "hESC-NP", "comparisons", "revealed", "a", "generally", "significant", "overlap", "from", "31%", "to", "85%", "of", "the", "smaller", "of", "two", "compared", "sets", "of", "enriched", "genes", "(see", "Figure", "S1).", "Thus", "for", "the", "purpose", "of", "identifying", "overall", "pluripotent", "and", "neural", "lineage-specific", "genes,", "the", "collective", "set", "of", "NPs", "(Cyt-NP,", "HUES6-NP,", "and", "hCNS-SCns)", "was", "compared", "to", "the", "collective", "set", "of", "hESCs", "(Cyt-ES", "and", "HUES6-ES).Oct4", "and", "Nanog,", "which", "are", "important", "in", "maintaining", "the", "pluripotent", "state", "of", "embryonic", "stem", "cells", "(ESCs),", "were", "highly", "expressed", "in", "hESCs", "but", "were", "significantly", "lower", "in", "NPs", "(Figure", "1B).", "RT-PCR", "of", "Oct4", "and", "Nanog", "mRNA", "levels", "accurately", "reflected", "the", "signal", "estimates", "from", "the", "array", "(Figure", "1C).", "Interestingly,", "Nestin", "was", "not", "significantly", "higher", "in", "NPs", "as", "compared", "to", "the", "hESC", "from", "the", "gene-level", "estimates", "(p-value", "0.065),", "which", "was", "further", "confirmed", "by", "RT-PCR", "(Figure", "1C).", "Notch", "was", "recently", "identified", "to", "be", "important", "in", "promoting", "the", "neural", "lineage", "entry", "in", "mouse", "ESCs", "[57]", "and", "was", "shown", "to", "regulate", "stem", "cell", "proliferation", "in", "somatic", "mouse", "and", "hESC", "[58].", "Gene-level", "signal", "estimates", "suggested", "that", "Notch", "was", "significantly", "higher", "in", "hCNS-SCns", "relative", "to", "hESCs,", "but", "levels", "of", "Notch", "were", "not", "significantly", "different", "in", "the", "derived", "NPs", "compared", "to", "hESCs.", "Delta/Notch-like", "EGF-related", "receptor", "(DNER),", "a", "neuron-specific", "transmembrane", "protein,", "was", "recently", "shown", "to", "bind", "to", "Notch", "at", "cell–cell", "contacts", "and", "activates", "Notch", "signaling", "in", "vitro", "[59].", "RT-PCR", "validation", "of", "DNER", "confirmed", "array-derived", "signal", "estimates,", "indicating", "an", "enrichment", "of", "DNER", "in", "NPs", "relative", "to", "hESCs", "(Figure", "1C).", "Finally,", "Sox1,", "a", "HMG-box", "protein", "related", "to", "SRY,", "was", "shown", "to", "be", "one", "of", "the", "earliest", "transcription", "factors", "expressed", "in", "cells", "committed", "to", "the", "neural", "fate", "[60].", "Here", "the", "gene-level", "estimates", "indicated", "that", "Sox1", "was", "expressed", "significantly", "higher", "in", "NPs", "relative", "to", "hESCs", "(p-value", "0.00013,", "Figure", "1B),", "a", "finding", "that", "was", "confirmed", "by", "RT-PCR", "(Figure", "1C).From", "these", "examples,", "we", "concluded", "that", "RT-PCR", "validation", "correlated", "well", "with", "gene-level", "estimates", "from", "the", "exon", "array.", "In", "addition,", "the", "derived", "NPs", "had", "decreased", "levels", "of", "pluripotent", "markers", "Oct4", "and", "Nanog", "but", "had", "levels", "of", "Sox1", "that", "were", "comparable", "to", "hCNS-SCns.", "This", "finding", "confirmed", "that", "the", "derived", "NPs", "were", "committed", "to", "a", "neural", "fate", "and", "validated", "the", "use", "of", "hCNS-SCns", "as", "a", "benchmark", "for", "NPs.Next", "we", "asked", "whether", "the", "highest", "enriched", "genes", "in", "hESCs", "relative", "to", "NPs", "reflected", "our", "existing", "knowledge", "in", "the", "literature.", "Using", "the", "above-mentioned", "groupings", "of", "hESCs", "(Cyt-ES,", "HUES6-ES)", "and", "NPs", "(Cyt-NP,", "HUES6-NP,", "and", "hCNS-SCns),", "2,945", "genes", "were", "enriched", "in", "hESCs", "relative", "to", "NPs;", "and", "552", "genes", "were", "enriched", "in", "the", "NPs", "relative", "to", "hESCs,", "at", "a", "p-value", "significance", "cutoff", "of", "0.01", "(correcting", "for", "multiple", "hypothesis", "testing", "using", "the", "Benjamini-Hochberg", "method).", "The", "15", "most", "enriched", "genes", "in", "hESCs", "included", "genes", "such", "as", "teratocarcinoma-derived", "growth", "factor", "1", "(TDGF1/cripto;", "p-value", "<", "10−12),", "zinc", "finger", "protein", "42", "(Zfp42/Rex1;", "p-value", "<", "10−12),", "Oct4", "(p-value", "<", "10−12),", "Nanog", "(p-value", "<", "10−10),", "lin-28", "homolog", "(p-value", "<", "10−10),", "cadherin", "1", "preprotein", "(p-value", "<", "10−10),", "claudin", "6", "(p-value", "<", "10−9),", "ephrin", "receptor", "EphA1", "(p-value", "<", "10−9),", "and", "erbB3", "(p-value", "<", "10−9).", "TDGF1/cripto", "was", "first", "shown", "to", "stimulate", "DNA", "synthesis", "and", "cell", "proliferation", "of", "both", "undifferentiated", "and", "differentiated", "embryonic", "carcinoma", "cells", "[61]", "and", "was", "later", "shown", "to", "be", "important", "for", "cardiomyocyte", "formation", "from", "mouse", "ESC", "[62].", "Oct4,", "reviewed", "in", "[63],", "and", "Nanog", "[64]", "are", "crucial", "for", "the", "pluripotency", "of", "hESCs.", "Recently,", "knockdown", "of", "Zfp42/Rex-1", "in", "mouse", "ESC", "caused", "the", "cells", "to", "differentiate", "[65].", "Our", "gene-level", "exon", "array", "analysis", "confirmed", "that", "the", "hESCs", "and", "NPs", "were", "molecularly", "distinct.To", "reveal", "global", "functional", "differences", "between", "the", "enriched", "genes", "in", "hESCs", "or", "NPs,", "the", "enriched", "genes", "were", "subjected", "to", "a", "Gene", "Ontology", "(GO,", "http://www.geneontology.org)", "analysis", "as", "described", "previously", "[55].", "Enriched", "genes", "in", "hESCs", "were", "more", "likely", "to", "be", "in", "molecular", "function", "categories,", "such", "as", "“RNA", "binding”", "(p-value", "<", "10−12),", "“structural", "constituent", "of", "ribosome”", "(p-value", "<", "10−51),", "“exonuclease", "activity”", "(p-value", "<", "10−6),", "“cytochrome-c", "oxidase", "activity”", "(p-value", "<", "10−5),", "and", "“ATP", "binding”", "(p-value", "<", "10−6),", "and", "in", "biological", "processes", "involved", "with", "“tRNA", "processing”", "(p-value", "<", "10−6)", "and", "“protein", "biosynthesis”", "(p-value", "<", "10−48),", "consistent", "with", "our", "knowledge", "of", "hESCs", "as", "a", "rapidly", "proliferating", "population", "of", "cells", "(Figure", "2A).", "Similar", "analysis", "of", "the", "enriched", "genes", "in", "NPs", "revealed", "an", "overrepresentation", "in", "molecular", "functional", "categories,", "such", "as", "“calcium", "ion", "binding”", "(p-value", "<", "10−8)", "and", "“structural", "molecule", "activity”", "(p-value", "<", "10−5),", "and", "in", "biological", "processes", "involved", "with", "“neurogenesis”", "(p-value", "<", "10−38),", "“cell", "adhesion”", "(p-value", "<", "10−13),", "“cell", "motility”", "(p-value", "<", "10−4),", "“development”", "(p-value", "<", "10−6),", "“neuropeptide", "signaling", "pathway”", "(p-value", "<", "10−4),", "and", "“endocytosis”", "(p-value", "<", "10−4)", "(Figure", "2B).", "Considering", "that", "these", "were", "the", "only", "categories", "that", "were", "significantly", "enriched", "out", "of", "more", "than", "18,000", "GO", "terms,", "and", "that", "randomly", "selected", "sets", "of", "similar", "numbers", "of", "genes", "did", "not", "reveal", "statistical", "differences", "in", "GO", "categories,", "our", "results", "confirmed", "that", "the", "global", "molecular", "profiles", "derived", "from", "exon", "array", "analysis", "were", "consistent", "with", "known", "differences", "between", "hESCs", "and", "NPs.Figure", "2Gene", "Ontology", "AnalysisDifferential", "gene", "expression", "of", "hESCs", "(Cyt-ES", "and", "HUES6-ES)", "and", "NPs", "(Cyt-NP,", "HUES6-NP,", "and", "hCNS-SCns)", "was", "computed", "from", "gene-level", "signal", "estimates.", "Statistical", "significance", "for", "differential", "gene", "expression", "was", "determined", "by", "using", "t-statistics", "with", "Benjamini-Hochberg", "correction", "for", "false", "discovery", "rate", "(p", "<", "0.01).", "Gene", "Ontology", "“molecular", "function,”", "“cellular", "component,”", "and", "“biological", "process”", "categories,", "which", "differed", "significantly", "(p", "<", "0.05)", "in", "the", "representation", "between", "significantly", "enriched", "genes", "(black", "bars)", "and", "all", "other", "genes", "(white", "bars),", "were", "shown.", "Statistical", "significance", "for", "GO", "analysis", "was", "assessed", "by", "using", "χ2", "statistics", "with", "Bonferroni", "correction", "for", "multiple", "hypothesis", "testing.", "GO", "categories", "are", "ordered", "from", "top", "to", "bottom", "in", "order", "of", "decreasingly", "significant", "bias", "toward", "enriched", "genes.(A)", "GO", "analysis", "of", "enriched", "genes", "in", "hESCs.(B)", "GO", "analysis", "of", "enriched", "genes", "in", "NPs.To", "summarize,", "firstly", "immunohistochemical", "and", "RT-PCR", "evidence", "validated", "that", "the", "cells", "exhibited", "expected", "characteristics;", "secondly,", "stage-specific", "marker", "gene", "differences", "by", "RT-PCR", "were", "reflected", "accurately", "by", "gene-level", "estimates", "from", "the", "exon", "arrays;", "thirdly,", "the", "hESC-enriched", "genes", "were", "coherent", "with", "known", "genes", "that", "controlled", "pluripotency", "and", "self-renewal;", "and", "lastly,", "the", "global", "functional", "profiles", "exemplified", "expected", "biological", "differences", "between", "hESC", "and", "NP", "cells.", "Description", "of", "the", "Regression-Based", "Exon", "Array", "ProtocolConvinced", "that", "the", "signal", "estimates", "from", "the", "exon", "arrays", "reflected", "expected", "molecular", "and", "biological", "differences", "between", "hESCs", "and", "NPs,", "we", "sought", "to", "identify", "AS", "events.", "We", "compared", "Cyt-ES", "to", "hCNS-SCns", "to", "illustrate", "our", "approach.", "First", "we", "normalized", "the", "data", "and", "generated", "signal", "estimates", "with", "Robust", "Multichip", "Analysis", "(RMA)", "and", "estimated", "the", "probability", "that", "each", "probeset", "was", "detected", "above", "background", "(DABG)", "using", "publicly", "available", "Affymetrix", "Power", "Tools", "(APT).", "We", "analyzed", "probesets", "that", "(i)", "comprised", "three", "or", "more", "individual", "probes;", "(ii)", "were", "localized", "within", "the", "exons", "of", "our", "gene", "models", "with", "evidence", "from", "at", "least", "three", "sources", "(mRNA,", "EST,", "or", "full-length", "cDNA);", "and", "(iii)", "were", "detected", "above", "background", "in", "at", "least", "one", "of", "the", "cell", "lines.", "In", "total,", "17,430", "gene", "models", "were", "represented", "by", "probesets", "that", "satisfied", "these", "criteria.Next", "we", "asked", "whether", "the", "probeset", "expression", "within", "each", "gene", "model", "was", "positively", "correlated", "for", "any", "two", "cell", "lines.", "To", "do", "this", "we", "calculated", "the", "Pearson", "correlation", "coefficient", "between", "the", "vectors", "of", "median", "signal", "estimates", "across", "replicates", "in", "Cyt-ES", "versus", "hCNS-SCns.", "The", "vast", "majority", "of", "genes", "(>80%)", "was", "found", "to", "have", "probeset-level", "Pearson", "correlation", "coefficients", "of", "greater", "than", "0.8", "(Figure", "3A).", "Next", "we", "randomly", "permuted", "the", "association", "between", "the", "median", "signal", "estimates", "and", "the", "probesets", "for", "each", "gene", "in", "hESCs", "(or", "hCNS-SCns)", "and", "observed", "that", "the", "distribution", "of", "Pearson", "correlation", "coefficients", "for", "the", "permuted", "sets", "was", "centered", "at", "zero,", "as", "expected", "(Figure", "3A).", "This", "indicated", "that", "the", "signal", "estimates", "for", "probesets", "between", "hESCs", "and", "hCNS-SCns", "were", "highly", "correlated", "and", "suggested", "that", "a", "scatter", "plot", "of", "probeset", "signal", "estimates", "between", "hESCs", "and", "hCNS-SCns", "would", "reveal", "a", "linear", "relationship", "for", "the", "majority", "of", "genes.", "We", "hypothesized", "that", "a", "linear", "regression", "to", "determine", "if", "some", "probesets", "behaved", "unexpectedly", "in", "one", "cell", "type", "compared", "to", "the", "other", "might", "be", "a", "reasonable", "approach", "to", "identify", "AS", "exons.Figure", "3Description", "of", "the", "REAP", "Algorithm", "Comparing", "Exon", "Array", "Signal", "Estimates", "from", "hCNS-SCns", "and", "Cyt-ES(A)", "Histogram", "of", "Pearson", "correlation", "coefficients", "computed", "from", "median", "signal", "estimates", "for", "probesets", "between", "Cyt-ES", "versus", "hCNS-SCns", "for", "genes", "(blue", "bars).", "Genes", "were", "required", "to", "have", "more", "than", "five", "probesets", "localized", "within", "the", "exons", "in", "the", "gene.", "Red", "bars", "represented", "Pearson", "correlation", "coefficients", "computed", "from", "exons", "with", "shuffled", "signal", "estimates.(B)", "Each", "probeset", "contained", "probeset-level", "estimates", "from", "three", "replicates", "each,", "(a,", "b,", "c)", "in", "Cyt-ES", "and", "(d,", "e,", "f)", "in", "hCNS-SCns.", "The", "five", "points", "summarizing", "the", "log2", "probeset-level", "estimates", "are", "indicated", "by", "black", "filled", "circles.(C)", "Each", "probeset", "was", "summarized", "by", "five", "points.", "Scatter", "plots", "of", "signal", "estimates", "for", "probesets", "that", "were", "present", "in", "at", "least", "one", "cell", "type", "(Cyt-ES", "or", "hCNS-SCns)", "for", "the", "EHBP1", "gene.", "Probesets", "were", "considered", "present", "if", "the", "DABG", "p-value", "was", "<0.05", "for", "all", "three", "replicates", "in", "the", "cell", "type.", "A", "regression", "line", "derived", "from", "robust", "linear", "regression", "with", "MM", "estimation", "is", "indicated.", "Points", "above", "the", "line", "represent", "probesets", "within", "exons", "that", "were", "enriched", "in", "Cyt-ES", "and", "points", "below", "represent", "exons", "that", "were", "enriched", "in", "hCNS-SCns.", "Points", "close", "to", "the", "regression", "line", "are", "not", "significantly", "different", "in", "Cyt-ES", "versus", "hCNS-SCns.", "Boxed", "points", "represented", "the", "five-point", "summary", "of", "a", "probeset", "that", "was", "significantly", "enriched", "in", "Cyt-ES", "but", "was", "skipped", "in", "hCNS-SCns.(D)", "Histogram", "of", "studentized", "residuals", "for", "points", "from", "the", "scatter", "plot", "in", "(C)", "in", "EHBP1.(E)", "The", "histogram", "of", "studentized", "residuals", "for", "all", "points", "for", "all", "analyzed", "probesets", "(100", "bins).(F)", "The", "scatter", "plot", "of", "studentized", "residuals", "generated", "from", "comparing", "Cyt-ES", "versus", "hCNS-SCns", "and", "hCNS-SCns", "versus", "Cyt-ES", "of", "5,000", "randomly", "chosen", "probesets.Here,", "a", "possible", "representation", "of", "the", "data", "was", "explored.", "If", "we", "had", "N", "replicates", "in", "one", "condition", "and", "M", "replicates", "in", "the", "other,", "we", "could", "consider", "N*M", "points", "if", "we", "analyzed", "every", "possible", "pairing.", "For", "instance,", "three", "replicate", "signal", "estimates", "for", "every", "probeset", "per", "cell", "line,", "such", "as", "signal", "estimates", "a,", "b,", "and", "c", "in", "hESCs", "and", "d,", "e,", "and", "f", "in", "hCNS-SCns,", "would", "translate", "to", "pairing", "every", "signal", "(d,a),", "(d,b),", "(d,c)", "…", "(f,a),", "(f,b),", "(f,c)", "for", "linear", "regression", "(Figure", "3B).", "Instead,", "pairing", "the", "signal", "estimates", "of", "all", "replicates", "in", "one", "condition", "to", "the", "median", "of", "the", "other", "would", "only", "require", "N", "+", "M", "−", "1", "points", "and", "would", "capture", "the", "variation", "of", "the", "signal", "estimates", "of", "each", "probeset.", "For", "example,", "we", "considered", "(d,b),", "(e,a),", "(e,b),", "(e,c),", "and", "(f,b)", "points", "where", "b", "and", "d", "were", "the", "median", "intensities", "for", "the", "replicates", "in", "Cyt-ES", "and", "hCNS-SCns,", "respectively", "(Figure", "3B).", "A", "scatter", "plot", "of", "all", "probesets", "of", "the", "EHBP1", "(EH", "domain", "binding", "protein,", "RefSeq", "identifier", "NM_015252)", "is", "shown", "in", "Figure", "3C", "in", "the", "format", "described.", "Each", "probeset", "was", "represented", "by", "5", "points", "of", "log-transformed", "(base", "2)", "values;", "and", "each", "point", "on", "the", "scatter", "plot", "reflected", "the", "extent", "of", "inclusion", "of", "an", "exon", "in", "hESCs", "and", "in", "hCNS-SCns", "(Figure", "3C).A", "classical", "linear", "regression", "model", "could", "be", "proposed", "to", "fit", "the", "response", "variable", "yij,", "the", "log2", "expression", "of", "probeset", "i", "in", "cell-type", "j", "(for", "example,", "j", "is", "Cyt-ESC)", "to", "explanatory", "variables", "xik,", "and", "the", "log2", "expression", "of", "probeset", "i", "in", "cell", "type", "k", "(for", "example,", "k", "is", "hCNS-SCns).", "However,", "classical", "linear", "regression", "by", "least-squares", "estimation", "is", "biased", "because", "the", "least", "squares", "predictions", "are", "strongly", "influenced", "by", "the", "outliers,", "leading", "to", "completely", "incorrect", "regression", "line", "estimates,", "masking", "of", "the", "outliers,", "and", "incorrect", "predictions", "of", "outliers.", "Therefore,", "we", "applied", "M-estimation", "robust", "regression", "to", "estimate", "the", "line,", "which", "is", "less", "sensitive", "to", "outliers.", "Fitting", "was", "performed", "using", "an", "iterated,", "re-weighted", "least", "squares", "analysis.", "Our", "assumption", "was", "that", "most", "of", "the", "points", "were", "“correct,”", "i.e.,", "that", "most", "of", "the", "exons", "were", "constitutively", "spliced.", "Thus,", "robust", "regression", "would", "find", "the", "line", "that", "was", "least", "dependent", "on", "outliers,", "which", "would", "be", "potential", "AS", "exons.", "This", "assumption", "was", "substantiated", "by", "our", "observation", "that,", "using", "publicly", "available", "ESTs", "and", "mRNAs,", "a", "minority", "of", "human", "exons", "(7%)", "have", "evidence", "for", "exon-skipping,", "the", "most", "common", "form", "of", "AS.", "Using", "robust", "regression,", "the", "regression", "line", "for", "Cyt-ESC", "versus", "hCNS-SCns", "in", "the", "EHBP1", "gene", "is", "illustrated", "in", "Figure", "3C.", "The", "boxed", "points", "belonged", "to", "a", "probeset", "that", "was", "enriched", "in", "hESCs", "but", "depleted", "in", "hCNS-SCns,", "which", "was", "suspected", "to", "be", "due", "to", "AS.", "The", "difference", "between", "the", "actual", "and", "regression-based", "predicted", "value,", "normalized", "by", "the", "estimate", "of", "its", "standard", "deviation,", "is", "called", "the", "studentized", "residuals.", "Studentized", "residuals", "were", "computed", "for", "all", "probeset", "pairs", "in", "EHBP1,", "and", "the", "histogram", "depicting", "their", "distribution", "is", "illustrated", "in", "Figure", "3D.", "As", "expected,", "the", "mean", "of", "the", "distribution", "was", "close", "to", "zero,", "and", "the", "distribution", "was", "approximated", "by", "a", "t-distribution", "with", "n-p-1", "degrees", "of", "freedom,", "where", "n", "was", "the", "number", "of", "points", "on", "the", "scatter", "plot,", "and", "the", "number", "of", "parameters", "p", "was", "2.", "The", "boxed", "points", "had", "studentized", "residuals", "of", "1.829,", "3.104,", "2.634,", "3.012,", "and", "2.125", "with", "p-values", "of", "0.034,", "0.00119,", "0.00477,", "0.00158,", "and", "0.01780,", "respectively,", "computed", "based", "on", "the", "t-distribution", "(Figure", "3C).", "At", "a", "stringent", "p-value", "cutoff", "of", "0.01,", "four", "of", "the", "five", "studentized", "residuals", "were", "designated", "as", "significant", "“outliers,”", "indicating", "that", "the", "probeset", "was", "“unusual.”", "RT-PCR", "confirmed", "that", "the", "exon,", "represented", "by", "the", "probeset,", "was", "indeed", "differentially", "included", "in", "hESCs", "and", "skipped", "in", "hCNS-SCns", "(Figure", "7B).", "Applying", "this", "approach", "to", "all", "gene", "models", "revealed", "that,", "as", "expected,", "the", "majority", "of", "studentized", "residuals", "are", "centered", "at", "zero", "(Figure", "3E).", "Thus", "far", "in", "the", "example,", "our", "analysis", "was", "based", "on", "regression", "of", "hESCs", "(y-axis)", "versus", "hCNS-SCns", "(x-axis)", "(Figure", "3B–3D).", "However,", "robust", "regression", "as", "described", "was", "not", "symmetrical,", "i.e.,", "parameter", "estimation", "of", "y", "as", "a", "function", "of", "x", "was", "not", "the", "same", "as", "that", "of", "x", "as", "a", "function", "of", "y.", "The", "negative", "slope", "revealed", "that", "probesets", "enriched", "in", "hESCs", "versus", "hCNS-SCns", "(positive", "valued),", "were", "expectedly", "depleted", "when", "hCNS-SCns", "was", "compared", "to", "hESCs", "(negative", "valued;", "Figure", "3F).", "As", "our", "method", "for", "predicting", "candidate", "alternative", "exons", "was", "based", "on", "identification", "of", "outliers", "using", "robust", "regression,", "we", "named", "the", "method", "REAP.", "Identification", "and", "Removal", "of", "False", "PositivesIn", "the", "process", "of", "experimentally", "validating", "our", "predictions,", "we", "encountered", "three", "main", "sources", "of", "false", "positives", "(FP)", "from", "robust", "regression.", "First,", "we", "identified", "genes", "with", "probeset", "signal", "estimates", "that", "were", "poorly", "correlated", "and", "were", "not", "amenable", "to", "our", "method.", "As", "an", "example,", "the", "median", "probeset", "signal", "estimates", "in", "hESCs", "and", "hCNS-SCns", "of", "the", "FIP1L1", "gene", "(gene", "identifiers", "BC011543,", "AL136910)", "had", "a", "Pearson", "correlation", "coefficient", "of", "0.38,", "and", "the", "distribution", "of", "points", "was", "not", "amenable", "to", "robust", "regression", "(Figure", "4A).", "To", "avoid", "inappropriate", "application", "of", "REAP", "and", "generating", "false", "predictions,", "we", "empirically", "determined", "that", "a", "gene", "had", "to", "have", "a", "Pearson", "correlation", "coefficient", "cutoff", "of", "0.6", "before", "being", "amenable", "to", "REAP", "analysis.", "Next,", "we", "managed", "two", "additional", "sources", "of", "FPs,", "namely", "“high-leverage”", "and", "“high-influence”", "points,", "which", "we", "were", "able", "to", "identify", "by", "computing", "the", "following", "metrics.", "For", "every", "point,", "we", "computed", "(i)", "the", "studentized", "residual", "(as", "described", "above),", "(ii)", "the", "influence,", "and", "(iii)", "the", "leverage", "(see", "Materials", "and", "Methods", "for", "more", "details).", "Leverage", "assessed", "how", "far", "away", "a", "value", "of", "the", "independent", "variable", "was", "from", "the", "mean", "value;", "the", "farther", "away", "the", "observation", "the", "more", "leverage", "it", "had.", "The", "influence", "of", "a", "point", "was", "related", "to", "its", "covariance", "ratio:", "a", "covariance", "ratio", "larger", "(or", "smaller)", "than", "1", "implied", "that", "the", "point", "was", "closer", "(or", "farther)", "than", "was", "typical", "to", "the", "regression", "line,", "so", "removing", "it", "would", "hurt", "(or", "help)", "the", "accuracy", "of", "the", "line", "and", "would", "increase", "(or", "decrease)", "the", "error", "term", "variance.", "Influence", "was", "computed", "as", "the", "absolute", "difference", "between", "the", "covariance", "ratio", "and", "unity.", "To", "illustrate", "further,", "a", "point", "was", "classified", "as", "an", "“outlier”", "if", "it", "had", "a", "large", "studentized", "residual", "(p", "<", "0.01)", "and", "low", "leverage", "(boxed", "point", "“a”);", "as", "a", "“high-leverage”", "point", "if", "it", "had", "a", "low", "studentized", "residual", "and", "high", "leverage", "(boxed", "point", "“b”);", "and", "as", "a", "“high-influence”", "point", "if", "it", "had", "a", "high", "studentized", "residual,", "high", "leverage,", "and", "high", "influence", "(boxed", "point", "“c”;", "Figure", "4B).", "Points", "that", "resembled", "boxed", "point", "“a”", "were", "designated", "as", "potential", "AS", "events.", "For", "example,", "four", "of", "the", "five", "boxed", "points", "in", "Figure", "3C", "were", "“outliers,”", "and", "RT-PCR", "validation", "indicated", "that", "the", "exon", "represented", "by", "the", "probeset", "was", "indeed", "skipped", "in", "hCNS-SCns", "(EHBP1,", "Figure", "7B).", "Points", "that", "were", "“high-leverage,”", "such", "as", "the", "five", "points", "in", "the", "CLCN2", "gene,", "were", "experimentally", "verified", "to", "be", "a", "FP", "(Figure", "4C;", "unpublished", "data).", "Points", "that", "were", "“high-influence,”", "such", "as", "the", "four", "of", "five", "boxed", "points", "in", "the", "ABCA3", "gene", "were", "also", "experimentally", "verified", "to", "be", "a", "FP", "(Figure", "4D;", "unpublished", "data).", "In", "conclusion,", "in", "order", "to", "reduce", "the", "FP", "rate,", "all", "points", "were", "evaluated", "according", "to", "the", "metrics", "described,", "and", "points", "that", "were", "significant", "“outliers”", "were", "considered", "putative", "AS", "events.Figure", "4Sources", "of", "False", "Positives(A)", "Scatter", "plot", "of", "points", "for", "the", "FIP1L1", "gene", "and", "the", "line", "representing", "the", "robust", "regression", "estimate.(B)", "Boxed", "point", "“a”", "represents", "a", "significant", "“outlier”", "(with", "a", "significantly", "different", "studentized", "residual", "and", "low", "leverage).", "Boxed", "point", "“b”", "represents", "a", "“high", "leverage”", "point", "(low", "studentized", "residual", "and", "a", "high", "leverage).", "Boxed", "point", "“c”", "represents", "a", "“high", "influence”", "point", "(high", "studentized", "residual,", "high", "leverage,", "and", "high", "influence).(C)", "Scatter", "plot", "of", "points", "for", "the", "CLCN2", "gene.", "Boxed", "points", "represent", "“high", "leverage”", "points.(D)", "Scatter", "plot", "of", "points", "for", "the", "ABCA3", "gene.", "Boxed", "points", "represent", "“high", "influence”", "points.", "Global", "Identification", "and", "Characterization", "of", "REAP[+]", "ExonsREAP", "was", "applied", "to", "identify", "AS", "events", "in", "NPs", "compared", "to", "hESCs:", "Cyt-NP", "versus", "Cyt-ES;", "HUES6-NP", "versus", "HUES6-ES;", "hCNS-SCns", "versus", "Cyt-ES,", "and", "hCNS-SCns", "versus", "HUES6-ES.", "After", "removing", "potential", "FPs,", "11,348", "genes", "containing", "158,657", "probesets", "were", "scored", "by", "REAP.As", "described", "above,", "for", "each", "pair", "of", "cell", "lines", "compared,", "each", "probset", "was", "represented", "by", "five", "points,", "where", "each", "point", "was", "defined", "a", "significant", "outlier", "if", "it", "had", "a", "high", "residual", "(p", "<", "0.01),", "low", "influence,", "and", "high", "leverage.", "Points", "per", "probeset", "should", "be", "correlated;", "in", "other", "words,", "if", "one", "point", "was", "a", "significant", "outlier,", "the", "other", "points", "were", "expected", "to", "be", "outliers", "as", "well.", "To", "ensure", "that", "this", "was", "the", "case,", "we", "counted", "the", "number", "of", "probesets", "with", "N", "significant", "outliers,", "where", "N", "was", "varied", "from", "0", "to", "5.", "Next,", "the", "identity", "of", "the", "probesets", "and", "points", "derived", "from", "them", "were", "exchanged", "with", "other", "probesets,", "keeping", "constant", "the", "total", "number", "of", "points", "that", "were", "considered", "significant", "outliers.", "At", "N", "=", "0,", "we", "observed", "approximately", "equal", "numbers", "of", "probesets", "in", "the", "actual", "versus", "shuffled", "controls.", "In", "contrast,", "we", "observed", "that", "there", "were", "1.5", "times", "more", "probesets", "with", "N", "=", "2", "significant", "outliers", "relative", "to", "shuffled", "controls;", "12–31", "times", "more", "probesets", "with", "N", "=", "3;", "and", "17–612", "times", "more", "probesets", "that", "had", "N", "=", "4", "significant", "outliers", "(Figure", "5A;", "see", "Table", "S1).", "For", "example,", "in", "hCNS-SCns", "compared", "to", "Cyt-ES,", "approximately", "0.39%", "(490", "of", "124,604)", "of", "probesets", "had", "three", "significant", "outliers", "and", "0.25%", "(308", "probesets)", "had", "four", "significant", "outliers,", "relative", "to", "0.02%", "and", "0%", "of", "shuffled", "controls,", "respectively.Figure", "5Correlation", "between", "“Outliers”(A)", "The", "number", "of", "probesets", "with", "N", "significant", "“outliers”", "was", "determined", "for", "hCNS-SCns", "versus", "Cyt-ES,", "hCNS-SCns", "versus", "HUES6-ES,", "Cyt-NPs", "versus", "Cyt-ES,", "and", "HUES6-NPs", "versus", "HUES6-ES", "(N", "=", "0,", "1,", "2,", "3,", "4,", "5).", "For", "comparison,", "points", "to", "probeset", "relationships", "were", "randomly", "permuted,", "retaining", "the", "same", "number", "of", "“outliers.”", "Vertical", "bars", "represent", "the", "ratio", "between", "the", "number", "of", "actual", "points", "and", "the", "randomly", "permutated", "sets.(B)", "Similar", "to", "(A),", "except", "points", "were", "counted", "as", "“outliers”", "only", "if", "they", "were", "“outliers”", "in", "both", "hCNS-SCns", "versus", "Cyt-ES", "and", "hCNS-SCns", "versus", "HUES6-ES", "(combined", "hCNS-SCns", "versus", "hESC;", "blue", "bars);", "in", "both", "HUES6-NP", "versus", "HUES6-ES", "and", "Cyt-NP", "versus", "Cyt-ES", "(combined", "derived", "NP", "versus", "hESC;", "red", "bars);", "and", "in", "all", "four", "comparisons", "(combined", "NP", "versus", "hESC;", "yellow", "bar).Next", "we", "asked", "whether", "the", "overlap", "between", "related", "comparisons", "was", "higher", "than", "expected.", "Comparing", "the", "significant", "probesets", "between", "hCNS-SCns", "versus", "Cyt-ES", "and", "hCNS-SCns", "versus", "HUES6-ES", "revealed", "672", "significant", "probesets", "(N", "≥", "2),", "whereas", "if", "we", "shuffled", "the", "associations", "between", "probeset", "identity", "and", "significant", "outliers,", "only", "four", "significant", "probesets", "(N", "≥", "2)", "were", "identified—a", "168-fold", "enrichment", "(Figure", "5B,", "Table", "S1).", "A", "total", "of", "236", "significant", "probesets", "overlapped", "when", "we", "compared", "the", "derived", "NPs", "to", "hESCs", "(Cyt-NP", "versus", "Cyt-ES", "and", "HUES6-NP", "versus", "HUES6-ES),", "relative", "to", "seven", "significant", "probesets", "(34-fold", "enrichment).At", "a", "cutoff", "of", "two", "significant", "outliers,", "1,737", "probesets", "contained", "in", "internal", "exons", "were", "defined", "as", "positive", "REAP", "predictions", "(hereafter", "called", "REAP[+])", "exons—candidate", "AS", "events", "that", "distinguished", "NP", "from", "hESC.", "Surprisingly,", "we", "observed", "that", "the", "majority", "of", "REAP[+]", "exons", "were", "specific", "to", "the", "pair", "of", "hESC", "and", "NP", "that", "was", "compared,", "likely", "reflecting", "differences", "in", "genetic", "origins", "and/or", "culturing", "and", "differentiation", "conditions", "of", "the", "cell", "lines:", "614", "REAP[+]", "events", "were", "unique", "to", "hCNS-SCns", "versus", "HUE6-ES;", "220", "were", "unique", "to", "hCNS-SCns", "versus", "Cyt-ES;", "439", "were", "unique", "to", "HUES6-NP", "versus", "HUES6-ES;", "and", "250", "were", "unique", "to", "Cyt-NP", "versus", "Cyt-ES.", "The", "shared", "events", "between", "pairs", "of", "comparisons", "made", "up", "a", "minority", "of", "the", "total", "number", "identified:", "102", "REAP[+]", "events", "were", "found", "to", "be", "in", "common", "between", "hCNS-SCns", "versus", "Cyt-ES", "and", "hCNS-SCns", "versus", "HUES6-ES;", "48", "between", "hCNS-SCns", "versus", "HUES6-ES", "and", "HUES6-NP", "versus", "HUES6-ES;", "and", "only", "17", "between", "hCNS-SCns", "versus", "Cyt-ES", "and", "Cyt-NP", "versus", "Cyt-ES", "(Table", "S2).", "Comparison", "of", "REAP", "to", "EST-Based", "Method", "and", "ACEScanTraditionally,", "AS", "exons", "were", "discovered", "by", "using", "EST", "alignments", "to", "genomic", "loci,", "and", "also", "more", "recently", "by", "computational", "algorithms", "that", "used", "sequence", "information", "extracted", "from", "multiple", "genomes.", "Here,", "we", "compared", "REAP", "predictions", "to", "both", "approaches.", "In", "the", "first", "comparison,", "publicly", "available", "ESTs", "and", "mRNA", "transcripts", "were", "aligned", "to", "the", "human", "genome", "sequence.", "13,934", "exons", "with", "evidence", "for", "exon-skipping", "and/or", "inclusion", "(EST-SE", "for", "EST-verified", "skipped", "exons)", "were", "generated,", "comprising", "∼7%", "of", "all", "internal", "exons.", "First", "we", "analyzed", "Cyt-ES", "versus", "hCNS-SCns.", "If", "we", "required", "that", "none", "of", "the", "points", "per", "probeset", "(exon)", "was", "significant,", "6%", "(4,402", "of", "71,731)", "of", "exons", "(after", "probeset", "mapping)", "had", "evidence", "for", "EST-SE", "(Figure", "6A).", "Shuffling", "the", "mapping", "between", "these", "probesets", "and", "exons", "resulted", "in", "8%", "(5,777", "of", "71,731)", "of", "exons", "with", "evidence", "for", "EST-SE", "(Figure", "6A).", "These", "percentages", "were", "not", "significantly", "different", "from", "the", "7%", "of", "exons", "with", "EST", "evidence", "for", "AS", "observed", "from", "using", "all", "exons.", "By", "raising", "the", "requirement", "that", "probesets", "had", "to", "contain", "at", "least", "one", "significant", "point", "to", "five", "significant", "points,", "the", "percentage", "of", "EST-SE", "increased", "dramatically", "from", "11%", "(531", "of", "4,898", "exons)", "to", "26%", "(33", "of", "126).", "In", "comparison,", "the", "shuffled", "probesets", "at", "the", "same", "requirements", "remained", "at", "∼8%,", "rising", "slightly", "to", "11%", "at", "five", "points,", "due", "to", "small", "sample", "sizes.", "Similar", "trends", "were", "observed", "with", "hCNS-SCns", "versus", "HUES6-ES", "and", "the", "derived", "NPs", "versus", "hESCs", "(Figure", "6A).", "Therefore,", "we", "concluded", "that", "REAP[+]", "exons", "were", "enriched", "for", "AS", "events", "independently", "identified", "by", "a", "transcript-based", "approach.Figure", "6Comparison", "of", "REAP", "Predictions", "for", "hCNS-SCns", "versus", "Cyt-hES,", "hCNS-SCns", "versus", "HUES6-ES,", "Cyt-NP", "versus", "Cyt-ES,", "and", "HUES6-NPs", "versus", "HUES6-ES", "with", "Alternative", "Exons", "Identified", "by", "an", "EST-Based", "Method", "and", "ACEScan(A)", "Black-filled", "squares", "represented", "the", "fraction", "of", "exons", "containing", "probesets", "with", "N", "significant", "points", "that", "had", "EST", "evidence", "for", "exon", "inclusion", "or", "exclusion", "(N", "=", "0,", "1,", "2,", "3,", "4", "and", "5).", "White-filled", "triangles", "represented", "similarly", "computed", "fractions", "with", "permuted", "probeset", "to", "exon", "mappings.(B)", "Black-filled", "squares", "represented", "the", "fraction", "of", "exons", "containing", "probesets", "with", "N", "significant", "points", "that", "had", "ACEScan", "positive", "scores,", "indicative", "of", "evolutionarily", "conserved", "alternative", "exons.", "White-filled", "triangles", "represented", "similarly", "computed", "fractions", "with", "permuted", "probeset", "to", "exon", "mappings.Next,", "we", "compared", "REAP", "predictions", "to", "a", "computational", "approach", "of", "identifying", "exons", "with", "AS", "conserved", "in", "human", "and", "mouse,", "ACEScan", "[55].", "ACEScan", "receives", "as", "input", "orthologous", "human–mouse", "exon", "pairs", "and", "flanking", "intronic", "regions", "and", "computes", "sequence", "features", "and", "integrates", "the", "features", "into", "a", "machine-learning", "algorithm", "to", "assign", "a", "real-valued", "score", "to", "the", "exon.", "A", "positive", "score", "indicated", "a", "higher", "likelihood", "of", "being", "AS", "in", "both", "human", "and", "mouse.", "ACEScan", "was", "updated", "in", "the", "following", "ways.", "Firstly,", "instead", "of", "relying", "on", "orthology", "information", "by", "Ensembl,", "and", "then", "aligning", "flanking", "introns", "in", "“orthologous”", "exons,", "conserved", "exonic", "and", "intronic", "regions", "in", "human", "and", "mouse", "from", "genome-wide", "multiple", "alignments", "were", "extracted.", "Secondly,", "whereas", "in", "our", "previous", "analysis", "exons", "from", "the", "longest", "transcript", "in", "Ensembl", "were", "utilized,", "now", "we", "collapsed", "all", "the", "transcripts", "available", "at", "the", "UCSC", "genome", "browser", "and", "analyzed", "all", "exons", "in", "the", "entire", "gene", "loci.", "ACEScan", "was", "utilized", "to", "assign", "ACEScan", "scores", "to", "all", "∼162,000", "internal", "exons", "in", "our", "genes.", "Exons", "annotated", "as", "first", "or", "last", "exons", "in", "Refseq", "mRNAs", "were", "excluded", "from", "our", "analysis,", "resulting", "in", "4,487", "positive-scoring", "exons,", "2-fold", "more", "exons", "than", "originally", "published.Here", "we", "repeated", "our", "analysis", "with", "exons", "with", "positive", "ACEScan", "scores", "(ACE[+])", "instead", "of", "EST-SEs.", "If", "we", "required", "that", "none", "of", "the", "points", "per", "probeset", "(exon)", "was", "significant,", "2%", "(1,645", "of", "71,731)", "of", "exons", "(after", "probeset", "mapping)", "were", "ACE[+]", "(Figure", "6B).", "Shuffling", "the", "mapping", "between", "these", "probesets", "and", "exons", "resulted", "in", "3%", "(2,044", "of", "71,731)", "of", "exons", "being", "ACE[+]", "(Figure", "6B).", "These", "percentages", "were", "not", "significantly", "different", "from", "the", "2.7%", "observed", "from", "all", "exons", "(4,487", "of", "the", "162,000", "exons", "that", "were", "scored", "by", "ACEScan).", "By", "raising", "the", "requirement", "that", "probesets", "had", "to", "contain", "five", "significant", "points,", "the", "percentage", "of", "ACE[+]", "exons", "increased", "from", "4%", "to", "11%.", "However,", "the", "sample", "sizes", "were", "small.", "In", "comparison,", "the", "shuffled", "probesets", "at", "the", "same", "requirements", "remained", "at", "∼4%.", "Similar", "overall", "trends", "were", "observed", "with", "hCNS-SCns", "versus", "HUES6-ES", "and", "the", "derived", "NPs", "versus", "hESCs", "(Figure", "6B).", "In", "total,", "7.5%", "(131", "of", "1,737)", "of", "REAP", "[+]", "exons", "were", "designated", "as", "ACEScan[+]", "compared", "to", "2.4%", "(2,328", "of", "97,437)", "of", "REAP[−]", "exons.", "This", "result", "suggested", "that", "a", "small", "but", "significantly", "enriched", "fraction", "of", "AS", "events", "in", "hESCs", "versus", "NPs", "was", "likely", "to", "be", "evolutionarily", "conserved", "in", "human", "and", "mouse.", "In", "conclusion,", "our", "results", "suggested", "that", "REAP", "predictions", "were", "congruent", "with", "predictions", "from", "two", "independent,", "orthogonal", "methods.", "Experimental", "Validation", "of", "Alternative", "ExonsThe", "sensitivity", "and", "specificity", "of", "REAP", "in", "the", "identification", "of", "REAP[+]", "exons", "was", "tested", "by", "RT-PCR.", "To", "validate", "REAP[+]", "alternative", "exons,", "RT-PCR", "primers", "were", "designed", "in", "the", "flanking", "exons", "to", "amplify", "both", "isoforms.", "To", "be", "a", "positively", "validated", "candidate,", "the", "PCR", "products", "on", "a", "gel", "had", "to", "satisfy", "all", "of", "the", "following", "criteria:", "(i)", "at", "least", "one", "isoform", "with", "the", "expected", "size", "must", "be", "visible", "in", "each", "cell", "type;", "(ii)", "the", "relative", "abundance", "of", "the", "two", "isoforms", "must", "be", "altered", "between", "two", "cell", "types", "and", "the", "direction", "of", "change", "have", "to", "be", "consistent", "with", "the", "REAP", "studentized", "residuals:", "in", "our", "study", "positive", "residuals", "implied", "inclusion", "in", "hESCs", "and", "skipping", "in", "NPs,", "and", "negative", "residuals", "implied", "inclusion", "in", "NP", "and", "skipping", "in", "hESCs;", "and", "(iii)", "the", "results", "were", "replicable", "in", "at", "least", "two", "experiments.For", "simplicity", "of", "design,", "we", "tested", "candidates", "predicted", "from", "Cyt-ES", "versus", "hCNS-SCns.", "Fifteen", "REAP[+]", "exons", "with", "at", "least", "two", "significant", "outliers", "(out", "of", "five)", "were", "randomly", "chosen", "as", "predicted", "alternative", "events", "and", "thirty-five", "exons", "with", "less", "than", "two", "significant", "outliers", "were", "randomly", "chosen", "as", "constitutive", "events", "(Table", "S3).", "Nine", "of", "the", "fifteen", "exons", "(60%)", "were", "validated", "as", "AS", "events", "by", "our", "criteria.", "The", "sensitivity", "and", "specificity", "of", "the", "algorithm", "at", "the", "cutoff", "of", "two", "is", "69%", "and", "77%.", "Increasing", "the", "cutoff", "to", "three", "increased", "the", "specificity", "to", "85%,", "with", "a", "slight", "decrease", "in", "sensitivity", "to", "67%", "(Figure", "7A).", "The", "patterns", "of", "AS", "in", "hESCs", "were", "similar", "in", "both", "Cyt-ES", "and", "HUES6-ES", "for", "all", "AS", "events", "validated,", "but", "the", "NPs", "(Cyt-NP,", "HUES6-NP,", "and", "hCNS-SCns)", "had", "more", "varied", "AS.", "The", "pattern", "of", "AS", "in", "the", "REAP[+]", "exons", "in", "the", "SLK", "(serine/threonine", "kinase", "2)", "and", "POT1", "(protection", "of", "telomeres", "1)", "genes", "showed", "remarkable", "agreement", "within", "derived", "NPs", "and", "hCNS-SCns", "(Figure", "7B).", "The", "AS", "exon", "in", "SLK", "was", "observed", "to", "be", "included", "in", "hESCs", "and", "completely", "excluded", "in", "NPs;", "the", "AS", "exon", "in", "the", "POT1", "gene", "was", "included", "more", "in", "hESCs", "and", "a", "smaller", "isoform", "persisted", "in", "NPs.", "The", "AS", "patterns", "of", "the", "other", "verified", "REAP[+]", "exons", "were", "consistently", "similar", "in", "hESCs", "but", "were", "more", "varied", "in", "the", "NP.", "Interestingly,", "the", "patterns", "of", "AS", "in", "the", "derived", "NPs", "(Cyt-NP", "and", "HUES6-NP)", "were", "not", "always", "identical", "to", "those", "of", "hCNS-SCns.", "For", "example,", "the", "AS", "exon", "in", "the", "EHBP1", "(EH", "domain", "binding", "protein", "1)", "gene", "was", "included", "in", "hESCs", "but", "skipped", "in", "hCNS-SCns,", "and", "both", "isoforms", "were", "present", "in", "the", "derived", "NPs", "(Figure", "7B).", "As", "another", "example,", "the", "AS", "exon", "in", "the", "SORBS1", "(sorbin", "and", "SH3", "domain", "containing", "1)", "gene", "was", "skipped", "in", "hESCs", "and", "included", "in", "hCNS-SCns,", "but", "exhibited", "an", "intermediate", "pattern", "in", "the", "derived", "NPs.", "However,", "in", "some", "cases,", "the", "AS", "patterns", "in", "the", "derived", "NPs", "were", "different", "from", "both", "hESCs", "and", "hCNS-SCns", "(such", "as", "in", "the", "AS", "exon", "in", "UNC84A,", "SIRT1,", "and", "MLLT10).Figure", "7RT-PCR", "Validation", "of", "REAP-Predicted", "Alternative", "Exons(A)", "Probesets", "(exons)", "were", "considered", "REAP[+]", "candidates", "if", "they", "contained", "at", "least", "N", "=", "2", "(white", "bars),", "3", "(gray", "bars),", "or", "4", "(black", "bars)", "significant", "outliers.", "True", "positive", "(TP),", "true", "negative", "(TN),", "false", "positive", "(FP),", "and", "false", "negative", "(FN)", "rates", "were", "calculated", "from", "RT-PCR-validated", "REAP[+]", "exons", "at", "the", "different", "cutoffs", "(N", "=", "2,", "3,", "4).(B)", "Nine", "RT-PCR", "validated", "REAP[+]", "AS", "events", "in", "hESCs", "(Cyt-ES", "and", "HUES6-ES),", "derived", "NPs", "(Cyt-NP", "and", "HUES6-NP),", "and", "hCNS-SCns.", "Arrows", "indicate", "the", "larger", "(exon-included)", "isoforms", "and", "smaller", "(exon-skipped)", "isoforms.(C)", "RT-PCR", "of", "REAP[+]", "alternative", "exons", "from", "EHBP1,", "SLK,", "and", "RAI14", "across", "a", "panel", "of", "human", "tissues.", "Arrows", "indicate", "the", "larger", "(exon-included)", "isoforms", "and", "smaller", "(exon-skipped)", "isoforms.First,", "given", "three", "independent", "samples", "each", "from", "two", "conditions,", "we", "concluded", "that", "REAP", "was", "able", "to", "identify", "AS", "events", "with", "high", "specificity", "but", "with", "moderate", "sensitivity.", "Second,", "AS", "events", "in", "hESCs", "were", "more", "similar,", "whereas", "the", "AS", "events", "in", "derived", "NPs", "were", "consistent", "with", "or", "intermediate", "to", "the", "benchmark", "hCNS-SCns,", "likely", "reflecting", "differences", "in", "the", "cell", "lines", "and/or", "differentiation", "protocols.", "In", "addition,", "we", "tested", "the", "AS", "patterns", "of", "REAP[+]", "exons", "from", "EHBP1,", "SLK,", "and", "RAI14", "in", "a", "panel", "of", "differentiated", "human", "tissues", "(Figure", "7C).", "The", "REAP[+]", "alternative", "exon", "in", "the", "RAI14", "(retinoic", "acid", "induced", "14)", "gene", "was", "observed", "to", "have", "the", "same", "AS", "pattern", "in", "NPs", "as", "in", "frontal", "and", "temporal", "cortex", "and", "in", "several", "other,", "non-brain", "adult", "tissues,", "such", "as", "heart", "and", "spleen.", "The", "AS", "pattern", "of", "the", "REAP[+]", "exon", "in", "the", "SLK", "gene", "in", "NPs", "was", "similar", "to", "most", "differentiated", "tissues;", "however,", "the", "relatively", "strong", "inclusion", "of", "the", "exon", "in", "hESCs", "was", "unique.", "Even", "in", "esophagus,", "kidney,", "liver,", "and", "prostate,", "both", "isoforms", "were", "present.", "The", "relative", "ratio", "of", "the", "exon-included", "to", "exon-skipped", "isoforms", "in", "SLK", "likely", "represents", "an", "ESC-specific", "AS", "signature.", "The", "alternative", "exon", "in", "the", "EHBP1", "gene", "was", "unusual.", "The", "exon", "was", "included", "in", "hESCs", "but", "also", "in", "frontal", "cortex", "and", "temporal", "cortex,", "a", "finding", "that", "was", "unexpected", "given", "the", "exclusion", "of", "the", "exon", "in", "hCNS-SCns", "(Figure", "7C).", "The", "AS", "pattern", "in", "hCNS-SCns", "may", "represent", "a", "transient,", "early", "neuronal", "molecular", "change.", "Functional", "and", "Expression", "Characteristics", "of", "REAP[+]", "GenesIn", "total,", "1,500", "genes", "were", "identified", "that", "contained", "1,737", "REAP[+]", "exons,", "68%", "of", "which", "lacked", "prior", "transcript", "(EST/cDNA)", "evidence", "for", "AS.", "To", "determine", "whether", "genes", "that", "contained", "REAP[+]", "exons,", "which", "we", "refer", "to", "as", "REAP[+]", "genes,", "are", "biased", "toward", "particular", "biological", "activities,", "REAP[+]", "genes", "were", "compared", "to", "a", "set", "of", "REAP", "analyzed", "genes", "not", "found", "to", "have", "REAP[+]", "exons", "(REAP[−]", "genes).", "A", "Gene", "Ontology", "analysis", "revealed", "that", "REAP[+]", "genes", "are", "enriched", "for", "GO", "molecular", "function", "categories", "“ATP", "binding,”", "“helicase", "activity,”", "“protein", "serine/theronine", "kinase", "activity,”", "“small", "GTPase", "regulatory/interacting", "protein", "activity,”", "and", "“thyroid", "hormone", "receptor", "binding”", "(Table", "1).", "In", "terms", "of", "GO", "biological", "process", "categories,", "REAP[+]", "genes", "were", "more", "frequently", "involved", "in", "“ubiquitin", "cycle.”", "Similar", "results", "were", "obtained", "when", "we", "compared", "REAP[+]", "genes", "to", "all", "human", "genes", "that", "did", "not", "contain", "REAP[+]", "exons", "(Table", "1)", "[55].Table", "1Significantly", "Enriched", "Gene", "Ontology", "Terms", "in", "REAP[+]", "Genes", "(Cutoff", "of", "Two", "Significant", "“Outliers”", "per", "Probeset)Next", "we", "asked", "if", "REAP[+]", "genes", "are", "differentially", "expressed", "in", "hESCs", "compared", "to", "NPs", "and", "vice", "versa.", "For", "this", "analysis,", "the", "t-statistics", "computed", "above", "measuring", "the", "enrichment", "of", "a", "gene", "in", "hESCs", "relative", "to", "NPs", "was", "utilized", "for", "only", "REAP-analyzed", "genes.", "At", "a", "defined", "absolute-valued", "cutoff,", "genes", "were", "divided", "into", "three", "categories:", "“enriched", "in", "hESCs,”", "“enriched", "in", "NP,”", "or", "“unchanged”", "(Figure", "8A).", "Increasing", "the", "t-statistic", "cutoff", "from", "one", "to", "five,", "the", "fraction", "of", "REAP[+]", "genes", "relative", "to", "REAP-analyzed", "genes", "remained", "constant", "in", "the", "“unchanged”", "categories", "(Figure", "8B).", "However,", "the", "fraction", "of", "REAP[+]", "exons", "decreased", "significantly", "in", "“enriched", "in", "hESCs”", "and", "“enriched", "in", "NPs”", "categories.", "If", "we", "increased", "the", "cutoffs", "on", "genes", "that", "were", "randomly", "assigned", "as", "REAP[+]", "and", "REAP[−],", "controlling", "for", "the", "same", "number", "of", "genes", "in", "each", "category,", "we", "observed", "that", "the", "fraction", "of", "REAP[+]", "exons", "remained", "unchanged", "for", "all", "three", "categories", "(Figure", "8C).", "To", "illustrate,", "at", "a", "cutoff", "of", "five,", "10%", "(29", "of", "267)", "of", "enriched", "NP", "genes", "were", "REAP[+]", "genes", "and", "8.8%", "(102", "of", "1,162)", "of", "enriched", "hESC", "genes", "were", "REAP[+],", "significantly", "different", "(p", "<", "0.000005)", "from", "the", "random", "control", "where", "∼14%", "of", "enriched", "NP", "and", "enriched", "hESC", "genes", "were", "REAP[+].", "At", "a", "cutoff", "of", "five,", "14%", "(1,368", "of", "9,636)", "of", "genes", "that", "were", "expressed", "at", "similar", "levels", "between", "hESCs", "and", "NPs", "were", "REAP[+].", "Our", "results", "suggested", "that", "a", "strategy", "of", "focusing", "on", "differentially", "expressed", "genes", "would", "miss", "at", "least", "14%", "of", "transcriptionally", "unchanged", "genes", "that", "may", "nevertheless", "have", "functional", "AS", "differences", "between", "hESCs", "and", "NPs.Figure", "8Analysis", "of", "REAP[+]", "Genes", "Relative", "to", "Transcriptional", "Differences(A)", "Histogam", "of", "t-statistics", "computed", "from", "gene-level", "signal", "estimates", "measuring", "the", "enrichment", "of", "genes", "in", "hESC", "and", "in", "NP.", "Genes", "on", "the", "right", "of", "the", "vertical", "line", "at", "5", "were", "designated", "enriched", "in", "hESC", "and", "genes", "on", "the", "left", "of", "the", "vertical", "line", "at", "−5", "were", "designated", "enriched", "in", "NP;", "genes", "in", "between", "−5", "and", "5", "were", "designated", "as", "“unchanged”", "or", "expressed", "similarly", "in", "hESC", "and", "NP.(B)", "Vertical", "bars", "representing", "the", "percentage", "of", "REAP[+]", "genes", "out", "of", "all", "genes", "in", "the", "different", "classifications", "(dashed", "bar:", "“enriched", "in", "hESC”;", "black", "filled", "bar:", "“unchanged”;", "white", "filled", "bar:", "“enriched", "in", "NP”),", "at", "different", "cutoffs", "of", "1", "to", "5.(C)", "Set", "of", "genes", "where", "REAP[+]", "designation", "was", "randomly", "chosen.", "Similar", "representation", "as", "in", "(B).", "Conserved", "Intronic", "Splicing", "Regulatory", "Elements", "Proximal", "to", "REAP[+]", "hESC", "and", "NP", "ExonsMany,", "if", "not", "most,", "alternative", "exons", "undergo", "cell", "type–specific", "regulation", "by", "the", "binding", "of", "trans-factors", "to", "splicing", "regulatory", "cis-elements", "located", "proximal", "to", "or", "within", "the", "exons.", "As", "many", "tissue-specific", "splicing", "cis-regulatory", "elements", "were", "localized", "in", "intronic", "regions", "of", "AS", "exons,", "we", "focused", "on", "the", "identification", "of", "intronic", "splicing", "regulatory", "elements", "(ISREs)", "proximal", "to", "REAP[+]", "exons.", "In", "addition,", "we", "wanted", "to", "identify", "both", "common", "and", "cell", "type–specific", "ISREs.", "Three", "sets", "of", "exons", "were", "generated:", "(i)", "REAP[+]", "exons", "that", "were", "predicted", "to", "be", "included", "in", "NPs", "and", "skipped", "in", "hESCs", "(REAP[+]NP);", "(ii)", "REAP[+]", "exons", "that", "were", "predicted", "to", "be", "included", "in", "hESCs", "and", "skipped", "in", "NPs", "(REAP[+]hESC);", "and", "(iii)", "all", "REAP[−]", "exons.", "Regions", "of", "400", "base", "pairs", "flanking", "the", "exons", "were", "targeted", "for", "search.", "Initially,", "5-mers", "that", "were", "significantly", "enriched", "between", "the", "upstream", "and", "downstream", "intronic", "regions", "of", "REAP[+]NP", "and", "REAP[+]ES", "relative", "to", "REAP[−]", "exons", "were", "enumerated.", "We", "were", "not", "able", "to", "identify", "5-mers", "that", "were", "statistically", "significantly", "different.Next,", "we", "focused", "on", "splicing", "signals", "that", "were", "conserved", "across", "mammalian", "genomes", "as", "a", "way", "of", "enhancing", "the", "signal", "of", "detecting", "functional", "splicing", "regulatory", "sequences", "[66].", "Exons", "that", "were", "orthologous", "across", "human,", "dog,", "rat,", "and", "mouse", "were", "obtained", "and", "the", "flanking", "intronic", "regions", "were", "aligned", "(400", "bases", "upstream", "and", "downstream", "separately;", "Figure", "9A).", "We", "enumerated", "k-mers", "that", "were", "perfectly", "conserved", "across", "all", "four", "genomes", "in", "the", "upstream", "(and", "downstream)", "intronic", "regions.", "Each", "conserved", "k-mer", "was", "attributed", "a", "χ", "score", "representing", "its", "enrichment", "in", "a", "set", "of", "exons", "relative", "to", "another", "set", "of", "exons.", "The", "higher", "the", "score,", "the", "more", "frequent", "the", "conserved", "k-mer", "was", "in", "the", "first", "set", "relative", "to", "the", "second", "set.", "As", "a", "negative", "control,", "the", "associations", "between", "REAP", "scores", "and", "exons", "were", "shuffled.", "The", "enrichment", "scores", "for", "all", "downstream", "intronic", "5-mers", "for", "shuffled", "REAP[+]NP", "versus", "set", "REAP[−]", "exons", "(x-axis),", "and", "for", "shuffled", "REAP[+]ES", "exons", "versus", "REAP[−]", "exons", "(y-axis)", "were", "displayed", "(Figure", "9B).", "At", "a", "χ", "cutoff", "of", "three,", "which", "corresponded", "to", "a", "p-value", "of", "0.0015,", "the", "majority", "of", "5-mers", "were", "not", "significantly", "enriched", "in", "either", "shuffled", "set.", "Confident", "that", "no", "association", "of", "k-mers", "with", "shuffled", "REAP", "exons", "were", "found;", "we", "repeated", "the", "analyses", "for", "upstream", "and", "downstream", "intronic", "5-mers", "for", "the", "original", "unshuffled", "sets.", "We", "identified", "68", "conserved", "5-mers", "enriched", "upstream", "of", "REAP[+]NP", "exons;", "and", "34", "5-mers", "enriched", "upstream", "of", "REAP[+]ES", "exons", "(Figure", "9C;", "Table", "S4).", "Of", "the", "5-mers", "that", "were", "significantly", "enriched", "upstream", "of", "REAP[+]NP", "exons,", "we", "identified", "a", "U-rich", "motif", "(UUUUU),", "a", "GU-rich", "motif", "(GUGUG),", "and", "a", "CU-rich", "motif", "(CCUCU,", "CUCUC,", "UCUCU,", "GCUCU).", "It", "is", "known", "that", "the", "heterogeneous", "ribonucleoprotein", "C", "(hnRNP", "C)", "binding", "site", "obtained", "by", "SELEX", "is", "five", "“U”s", "[67].", "GU-rich", "sequences", "in", "flanking", "intronic", "regions", "were", "shown", "to", "bind", "to", "splicing", "factor", "ETR-3", "to", "regulate", "AS", "[68].", "CU-rich", "sequences", "were", "shown", "to", "bind", "the", "splicing", "factor", "PTB", "[69].", "Of", "the", "5-mers", "enriched", "upstream", "of", "REAP[+]ES", "exons,", "we", "observed", "CUAAC,", "which", "resembled", "the", "splicing", "branch-signal.", "Of", "the", "six", "5-mers", "that", "were", "enriched", "upstream", "of", "both", "REAP[+]NP", "and", "REAP[+]ES", "exons,", "we", "identified", "GCAUG,", "which", "was", "previously", "shown", "to", "be", "an", "intronic", "splicing", "cis-element", "for", "the", "mammalian", "fibronectin", "and", "calcitonin/CGRP", "genes", "[70–72].", "More", "recently,", "both", "mammalian", "Fox1", "and", "2", "have", "been", "demonstrated", "to", "regulate", "alternatively", "spliced", "exons", "via", "UGCAUG", "binding", "sites", "in", "neighboring", "introns", "in", "neuronal", "cell", "cultures", "[73].Figure", "9Conserved", "Intronic", "cis-Elements", "Enriched", "Proximal", "to", "REAP[+]", "Alternative", "Exons(A)", "Schematic", "describing", "the", "enumeration", "of", "intronic", "elements", "across", "400", "bases", "of", "flanking", "mammalian", "introns", "(human,", "dog,", "rat,", "and", "mouse).", "Red", "and", "green", "horizontal", "bars", "represent", "conserved", "intronic", "elements", "and", "nonconserved", "elements,", "respectively.", "Internal", "exons", "were", "divided", "into", "REAP[+]NP,", "REAP[+]ES,", "and", "REAP[−]", "exons.", "The", "χ", "statistic", "was", "computed", "to", "represent", "the", "enrichment", "of", "conserved", "elements", "in", "intronic", "regions", "flanking", "REAP[+]NP", "versus", "REAP[−]", "exons", "(x-axis),", "and", "REAP[+]ES", "versus", "REAP[−]", "exons", "(y-axis).", "The", "sign", "represented", "the", "direction", "of", "change,", "i.e.,", "positive", "if", "enriched", "in", "introns", "flanking", "REAP[+]", "versus", "REAP[−]", "exon.", "Each", "conserved", "5-mer", "was", "associated", "with", "two", "numbers:", "the", "enrichment", "in", "introns", "proximal", "to", "REAP[+]NP", "versus", "REAP[−]", "exons", "(x-axis),", "and", "REAP[+]ES", "versus", "REAP[−]", "exons", "(y-axis).(B)", "Downstream", "intronic", "regions,", "where", "the", "association", "between", "REAP[+]", "designation", "and", "the", "exons", "was", "shuffled.(C)", "Upstream", "intronic", "regions.", "Circled", "5-mers", "in", "the", "upper", "right", "quadrant", "represent", "conserved", "5-mers", "enriched", "in", "the", "upstream", "intronic", "regions", "of", "REAP[+]NP", "and", "REAP[+]ES", "exons.(D)", "Downstream", "intronic", "regions.", "Circled", "5-mers", "in", "the", "upper", "right", "quadrant", "represent", "conserved", "5-mers", "enriched", "in", "the", "downstream", "intronic", "regions", "of", "REAP[+]NP", "and", "REAP[+]ES", "exons.Eighteen", "conserved", "5-mers", "were", "significantly", "enriched", "in", "the", "downstream", "introns", "of", "REAP[+]ES", "exons;", "and", "76", "5-mers", "were", "enriched", "downstream", "of", "REAP[+]NP", "exons", "(Table", "S4,", "Figure", "9D).", "We", "identified", "a", "motif", "CUCAU", "resembling", "the", "Nova", "binding", "site", "YCAY", "[74],", "and", "a", "G-rich", "motif", "(AGGGG,", "GGGGA,", "GGGGC,", "GGGGG,", "GGGGU)", "enriched", "in", "the", "introns", "downstream", "of", "REAP[+]ES", "exons.", "G-rich", "motifs", "had", "previously", "been", "shown", "to", "be", "part", "of", "a", "bipartite", "signal", "that", "silences", "AS", "exons", "[75].", "Of", "the", "five", "5-mers", "that", "were", "enriched", "downstream", "of", "both", "REAP[+]NP", "and", "REAP[+]ES", "exons,", "GCAUG", "and", "a", "U-rich", "motif", "(UUUUU)", "were", "identified.", "We", "concluded", "that", "potential", "ISREs", "were", "enriched", "proximal", "to", "a", "subset", "of", "REAP[+]", "exons;", "in", "particular,", "the", "Fox1/2", "binding", "site", "GCUAG", "may", "play", "a", "regulatory", "role", "in", "controlling", "AS", "events", "in", "hESCs", "and", "NPs.", "The", "ability", "of", "ESCs", "to", "generate", "all", "three", "embryonic", "germ", "layers", "has", "raised", "the", "exciting", "possibility", "that", "hESCs", "may", "become", "an", "unlimited", "source", "of", "cells", "for", "transplantation", "therapies", "involving", "organs", "or", "tissues", "such", "as", "the", "liver,", "pancreas,", "blood,", "and", "nervous", "system,", "and", "become", "tools", "to", "explore", "the", "molecular", "mechanisms", "of", "human", "development.", "Despite", "such", "interests,", "relatively", "little", "is", "understood", "about", "the", "molecular", "mechanisms", "defining", "their", "pluripotency", "and", "the", "molecular", "changes", "important", "for", "hESCs", "to", "differentiate", "into", "specific", "cell", "types.", "To", "understand", "these", "events,", "protocols", "are", "still", "being", "developed", "to", "differentiate", "ESCs", "into", "a", "variety", "of", "lineages.", "Of", "particular", "biomedical", "interest", "is", "in", "the", "capacity", "of", "hESCs", "to", "be", "differentiated", "into", "a", "self-renewing", "population", "of", "NPs", "that", "can", "be", "then", "further", "coaxed", "into", "a", "variety", "of", "neuronal", "subtypes,", "such", "as", "dopaminergic", "neurons", "that", "are", "important", "in", "the", "treatment", "of", "Parkinson", "disease", "or", "cholinergic", "neurons", "for", "ALS", "(amyotrophic", "lateral", "sclerosis).", "While", "many", "microarray", "studies", "have", "explored", "molecular", "differences", "between", "hESCs", "and", "derived", "NPs,", "most,", "if", "not", "all,", "have", "focused", "on", "transcriptional", "changes.", "These", "studies", "have", "largely", "ignored", "intermediate", "RNA", "processing", "events", "prior", "to", "and", "during", "translation.", "In", "recent", "years,", "AS", "has", "gained", "momentum", "as", "being", "important", "in", "development,", "apoptosis,", "and", "cancer.", "REAP,", "a", "regression-based", "method", "for", "analyzing", "exon", "array", "data", "was", "introduced,", "and", "was", "applied", "to", "discover", "AS", "events", "in", "hESCs,", "their", "derived", "NPs,", "and", "in", "hCNS-SCns.", "REAP", "was", "based", "on", "the", "assumptions", "that", "most", "exons", "in", "the", "gene", "of", "interest", "and", "in", "the", "genome", "are", "constitutively", "spliced", "and", "that", "outliers", "in", "a", "linear", "pairwise", "comparison", "of", "the", "signal", "estimates", "for", "probesets", "in", "a", "gene", "could", "be", "detected", "using", "a", "robust", "regression-based", "approach.", "REAP", "predictions", "were", "found", "to", "correlate", "well", "with", "transcript-based", "methods", "for", "identifying", "alternative", "exons,", "which", "interestingly", "suggested", "that", "current", "databases", "of", "transcript", "information,", "albeit", "not", "specifically", "enriched", "for", "hESC", "or", "NPs,", "in", "aggregate", "are", "nevertheless", "predictive", "of", "AS", "events", "in", "hESC", "and", "NP.", "In", "addition,", "REAP[+]", "exons", "were", "also", "enriched", "for", "ACEScan-predicted", "evolutionarily", "conserved", "exons", "[55].", "As", "ACEScan", "utilized", "a", "different", "set", "of", "information", "from", "REAP,", "the", "agreement", "between", "both", "algorithms", "served", "to", "further", "validate", "the", "predicted", "alternative", "exons.", "Additional", "studies", "in", "mouse", "ESCs", "and", "neural", "derivatives", "will", "be", "necessary", "to", "determine", "if", "these", "AS", "events", "are", "indeed", "preserved", "in", "these", "analogous", "and", "orthologous", "cell", "types.", "Our", "finding", "that", "only", "a", "minority", "of", "AS", "events", "was", "common", "between", "various", "hESC", "to", "NP", "comparisons", "is", "intriguing.", "A", "possible", "explanation", "is", "that", "the", "cell", "lines", "were", "not", "only", "genetically", "different,", "but", "were", "also", "exposed", "to", "different", "isolation", "and", "culture", "conditions.", "In", "addition,", "the", "different", "differentiation", "protocols", "established", "as", "optimal", "for", "generating", "Nestin", "and", "Sox1", "positive", "neural", "precursors", "may", "lead", "to", "vastly", "different", "molecular", "changes.", "It", "is", "likely", "that", "post-transcriptional", "changes", "such", "as", "AS", "may", "be", "more", "variable", "despite", "the", "cells", "being", "at", "acknowledged", "“end-points”", "defined", "by", "a", "limited", "set", "of", "immunohistochemical", "markers.", "Our", "results", "are", "consistent", "with", "a", "recent", "study", "that", "showed", "that", "while", "two", "well-established", "hESC", "lines", "differentiate", "into", "functional", "neurons,", "the", "two", "lines", "exhibited", "distinct", "differentiation", "potentials,", "suggesting", "that", "some", "preprogramming", "had", "occurred", "[76].", "In", "particular,", "microRNA", "profiling", "revealed", "significant", "expression", "differences", "between", "the", "two", "hESC", "lines,", "suggesting", "that", "microRNAs,", "known", "post-transcriptional", "regulators,", "may", "sway", "the", "differentiation", "properties", "of", "the", "cell", "lines", "[76].", "We", "postulated", "that", "AS", "events", "may", "serve", "also", "to", "bias", "the", "differentiation", "spectrum", "of", "the", "cells,", "an", "important", "avenue", "for", "future", "work.", "Experimental", "validation", "of", "REAP[+]", "exons", "suggested", "a", "high", "specificity", "at", "the", "expense", "of", "relatively", "moderate", "sensitivity.", "We", "believe", "that", "the", "high", "FP", "rates", "may", "arise", "from", "cross-hybridization", "effects", "that", "remained", "unaccounted", "for.", "However,", "our", "specificity", "of", "77%", "at", "the", "cutoff", "of", "two", "significant", "outliers", "per", "probeset", "allowed", "us", "to", "estimate", "that", "at", "least", "1,336", "of", "1,737", "REAP[+]", "exons", "were", "true", "AS", "events", "that", "changed", "during", "neuronal", "differentiation", "of", "hESC", "cells,", "and/or", "were", "different", "between", "endogeneous", "NPs", "and", "hESC.", "On", "average,", "7%", "of", "all", "human", "exons", "have", "been", "estimated", "by", "transcript", "data", "to", "undergo", "AS;", "thus", "REAP's", "validation", "rate", "of", "60%", "at", "the", "cutoff", "of", "two", "is", "73-fold", "(60/7)", "higher", "than", "expected.", "In", "addition,", "we", "validated", "nine", "novel", "AS", "events", "that", "distinguish", "hESCs", "and", "NPs.", "Consistent", "with", "our", "computational", "results,", "we", "observed", "that", "the", "AS", "patterns", "in", "hCNS-SCns", "were", "not", "always", "similar", "to", "those", "of", "the", "derived", "NPs.", "It", "was", "important", "to", "point", "out", "that", "while", "transcriptional", "expression", "of", "these", "genes", "did", "not", "distinguish", "these", "cells", "from", "one", "another,", "in", "several", "instances", "the", "REAP-predicted", "AS", "event", "was", "able", "to", "separate", "derived", "NPs", "and", "hCNS-SCns.", "A", "notable", "exception", "was", "the", "alternative", "exon", "in", "the", "SLK", "gene,", "encoding", "a", "serine/threonine", "kinase", "protein,", "which", "was", "commonly", "included", "in", "both", "hESCs,", "i.e.,", "the", "exon-excluded", "isoform", "was", "not", "present", "in", "hESCs", "compared", "to", "NPs,", "as", "well", "as", "in", "a", "variety", "of", "differentiated", "tissues.", "Closer", "inspection", "of", "the", "REAP[+]-validated", "AS", "exon", "in", "the", "SLK", "gene", "revealed", "strong", "conservation", "in", "the", "intronic", "region", "flanking", "the", "exon,", "a", "hallmark", "feature", "of", "evolutionarily", "conserved", "AS", "exons", "[55,77,78].", "A", "study", "analyzing", "the", "expression", "patterns", "of", "the", "SLK", "gene", "suggested", "a", "potential", "functional", "role", "during", "embryonic", "development", "and", "in", "the", "adult", "central", "nervous", "system", "[79];", "however,", "to", "our", "knowledge,", "our", "identification", "of", "the", "SLK", "alternative", "exon", "is", "the", "first", "report", "of", "a", "hESC-biased", "AS", "pattern", "during", "neuronal", "differentiation", "and", "across", "a", "myriad", "of", "differentiated", "tissues.", "In", "agreement,", "GO", "analysis", "suggested", "that", "genes", "containing", "REAP[+]", "exons", "were", "enriched", "in", "serine/threonine", "kinase", "activity,", "of", "which", "SLK", "is", "a", "family", "member.", "Future", "work", "will", "be", "required", "to", "study", "the", "impact", "of", "AS", "in", "these", "genes", "in", "hESCs", "and", "NPs.", "We", "predict", "it", "is", "unlikely", "that", "the", "alternative", "exon", "in", "the", "SLK", "gene", "is", "the", "only", "case", "common", "across", "hESC", "and", "different", "from", "differentiated", "tissues,", "but", "further", "studies", "will", "be", "necessary", "to", "identify", "other", "hESC-specific", "exons.", "REAP[+]", "exons", "were", "underrepresented", "in", "genes", "that", "were", "differentially", "transcriptionally", "regulated", "in", "hESCs", "and", "NPs.", "Our", "results", "act", "as", "a", "reminder", "that", "focusing", "only", "on", "genes", "that", "are", "differentially", "expressed", "will", "overlook", "RNA", "processing", "events", "that", "may", "be", "biologically", "relevant", "to", "the", "system", "of", "interest.", "Finally,", "we", "identified", "potential", "cis-regulatory", "intronic", "elements", "conserved", "and", "enriched", "proximal", "to", "the", "REAP[+]", "exons.", "In", "particular,", "the", "FOX1/2", "binding", "site,", "GCUAG,", "was", "conserved", "and", "enriched", "in", "the", "flanking", "introns", "of", "a", "subset", "of", "REAP[+]", "exons.", "Further", "studies", "will", "be", "required", "to", "explore", "the", "importance", "of", "FOX1", "family", "members", "in", "early", "neuronal", "differentiation.", "In", "conclusion,", "our", "introduction", "of", "REAP", "and", "its", "application", "to", "identifying", "AS", "events", "has", "revealed", "new", "and", "unanticipated", "insights", "into", "hESC", "biology", "and", "their", "transition", "to", "NP", "cells.", "Collectively,", "these", "exons", "represent", "a", "set", "of", "molecular", "changes", "that", "are", "likely", "to", "be", "important", "for", "studying", "human", "neural", "differentiation", "with", "applications", "in", "neuronal", "regenerative", "medicine.", "Maintenance", "and", "differentiation", "of", "hESCs", "and", "hCNS-SCns.hESC", "line", "Cy203", "(Cythera)", "was", "cultured", "as", "previously", "described", "[12].", "To", "differentiate", "into", "neuroepithelial", "precursor", "cells,", "colonies", "were", "manually", "isolated", "from", "mouse", "embryonic", "fibroblasts", "(MEFs)", "and", "cut", "in", "small", "pieces.", "These", "pieces", "were", "transferred", "to", "a", "T75", "flask", "with", "hESCs", "differentiation", "media", "(same", "hESC", "medium", "but", "10%", "KSR", "and", "no", "FGF-2).", "Medium", "was", "changed", "the", "next", "day", "by", "transferring", "the", "floating", "hESC", "aggregates", "to", "a", "new", "flask.", "After", "culturing", "for", "a", "week,", "the", "hESC", "cell", "aggregates", "formed", "mature", "embroid", "bodies", "(EBs;", "∼10", "um", "round", "clusters", "with", "dark", "centers).", "EBs", "were", "plated", "on", "a", "coated", "10-cm", "dish", "in", "hESC", "differentiation", "media.", "The", "next", "day,", "the", "medium", "was", "changed", "to", "DMEM/F12", "supplemented", "with", "ITS", "and", "fibronectin.", "Medium", "was", "changed", "every", "other", "day", "for", "a", "week", "or", "until", "the", "cells", "formed", "rosette-like", "columnar", "structures", "that", "were", "isolated", "manually.", "These", "structures", "were", "then", "transferred", "to", "coated", "dishes", "in", "neural", "induction", "medium", "(DMEM/F12", "supplemented", "with", "N2", "and", "FGF-2)", "for", "a", "week.", "Elongated", "single", "cells", "were", "separated", "from", "leftover", "aggregates", "using", "non-enzymatic", "dissociation.", "After", "one", "to", "two", "passages,", "the", "cells", "formed", "a", "monolayer", "of", "homogeneous", "NPs", "(negative", "for", "Sox1", "immunostaining).", "Upon", "confluence,", "cells", "will", "form", "neurospheres", "that", "can", "also", "be", "isolated", "from", "the", "neuroepithelial", "precursor", "cells", "(positive", "for", "Sox1", "immunostaining).", "At", "any", "of", "these", "two", "stages,", "pan-neuronal", "differentiation", "can", "be", "achieved", "after", "three", "to", "four", "weeks.", "hESC", "line", "HUES6", "was", "cultured", "on", "MEF", "feeders", "as", "previously", "described", "(http://www.mcb.harvard.edu/melton/hues/)", "or", "on", "GFR", "matrigel", "coated", "plates.", "Cells", "grown", "on", "matrigel", "were", "grown", "in", "MEF-conditioned", "medium", "and", "FGF-2", "was", "used", "at", "20", "ng/mL", "instead", "of", "10", "ng/mL", "for", "cells", "grown", "on", "MEFs.", "To", "differentiate", "neuroepithelial", "precursors,", "colonies", "were", "removed", "by", "treatment", "with", "collagenase", "IV", "(Sigma)", "and", "washed", "three", "times", "in", "growth", "media.", "The", "pieces", "of", "colonies", "were", "resuspended", "in", "HUES", "growth", "media", "without", "FGF2", "in", "an", "uncoated", "bacterial", "Petri", "dish", "to", "form", "EBs.", "After", "one", "week,", "EBs", "were", "plated", "on", "polyornathine/laminin", "coated", "plates", "in", "DMEM/F12", "supplemented", "with", "N2", "and", "FGF2.", "Rosette", "structures", "were", "manually", "collected", "and", "enzymatically", "dissociated", "with", "TryPLE", "(Invitrogen),", "plated", "on", "polyornathine/laminin", "coated", "plates,", "and", "grown", "in", "DMEM/F12", "supplemented", "with", "N2", "and", "B27-RA", "and", "20", "ng/mL", "FGF-2.", "Cells", "could", "be", "grown", "as", "a", "monolayer", "for", "up", "to", "at", "least", "ten", "passages.", "Cells", "were", "Sox1", "and", "nestin", "positive", "and", "readily", "differentiated", "into", "neurons", "upon", "withdrawal", "of", "FGF-2.", "Human", "central", "nervous", "system", "stem", "cell", "line", "FBR1664", "(StemCells)", "which", "is", "referred", "to", "as", "hCNS-SCns", "in", "the", "main", "text", "was", "cultured", "as", "previously", "described", "[23].", "The", "cells", "were", "cultured", "in", "medium", "consisting", "of", "Ex", "Vivo", "15", "(BioWhittaker)", "medium", "with", "N2", "supplement", "(GIBCO),", "FGF2", "(20", "ng/mL),", "epidermal", "growth", "factor", "(20", "ng/mL),", "lymphocyte", "inhibitory", "factor", "(10", "ng/mL),", "0.2", "mg/ml", "heparin,", "and", "60", "ug/mL", "N-acetylcysteine.", "Cultures", "were", "fed", "weekly", "and", "passaged", "at", "∼two", "to", "three", "weeks", "using", "collagenases", "(Roche).", "The", "following", "antibodies", "and", "corresponding", "dilutions", "were", "utilized", "for", "the", "immunohistochemical", "analysis", "of", "marker", "genes", "in", "Cyt-ES", "and", "HUES6-ES:", "Sox2", "(Chemicon,", "1:500),", "Oct4", "(Santa", "Cruz,", "1:500),", "Sox1", "(Chemicon,", "1:500),", "Nestin", "(Pharmingen,", "1:250);", "hCNS-SCns:", "Sox2", "(Chemicon,", "1:200),", "Nestin", "(Chemicon,", "1:200).", "RNA", "preparation", "and", "array", "hybridization.Total", "RNA", "from", "cells", "was", "processed", "as", "follows.", "Cells", "were", "lysed", "in", "1", "mL", "of", "RNA-bee", "(Teltest).", "The", "RNA", "was", "isolated", "by", "chloroform", "extraction", "of", "the", "aqueous", "phase,", "followed", "by", "isopropanol", "precipitation", "as", "per", "the", "manufacturer's", "instructions.", "The", "precipitated", "RNA", "was", "washed", "in", "75%", "ethanol", "and", "eluted", "with", "DEPC-treated", "water.", "Five", "ug", "of", "RNA", "was", "treated", "with", "RQ1", "DNAase", "(Promega)", "according", "to", "the", "manufacturer's", "instructions.", "One", "ug", "of", "total", "RNA", "for", "each", "sample", "was", "processed", "using", "the", "Affymetrix", "GeneChip", "Whole", "Transcript", "Sense", "Target", "Labeling", "Assay", "(Affymetrix).", "Ribosomal", "RNA", "was", "reduced", "with", "the", "RiboMinus", "Kit", "(Invitrogen).", "Target", "material", "was", "prepared", "using", "commercially", "available", "Affymetrix", "GeneChip", "WT", "cDNA", "Synthesis", "Kit,", "WT", "cDNA", "Amplification", "Kit,", "and", "WT", "Terminal", "Labeling", "Kit", "(Affymetrix)", "as", "per", "manufacturer's", "instructions.", "Hybridization", "cocktails", "containing", "∼5", "ug", "of", "fragmented", "and", "labeled", "DNA", "target", "were", "prepared", "and", "applied", "to", "GeneChip", "Human", "Exon", "1.0", "ST", "arrays.", "Hybridization", "was", "performed", "for", "16", "hours", "using", "the", "Fluidics", "450", "station.", "Arrays", "were", "scanned", "using", "the", "Affymetrix", "3000", "7G", "scanner", "and", "GeneChip", "Operating", "Software", "version", "1.4", "to", "produce", ".CEL", "intensity", "files.", "Detection", "of", "AS", "by", "RT-PCR.cDNAs", "were", "generated", "from", "total", "RNA", "with", "Superscript", "III", "reverse", "transcriptase", "(Invitrogen).", "PCR", "reactions", "were", "performed", "with", "primer", "pairs", "designed", "for", "AS", "targets", "(annealing", "at", "58", "°C", "and", "amplification", "for", "30", "or", "35", "cycles).", "PCR", "products", "were", "resolved", "on", "either", "1.5%", "or", "3%", "agarose", "gel", "in", "TBE.", "The", "Ethidium", "Bromide-stained", "gels", "were", "scanned", "with", "Typhoon", "8600", "scanner", "(Molecular", "Dynamics)", "for", "quantification.", "The", "number", "of", "true", "positives", "(TP;", "false", "negatives,", "FN)", "was", "computed", "as", "the", "number", "of", "REAP[+]", "(REAP[−])", "exons", "that", "were", "validated", "by", "RT-PCR", "as", "AS.", "The", "number", "of", "true", "negatives", "(TN;", "or", "FPs)", "was", "computed", "as", "the", "number", "of", "REAP[−]", "(REAP[+])", "exons", "that", "were", "validated", "by", "RT-PCR", "as", "constitutively", "spliced.", "The", "true", "(false)", "positive", "rate", "was", "computed", "as", "TP", "(FP)", "divided", "by", "the", "total", "number", "of", "REAP[+]", "exons", "in", "the", "experimentally", "validated", "set.", "The", "true", "(false)", "negative", "rate", "was", "computed", "as", "the", "TN", "(FN)", "divided", "by", "the", "total", "number", "of", "REAP[−]", "exons", "in", "the", "experimentally", "validated", "set.", "Sensitivity", "was", "computed", "as", "TP/(TP+FN)", "and", "specificity", "was", "computed", "as", "TN/(FP+TN).", "Sequence", "databases.Genome", "sequences", "of", "human", "(hg17),", "dog", "(canFam1),", "rat", "(rn3),", "and", "mouse", "(mm5)", "were", "obtained", "from", "UCSC,", "as", "were", "the", "whole-genome", "MULTIZ", "alignments", "[80].", "The", "lists", "of", "known", "human", "genes", "(knownGene", "containing", "43,401", "entries)", "and", "known", "isoforms", "(knownIsoforms", "containing", "43,286", "entries", "in", "21,397", "unique", "isoform", "clusters)", "with", "annotated", "exon", "alignments", "to", "human", "hg17", "genomic", "sequence", "were", "processed", "as", "follows.", "Known", "genes", "that", "were", "mapped", "to", "different", "isoform", "clusters", "were", "discarded.", "All", "mRNAs", "aligned", "to", "hg17", "that", "were", "greater", "than", "300", "bases", "long", "were", "clustered", "together", "with", "the", "known", "isoforms.", "Genes", "containing", "less", "than", "three", "exons", "were", "removed", "from", "further", "consideration.", "A", "total", "of", "2.7", "million", "spliced", "ESTs", "were", "mapped", "onto", "the", "17,478", "high-quality", "genes", "to", "infer", "AS.", "Exons", "with", "canonical", "splice", "signals", "(GT-AG,", "AT-AC,", "GC-AG)", "were", "retained,", "resulting", "in", "a", "total", "of", "213,736", "exons.", "Of", "these,", "197,262", "(92%", "of", "all", "exons)", "were", "constitutive", "exons,", "13,934", "exons", "(7%)", "had", "evidence", "of", "exon-skipping,", "1,615", "(1%)", "exons", "were", "mutually", "exclusive", "alternative", "events,", "5,930", "(3%)", "exons", "had", "alternative", "3′", "splice", "sites,", "5,181", "(2%)", "exons", "had", "alternative", "5′", "splice", "sites,", "and", "175", "(<1%)", "exons", "overlapped", "another", "exon,", "but", "did", "not", "fall", "into", "the", "above", "classifications.", "A", "total", "of", "324,139", "probesets", "from", "the", "Affymetrix", "Human", "Exon", "1.0", "ST", "array", "were", "mapped", "to", "208,422", "human", "exons,", "representing", "17,431", "genes.", "These", "probesets", "were", "used", "to", "derive", "gene", "and", "exon-level", "signal", "estimates", "from", "the", "CEL", "files.", "The", "four-way", "mammalian", "(four-mammal)", "whole-genome", "alignment", "(hg17,", "canFam1,", "mm5,", "rn3)", "was", "extracted", "from", "the", "eight-way", "vertebrate", "MULTIZ", "alignments", "(hg17,", "panTrol1,", "mm5,", "rn3,", "canFam1,", "galGal2,", "fr1,", "danRer1)", "obtained", "from", "the", "UCSC", "Genome", "Browser.", "Four-way", "mammal", "alignments", "were", "extracted", "for", "all", "internal", "exons,", "and", "400", "bases", "of", "flanking", "intronic", "sequence,", "resulting", "in", "a", "total", "of", "161,731", "conserved", "internal", "exons.", "A", "total", "of", "145,613", "(90%", "of", "total)", "conserved", "internal", "exons", "were", "constitutive", "exons,", "13,653", "exons", "(8%)", "had", "evidence", "of", "exon-skipping,", "1,576", "exons", "were", "mutually", "exclusive", "alternative", "events,", "5,818", "exons", "had", "alternative", "3′", "splice", "sites,", "5,046", "exons", "had", "alternative", "5′", "splice", "sites,", "and", "168", "exons", "overlapped", "another", "exon.", "Exon", "array", "analysis.The", "Affymetrix", "Power", "Tools", "(APT)", "suite", "of", "programs", "was", "obtained", "from", "http://www.affymetrix.com/support/developer/powertools/index.affx.", "Exon", "(probeset)", "and", "gene-level", "signal", "estimates", "were", "derived", "from", "the", "CEL", "files", "by", "RMA–sketch", "normalization", "as", "a", "method", "in", "the", "apt-probeset-summarize", "program.", "To", "determine", "if", "the", "signal", "intensity", "for", "a", "given", "probeset", "is", "above", "the", "expected", "level", "of", "background", "noise,", "we", "utilized", "the", "DABG", "(detection", "above", "background)", "quantification", "method", "available", "in", "the", "apt-probeset-summarize", "program", "as", "part", "of", "Affymetrix", "Power", "Tools", "(APT).", "Briefly,", "DABG", "compared", "the", "signal", "for", "each", "probe", "to", "a", "background", "distribution", "of", "signals", "from", "anti-genomic", "probes", "with", "the", "same", "GC", "content.", "The", "DABG", "algorithm", "generated", "a", "p-value", "representing", "the", "probability", "that", "the", "signal", "intensity", "of", "a", "given", "probe", "was", "part", "of", "the", "background", "distribution.", "We", "considered", "a", "probeset", "with", "a", "DABG", "p-value", "lower", "than", "0.05", "as", "detected", "above", "background.", "The", "statistic", "thCNS-SCns,ESC", "=", "(μhCNS-SCns", "−", "μESC)", "/", "sqrt", "(((nhCNS-SCns", "−", "1)σ2hCNS-SCns", "+", "(nESC", "−", "1)σ2ESC)(nhCNS-SCns", "+", "nESC))", "/", "((nhCNS-SCnsnESC)", "(nhCNS-SCns", "+", "nESC", "−", "2))),", "where", "nhCNS-SCns", "and", "nESC", "were", "the", "number", "of", "replicates,", "μhCNS-SCns", "and", "μESC", "were", "the", "mean,", "and", "σ2hCNS-SCns", "and", "σ2ESC", "were", "the", "variances", "of", "the", "expression", "values", "for", "the", "two", "datasets", "used", "to", "represent", "the", "differential", "enrichment", "of", "a", "gene", "using", "gene-level", "estimates", "in", "hCNS-SCns", "relative", "to", "hESCs.", "Multiple", "hypothesis", "testing", "was", "corrected", "by", "controlling", "for", "the", "false", "discovery", "rate", "(Benjamini-Hochberg).", "AS", "detection", "by", "REAP.The", "log2", "signal", "estimate", "xij", "for", "probeset", "i", "in", "cell-type", "j", "had", "to", "satisfy", "two", "conditions,", "otherwise", "the", "probeset", "was", "discarded:", "(i)", "2", "<", "xij", "<", "10,000", "for", "all", "conditions/cell-types", "j;", "and", "(ii)", "DABG", "p-value", "<", "0.05", "for", "all", "replicates", "in", "at", "least", "one", "condition/cell-type", "j.", "A", "gene", "had", "to", "have", "five", "probesets", "that", "satisfied", "the", "two", "conditions", "above", "in", "order", "to", "be", "considered", "for", "robust", "regression", "analysis.", "After", "generating", "the", "points", "(as", "described", "in", "the", "Results", "section),", "we", "utilized", "the", "robust", "regression", "method", "rlm", "in", "R-package", "“MASS”", "(version", "6.1–2)", "with", "M-estimation", "and", "a", "maximum", "iteration", "setting", "of", "30", "to", "estimate", "the", "linear", "function", "yi", "=", "αxi", "+", "β.", "For", "each", "probeset,", "we", "computed", "the", "error", "term", "ei,,", "which", "was", "the", "difference", "between", "the", "actual", "value", "yi", "and", "the", "estimated", "value", "ξi,", "from", "the", "estimated", "function", "ξi", "=", "Axi", "+", "B,", "where", "A", "and", "B", "were", "estimates", "of", "α", "and", "β.", "The", "error", "term", "variance", "was", "estimated", "by", "se2", "=", "Σei2/(n", "−", "p),", "which", "was", "used", "to", "estimate", "the", "variance", "of", "the", "predicted", "value,", "sξi2", "=", "se2(n−1", "+", "(xi", "−", "μx)2", "/", "sx2(n", "−", "1)).", "Here,", "n", "referred", "to", "the", "number", "of", "points", "(generated", "for", "each", "gene),", "and", "p", "referred", "to", "the", "number", "of", "independent", "variables", "(p", "=", "2", "in", "our", "method);", "and", "μx", "=", "Σxi2/n;", "sx2", "=", "n−1", "Σ(xi", "−", "μx)2.", "Following", "Belsley", "et", "al.", "[81],", "we", "defined", "the", "leverage", "hi", "of", "the", "ith", "point", "as", "hi", "=", "n−1", "+", "(xi", "−", "μx)2", "/", "sx2(n", "−", "1).", "Here", "we", "considered", "a", "point", "to", "have", "high", "leverage", "if", "hi", ">", "3p/n.", "Next,", "we", "calculated", "the", "covariance", "ratio,", "covi", "=", "(si2/sr2)p/(1", "−", "hi),", "which", "is", "the", "ratio", "of", "the", "determinant", "of", "the", "covariance", "matrix", "after", "deleting", "the", "ith", "observation", "to", "the", "determinant", "of", "the", "covariance", "matrix", "with", "the", "entire", "sample.", "We", "considered", "a", "point", "to", "have", "high", "influence", "if", "|covi", "−", "1|", ">", "3p/n.", "Lastly,", "we", "computed", "the", "studentized", "residuals,", "rstudenti", "=", "ei", "/", "(s(i)2", "(1", "−", "hi)0.5),", "where", "s(i)2", "=", "(n-p)se2", "/", "(n-p-1)", "–", "ei2", "/", "(n-p-1)(1", "−", "hi),", "the", "error", "term", "variance", "after", "deleting", "the", "ith", "point.", "As", "rstudenti", "was", "distributed", "as", "Student's", "t-distribution", "with", "n-p-1", "degrees", "of", "freedom,", "each", "rstudenti", "value", "was", "associated", "with", "a", "p-value.", "We", "considered", "a", "point", "to", "be", "an", "“outlier”", "if", "p", "<", "0.01.", "Identification", "of", "motifs.The", "enrichment", "score", "of", "a", "sequence", "element", "of", "length", "k", "(k-mer)", "in", "one", "set", "of", "sequences", "(set", "1)", "versus", "another", "set", "of", "sequences", "(set", "2)", "was", "represented", "by", "the", "nonparametric", "χ2", "statistic", "with", "Yates", "correction,", "computed", "from", "the", "two", "by", "two", "contingency", "table,", "T", "(T11:", "number", "of", "occurrences", "of", "the", "element", "in", "set", "1;", "T12:", "number", "of", "occurrences", "of", "all", "other", "elements", "of", "similar", "length", "in", "set", "1;", "T21:", "number", "of", "occurrences", "of", "element", "in", "set", "2;", "T22:", "number", "of", "occurrences", "of", "all", "other", "elements", "of", "similar", "length", "in", "set", "2.", "All", "elements", "had", "to", "be", "greater", "than", "5.", "To", "correct", "for", "multiple", "hypothesis", "testing,", "p-values", "were", "multiplied", "by", "the", "total", "number", "of", "comparisons." ]
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9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9 ]
[ "BackgroundHuman", "embryonic", "stem", "cells", "(hESCs)", "offer", "a", "virtually", "unlimited", "source", "of", "neural", "cells", "for", "structural", "repair", "in", "neurological", "disorders,", "such", "as", "stroke.", "Neural", "cells", "can", "be", "derived", "from", "hESCs", "either", "by", "direct", "enrichment,", "or", "by", "isolating", "specific", "growth", "factor-responsive", "and", "expandable", "populations", "of", "human", "neural", "stem", "cells", "(hNSCs).", "Studies", "have", "indicated", "that", "the", "direct", "enrichment", "method", "generates", "a", "heterogeneous", "population", "of", "cells", "that", "may", "contain", "residual", "undifferentiated", "stem", "cells", "that", "could", "lead", "to", "tumor", "formation", "in", "vivo.Methods/Principal", "FindingsWe", "isolated", "an", "expandable", "and", "homogenous", "population", "of", "hNSCs", "(named", "SD56)", "from", "hESCs", "using", "a", "defined", "media", "supplemented", "with", "epidermal", "growth", "factor", "(EGF),", "basic", "fibroblast", "growth", "factor", "(bFGF)", "and", "leukemia", "inhibitory", "growth", "factor", "(LIF).", "These", "hNSCs", "grew", "as", "an", "adherent", "monolayer", "culture.", "They", "were", "fully", "neuralized", "and", "uniformly", "expressed", "molecular", "features", "of", "NSCs,", "including", "nestin,", "vimentin", "and", "radial", "glial", "markers.", "These", "hNSCs", "did", "not", "express", "the", "pluripotency", "markers", "Oct4", "or", "Nanog,", "nor", "did", "they", "express", "markers", "for", "the", "mesoderm", "or", "endoderm", "lineages.", "The", "self-renewal", "property", "of", "the", "hNSCs", "was", "characterized", "by", "a", "predominant", "symmetrical", "mode", "of", "cell", "division.", "The", "SD56", "hNSCs", "differentiated", "into", "neurons,", "astrocytes", "and", "oligodendrocytes", "throughout", "multiple", "passages", "in", "vitro,", "as", "well", "as", "after", "transplantation.", "Together,", "these", "criteria", "confirm", "the", "definitive", "NSC", "identity", "of", "the", "SD56", "cell", "line.", "Importantly,", "they", "exhibited", "no", "chromosome", "abnormalities", "and", "did", "not", "form", "tumors", "after", "implantation", "into", "rat", "ischemic", "brains", "and", "into", "naïve", "nude", "rat", "brains", "and", "flanks.", "Furthermore,", "hNSCs", "isolated", "under", "these", "conditions", "migrated", "toward", "the", "ischemia-injured", "adult", "brain", "parenchyma", "and", "improved", "the", "independent", "use", "of", "the", "stroke-impaired", "forelimb", "two", "months", "post-transplantation.Conclusions/SignificanceThe", "SD56", "human", "neural", "stem", "cells", "derived", "under", "the", "reported", "conditions", "are", "stable,", "do", "not", "form", "tumors", "in", "vivo", "and", "enable", "functional", "recovery", "after", "stroke.", "These", "properties", "indicate", "that", "this", "hNSC", "line", "may", "offer", "a", "renewable,", "homogenous", "source", "of", "neural", "cells", "that", "will", "be", "valuable", "for", "basic", "and", "translational", "research.", "To", "date", "there", "have", "been", "no", "effective", "treatments", "for", "improving", "residual", "structural", "and", "functional", "deficits", "resulting", "from", "stroke.", "Current", "therapeutic", "approaches,", "such", "as", "the", "use", "of", "thrombolytics,", "benefit", "only", "1", "to", "4%", "of", "patients", "[1].", "Consequently,", "the", "majority", "of", "stroke", "patients", "experience", "progression", "of", "ischemia", "associated", "with", "debilitating", "neurological", "deficits.", "Recent", "evidence", "has", "suggested", "that", "the", "transplantation", "of", "cells", "derived", "from", "cord", "blood,", "bone", "marrow", "or", "brain", "tissue", "(fetal", "and", "adult)", "enhances", "sensorimotor", "function", "in", "experimental", "models", "of", "stroke", "[2],", "[3].", "However,", "the", "normal", "human-derived", "somatic", "stem", "cells", "used", "in", "these", "studies", "have", "a", "limited", "capacity", "to", "differentiate", "into", "the", "diverse", "neural", "cell", "types", "optimal", "for", "structural", "and", "physiological", "tissue", "repair", "and", "are", "not", "amenable", "for", "large-scale", "cell", "production.", "Unlike", "other", "sources", "of", "stem", "cells,", "hESC", "lines", "possess", "a", "nearly", "unlimited", "self-renewal", "capacity", "and", "the", "developmental", "potential", "to", "differentiate", "into", "virtually", "any", "cell", "type", "of", "the", "organism.", "As", "such,", "they", "constitute", "an", "ideal", "source", "of", "cells", "for", "regenerative", "medicine.", "The", "successful", "derivation", "of", "hESC", "lines", "from", "the", "inner", "cell", "mass", "of", "preimplantation", "embryos", "and", "their", "long-term", "maintenance", "in", "vitro", "over", "multiple", "passages", "has", "been", "demonstrated", "[4]", "and", "standardized.", "Differentiation", "and", "enrichment", "processes", "that", "direct", "hESCs", "towards", "a", "neural", "lineage", "have", "also", "been", "achieved.", "To", "promote", "neuralization,", "ESCs", "were", "cultured", "in", "a", "defined", "media", "supplemented", "with", "morphogens", "or", "growth", "factors", "[5],", "[6],", "[7]", "or", "cultured", "under", "conditions", "that", "promote", "“rosettes”,", "structures", "morphologically", "similar", "to", "the", "developing", "neural", "tube", "[8],", "[9].", "This", "neuralization", "process", "has", "proven", "invaluable", "in", "understanding", "the", "specification", "of", "hESC-derived", "neural", "tissue", "[10],", "[11],", "[12].", "However,", "the", "enriched", "neural", "progeny", "derived", "displayed", "overgrowth", "and", "limited", "migration", "after", "grafting", "into", "normal", "newborn", "mice", "[13]", "and", "lesioned", "adult", "rat", "striatum", "[12],", "[14],", "[15],", "[16].", "The", "inner", "cores", "of", "these", "grafts", "contained", "tumorigenic", "precursor", "cells", "(reviewed", "in", "[17]).", "These", "findings", "suggest", "that", "neural", "cells", "generated", "by", "acute", "exposure", "to", "growth", "factors", "and/or", "morphogens", "may", "still", "be", "heterogeneous", "and", "potentially", "tumorigenic.", "We", "report", "an", "alternative", "method", "for", "the", "isolation", "and", "the", "perpetuation", "of", "a", "multipotent", "hNSC", "line", "from", "the", "hESCs", "with", "a", "primitive", "mode", "of", "self-renewal.", "We", "also", "demonstrate", "their", "long-term", "expansion,", "non-tumorigenic", "properties", "and", "functional", "engraftability", "in", "an", "experimental", "model", "of", "stroke.", "1.", "In", "vitro", "isolation,", "growth", "and", "differentiation", "of", "hESC-derived", "hNSCsThe", "hESCs", "were", "maintained", "and", "expanded", "on", "mouse", "feeder", "layer", "in", "media", "supplemented", "with", "bFGF", "(Figure", "1A).", "After", "cell", "dissociation,", "a", "portion", "of", "the", "hESCs", "was", "cultured", "in", "serum", "free", "medium", "containing", "EGF,", "bFGF", "and", "LIF.", "These", "factors", "are", "known", "to", "stimulate", "the", "proliferation", "of", "human", "fetal-derived", "NSCs", "[18],", "[19].", "After", "3", "days", "in", "vitro", "(DIV),", "there", "was", "selective", "survival", "and", "growth", "of", "cells", "that", "aggregated", "in", "clusters", "or", "spheres", "(Figure", "1B).", "These", "primary", "spheres", "were", "harvested", "and", "replated", "in", "fresh", "media.", "During", "the", "following", "week,", "the", "spheres", "attached", "to", "the", "flask", "and", "a", "fibroblast-like", "cell", "population", "began", "to", "migrate", "out", "(Figure", "1C).", "Secondary", "spheres", "(2°", "spheres)", "were", "generated", "from", "these", "cultures", "and", "lifted", "off", "by", "the", "end", "of", "the", "week", "leaving", "a", "hollow", "in", "the", "middle", "of", "the", "attached", "cells", "(Figure", "1D).", "The", "floating", "2°", "spheres", "were", "collected", "and", "replated", "in", "fresh", "growth", "medium", "for", "2", "weeks.", "The", "cultures", "were", "then", "passaged", "by", "collagenase", "cell", "dissociation", "every", "7", "DIV", "for", "an", "additional", "4", "passages", "(Figure", "S1).", "At", "the", "5th", "and", "6th", "passages,", "spheres", "were", "dissociated", "into", "a", "single-cell", "suspension", "using", "trypsin-EDTA.", "At", "this", "stage", "there", "was", "a", "change", "in", "the", "hNSCs'", "adherent", "properties,", "and", "the", "cells", "began", "to", "grow", "as", "a", "monolayer", "with", "multiple", "foci", "of", "cells", "throughout", "the", "culture", "(Figure", "1F).", "The", "adherent", "hNSC", "culture", "stained", "uniformly", "for", "nestin", "(Figure", "1K),", "vimentin", "(Figure", "1L)", "and", "with", "the", "radial", "glial", "marker", "3CB2", "(Figure", "1M)", "indicating", "their", "homogeneity", "and", "NSC", "identity.", "Under", "these", "culture", "conditions,", "it", "is", "noteworthy", "that", "we", "did", "not", "observe", "the", "formation", "of", "rosettes", "which", "has", "been", "previously", "reported", "to", "occur", "under", "certain", "conditions", "during", "neuralization", "of", "hESCs", "[8],", "[20],", "[21].", "RT-PCR", "analysis", "confirmed", "that", "these", "hNSCs", "did", "not", "express", "the", "pluripotency", "transcripts", "Oct-4", "and", "Nanog", "(Figure", "1I).", "Furthermore,", "the", "hNSCs", "did", "not", "express", "transcripts", "for", "brachyury", "and", "foxa2,", "marker", "genes", "for", "mesoderm", "and", "endoderm", "respectively", "(negative", "result,", "data", "not", "shown).10.1371/journal.pone.0001644.g001Figure", "1Isolation", "and", "purification", "of", "hNSCs", "from", "the", "hESCs.The", "hESCs", "were", "grown", "on", "a", "mouse", "feeder", "layer", "(A).", "Primary", "neurospheres", "(B)", "were", "isolated", "and", "replated", "to", "eliminate", "other", "non-neural", "cells.", "The", "selectively", "harvested", "secondary", "neurospheres", "(arrow", "in", "C),", "left", "behind", "hollow", "cores", "in", "the", "surface", "area", "(star", "in", "D)", "where", "they", "attached", "earlier.", "They", "were", "perpetuated", "for", "an", "additional", "5", "passages", "(E).", "These", "2°", "spheres", "were", "then", "passaged", "using", "a", "single", "cell", "dissociation", "protocol", "(F).", "Arrow", "in", "F", "shows", "an", "example", "of", "a", "focus", "of", "proliferating", "cells.", "(G,", "H)", "The", "hNSCs", "were", "passaged", "every", "5–7", "days,", "as", "described", "in", "the", "Methods", "section.", "Starting", "from", "an", "initial", "population", "of", "1", "million", "cells,", "the", "cumulative", "cell", "number", "was", "calculated", "at", "each", "passage", "as", "the", "fold", "of", "increase×the", "total", "cell", "number", "and", "plotted", "as", "logarithm", "with", "base", "2", "in", "function", "of", "time", "(G).", "The", "cell", "perpetuation", "(G)", "and", "population", "doubling", "(H)", "analysis", "demonstrated", "the", "continuous", "and", "stable", "growth", "of", "the", "hNSCs.", "(I)", "RT-PCR", "analysis", "showing", "the", "down", "regulation", "of", "the", "pluripotency", "transcripts", "Oct4", "and", "Nanog", "in", "secondary", "neurospheres", "and", "in", "expanded", "hNSCs", "at", "passage", "8", "(P8).", "(J)", "Cytogenetic", "evaluation", "of", "the", "SD56", "hNSCs", "line", "at", "passage", "12", "by", "standard", "G-banding", "was", "performed.", "Twenty", "metaphase", "cells", "were", "analyzed", "and", "showed", "a", "normal", "female", "chromosome", "complement", "(46,XX).", "Isolated", "and", "expanded", "hNSCs", "expressed", "the", "neural", "precursor", "cell", "markers", "nestin", "(K),", "Vimentin", "(L)", "and", "the", "radial", "glial", "cell", "marker", "3CB2", "(M)", "in", "virtually", "all", "the", "progeny.", "(N-P)", "Clonal", "self-renewal", "ability", "of", "the", "isolated", "hNSCs", "through", "symmetrical", "cell", "division.", "Single", "(N),", "two-cell", "stage", "(O)", "and", "four-cell", "stage", "(P)", "of", "a", "hNSC", "proliferating", "over", "a", "3-day", "culture", "period.", "Note", "the", "symmetrical", "segregation", "of", "BrdU", "and", "nestin", "in", "the", "progeny.", "Bars:", "(A,", "B,", "C,", "D)", "200", "µm;", "(E,", "F)", "100", "µm;", "(K–M)", "20", "µm;", "(N–P)", "10", "µm.To", "ascertain", "self-renewal", "ability", "under", "clonal", "conditions,", "a", "single", "cell", "suspension", "was", "plated", "at", "clonal", "density", "(1–2", "cell/10", "µl).", "To", "determine", "if", "the", "hNSCs", "divide", "symmetrically,", "we", "pulsed", "cultures", "with", "the", "thymidine", "analog,", "bromodeoxyuridine", "(BrdU),", "after", "plating", "and", "looked", "for", "the", "expression", "of", "nestin", "in", "the", "progeny.", "Cultures", "were", "fixed", "after", "1,", "2", "or", "3", "DIV", "(Figure", "1N–P).", "After", "2", "days,", "plated", "single", "cells", "first", "underwent", "a", "symmetric", "cell", "division", "and", "gave", "rise", "to", "daughter", "cells", "that", "were", "both", "positive", "for", "BrdU", "and", "nestin.", "The", "clone", "of", "cells", "continued", "to", "grow", "over", "the", "3", "DIV", "and", "all", "the", "progeny", "expressed", "nestin.", "BrdU", "labeling", "demonstrated", "that", "it", "was", "rare", "for", "only", "one", "daughter", "cell", "to", "inherit", "the", "BrdU", "and", "thus", "had", "undergone", "asymmetric", "segregation", "of", "the", "chromatids", "(Figure", "S2).", "G-band", "karyotyping", "of", "these", "hNSCs", "confirmed", "the", "normal,", "non-transformed", "nature", "of", "cells", "after", "12", "passages", "(Figure", "1J).", "We", "named", "the", "derived", "hNSCs", "SD56", "(intermittently", "referred", "to", "as", "SD56", "hNSCs", "or", "hNSCs).Under", "these", "defined", "growth", "conditions,", "the", "hNSCs", "showed", "stable", "growth", "with", "a", "2.7±0.2", "fold", "increase", "every", "5", "to", "7", "days", "(Figure", "1G).", "The", "population", "doubling", "at", "each", "passage", "averaged", "at", "1.4±0.1", "(Figure", "1H).", "The", "viability", "of", "hNSCs", "at", "each", "passage", "was", "consistent", "at", "the", "approximate", "value", "of", "98%.", "The", "projected", "cumulative", "cell", "numbers", "demonstrated", "that", "trillions", "of", "cells", "could", "be", "generated", "over", "a", "period", "of", "5", "months", "(Figure", "1G).", "We", "expanded", "the", "isolated", "hNSCs", "lines", "for", "over", "20", "passages", "with", "a", "stable", "phenotype.", "An", "initial", "cell", "bank", "of", "75", "vials", "containing", "2", "to", "5", "million", "cells", "each", "was", "generated", "and", "cryopreserved.Upon", "removal", "of", "the", "mitogenic", "factors", "and", "plating", "on", "a", "coverslip", "pre-coated", "with", "poly-L-ornithine", "(PLO)", "substrate,", "the", "hNSCs", "spontaneously", "differentiated", "into", "neurons,", "astrocytes", "and", "oligodendrocytes,", "a", "property", "that", "is", "consistent", "with", "normal", "multipotent", "hNSCs", "(Figure", "2).", "After", "2", "DIV,", "hNSCs", "expressed", "transcripts", "for", "the", "neural-specific", "genes", "nestin,", "Notch1", "and", "neural", "cell", "adhesion", "molecule", "(N-CAM)", "(Figure", "2A)", "and", "for", "the", "lineage", "specific", "markers", "β-tubulin", "class", "III,", "medium-size", "neurofilament", "(NF-M)", "and", "microtubule-associated", "protein", "2", "(MAP-2)", "for", "neurons,", "GFAP", "for", "astrocytes", "and", "myelin", "basic", "protein", "(MBP)", "for", "oligodendrocytes", "(Figure", "2A).", "Transcripts", "for", "the", "GABAergic", "cell", "marker", "glutamic", "acid", "decarboxylase", "(GAD)", "were", "expressed,", "but", "transcripts", "for", "the", "tyrosine", "hydroxylase", "(TH),", "a", "marker", "for", "dopaminergic", "neurons,", "were", "undetectable.", "Immunocytochemical", "analysis", "(Figure", "2B–F)", "of", "10", "day-old", "cultures", "demonstrated", "that", "the", "proportion", "of", "nestin+", "cells", "was", "36.6±2.7%,", "62.5±2.8%", "expressed", "the", "neuronal", "marker", "TuJ1,", "1.9±0.3%", "expressed", "the", "astrocytic", "marker", "GFAP", "and", "7.1±0.4%", "were", "oligodendrocytes", "and", "expressed", "galactocerebrocide", "(GC)", "(Figure", "2F).", "A", "subset", "(9.8±1.6%)", "of", "the", "GFAP+", "astrocytes", "co-expressed", "nestin.10.1371/journal.pone.0001644.g002Figure", "2hESC-derived", "hNSCs", "spontaneously", "differentiated", "into", "the", "3", "principal", "central", "nervous", "system", "cell", "types.Dissociated", "hNSCs", "were", "washed", "free", "of", "growth", "factors", "and", "plated", "on", "poly-L-onithine", "coated", "glass", "coverslips.", "Differentiated", "cultures", "were", "either", "harvested", "after", "2", "DIV", "for", "total", "RNA", "extraction", "and", "RT-PCR", "analysis", "or", "fixed", "after", "10", "DIV", "and", "processed", "for", "indirect", "immunocytochemistry.", "(A)", "Differentiated", "hNSCs", "expressed", "the", "neural-specific", "transcripts", "nestin", "and", "Notch1", "as", "well", "as", "transcripts:", "for", "neurons", "(β-tubulin", "class", "III,", "MAP-2,", "NCAM", "and", "medium-size", "neurofilament,", "NF-M),", "for", "astrocytes", "(GFAP)", "and", "for", "oligodendrocytes", "(MBP).", "The", "hNSCs", "expressed", "transcripts", "for", "GAD,", "but", "not", "for", "TH.", "B,", "C", "&", "D,", "morphology", "of", "NSC-derived", "progeny", "differentiated", "into", "GFAP+", "astrocytes", "(B),", "GC-expressing", "oligodendrocytes", "(C)", "and", "TuJ1+", "neurons", "(D),", "DAPI", "(blue)", "show", "life", "cell", "nuclei.", "(E)", "Photo", "showing", "cultures", "double-immunostained", "for", "TuJ1", "(green)", "and", "nestin", "(red)", "(DAPI,", "blue).", "(F)", "Quantitative", "analysis", "of", "immunostained", "10", "day-old", "cultures", "for", "the", "3", "neural", "cell", "types.", "Results", "are", "mean±s.e.m.", "of", "three", "independent", "experiments,", "each", "performed", "in", "duplicate.", "Bars:", "(c)", "20", "µm;", "(d,", "e)", "10", "µm.", "2.", "The", "isolated", "hNSCs", "are", "normal", "and", "do", "not", "form", "tumors", "in", "normal", "nude", "ratsThe", "self-renewal", "and", "pluripotent", "abilities", "of", "the", "hESCs", "are", "also", "associated", "with", "tumorigenic", "properties.", "Therefore,", "the", "first", "critical", "step", "toward", "developing", "therapeutic", "hNSCs", "is", "to", "verify", "that", "they", "are", "non-tumorigenic.", "The", "monolayer", "culture", "of", "SD56", "hNSCs", "clearly", "demonstrated", "contact", "inhibition", "of", "growth,", "a", "normal", "karyotype", "and", "did", "not", "express", "the", "pluripotency", "transcripts", "Oct-4", "and", "Nanog.", "Removal", "of", "mitogenic", "factors", "and", "continued", "culture", "on", "plastic", "resulted", "in", "cell", "senescence", "that", "is", "characteristic", "of", "non-transformed", "cells.", "To", "determine", "whether", "SD56", "hNSCs", "form", "tumors", "in", "vivo,", "we", "transplanted", "them", "at", "high", "density", "(see", "Methods)", "into", "the", "forebrain", "and", "subcutaneously", "into", "the", "flank", "of", "nude", "rats.", "The", "animals", "were", "kept", "for", "a", "two-month", "post-transplant", "survival", "period.", "To", "label", "mitotically", "active", "cells", "in", "vivo", "during", "S-phase,", "the", "rats", "were", "injected", "IP", "with", "BrdU", "(50", "mg/kg)", "every", "8", "hours", "during", "the", "last", "24", "hours", "before", "euthanasia.", "The", "transit", "amplifying", "endogenous", "precursors", "located", "in", "the", "subventricular", "zone", "(SVZ)", "were", "labeled", "(Figure", "S3);", "however,", "we", "were", "unable", "to", "detect", "grafted", "SD56", "hNSCs", "co-localizing", "the", "human-specific", "nuclear", "marker", "hNuc", "and", "BrdU", "(Figure", "S3).", "No", "surviving", "SD56", "hNSCs", "were", "detected", "in", "the", "flank", "of", "the", "transplanted", "animals", "suggesting", "that", "the", "grafted", "cells", "are", "not", "tumorigenic.", "3.", "Transplanted", "cells", "survived,", "migrated", "toward", "and", "engrafted", "into", "the", "stroke-damaged", "host", "tissueTo", "investigate", "the", "survival", "and", "functional", "engraftment", "in", "an", "injury", "environment,", "hNSCs", "(4×105)", "were", "transplanted", "into", "the", "ischemic", "boundary", "zone", "in", "the", "rat", "striatum", "one", "week", "after", "the", "middle", "cerebral", "artery", "occlusion", "(MCAO)", "was", "performed.", "Animals", "were", "euthanized", "two", "months", "later", "and", "the", "brains", "processed", "for", "histo-pathology", "and", "immunocytochemistry.", "Grafted", "SD56", "hNSCs,", "identified", "with", "hNuc,", "demonstrated", "a", "37.0±15.8%", "survival", "rate", "and", "a", "remarkable", "dispersion", "toward", "the", "stroke-damaged", "tissue", "with", "no", "sign", "of", "overgrowth", "or", "tumorigenesis.", "The", "majority", "of", "grafted", "cells", "(61.2±4.7%)", "migrated", "at", "least", "200", "µm", "away", "from", "the", "injection", "site", "and", "penetrated", "an", "average", "distance", "of", "806.4±49.3", "µm", "into", "the", "stroke-damaged", "tissue", "(Figure", "3A–C).", "Immunostaining", "with", "the", "blood", "vessel", "marker,", "GluT1,", "revealed", "dilated", "vessels", "in", "the", "infarcted", "striatum", "and", "a", "close", "association", "between", "vessels", "and", "the", "grafted", "hNSCs", "(Figure", "3B,", "3C).", "The", "grafted", "cells", "rarely", "expressed", "the", "proliferation", "marker", "Ki67", "(Figure", "3D),", "29.8±4.4%", "expressed", "nestin", "(Figure", "3E),", "6.5±0.9%", "expressed", "doublecortin", "(DCX)", "and", "60.8±8.1%", "were", "TuJ1+", "(Figure", "3F,", "G).", "Grafted", "cells", "rarely", "co-expressed", "the", "astroglial", "marker", "GFAP", "(Figure", "3H)", "or", "differentiated", "into", "CNPase-expressing", "oligodendrocytes", "(Figure", "3I).", "Immunostaining", "for", "GAD", "demonstrated", "that", "25.1±2.3%", "of", "grafted", "hNSCs", "differentiated", "into", "GABAergic", "neurons", "while", "less", "than", "2%", "were", "positive", "for", "glutamate", "(Figure", "3J,", "K).10.1371/journal.pone.0001644.g003Figure", "3Dispersion,", "engraftment", "and", "differentiation", "of", "the", "hNSCs", "in", "stroke-lesioned", "animals.(A)", "Schematic", "drawing", "of", "a", "frontal", "section", "through", "the", "striatum", "illustrating", "the", "dispersion", "of", "grafted", "hNSCs", "in", "the", "focal", "ischemia-lesioned", "parenchyma", "(shaded", "area).", "(B,", "C)", "Photos", "show", "frontal", "sections", "through", "the", "graft", "in", "the", "striatum", "immunostained", "with", "the", "human", "specific", "antibodies:", "anti-hNuc", "(green", "in", "B", "&", "C)", "and", "anti-GluT1", "(red,", "B", "&", "C)", "showing", "blood", "vessels", "and", "dispersed", "hNSCs", "in", "the", "graft", "zone.", "C:", "higher", "magnification", "of", "the", "inset", "in", "B.", "(D–I)", "Photos", "taken", "from", "frontal", "sections", "through", "the", "graft", "in", "the", "striatum", "double", "immunoprocessed", "for", "cell", "proliferation", "and", "neural", "lineage", "markers.", "(D)", "Note", "the", "endogenous", "Ki67+", "cells", "(red", "cells,", "arrow)", "in", "the", "stroke", "damaged", "area", "and", "the", "hNuc+", "(green)/Ki67-", "grafted", "hNSCs", "(arrowheads).", "(E)", "Examples", "of", "grafted", "SD56", "hNSCs", "showing", "co-expression", "of", "hNuc", "(green)", "and", "nestin", "(red).", "(F)", "Confocal", "3D", "reconstructed", "orthogonal", "images", "of", "the", "hNuc+(green)/DCX+(red)", "NSCs", "(arrowheads)", "viewed", "in", "the", "x-z", "plan", "on", "the", "top", "and", "y-z", "plan", "on", "the", "right.", "(G)", "Examples", "show", "the", "majority", "of", "grafted", "NSC", "progeny", "co-expressing", "hNuc", "(red)", "and", "the", "neuronal", "marker", "TuJ1", "(green).", "Grafted", "NSCs", "rarely", "differentiate", "into", "GFAP+", "astrocytes", "(H).", "In", "I,", "rare", "example", "of", "grafted", "NSC", "progeny", "becoming", "an", "oligodendrocyte", "identified", "by", "the", "expression", "of", "CNPase", "(green).", "Grafted", "NSCs", "expressed", "the", "GABAergic", "marker", "GAD65/67", "(J)", "and", "rarely", "expressed", "glutamate", "(K).", "(Abbreviations:", "Cx:", "cortex,", "Str:", "striatum).", "Bars:", "(B,", "C)", "100", "µm;", "(D,", "F)", "20", "µm;", "(E,", "G–K)", "10", "µm.", "4.", "Transplanted", "cells", "improve", "sensorimotor", "function", "of", "the", "stroke-disabled", "forelimbWe", "asked", "whether", "transplanted", "SD56", "hNSCs", "could", "enhance", "the", "recovery", "of", "sensorimotor", "function", "that", "is", "compromised", "in", "the", "stroke-injured", "rats.", "We", "used", "the", "cylinder", "test", "to", "measure", "the", "sensorimotor", "asymmetry", "in", "forelimb", "use", "during", "spontaneous", "exploration", "[22].", "To", "establish", "the", "baseline", "of", "the", "stroke-induced", "sensorimotor", "deficit,", "spontaneous", "behavior", "of", "rats", "in", "a", "transparent", "cylinder", "was", "videotaped", "one", "week", "after", "stroke", "(pre-transplant,", "Figure", "4).", "Tests", "were", "then", "conducted", "4", "and", "8", "weeks", "after", "vehicle", "and", "SD56", "hNSCs", "transplantation.", "Stable", "asymmetry", "in", "forelimb", "use", "was", "observed", "7", "days", "post-stroke", "(pre-transplant,", "Figure", "4).", "Ischemic", "rats", "used", "their", "impaired", "forelimbs", "(contralateral", "to", "lesion)", "during", "lateral", "exploration", "less", "than", "they", "did", "before", "stroke.", "Transplantation", "of", "SD56", "hNSCs", "significantly", "enhanced", "the", "independent", "use", "of", "the", "impaired", "contralateral", "forelimb", "4", "weeks", "post", "transplantation", "(P<0.05", "vs", "pre-transplant).", "Eight", "weeks", "after", "transplantation", "the", "improvement", "in", "the", "use", "of", "the", "impaired", "forelimb", "was", "stable", "and", "significant", "when", "compared", "to", "the", "pre-transplant", "group", "and", "significantly", "improved", "in", "comparison", "to", "vehicle", "treated", "group", "at", "8", "weeks", "(Figure", "4).", "In", "the", "vehicle", "treated", "group,", "the", "independent", "use", "of", "the", "contralateral", "forelimb", "remained", "impaired", "4", "and", "8", "weeks", "post-injection.", "In", "an", "independent", "study", "and", "using", "the", "same", "MCAO", "rat", "animal", "model,", "we", "found", "that", "transplantation", "of", "dermal", "fibroblasts", "did", "not", "improve", "the", "stroke-induced", "motor", "deficits", "(unpublished", "data).10.1371/journal.pone.0001644.g004Figure", "4Transplantation", "of", "NSCs", "improves", "sensorimotor", "function", "of", "the", "stroke-disabled", "forelimb.Forelimb", "use", "during", "spontaneous", "lateral", "exploration", "was", "measured", "in", "the", "cylinder", "test", "(see", "Method", "and", "Results", "sections", "for", "details).", "Groups", "of", "vehicle", "injected", "(n", "=", "7)", "and", "hNSCs", "(n", "=", "10)", "transplanted", "are", "represented.", "The", "animals", "were", "tested", "as", "described", "in", "Method", "section.", "Note", "the", "significant", "increase", "in", "the", "independent", "use", "of", "the", "impaired", "contralateral", "forelimb", "at", "4", "and", "8", "weeks", "post", "transplantation", "(P<0.05", "vs", "pre-transplant", "group).", "The", "contralateral", "forelimb", "remained", "impaired", "in", "the", "vehicle", "treated", "group", "at", "4", "and", "8", "weeks", "post-injection.", "Bars", "represent", "percentages±s.e.m.", "of", "steps", "taken", "by", "the", "ipsilateral,", "contralateral", "and", "both", "forelimbs", "simultaneously.", "*P<0.05", "vs", "pre-transplant", "group;", "#P<0.05", "vs", "vehicle", "groups.", "Our", "results", "indicate", "that", "a", "self-renewable", "and", "homogenous", "population", "of", "hNSCs,", "SD56,", "was", "derived", "from", "hESCs", "using", "defined", "media", "supplemented", "with", "a", "specific", "combination", "of", "growth", "factors.", "The", "SD56", "hNSCs", "grew", "as", "an", "adherent", "monolayer", "culture,", "uniformly", "expressed", "molecular", "features", "of", "hNSCs", "including", "nestin,", "vimentin", "and", "the", "radial", "glial", "marker", "3CB2,", "and", "did", "not", "express", "the", "pluripotency", "markers", "Oct4", "or", "Nanog.", "The", "self-renewal", "property", "of", "the", "hNSCs", "was", "characterized", "by", "a", "predominant", "symmetrical", "mode", "of", "cell", "division.", "They", "exhibited", "no", "chromosomal", "abnormalities", "and", "demonstrated", "non-tumorigenic", "properties", "after", "implantation", "into", "ischemic", "brains", "and", "into", "naïve", "nude", "rat", "brains", "and", "flanks.", "Furthermore,", "the", "transplanted", "SD56", "hNSCs", "migrated", "toward", "the", "stroke-damaged", "adult", "brain", "parenchyma,", "engrafted", "and", "improved", "the", "independent", "use", "of", "the", "stroke-impaired", "forelimb.", "Maintenance", "of", "stem", "cells", "requires", "symmetrical", "and", "asymmetrical", "cell", "divisions", "to", "both", "expand", "and", "to", "give", "rise", "to", "specialized", "progeny", "of", "a", "specific", "tissue", "(reviewed", "in", "[23]).", "In", "vivo,", "a", "complex", "cellular", "micro-environment", "or", "niche", "ensures", "the", "self-maintenance", "property", "of", "NSCs", "[24],", "[25],", "[26],", "[27].", "In", "vitro,", "defined", "growth", "factors", "and", "extracellular", "matrices", "support", "stem", "cell", "self-renewal", "[28],", "[29].", "The", "embryonic", "stem", "cells", "can", "propagate", "in", "a", "predominantly", "proliferative", "symmetrical", "mode,", "leading", "to", "homogeneous", "cell", "cultures", "growing", "relatively", "quickly", "with", "minimal", "cell", "differentiation", "[30],", "[31],", "[32],", "[33],", "[34].", "Tissue", "specific", "stem", "cells,", "however,", "self-renew", "in", "a", "predominant", "asymmetric", "mode", "to", "maintain", "them", "selves", "and", "compensate", "for", "the", "loss", "of", "differentiated", "cells", "due", "to", "disease", "or", "injury.", "Thus,", "NSCs", "isolated", "from", "developing", "or", "adult", "brain", "grow", "as", "a", "mixture", "of", "undifferentiated", "and", "differentiated", "cells", "due", "the", "predominant", "asymmetrical", "mode", "of", "cell", "division", "[35],", "[36],", "[37],", "[38],", "[39],", "[40],", "[41].", "A", "recent", "study", "has", "reported", "that", "a", "murine", "ESC-derived", "NSC", "line", "(LC1)", "is", "propagated", "as", "an", "adherent", "homogenous", "culture", "with", "a", "dominant", "mode", "of", "symmetrical", "self-renewal", "[21].", "A", "combination", "of", "EGF", "and", "FGF2", "was", "sufficient", "to", "propagate", "these", "NSCs", "as", "an", "adherent", "monolayer.", "However,", "the", "SD56", "hNSC", "line", "described", "here", "required", "the", "combination", "of", "EGF,", "bFGF", "and", "LIF", "for", "self-maintenance.", "Although", "there", "are", "morphological", "and", "molecular", "similarities", "between", "our", "hNSCs", "and", "the", "NSCs", "previously", "described", "[21],", "the", "methods", "of", "isolation", "and", "growth", "are", "different.", "In", "addition", "to", "the", "different", "combination", "of", "growth", "factors", "used,", "the", "hNSC", "line", "we", "have", "isolated", "did", "not", "go", "through", "the", "rosette-structure", "stage.", "The", "in", "vitro", "analysis", "of", "BrdU", "incorporation", "and", "nestin", "expression", "indicated", "that", "our", "hNSCs", "divide", "predominantly", "symmetrically.", "This", "type", "of", "growth", "pattern", "is", "characteristic", "of", "primitive", "normal", "stem", "cells", "undergoing", "mostly", "symmetric", "cell", "division", "to", "increase", "the", "stem", "cell", "pool", "at", "the", "early", "stage", "of", "development", "or", "during", "tissue", "regeneration", "after", "injury", "[23].", "RT-PCR", "and", "immunocytochemistry", "analysis", "demonstrated", "that", "these", "undifferentiated", "SD56", "cells", "did", "not", "express", "any", "pluripotency,", "endodermal", "or", "mesodermal", "markers.", "Furthermore,", "the", "SD56", "hNSCs", "described", "here", "exhibited", "the", "multipotential", "characteristic", "to", "differentiate", "into", "neurons,", "astrocytes", "and", "oligodendrocytes", "both", "in", "vitro", "and", "upon", "transplantation.", "Together", "these", "findings", "suggest", "that", "the", "hNSC", "line", "we", "isolated", "are", "appropriately", "programmed", "and", "share", "similar", "characteristics", "with", "the", "definitive", "NSCs", "of", "the", "developing", "brain.", "The", "SD56", "hNSCs", "demonstrated", "a", "remarkable", "ability", "to", "migrate", "toward", "the", "stroke-damaged", "parenchyma", "of", "the", "adult", "rat", "brain.", "This", "directed", "migration", "by", "the", "majority", "of", "the", "grafted", "cells", "could", "be", "due", "to", "their", "uniform", "cellular", "composition,", "which", "results", "in", "an", "equal", "response", "to", "the", "host", "microenvironment.", "In", "previous", "studies,", "subpopulations", "of", "transplanted", "hESCs", "that", "were", "enriched", "in", "neural", "cells", "migrated", "in", "host", "microenvironments", "conducive", "to", "cell", "migration,", "such", "as", "the", "developing", "brain", "or", "in", "structures", "such", "as", "the", "rostral", "migratory", "stream", "[13],", "[20].", "In", "the", "adult", "lesioned", "brain,", "the", "grafted", "hESC-derived", "neural", "cells", "proliferated", "and", "formed", "rosettes", "[14],", "teratomas", "[12],", "[15]", "or", "a", "cellular", "mass", "that", "induced", "a", "gliotic", "host", "response", "whereby", "local", "astrocytes", "demarcated", "the", "grafts", "[16].", "Enriched", "neural", "cultures", "derived", "from", "mouse", "[42]", "and", "monkey", "ESCs", "[43]", "have", "produced", "behavioral", "improvements", "when", "transplanted", "into", "animal", "models", "of", "stroke", "and", "brain", "injury.", "However,", "in", "these", "cases,", "the", "transplanted", "non-human", "ESCs", "also", "formed", "a", "mass", "with", "signs", "of", "overgrowth", "in", "the", "core,", "as", "well", "as", "deformations", "[44],", "[45],", "[46].", "ESCs", "plated", "at", "low", "density", "acquire", "neural", "identity", "within", "few", "hours", "after", "plating", "[47].", "Interestingly,", "nearly", "all", "viable", "cells", "expressed", "nestin,", "the", "early", "neural", "fate", "marker", "Sox1,", "and", "the", "pluripotency", "marker", "Oct4.", "Together,", "these", "studies", "are", "seminal", "and", "suggest", "that", "complete", "neuralization", "may", "not", "be", "achieved", "through", "certain", "enrichment", "processes,", "consequently", "the", "neural", "cells", "could", "revert", "to", "a", "pluripotent", "stage", "[17].", "The", "dispersion", "of", "the", "grafted", "hNSCs", "within", "host", "parenchyma", "may", "allow", "for", "more", "graft-host", "interactions", "that", "could", "stabilize", "differentiation,", "inhibit", "growth", "and", "prevent", "gliotic", "host", "response.", "In", "the", "present", "study,", "SD56", "hNSC-transplanted", "animals", "demonstrated", "a", "stable", "improvement", "in", "the", "sensorimotor", "function", "when", "evaluated", "for", "spontaneous", "exploratory", "activity", "in", "the", "cylinder", "test", "that", "detects", "long-lasting", "deficits", "in", "forelimb", "use", "in", "the", "experimental", "models", "of", "stroke", "[22].", "The", "transplantation", "of", "hNSCs", "significantly", "enhanced", "the", "independent", "use", "of", "the", "impaired", "contralateral", "forelimb", "8", "weeks", "post", "transplantation.", "This", "is", "the", "first", "report", "demonstrating", "that", "the", "transplantation", "of", "hNSCs", "derived", "from", "hESCs", "can", "improve", "neurologic", "behavior", "after", "experimental", "stroke.", "Together,", "these", "findings", "are", "encouraging", "and", "suggest", "that", "these", "cells", "are", "promising", "for", "future", "development.", "In", "addition", "to", "their", "therapeutic", "application,", "the", "hNSCs", "isolated", "under", "the", "reported", "conditions", "offer", "a", "means", "to", "interrogate", "host", "environments", "and", "unravel", "mechanistic", "features", "of", "self-renewal,", "non-tumorigenicity", "and", "functional", "engraftability", "in", "animal", "models", "of", "neurological", "disorders.", "hESC", "and", "NSC", "CulturesThe", "hESC", "line", "H9", "(WiCell", "Research", "Institute)", "was", "propagated", "every", "5", "to", "7", "days", "on", "irradiated", "mouse", "embryonic", "fibroblasts.", "The", "cell", "culture", "media", "consisted", "of", "a", "1∶1", "mixture", "of", "Dulbecco's", "modified", "Eagle's", "medium", "(DMEM)", "and", "F12", "nutrient,", "20%", "serum", "replacement", "(Invitrogen),", "0.1", "mM", "β-mercaptoethanol,", "2", "µg/ml", "heparin", "and", "4", "ng/ml", "bFGF", "(R&D", "Systems).", "To", "generate", "the", "NSCs,", "dissociated", "hESCs", "were", "cultured", "in", "a", "chemically", "defined", "medium", "composed", "of", "DMEM/F12", "(1∶1)", "including", "glucose", "(0.6%),", "glutamine", "(2", "mM),", "sodium", "bicarbonate", "(3", "mM),", "and", "HEPES", "buffer", "(5", "mM)", "[all", "from", "Sigma", "except", "glutamine", "(Invitrogen)].", "A", "defined", "hormone", "mix", "and", "salt", "mixture", "(Sigma),", "including", "insulin", "(25", "mg/ml),", "transferrin", "(100", "mg/ml),", "progesterone", "(20", "nM),", "putrescine", "(60", "mM),", "and", "selenium", "chloride", "(30", "nM)", "was", "used", "in", "place", "of", "serum.", "The", "medium", "was", "supplemented", "with", "EGF", "(20", "ng/ml),", "bFGF", "(10", "ng/ml)", "and", "LIF", "(10", "ng/ml).", "Dissociated", "hNSCs", "were", "plated", "at", "a", "density", "of", "100,000", "cell/ml", "in", "Corning", "T75", "(Invitrogen)", "culture", "flasks", "in", "the", "defined", "media", "together", "with", "the", "growth", "factors.", "After", "5–7", "DIV,", "the", "adherent", "culture", "was", "incubated", "in", "0.025%trypsin/0.01%", "EDTA", "(w/v)", "for", "1", "min", "followed", "by", "the", "addition", "of", "trypsin", "inhibitor", "(Invitrogen)", "then", "gently", "triturated", "to", "achieve", "single", "cell", "suspension.", "The", "cells", "were", "then", "washed", "twice", "with", "fresh", "medium", "and", "reseeded", "in", "fresh", "growth", "factor-containing", "media", "at", "100,000", "cells/ml.", "This", "procedure", "was", "performed", "for", "21", "passages", "and", "the", "fold", "of", "increase", "and", "population", "doubling", "were", "calculated", "at", "each", "passage.", "For", "clonal", "analysis,", "single", "spheres", "or", "confluent", "hNSC", "cultures", "were", "single", "cell", "dissociated", "and", "serially", "diluted", "to", "yield", "1–2", "cell/10", "µl.", "A", "10-µl-cell", "suspension", "was", "then", "added", "to", "each", "of", "96", "or", "24", "well", "plates", "containing", "200–300", "µl", "of", "growth", "media.", "Wells", "containing", "one", "viable", "cell", "were", "marked", "the", "next", "day", "and", "re-scored", "5", "to", "7", "days", "later", "for", "cell", "proliferation.", "The", "differentiation", "of", "the", "hNSCs", "was", "performed", "as", "previously", "described", "[48].", "Dissociated", "hNSCs", "were", "plated", "at", "a", "density", "of", "106", "cells/ml", "in", "control", "(media/hormone", "mix)", "medium", "devoid", "of", "any", "growth", "factors", "and", "supplemented", "with", "1%", "fetal", "bovine", "serum", "(FBS)", "on", "poly-L-ornithine-coated", "(15", "mg/ml;", "Sigma)", "glass", "coverslips", "in", "24-well", "Nunclon", "culture", "dishes", "with", "0.5", "ml/well.", "After", "2,", "7–15", "DIV", "cultures", "were", "fixed", "and", "processed", "for", "immunocytochemistry", "or", "used", "for", "RT-PCR", "analysis.", "Karyotype", "analysisLong-term", "cultures", "of", "hNSCs", "were", "incubated", "at", "37°C", "and", "harvested", "for", "metaphase", "chromosomes", "when", "the", "cultures", "were", "75%", "confluent.", "Metaphase", "chromosomes", "were", "obtained", "by", "standard", "chromosome", "harvest", "methods", "by", "exposure", "to", "Colcemid", "at", "0.1", "µg/ml", "for", "1", "hour", "at", "37°C,", "a", "2-minute", "exposure", "to", "trypsin/EDTA,", "hypotonized", "with", "0.057", "M", "KCl", "and", "fixed", "with", "3∶1", "methanol:acetic", "acid.", "Slide", "preparations", "were", "made", "by", "dropping", "the", "fixed", "cell", "pellet", "onto", "cold,", "wet", "slides", "and", "air-dried.", "After", "incubating", "the", "slides", "at", "90°C", "for", "30", "minutes,", "chromosomes", "were", "trypsin", "banded", "and", "then", "Wright/Giemsa", "stained", "for", "G-banding", "analysis.", "Twenty", "metaphase", "cells", "were", "completely", "analyzed", "and", "a", "normal", "female", "chromosome", "complement", "was", "found", "(46,XX).", "Tumorigenicity", "in", "nude", "ratsAll", "animal", "experiments", "were", "conducted", "according", "to", "the", "National", "Institute", "of", "Health", "(NIH)", "guidelines", "and", "approved", "by", "the", "IACUC.", "Normal", "adult", "NIH-Nude", "rats", "(n", "=", "5,", "8", "week-old,", "Taconic,", "Germantown,", "New", "York,", "United", "States)", "were", "used", "to", "test", "the", "tumorigenic", "potential", "of", "the", "SD56", "hNSCs.", "Undifferentiated", "hNSCs", "from", "passage", "9", "were", "single", "cell", "dissociated", "using", "trypsin-EDTA", "and", "suspended", "at", "the", "concentration", "of", "125,000", "cell/µl", "in", "preparation", "for", "cell", "transplantation.", "Two", "µl", "of", "the", "cell", "suspension", "were", "stereotaxically", "transplanted", "into", "4", "sites", "within", "the", "striatum", "at", "the", "following", "coordinates:", "AP:", "+1.0", "mm,", "ML:", "+3.2", "mm,", "DV:", "−5.0;", "AP:", "+0.5", "mm,", "ML:", "+3.0", "mm,", "DV:", "−5.0;", "AP:", "−0.5", "mm,", "ML:", "+3.0", "mm,", "DV:", "−5.0;", "AP:", "−1.0", "mm,", "ML:", "+3.5", "mm,", "DV:", "−5.0", "mm", "with", "the", "incisor", "bar", "set", "at", "3.4", "mm.", "The", "injection", "rate", "was", "1", "µl/min,", "and", "the", "cannula", "was", "left", "in", "place", "for", "an", "additional", "5", "min", "before", "retraction.", "For", "the", "flank", "tumor", "assay,", "2×106", "cells", "(125,000", "cell/µl)", "were", "injected", "subcutaneously", "to", "the", "side", "of", "the", "adult", "nude", "rats.", "To", "label", "mitotically", "active", "cells", "in", "vivo", "during", "S-phase,", "the", "rats", "were", "injected", "IP", "with", "the", "BrdU", "(50", "mg/kg,", "Sigma)", "every", "8", "hours", "during", "the", "last", "24", "hours", "before", "euthanasia.", "After", "2-month", "survival", "time,", "rats", "were", "euthanized", "and", "a", "postmortem", "examination", "for", "tumor", "formation", "was", "performed.", "Induction", "of", "Focal", "Ischemia", "and", "Cell", "TransplantationAll", "animal", "experimentations", "were", "conducted", "according", "to", "the", "National", "Institute", "of", "Health", "(NIH)", "guidelines", "and", "approved", "by", "the", "IACUC.", "Sprague", "Dawley", "adult", "male", "rats", "(n", "=", "17,", "275g–310g,", "Charles", "River", "Laboratories,", "Wilmington,", "Massachusetts,", "United", "States)", "were", "subjected", "to", "one", "and", "a", "half", "hour", "suture", "occlusion", "of", "the", "middle", "cerebral", "artery", "(MCAO),", "as", "previously", "described", "[49]", "and", "immunosuppressed", "2", "days", "before", "cell", "transplantation", "and", "daily", "thereafter", "for", "one", "week", "with", "i.p.", "injections", "of", "cyclosporine", "A", "(20", "mg/ml,", "Sandimmune,", "Novartis", "Pharmaceuticals).", "Thereafter", "oral", "cyclosporine", "was", "used", "at", "210", "µg/ml", "in", "drinking", "water", "until", "euthanasia.", "Undifferentiated", "SD56", "hNSCs", "from", "passages", "between", "P9", "and", "P13", "were", "single", "cell", "dissociated", "using", "trypsin-EDTA", "in", "preparation", "for", "cell", "transplantation.", "One", "week", "after", "the", "stroke", "lesion,", "2", "µl", "of", "the", "hNSCs,", "at", "a", "concentration", "of", "50,000", "cell/µl,", "were", "stereotaxically", "transplanted", "into", "4", "sites", "within", "the", "lesioned", "striatum", "(n", "=", "10)", "at", "the", "following", "coordinates:", "AP:", "+1.0", "mm,", "ML:", "+3.2", "mm,", "DV:", "−5.0;", "AP:", "+0.5", "mm,", "ML:", "+3.0", "mm,", "DV:", "−5.0;", "AP:", "−0.5", "mm,", "ML:", "+3.0", "mm,", "DV:", "−5.0;", "AP:", "−1.0", "mm,", "ML:", "+3.5", "mm,", "DV:", "−5.0", "mm", "with", "the", "incisor", "bar", "set", "at", "3.4", "mm.", "The", "injection", "rate", "was", "1", "µl/min,", "and", "the", "cannula", "was", "left", "in", "place", "for", "an", "additional", "5", "min", "before", "retraction.", "As", "a", "control", "group,", "we", "used", "rats", "subjected", "to", "ischemia", "and", "injected", "with", "the", "vehicle", "(n", "=", "7).", "All", "animals", "underwent", "baseline", "motor", "behavioral", "assessment", "before", "and", "after", "the", "ischemic", "lesion,", "and", "4", "&", "8", "weeks", "after", "cell", "transplantation.", "The", "animals", "were", "killed", "after", "2-month", "survival", "time", "by", "transcardial", "perfusion", "with", "phosphate", "buffered", "saline", "(PBS)", "followed", "by", "4%", "paraformaldehyde.", "The", "brains", "were", "cryoprotected", "in", "an", "increasing", "gradient", "of", "10,", "20", "and", "30%", "sucrose", "solution", "and", "cryostat", "sectioned", "at", "40", "µm", "and", "processed", "for", "immunocytochemistry.", "ImmunocytochemistryCultures", "were", "fixed", "with", "4%", "paraformaldehyde", "for", "15", "min.", "Both", "cells", "and", "brain", "sections", "were", "rinsed", "in", "PBS", "for", "3×5", "min", "then", "incubated", "for", "2", "hrs", "(cultures)", "or", "overnight", "(brain", "sections)", "with", "the", "appropriate", "primary", "antibodies", "for", "multiple", "labeling.", "Secondary", "antibodies", "raised", "in", "the", "appropriate", "hosts", "and", "conjugated", "to", "FITC,", "RITC,", "AMCA,", "CY3", "or", "CY5", "chromogenes", "(Jackson", "ImmunoResearch)", "were", "used.", "Cells", "and", "sections", "were", "counterstained", "with", "the", "nuclear", "marker", "4′,6-diamidine-2′-phenylindole", "dihydrochloride", "(DAPI).", "Positive", "and", "negative", "controls", "were", "included", "in", "each", "run.", "Immunostained", "sections", "were", "coverslipped", "using", "fluorsave", "(Calbiochem)", "as", "the", "mounting", "medium.", "The", "following", "antibodies", "were", "used:", "Anti-human", "Nuclei", "(hNuc,", "monoclonal", "1∶100,", "Chemicon),", "Anti-TuJ1", "(monoclonal", "1∶100,", "Covance;", "Polyclonal", "1∶200,", "Aves", "Labs);", "anti-GAD65/67", "(polyclonal", "1∶1000,", "Chemicon);", "Anti-glial", "fibrillary", "acidic", "protein", "(GFAP,", "monoclonal", "1∶1000,", "Chemicon;", "polyclonal", "1∶200,", "Aves", "Labs);", "Anti-galactocerebrocide", "(GC,", "monoclonal", "1∶250,", "Chemicon);", "Anti-CNPase", "(polyclonal", "1∶200,", "Aves", "Labs);", "Anti-Glucose", "Transporter", "type", "1", "(Glut-1", "polyclonal,", "1∶500,", "Chemicon);", "Anti-Nestin", "(polyclonal", "1∶1000,", "Chemicon);", "Anti-vimentin", "(monoclonal", "1∶500,", "Calbiochem);", "Anti-3CB2", "(monoclonal", "1∶500,", "Developmental", "Studies", "Hybridoma", "Bank);", "Anti-doublecortin", "(DCX,", "polyclonal", "1∶250,", "SantaCruz", "Biotechnology);", "Anti-Ki67", "(polyclonal", "1∶250,", "SantaCruz", "Biotechnology).", "Fluorescence", "was", "detected,", "analyzed", "and", "photographed", "with", "a", "Zeiss", "LSM550", "laser", "scanning", "confocal", "photomicroscope.", "For", "each", "animal,", "quantitative", "estimates", "of", "the", "total", "number", "of", "grafted", "cells", "were", "stereologically", "determined", "using", "the", "optical", "fractionator", "procedure", "[50].", "A", "computer-assisted", "image", "analysis", "system", "was", "performed", "using", "Stereo", "Investigator", "software", "(MicroBrightField,", "Inc.).", "The", "rostral", "and", "caudal", "limits", "of", "the", "reference", "volume", "were", "determined", "by", "first", "and", "last", "frontal", "sections", "containing", "grafted", "cells.", "The", "striatum", "and", "cortex", "were", "accurately", "outlined", "at", "low", "magnification", "(2.5×", "objective).", "The", "optical", "fractionator", "probe", "was", "selected", "to", "perform", "systematic", "sampling", "of", "the", "immunoreactive", "cell", "population", "distributed", "within", "the", "serial", "sections", "to", "estimate", "the", "population", "number", "in", "the", "volume", "of", "tissue.", "The", "counting", "frame", "of", "the", "optical", "fractionator", "was", "defined", "at", "50×50", "µm", "squares", "and", "the", "systematic", "sampling", "was", "performed", "by", "translating", "a", "grid", "with", "200×200", "µm", "squares", "onto", "the", "sections", "of", "interest", "using", "the", "Stereo", "Investigator", "software.", "The", "sample", "sites", "were", "systematically", "and", "automatically", "generated", "by", "the", "computer", "and", "examined", "using", "a", "60×", "objective", "of", "a", "Nikon", "Eclipse", "TE", "300", "microscope.", "The", "counting", "frame", "displayed", "inclusion", "and", "exclusion", "lines", "and", "only", "immunoreactive", "cell", "bodies", "falling", "within", "the", "counting", "frame", "with", "no", "contact", "with", "the", "exclusion", "lines", "were", "counted.", "The", "cell", "dispersion", "was", "measured", "by", "counting", "the", "number", "of", "cells", "within", "200", "µm", "distance", "from", "the", "graft", "site.", "The", "number", "and", "distance", "in", "µm", "of", "cells", "dispersed", "beyond", "200", "µm", "was", "also", "measured.", "An", "average", "of", "2,000", "cells", "was", "counted", "per", "animal.", "Double", "labeling", "was", "determined", "using", "the", "confocal", "laser", "scanning", "microscope", "by", "random", "sampling", "of", "100", "or", "more", "cells", "per", "marker", "for", "each", "animal,", "scoring", "first", "for", "hNuc+,", "followed", "by", "DAPI+", "nuclei", "and", "then", "the", "marker", "of", "choice.", "The", "double", "labeling", "was", "always", "confirmed", "in", "x-z", "and", "y-z", "cross-sections", "produced", "by", "the", "orthogonal", "projections", "of", "z-series.", "Reverse", "Transcription-Polymerase", "Chain", "Reaction", "(RT-PCR)", "analysisTotal", "RNA", "was", "extracted", "from", "cultured", "cells", "using", "RNAeasy", "kit", "(Quiagen).", "Aliquots", "(1", "µg)", "of", "total", "RNA", "from", "the", "cells", "were", "reverse", "transcribed", "in", "the", "presence", "of", "50", "mM", "Tris-HCl,", "pH", "8.3,", "75", "mM", "KCl,", "3", "mM", "MgCl2,", "10", "mM", "DTT,", "0.5", "µM", "dNTPs,", "and", "0.5", "µg", "oligo-dT(12–18)", "with", "200", "U", "Superscript", "RNase", "H-Reverse", "Transcriptase", "(Invitrogen).", "PCR", "amplification", "was", "performed", "using", "standard", "procedure", "with", "Taq", "Polymerase.", "Aliquots", "of", "cDNA", "equivalent", "to", "50", "ng", "of", "total", "RNA", "were", "amplified", "in", "25", "µl", "reactions", "containing", "10", "mM", "Tris-HCl,", "pH", "8.3,", "50", "mM", "KCl,", "1.5", "mM", "MgCl2", ",", "50", "pmol", "of", "each", "primer,", "400", "µM", "dNTPs,", "and", "0.5", "U", "AmpliTaq", "DNA", "polymerase", "(Perkin-Elmer).", "PCR", "was", "performed", "using", "the", "following", "thermal", "profile:", "4", "min", "at", "94°C;", "1", "min", "at", "94°C,", "1", "min", "at", "60°C,", "1.5", "min", "at", "72°C,", "for", "30–40", "cycles;", "7", "min", "at", "72°C,", "and", "finally", "a", "soak", "at", "4°C", "overnight.", "The", "following", "day,", "10", "µl", "aliquots", "of", "the", "amplified", "products", "were", "run", "on", "a", "2%", "agarose", "Tris–acetate", "gel", "containing", "0.5", "mg/ml", "ethidium", "bromide.", "The", "products", "were", "visualized", "through", "a", "UV", "transilluminator,", "captured", "in", "a", "digital", "format", "using", "Quantify", "One", "Gel", 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"remains", "unknown,", "their", "unique", "expression", "pattern", "and", "timing", "of", "expression", "provide", "a", "useful", "tool", "for", "scientists", "to", "identify", "as", "well", "as", "isolate", "stem", "cells.", "Embryonic", "stem", "cells", "(ESC),", "derived", "from", "the", "inner", "cell", "mass", "of", "pre-implantation", "embryos,", "have", "been", "recognized", "as", "the", "earliest", "stem", "cell", "population", "[1,2].", "This", "pluripotent", "population", "can", "differentiate", "into", "all", "somatic", "tissue", "including", "germ", "cells.", "In", "the", "case", "of", "human", "ESC,", "they", "can", "differentiate", "into", "some", "extra-embryonic", "derivatives", "as", "well.", "Like", "mouse", "ESC,", "human", "ES", "cells", "can", "be", "maintained", "and", "propagated", "on", "mouse", "fibroblast", "feeders", "for", "extended", "periods", "in", "media", "containing", "basic", "fibroblast", "growth", "factor", "(bFGF)", "[3].", "Gene", "expression", "of", "undifferentiated", "human", "ES", "cells", "has", "been", "investigated", "among", "several", "ES", "cell", "lines", "by", "a", "variety", "of", "techniques.", "They", "include", "comparison", "with", "databases,", "reverse", "transcriptase-polymerase", "chain", "reaction,", "focused", "cDNA", "microarrays,", "and", "immunocytochemistry.", "A", "list", "of", "molecules", "comprised", "of", "known", "ES-specific", "or", "-highly", "expressed", "genes", "and", "candidates", "that", "can", "serve", "as", "markers", "for", "human", "ESCs", "and", "may", "also", "contribute", "to", "the", "\"stemness\"", "phenotype", "has", "been", "established", "[3-11].", "For", "example,", "pluripotent", "ESC", "can", "be", "characterized", "by", "high", "level", "expression", "of", "Oct3/4", "(POU", "domain,", "class", "5,", "transcription", "factor", "1,", "Pou5f1)", "and", "Nanog,", "which", "are", "a", "member", "of", "POU", "domain", "and", "homeobox", "transcription", "factors", "respectively.", "A", "critical", "amount", "of", "Oct3/4", "and", "Nanog", "expression", "is", "required", "to", "sustain", "stem-cell", "pluripotency", "and", "both", "of", "these", "markers", "are", "downregulated", "as", "cells", "differentiate", "in", "vitro", "and", "in", "vivo", "[4-9].", "Antibodies", "to", "Oct3/4", "which", "cross", "react", "with", "human", "Oct", "3/4", "have", "been", "widely", "used", "to", "monitor", "the", "presence", "of", "undifferentiated", "ESC.", "No", "single", "marker", "however", "is", "sufficient", "or", "unique", "for", "identifying", "ESCs.", "Oct3/4", "for", "example", "is", "expressed", "by", "germ", "cells", "and", "may", "be", "expressed", "by", "specific", "populations", "later", "in", "development.", "Likewise,", "Nanog", "has", "been", "shown", "to", "express", "in", "other", "tissues.", "We", "and", "other", "have", "noted", "however,", "that", "while", "no", "single", "marker", "is", "sufficient", "a", "constellation", "of", "positive", "and", "negative", "markers", "can", "in", "concert", "unambiguously", "allow", "one", "to", "define", "the", "state", "of", "ESC", "cultures", "and", "that", "surface", "markers", "in", "combination", "can", "be", "used", "to", "prospectively", "sort", "for", "ESC.", "Based", "on", "published", "data", "at", "the", "level", "of", "gene", "expression,", "we", "have", "cloned", "a", "number", "of", "candidate", "marker", "genes.", "We", "have", "also", "expressed", "the", "recombinant", "protein", "and", "generated", "a", "panel", "of", "monoclonal", "or", "polyclonal", "antibodies", "to", "these", "proteins.", "Using", "these", "antibodies", "we", "have", "confirmed", "the", "specificity", "and", "selectivity", "of", "these", "antibodies", "on", "several", "ESC", "lines", "and", "established", "their", "utility", "as", "stem", "cells", "markers.", "Our", "results", "confirm", "the", "expression", "pattern", "and", "timing", "of", "these", "cell", "markers", "at", "the", "protein", "level,", "whereas", "previous", "data", "reported", "at", "the", "level", "of", "gene", "expression.", "Characterization", "of", "undifferentiated", "human", "ES", "cells", "and", "differentiated", "EBs", "by", "antibodiesAll", "monoclonal", "antibodies", "were", "initially", "selected", "for", "their", "abilities", "to", "recognize", "recombinant", "proteins", "in", "direct", "ELISAs.", "A", "subset", "were", "also", "tested", "by", "Western", "Blot", "analysis", "using", "recombinant", "proteins", "and", "cell", "lysate", "to", "confirm", "binding", "to", "a", "single", "epitope.", "The", "best", "clone", "was", "later", "screened", "for", "its", "applications", "for", "immunocytochemistry", "and", "flow", "cytometry", "using", "various", "cell", "lines.", "Human", "peripheral", "blood", "platelets", "were", "used", "for", "screening", "mouse", "anti-human", "CD9", "antibody.", "MCF-7", "cells", "were", "used", "for", "screening", "mouse", "anti-human", "E-Cadherin", "and", "PODXL", "(podocalyxin-like)", "antibodies.", "MG-63", "cells", "were", "used", "for", "screening", "mouse", "anti-human", "GATA1", "(GATA", "binding", "protein", "1)", "antibody.", "Beta-TC6", "cells", "were", "used", "for", "screening", "for", "mouse", "anti-human/mouse", "PDX-1", "(pancreatic", "duodenal", "homeobox-1)", "antibody.", "NTERA-2", "cells", "were", "used", "for", "screening", "mouse", "anti-human", "Oct3/4", "and", "SOX2", "(sex-determining", "region", "Y-box", "2)", "antibodies.", "All", "polyclonal", "antibodies", "were", "affinity-purified", "using", "recombinant", "proteins", "and", "validated", "by", "direct", "ELISAs", "and", "Western.", "Caco-2", "cells", "were", "used", "for", "validation", "of", "goat", "anti-human", "GATA6", "antibody", "and", "NTERA-2", "cells", "were", "used", "for", "validation", "of", "goat", "anti-human", "Nanog", "and", "anti-human", "Oct3/4", "antibodies", "(Summarized", "in", "Table", "1).Table", "1Summary", "list", "of", "antibody", "verification", "by", "western", "blot.AntibodySample", "used", "for", "analysisMol.", "Wt.", "(KD)Gt", "×", "hBrachyurymouse", "ES-derived", "EB", "lysate48Ms", "×", "hDPPA5N/AN/AGt", "×", "hGATA6Caco2", "cell", "lysate65Gt", "×", "hNanogNTERA-2", "cell", "lysate33Gt", "×", "hOct", "3/4NTERA-2", "cell", "lysate39Gt", "×", "hPDX1beta-TC", "6", "cell", "lysate32Gt", "×", "hSOX17mouse", "ES-derived", "EB", "lysate45Ms", "×", "hCD9PBMC25Rt", "×", "hGATA-1N/AN/AMs", "×", "hE-CadherinMCF-7", "cell", "lysate97Ms", "×", "hPODXLMCF-7", "cell", "lysate57Ms", "×", "hSOX2NTERA-2", "cell", "lysate36N/A:", "1.", "DPPA5", "is", "still", "being", "subcloned.", "Only", "Elisa", "verification", "is", "available.2.", "The", "clone", "for", "GATA-1", "(MAB1779)", "does", "not", "work", "for", "Western", "blot", "application", "but", "is", "useful", "for", "IHC,", "The", "clone", "picked", "for", "Western", "blot", "analysis", "does", "not", "work", "for", "IHC", "(MAB17791,", "see", "data", "in", ").After", "antibodies", "were", "validated", "in", "direct", "ELISAs,", "Western", "blot", "or", "cell", "lines", "(Fig.", "1", "and", "data", "not", "shown),", "they", "were", "used", "to", "examine", "the", "expression", "of", "individual", "molecules", "in", "undifferentiated", "human", "ES", "cells", "and", "differentiated", "EBs.", "When", "examined", "by", "immunohistochemistry,", "high", "level", "of", "expressions", "of", "Oct3/4,", "SOX2,", "E-Cadherin,", "PODXL", "and", "Nanog", "were", "observed", "in", "undifferentiated", "human", "ES", "cells", "(Fig.", "2A,", "2B", "and", "2C).", "DPPA5", "(developmental", "pluripotency", "associated", "5)", "expression", "was", "also", "observed", "in", "undifferentiated", "human", "ES", "cells", "(data", "not", "shown).", "We", "noted", "that", "a", "subset", "of", "the", "proteins", "used", "were", "membrane", "bound", "proteins.", "To", "test", "if", "any", "of", "the", "antibodies", "generated", "could", "recognize", "an", "extracellular", "epitope", "and", "thus", "be", "used", "for", "live", "cell", "sorting,", "we", "repeated", "staining", "of", "live", "cells", "as", "previously", "described.", "The", "CD9,", "E-Cadherin", "and", "PODXL", "antibodies", "recognized", "an", "extracellular", "epitope", "and", "their", "ability", "to", "select", "cells", "by", "FACS", "was", "confirmed", "(Fig.", "3).", "Minimal", "or", "no", "expressions", "of", "Oct3/4,", "E-Cadherin,", "PODXL", "and", "Nanog", "were", "detected", "in", "the", "differentiated", "EBs", "(Fig.", "2D,", "2E", "and", "2F).", "However,", "SOX2", "expression,", "which", "is", "observed", "in", "neural", "progenitor", "cells,", "is", "persistent", "in", "subsets", "of", "EBs.Figure", "1Western", "blot", "analysis", "for", "Gt", "×", "hOct3/4", "(A),", "Gt", "×", "hNanog", "(B)", "and", "Ms", "×", "hSOX2", "(C)", "in", "NTERA-2", "cell", "lysate,", "Ms", "×", "hE-Cadherin", "(D)", "in", "MCF-7", "cell", "lysate,", "Ms", "×", "hCD9", "(E)", "in", "PBMC", "lysate", "and", "Ms", "×", "hPDX-1(F)", "in", "β-TC-6", "cell", "lysate.", "Numbers", "indicate", "the", "positions", "of", "molecular", "weight", "markers.Figure", "2Undifferentiated", "human", "ES", "cells", "(A,", "B,", "and", "C)", "and", "differentiated", "EBs", "(D,", "E", "and", "F)", "were", "analyzed", "using", "antibodies", "to", "indicated", "molecular", "markers.", "Immunostaining", "with", "goat", "anti-human", "Oct3/4", "(Red", "in", "A", "and", "D),", "mouse", "anti-human", "SOX2", "(Green", "in", "A", "and", "D),", "goat", "anti-human", "E-Cadherin", "(Red", "in", "B", "and", "E),", "mouse", "anti-human", "PODXL", "(Green", "in", "B", "and", "E),", "and", "goat", "anti-human", "Nanog", "(Red", "in", "C", "and", "F),", "are", "contrasted", "with", "DAPI", "nuclear", "staining", "(Blue", "in", "C-F).", "Note", "the", "dramatic", "downregulation", "of", "ESC", "specific", "markers", "(Oct3/4,", "E-Cadherin,", "PODXL,", "and", "Nanog)", "in", "EBs.", "However,", "SOX2", "expression", "is", "persistent", "in", "subsets", "of", "EB", "cells.", "Scale", "bars", "=", "100", "μm.Figure", "3Human", "embryonic", "stem", "cells", "stained", "with", "anti-CD9", "(A),", "anti-E-Cadherin", "(B),", "and", "anti-PODXL", "(C)", "and", "antigen", "expression", "detected", "by", "a", "flow", "cytometer.", "The", "specific", "staining", "is", "indicated", "by", "green", "histogram", "and", "corresponding", "isotype", "control", "is", "indicated", "by", "black", "histogram.Suspension", "culture", "with", "FGF", "withdrawal", "is", "known", "to", "induce", "differentiation", "of", "ES", "cells", "to", "all", "three", "germ", "layer", "precursors", "[12].", "The", "differentiation", "status", "of", "the", "EB", "used", "here", "was", "detected", "to", "contain", "all", "germ", "cell", "markers", "by", "RT-PCR", "(Fig.", "4).", "In", "order", "to", "examine", "how", "more", "antibodies", "can", "be", "used", "for", "characterization", "of", "early", "differentiation", "events", "from", "human", "ES", "cells,", "we", "examined", "the", "expressions", "of", "endodermal", "markers,", "SOX17,", "GATA6", "and", "PDX-1,", "and", "mesodermal", "markers,", "Brachyury", "and", "GATA1,", "in", "the", "undifferentiated", "human", "ES", "cells", "and", "differentiated", "EBs.", "Expressions", "of", "SOX17,", "GATA6,", "PDX-1,", "Brachyury", "and", "GATA1", "were", "not", "detected", "in", "undifferentiated", "human", "ES", "cells", "(data", "not", "shown).", "In", "contrast", "to", "the", "undifferentiated", "ES", "cells,", "subpopulations", "of", "SOX17-,", "GATA6-,", "Brachyury-", "and", "GATA1-positive", "cells", "were", "observed", "(Fig", "4).", "These", "results", "suggest", "that", "both", "endodermal", "and", "mesodermal", "precursors", "exist", "in", "EBs", "with", "FGF", "withdrawal", "for", "8", "days.", "However,", "no", "PDX-1-positive", "cells", "were", "seen", "in", "EBs", "differentiated", "with", "the", "same", "treatment", "(data", "not", "shown).Figure", "4Differentiated", "EBs", "were", "analyzed", "by", "either", "immunocytochemistry", "or", "RT-PCR", "to", "the", "indicated", "molecular", "markers.", "(A)", "Immunostaining", "with", "goat", "anti-human", "SOX17", "(Red),", "is", "contrasted", "with", "Fluoro", "Nissl", "nuclear", "staining", "(Green).", "(B)", "Immunostaining", "with", "goat", "anti-human", "GATA6", "(Red),", "is", "contrasted", "with", "DAPI", "nuclear", "staining", "(Blue).", "(C)", "Immunostaining", "with", "goat", "anti-human", "brachyury", "(Red),", "is", "contrasted", "with", "DAPI", "nuclear", "staining", "(Blue).", "(D)", "Immunostaining", "with", "mouse", "anti-human", "GATA1", "(Red).", "Note", "that", "each", "antibody", "recognizes", "subsets", "of", "EB", "cells.", "Scale", "bars", "=", "100", "μm.", "(E)", "The", "differentiation", "status", "of", "EB", "is", "detected", "by", "RT-PCR", "using", "different", "germ", "layer", "cell", "markers.", "Selected", "endoderm", "markers", "AFP,", "FoxA2;", "mesoderm", "markers", "Hand1,", "MSX1", "and", "ectoderm", "marker", "Msl1", "were", "all", "highly", "expressed", "in", "the", "EB", "samples", "while", "their", "expression", "was", "either", "undetectable", "or", "at", "low", "level", "in", "the", "ES", "samples.", "G3PDH", "was", "a", "positive", "control", "showing", "similar", "amount", "of", "RNA", "samples", "were", "used", "for", "analysis.", "Examination", "of", "cross-reactivity", "of", "antibodies", "on", "mouse", "ES", "and", "differentiated", "cellsWe", "have", "also", "examined", "the", "cross-reactivities", "of", "these", "antibodies", "to", "mouse", "ES", "cells", "using", "mouse", "D3", "ES", "cell", "line", "and", "mouse", "fetal", "endodermal", "tissue.", "Cross-reactivity", "to", "mouse", "of", "goat", "anti-Oct3/4,", "goat", "anti-PDX-1,", "goat", "anti-SOX17", "and", "mouse", "anti-SOX2", "was", "detected.", "Minimal", "cross-reactivity", "to", "mouse,", "measured", "by", "10%", "intensity", "to", "human", "by", "higher", "than", "control", "cells,", "was", "observed", "in", "mouse", "anti-CD9", "and", "mouse", "anti-E-cadherin", "antibodies.", "Goat", "anti-Nanog", "and", "mouse", "anti-PODXL", "antibodies", "appear", "to", "be", "human-specific", "as", "well", "(data", "not", "shown).", "The", "subtypes", "of", "monoclonal", "antibodies", "were", "also", "identified", "in", "the", "best", "clones.", "These", "results", "are", "summarized", "in", "Table", "2.Table", "2Summary", "of", "antibodies", "detection", "in", "ES", "and", "EB", "samples.AntibodyESEBReactivity", "to", "mouseIsotype", "of", "monoclonal", "antibody", "(Clone", "No.)Gt", "×", "hBrachyuryNoYesNT*Ms", "×", "hDPPA5YesNT*NT*ND*Gt", "×", "hGATA6NoYesNT*Gt", "×", "hNanogYesDownNoGt", "×", "hOct", "3/4YesDownYesGt", "×", "hPDX-1NoNoYesGt", "×", "hSOX17NoYesYesMs", "×", "hCD9YesNoMinimalMouse", "IgG2B", "(clone", "209306)Ms", "×", "hE-cadherinYesNoMinimalMouse", "IgG2B", "(clone", "180224)Ms", "×", "hGATA1NoYesNT*Rat", "IgG2B", "(clone", "234732)Ms", "×", "hPODXLYesNoNoMouse", "IgG2A", "(clone", "222328)Ms", "×", "hSOX2YesYesYesMouse", "IgG2A", "(clone", "245610)*NT,", "Not", "tested;", "ND,", "Not", "determined.", "The", "expression", "patterns", "detected", "using", "antibodies", "developed", "in", "our", "facility", "are", "consistent", "with", "data", "reported", "using", "reverse", "transcriptase-polymerase", "chain", "reaction", "or", "cDNA", "microarrays.", "Moreover", "several", "of", "the", "monoclonal", "antibodies", "have", "differing", "heavy", "chain", "subunits", "allowing", "double", "labeling", "using", "subtype", "specific", "markers", "to", "be", "performed.", "In", "summary,", "we", "have", "developed", "a", "useful", "collection", "of", "antibodies", "that", "would", "be", "useful", "for", "identification", "of", "stem", "cell", "characteristics", "and", "assessment", "of", "differentiation.", "Several", "additional", "antibodies", "to", "the", "molecules", "that", "have", "been", "identified", "as", "potential", "cell", "lineage", "markers", "[13]", "are", "currently", "under", "development", "using", "the", "same", "approach.", "Cloning", "and", "expression", "of", "Brachyury,", "DPPA5,", "CD9,", "E-Cadherin,", "GATA1,", "GATA6,", "Nanog,", "Oct3/4,", "PDX-1,", "PODXL,", "SOX2", "and", "SOX17Brachyury", "(aa.", "1–202),", "DPPA5", "(a.a.", "1–116),", "GATA1", "(a.a.", "1–413),", "GATA6", "(aa.", "1–449),", "Nanog", "(aa.", "153–305),", "Oct3/4", "(aa.", "1–265),", "PDX-1", "(aa.", "1–283),", "SOX2", "(aa.", "135–317)", "and", "SOX17", "(aa.", "177–414)", "were", "expressed", "in", "E.", "Coli", "and", "extracellular", "domains", "of", "CD9,", "E-Cadherin,", "PODXL", "were", "expressed", "in", "mouse", "NSO", "cells.", "All", "proteins", "were", "purified", "and", "sequenced", "before", "they", "were", "used", "as", "antigens", "for", "immunizations", "and", "as", "substrate", "for", "antibody", "screening", "and", "subcloning.", "Production", "and", "purification", "of", "antibodiesAll", "monoclonal", "antibodies", "were", "derived", "from", "fusions", "of", "mouse", "myeloma", "with", "B", "cells", "obtained", "from", "BALB/c", "mice", "which", "had", "been", "immunized", "with", "purified", "antigen.", "The", "IgG", "fraction", "of", "the", "culture", "supernatant", "was", "purified", "by", "Protein", "G", "affinity", "chromatography", "(Sigma).", "Each", "panel", "of", "antibodies", "was", "screened", "and", "selected", "for", "their", "abilities", "to", "detect", "purified", "recombinant", "antigen", "in", "direct", "ELISA", "and", "Western", "blot.", "All", "polyclonal", "antibodies", "were", "derived", "from", "sera", "of", "goats", "which", "had", "been", "immunized", "and", "boost", "it", "with", "purified", "antigen.", "Antibody", "was", "purified", "from", "the", "sera", "by", "an", "antigen-affinity", "chromatography.", "Cells", "and", "cell", "cultureHuman", "Caco-2,", "MG-63,", "MCF-7,", "NTERA-2", "and", "mouse", "D3", "cells", "were", "purchased", "from", "American", "Type", "Culture", "Collection", "(ATCC).", "Cells", "were", "cultured", "according", "to", "the", "ATCC", "instructions.", "Information", "regarding", "human", "ES", "cell", "line", "HSF-6", "(NIH", "code", "UC06)", "can", "be", "obtained", "at", "the", "website", "[14].", "Undifferentiated", "human", "ES", "cells", "were", "cultured", "according", "to", "the", "protocol", "provided", "by", "the", "University", "of", "California,", "San", "Francisco", "in", "human", "ES", "culture", "medium", "[DMEM", "supplemented", "with", "20%", "KnockOut", "Serum", "Replacement", "(Invitrogen)", "and", "5", "ng/mL", "of", "bFGF", "(R&D", "Systems)].", "To", "induce", "formation", "of", "embryoid", "bodies", "(EBs),", "ES", "colonies", "were", "harvested,", "separated", "from", "the", "MEF", "feeder", "cells", "by", "gravity,", "gently", "resuspended", "in", "ES", "culture", "medium", "and", "transferred", "to", "non-adherent", "suspension", "culture", "dishes", "(Corning).", "Unless", "otherwise", "noted,", "EBs", "derived", "from", "human", "ES", "cell", "aggregates", "were", "cultured", "for", "8", "days", "in", "ES", "culture", "medium", "deprived", "of", "bFGF", "and", "used", "for", "analysis", "by", "immunohistochemistry", "as", "described.", "Western", "blotCells", "are", "solubilized", "in", "hot", "2×", "SDS", "gel", "sample", "buffer", "(20", "mM", "dithiothreitol,", "6%", "SDS,", "0.25", "M", "Tris,", "pH", "6.8,", "10%", "glycerol,", "10", "mM", "NaF", "and", "bromophenyl", "blue)", "at", "2", "×", "106", "per", "mL.", "The", "extracts", "are", "heated", "in", "a", "boiling", "water", "bath", "for", "5", "minutes", "and", "sonicated", "with", "a", "probe", "sonicator", "with", "3–4", "bursts", "of", "5–10", "seconds", "each.", "Samples", "are", "diluted", "with", "1×", "SDS", "sample", "buffer", "to", "the", "desired", "loading", "of", "1–5", "×", "103", "per", "lane.", "Lysates", "were", "resolved", "by", "SDS-PAGE,", "transferred", "to", "Immobilon-P", "membrane,", "and", "immunoblotted", "with", "0.5", "μg/mL", "primary", "Abs", "as", "described", "in", "R&D", "Systems", "Website", "[15].", "ImmunohistochemistryAntibodies", "were", "used", "with", "the", "appropriate", "secondary", "reagents", "at", "a", "concentration", "of", "5", "to", "10", "μg/ml.", "Cells", "or", "sections", "of", "EBs", "were", "fixed", "with", "4%", "paraformaldehyde", "in", "PBS", "at", "room", "temperature", "for", "20", "min,", "then", "blocked", "and", "permeabilized", "with", "0.1%", "Triton", "X-100,", "1%", "BSA,", "10%", "normal", "donkey", "serum", "in", "PBS", "at", "room", "temperature", "for", "45", "min.", "After", "blocking,", "cells", "were", "incubated", "with", "diluted", "primary", "antibody", "overnight", "at", "4°C", "followed", "by", "coupled", "anti-mouse", "or", "anti-goat", "IgG", "(Molecular", "Probes)", "at", "room", "temperature", "in", "the", "dark", "for", "an", "hour.", "Between", "each", "step", "cells", "were", "washed", "with", "PBS", "with", "0.1%", "BSA.", "RT-PCRTotal", "RNA", "was", "extracted", "from", "EBs", "using", "Trizol", "LS", "(Invitrogen).", "cDNA", "was", "synthesized", "by", "using", "Superscript", "II", "reverse", "transcriptase", "(Invitrogen)", "according", "to", "the", "manufacturer's", "recommendations.", "The", "PCR", "primers", "are", "available", "upon", "request.", "Flow", "cytometryAntibodies", "were", "prepared", "at", "the", "concentration", "of", "0.1", "mg/mL.", "10", "μL", "of", "the", "stock", "solution", "was", "added", "to", "1", "–", "2.5", "×", "105", "cells", "in", "a", "total", "reaction", "volume", "not", "exceeding", "200", "μL.", "The", "sample", "was", "then", "incubated", "for", "20", "min", "at", "2–8", "°C.", "Following", "incubation,", "excess", "antibody", "was", "removed", "by", "washing", "cells", "twice", "with", "FACS", "buffer", "(2%", "FCS", "and", "0.1%", "sodium", "azide", "in", "Hank's", "buffer).", "After", "wash,", "cells", "were", "resuspend", "in", "200", "μL", "of", "FACS", "buffer", "and", "the", "binding", "of", "unlabeled", "monoclonal", "antibodies", "was", "visualized", "by", "adding", "10", "μL", "of", "a", "25", "μg/mL", "stock", "solution", "of", "a", "secondary", "developing", "reagent", "such", "as", "goat", "anti-mouse", "IgG", "conjugated", "to", "a", "fluorochrome", "for", "20", "min", "at", "2–8°C.", "Following", "incubation,", "cells", "were", "washed", "once", "with", "FACS", "buffer,", "once", "with", "PBS.", "After", "wash,", "cells", "were", "resuspend", "in", "400", "μL", "of", "PBS", "and", "analyzed", "on", "a", "FACScant", "flow", "cytometer", "(Becton-Dickinson,", "Mountain", "View,", "CA).", "Five", "thousand", "events", "were", "collected", "and", "analyzed", "using", "CELL", "Quest", "software." ]
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[ "BackgroundMany", "novel", "studies", "and", "therapies", "are", "possible", "with", "the", "use", "of", "human", "embryonic", "stem", "cells", "(hES", "cells)", "and", "their", "differentiated", "cell", "progeny.", "The", "hES", "cell", "derived", "CD34", "hematopoietic", "stem", "cells", "can", "be", "potentially", "used", "for", "many", "gene", "therapy", "applications.", "Here", "we", "evaluated", "the", "capacity", "of", "hES", "cell", "derived", "CD34", "cells", "to", "give", "rise", "to", "normal", "macrophages", "as", "a", "first", "step", "towards", "using", "these", "cells", "in", "viral", "infection", "studies", "and", "in", "developing", "novel", "stem", "cell", "based", "gene", "therapy", "strategies", "for", "AIDS.ResultsUndifferentiated", "normal", "and", "lentiviral", "vector", "transduced", "hES", "cells", "were", "cultured", "on", "S17", "mouse", "bone", "marrow", "stromal", "cell", "layers", "to", "derive", "CD34", "hematopoietic", "progenitor", "cells.", "The", "differentiated", "CD34", "cells", "isolated", "from", "cystic", "bodies", "were", "further", "cultured", "in", "cytokine", "media", "to", "derive", "macrophages.", "Phenotypic", "and", "functional", "analyses", "were", "carried", "out", "to", "compare", "these", "with", "that", "of", "fetal", "liver", "CD34", "cell", "derived", "macrophages.", "As", "assessed", "by", "FACS", "analysis,", "the", "hES-CD34", "cell", "derived", "macrophages", "displayed", "characteristic", "cell", "surface", "markers", "CD14,", "CD4,", "CCR5,", "CXCR4,", "and", "HLA-DR", "suggesting", "a", "normal", "phenotype.", "Tests", "evaluating", "phagocytosis,", "upregulation", "of", "the", "costimulatory", "molecule", "B7.1,", "and", "cytokine", "secretion", "in", "response", "to", "LPS", "stimulation", "showed", "that", "these", "macrophages", "are", "also", "functionally", "normal.", "When", "infected", "with", "HIV-1,", "the", "differentiated", "macrophages", "supported", "productive", "viral", "infection.", "Lentiviral", "vector", "transduced", "hES", "cells", "expressing", "the", "transgene", "GFP", "were", "evaluated", "similarly", "like", "above.", "The", "transgenic", "hES", "cells", "also", "gave", "rise", "to", "macrophages", "with", "normal", "phenotypic", "and", "functional", "characteristics", "indicating", "no", "vector", "mediated", "adverse", "effects", "during", "differentiation.ConclusionPhenotypically", "normal", "and", "functionally", "competent", "macrophages", "could", "be", "derived", "from", "hES-CD34", "cells.", "Since", "these", "cells", "are", "susceptible", "to", "HIV-1", "infection,", "they", "provide", "a", "uniform", "source", "of", "macrophages", "for", "viral", "infection", "studies.", "Based", "on", "these", "results,", "it", "is", "also", "now", "feasible", "to", "transduce", "hES-CD34", "cells", "with", "anti-HIV", "genes", "such", "as", "inhibitory", "siRNAs", "and", "test", "their", "antiviral", "efficacy", "in", "down", "stream", "differentiated", "cells", "such", "as", "macrophages", "which", "are", "among", "the", "primary", "cells", "that", "need", "to", "be", "protected", "against", "HIV-1", "infection.", "Thus,", "the", "potential", "utility", "of", "hES", "derived", "CD34", "hematopoietic", "cells", "for", "HIV-1", "gene", "therapy", "can", "be", "evaluated.", "Human", "embryonic", "stem", "cells", "(hES", "cells)", "show", "great", "promise", "for", "many", "novel", "cellular", "therapies", "due", "to", "their", "pluripotent", "nature", "[1].", "These", "cells", "have", "the", "capacity", "to", "give", "rise", "to", "mature", "cells", "and", "tissues", "that", "arise", "from", "all", "three", "germ", "layers", "during", "embryonic", "development", "[2-4].", "Several", "pluripotent", "hES", "cell", "lines", "have", "so", "far", "been", "derived", "from", "the", "inner", "cell", "mass", "of", "human", "blastocysts", "and", "can", "be", "cultured", "indefinitely", "in", "an", "undifferentiated", "state", "[5-7].", "Thus,", "these", "cells", "provide", "a", "renewable", "source", "of", "pluripotent", "stem", "cells", "from", "which", "many", "types", "of", "differentiated", "cells", "could", "be", "produced", "for", "experimental", "and", "therapeutic", "purposes.", "Cell", "differentiation", "protocols", "currently", "exist", "for", "the", "derivation", "of", "neurons,", "cardiomyocytes,", "endothelial", "cells,", "hematopoietic", "progenitor", "cells,", "keratinocytes,", "osteoblasts,", "and", "hepatocytes", "to", "name", "a", "few", "[2,3,8,9].", "In", "addition", "to", "providing", "for", "potential", "cellular", "replacement", "therapies,", "opportunities", "exist", "in", "programming", "hES", "cells", "to", "correct", "a", "genetic", "defect", "and/or", "to", "express", "a", "therapeutic", "transgene", "of", "interest.", "Using", "such", "approaches,", "many", "possibilities", "exist", "for", "treating", "a", "number", "of", "genetic", "and", "immune", "system", "disorders", "[1].", "Many", "novel", "applications", "can", "be", "foreseen", "for", "hES", "cells", "in", "infectious", "disease", "research.", "AIDS", "is", "a", "potential", "disease", "that", "can", "benefit", "from", "exploiting", "hES", "cells", "for", "cell", "replacement", "therapy", "as", "they", "have", "the", "capacity", "to", "differentiate", "into", "various", "hematopoietic", "cells.", "HIV", "continues", "to", "be", "a", "major", "global", "public", "health", "problem", "with", "infections", "increasing", "at", "an", "alarming", "rate", "[10,11].", "Given", "the", "present", "lack", "of", "effective", "vaccines", "and", "the", "ineffectiveness", "of", "drug", "based", "therapies", "for", "a", "complete", "cure,", "new", "and", "innovative", "approaches", "are", "essential.", "Gene", "therapy", "through", "intracellular", "immunization", "offers", "a", "promising", "alternative", "approach", "and", "possible", "supplement", "to", "current", "HAART", "therapy", "[12-14].", "HIV", "mainly", "targets", "cells", "of", "the", "hematopoietic", "system,", "namely,", "T", "cells,", "macrophages,", "and", "dendritic", "cells", "[15].", "As", "infection", "progresses,", "the", "immune", "system", "is", "rendered", "defenseless", "against", "other", "invading", "pathogens", "and", "succumbs", "to", "opportunistic", "infections.", "There", "is", "a", "great", "deal", "of", "progress", "in", "the", "area", "of", "stem", "cell", "gene", "therapy", "for", "AIDS", "[12].", "A", "primary", "goal", "of", "many", "ongoing", "studies", "is", "to", "introduce", "an", "effective", "anti-HIV", "gene", "into", "hematopoietic", "stem", "cells", "[16-18].", "As", "these", "cells", "possess", "the", "ability", "to", "self", "renew,", "they", "have", "the", "potential", "to", "continually", "produce", "HIV", "resistant", "T", "cells", "and", "macrophages", "in", "the", "body", "thus", "providing", "long", "term", "immune", "reconstitution.", "These", "approaches", "use", "CD34", "hematopoietic", "stem", "cells", "for", "anti-HIV", "gene", "transduction", "via", "integrating", "viral", "vectors", "such", "as", "lentiviral", "vectors", "[16-18].", "Lentiviral", "vectors", "have", "several", "advantages", "over", "conventional", "retroviral", "vectors", "since", "higher", "transduction", "efficiencies", "can", "be", "obtained", "and", "there", "is", "less", "gene", "silencing.", "The", "CD34", "cells", "currently", "used", "for", "many", "therapies", "are", "primarily", "obtained", "from", "bone", "marrow", "or", "mobilized", "peripheral", "blood", "[1,19].", "Thus,", "CD34", "progenitor", "cells", "are", "an", "essential", "ingredient", "for", "HIV", "gene", "therapy.", "In", "view", "of", "the", "need", "for", "CD34", "cells", "for", "HIV", "gene", "therapy", "as", "well", "as", "for", "other", "hematopoietic", "disorders,", "if", "one", "can", "produce", "these", "cells", "in", "unlimited", "quantities", "from", "a", "renewable", "source,", "it", "will", "overcome", "the", "limitations", "of", "securing", "large", "numbers", "of", "CD34", "cells", "for", "therapeutic", "purposes.", "In", "this", "regard,", "progress", "has", "been", "made", "in", "deriving", "CD34", "cells", "from", "hES", "cells", "(hES-CD34).", "Different", "methods", "currently", "exist", "to", "derive", "CD34", "cells", "from", "hES", "cells", "with", "varying", "efficiencies", "[20-27].", "Recent", "reports", "have", "indicated", "the", "capacity", "of", "hES", "cell", "derived", "CD34", "cells", "to", "give", "rise", "to", "lymphoid", "and", "myeloid", "lineages", "thus", "paving", "the", "way", "for", "utilization", "of", "these", "cells", "for", "hematopoietic", "cell", "therapy", "[20,27-29].", "For", "the", "effective", "utilization", "of", "hES-CD34", "cells", "for", "HIV", "gene", "therapy,", "a", "number", "of", "parameters", "need", "to", "be", "examined.", "First,", "one", "has", "to", "demonstrate", "that", "hES-CD34", "cells", "can", "give", "rise", "to", "macrophages", "and", "helper", "T", "cells", "which", "are", "the", "main", "cells", "that", "need", "to", "be", "protected", "against", "HIV", "infection.", "Recent", "evidence", "has", "shown", "that", "hES-CD34", "cells", "can", "give", "rise", "to", "myelomonocytic", "cells", "[21].", "However,", "thorough", "phenotypic", "or", "functional", "characterization", "of", "these", "cells", "is", "lacking.", "It", "is", "also", "not", "clear", "if", "these", "cells", "are", "susceptible", "to", "HIV", "infection.", "Similarly,", "although", "the", "hES-CD34", "cells", "were", "shown", "to", "have", "lymphoid", "progenitor", "capacity,", "only", "B", "cell", "and", "natural", "killer", "(NK)", "cell", "differentiation", "has", "been", "examined", "so", "far", "[21,28].", "The", "capacity", "to", "generate", "T", "cells", "remains", "to", "be", "evaluated.", "With", "this", "background,", "as", "a", "first", "step,", "our", "primary", "goal", "in", "these", "studies", "is", "to", "examine", "the", "capacity", "of", "hES-CD34", "cells", "to", "give", "rise", "to", "phenotypically", "and", "functionally", "normal", "macrophages", "and", "whether", "such", "cells", "are", "susceptible", "to", "productive", "HIV", "infection.", "Since", "lentiviral", "vectors", "have", "been", "shown", "to", "successfully", "transduce", "hES", "cells", "[30-33],", "we", "further", "investigated", "the", "ability", "of", "transduced", "hES", "cells", "to", "differentiate", "into", "transgenic", "macrophages", "that", "can", "support", "HIV-1", "infection.", "Demonstration", "of", "HIV-1", "productive", "infection", "in", "these", "cells", "will", "permit", "future", "efficacy", "evaluations", "of", "anti-HIV", "genes", "in", "this", "system.", "Here", "we", "show", "that", "normal", "and", "lentiviral", "vector", "transduced", "hES-CD34", "cells", "can", "give", "rise", "to", "phenotypically", "and", "functionally", "normal", "macrophages", "that", "support", "HIV", "infection", "thus", "paving", "the", "way", "for", "many", "novel", "approaches", "to", "evaluate", "their", "potential", "for", "HIV", "gene", "therapy.", "Derivation", "of", "macrophages", "from", "hES", "cellsUndifferentiated", "hES", "cell", "colonies", "grown", "in", "media", "supplemented", "with", "4", "ng/ml", "bFGF", "displayed", "normal", "morphology", "of", "pluripotent", "human", "embryonic", "stem", "cells", "with", "tight", "and", "discreet", "borders", "on", "the", "MEF", "feeder", "layers", "(Fig", "1A).", "Similarly,", "lentiviral", "vector", "transduced", "hES", "cell", "colonies,", "also", "displayed", "normal", "morphology", "and", "growth", "characteristics", "(Fig", "1A).", "As", "expected,", "the", "vector", "transduced", "colonies", "displayed", "green", "fluorescence", "due", "to", "the", "presence", "of", "the", "GFP", "reporter", "gene.", "When", "cultured", "on", "irradiated", "S17", "mouse", "bone", "marrow", "stromal", "cells,", "both", "nontransduced", "and", "transduced", "hES", "cells", "developed", "into", "embryonic", "cystic", "bodies", "(Fig", "1A).", "FACS", "analysis", "of", "single", "cell", "suspensions", "of", "the", "cystic", "bodies", "showed", "levels", "of", "CD34", "cells", "which", "ranged", "from", "7–15%.", "Figure", "1B", "displays", "a", "representative", "FACS", "profile", "of", "hES-CD34", "cells.", "Purified", "CD34", "cells", "were", "later", "cultured", "in", "semi-solid", "methylcellulose", "medium", "to", "derive", "myeloid", "colonies.", "Both", "nontransduced", "(denoted", "as", "ES", "in", "figures)", "and", "vector", "transduced", "(denoted", "as", "GFP", "ES", "in", "figures)", "hES", "cell", "derived", "CD34", "cells", "gave", "rise", "to", "normal", "myelomonocytic", "colonies", "similar", "to", "human", "fetal", "liver", "derived", "CD34", "cells", "(denoted", "as", "CD34", "in", "figures)", "(Fig", "1A).", "When", "pooled", "colonies", "were", "cultured", "further", "in", "liquid", "cytokine", "media", "for", "12–15", "days", "for", "differentiation,", "the", "cells", "developed", "into", "morphologically", "distinct", "macrophages", "(Fig", "1A).", "When", "compared,", "the", "morphology", "of", "macrophages", "derived", "from", "all", "stem", "cell", "progenitor", "populations", "appeared", "similar.", "These", "results", "were", "found", "to", "be", "consistent", "in", "replicative", "experiments.", "The", "transgene", "GFP", "expression", "was", "also", "maintained", "during", "the", "differentiation", "of", "hES", "cells", "into", "mature", "macrophages.", "GFP", "expression", "in", "cystic", "body", "derived", "CD34", "cells", "was", "around", "80%", "(data", "not", "shown)", "with", "similar", "levels", "seen", "in", "differentiated", "macrophages", "(Fig", "2).Figure", "1Derivation", "of", "macrophages", "from", "lentiviral", "vector", "transduced", "and", "normal", "hES", "cells.", "A)", "Transduced", "and", "non-transduced", "H1", "hES", "cells", "were", "cultured", "on", "mouse", "S17", "bone", "marrow", "stromal", "cell", "layers", "to", "derive", "cystic", "bodies.", "Cystic", "body", "derived", "CD34", "cells", "were", "purified", "by", "positive", "selection", "with", "antibody", "conjugated", "magnetic", "beads", "and", "placed", "in", "methocult", "media", "to", "obtain", "myelomonocytic", "colonies.", "Pooled", "colonies", "were", "cultured", "in", "liquid", "cytokine", "media", "supplemented", "with", "GM-CSF", "and", "M-CSF", "to", "promote", "macrophage", "growth.", "For", "comparison,", "fetal", "liver", "derived", "CD34", "cells", "were", "cultured", "similarly", "to", "derive", "macrophages.", "Representative", "ES", "cell", "colonies,", "cystic", "bodies,", "methocult", "colonies,", "and", "derivative", "macrophages", "are", "shown", "with", "GFP", "expressing", "cells", "fluorescing", "green", "under", "UV", "illumination.", "B)", "Representative", "FACS", "profile", "of", "hES", "cell", "derived", "CD34", "cells", "stained", "with", "PE", "conjugated", "antibodies.", "Percent", "positive", "CD34", "cells", "are", "shown", "with", "isotype", "control", "shown", "in", "the", "left", "panel.Figure", "2Phenotypic", "FACS", "analysis", "of", "hES", "cell", "derived", "macrophages.", "A)", "Macrophages", "derived", "from", "transduced", "and", "nontransduced", "hES", "CD34", "and", "fetal", "liver", "CD34", "cells", "were", "stained", "with", "antibodies", "to", "CD14,", "HLA-DR,", "CD4,", "CCR5,", "and", "CXCR4", "and", "the", "expression", "of", "these", "surface", "markers", "was", "analyzed", "by", "FACS.", "B)", "Isotype", "controls", "for", "PE", "and", "PE-CY5", "antibodies.", "Percent", "positive", "cells", "are", "displayed", "in", "the", "plots", "for", "each", "respective", "cell", "surface", "marker", "staining.", "Dot", "plots", "are", "representative", "of", "triplicate", "experiments.", "hES", "cell", "derived", "macrophages", "display", "a", "normal", "phenotypic", "profileMacrophages", "play", "a", "critical", "role", "in", "immune", "system", "function", "and", "are", "also", "major", "target", "cells", "for", "many", "viral", "infections", "including", "HIV-1.", "Distinct", "surface", "phenotypic", "markers", "exist", "on", "these", "cells", "and,", "thus", "far,", "there", "has", "been", "no", "thorough", "evaluation", "of", "hES", "cell", "derived", "macrophages.", "Therefore", "we", "analyzed", "hES", "cell", "derived", "macrophages", "for", "the", "presence", "of", "characteristic", "cell", "surface", "markers", "and", "compared", "these", "to", "the", "phenotypic", "profile", "displayed", "on", "fetal", "CD34", "cell", "derived", "macrophages.", "The", "surface", "markers", "analyzed", "were", "CD14,", "a", "monocyte/macrophage", "specific", "marker,", "HLA-DR", "(a", "class", "II", "MHC", "molecule", "found", "on", "antigen", "presenting", "cells),", "CD4,", "the", "major", "receptor", "for", "HIV-1", "infection,", "and", "CCR5", "and", "CXCR4,", "chemokine", "receptors", "which", "are", "critical", "coreceptors", "essential", "for", "HIV-1", "entry.", "EGFP", "expression", "was", "also", "analyzed", "to", "determine", "the", "levels", "of", "transduction", "and", "any", "transgene", "silencing", "that", "may", "occur", "during", "differentiation.", "Fetal", "liver", "(CD34),", "nontransduced", "(ES),", "and", "vector", "transduced", "(GFP", "ES)", "hES", "cell", "derived", "macrophages", "were", "all", "positive", "for", "the", "monocyte/macrophage", "marker", "CD14", "(99.3%,", "88.7%,", "and", "99.2%,", "respectively)", "(Fig", "2A).", "However,", "the", "mean", "fluorescent", "intensity", "(MFI)", "was", "found", "to", "be", "lower", "on", "hES", "cell", "derived", "macrophages.", "Surface", "expression", "of", "HLA-DR", "was", "observed", "at", "similar", "levels", "between", "macrophages", "derived", "from", "fetal", "liver", "CD34", "cells", "(99.6%),", "nontransduced", "hES", "cells", "(92.8%),", "and", "transduced", "hES", "cells", "(98.2%)", "(Fig", "2A).", "CD4", "levels", "were", "comparable", "for", "all", "stem", "cell", "derived", "macrophages", "(99.2%,", "83.3%,", "and", "88.7%,", "respectively)", "(Fig", "2A).", "CCR5", "and", "CXCR4", "cell", "surface", "expression", "was", "also", "observed", "for", "fetal", "liver", "CD34", "cell", "(99.6%", "and", "99.3%),", "nontransduced", "hES", "cell", "(91.9%", "and", "92.6%),", "and", "transduced", "hES", "cell", "(98.9%", "and", "99.3%)", "derived", "macrophages", "(Fig", "2A).", "As", "compared", "to", "fetal", "liver", "CD34", "cell", "derived", "macrophages,", "hES", "cell", "derived", "macrophages", "displayed", "a", "higher", "level", "of", "expression", "of", "CXCR4.", "Isotype", "controls", "for", "both", "PE", "and", "PECY5", "stains", "are", "shown", "in", "Fig", "2B.", "The", "above", "phenotypic", "data", "are", "representative", "of", "triplicate", "experiments.", "Transgenic", "hES", "cell", "derived", "macrophages", "are", "functionally", "normalThe", "antigen", "presenting", "cell", "surface", "specific", "marker", "HLA-DR", "(MHC", "II)", "on", "normal", "macrophages", "is", "critical", "for", "presenting", "antigen", "to", "CD4", "T", "cells.", "A", "second", "co-stimulatory", "molecule,", "B7.1", "is", "present", "at", "low", "basal", "levels", "on", "resting", "macrophages", "and", "is", "necessary", "to", "activate", "T", "cells.", "Its", "expression", "is", "elevated", "upon", "activation", "with", "certain", "stimuli", "such", "as", "LPS.", "Our", "results", "of", "LPS", "stimulation", "of", "respective", "macrophages", "have", "shown", "upregulation", "of", "B7.1", "with", "values", "for", "fetal", "liver", "CD34", "cell", "(CD34)", "(27.9%", "to", "75.4%)", "nontransduced", "(ES)", "(17.8%", "to", "49.4%)", "and", "transduced", "(GFP", "ES)", "(35.6%", "to", "65.7%)", "hES", "cell", "derived", "macrophages", "(Fig", "3A).", "These", "values", "represent", "a", "significant", "upregulation", "of", "B7.1", "for", "all", "three", "macrophage", "populations.Figure", "3Functional", "analysis", "of", "hES", "cell", "derived", "macrophages", "for", "B7.1", "costimulatory", "molecule", "upregulation", "and", "phagocytosis", "of", "E.", "coli", "particles:", "A)", "Mature", "macrophages", "were", "stimulated", "with", "LPS", "to", "determine", "B7.1", "upregulation.", "Twenty-four", "hours", "post-stimulation,", "macrophages", "were", "labeled", "with", "a", "PE-CY5", "conjugated", "anti-B7.1", "antibody", "and", "analyzed", "by", "FACS.", "B7.1", "upregulation", "data", "are", "representative", "of", "triplicate", "experiments.", "Isotype", "control", "is", "shown", "in", "the", "left", "panel.", "B)", "To", "assess", "phagocytic", "function,", "E.", "coli", "Bioparticles®", "were", "added", "directly", "to", "the", "cultured", "macrophages.", "Twenty", "four", "hours", "post-addition,", "cells", "were", "analyzed", "by", "FACS.", "Percent", "positive", "cells", "are", "displayed", "in", "the", "plots", "for", "each", "experiment.", "These", "data", "are", "representative", "of", "triplicate", "experiments.Another", "important", "function", "of", "macrophages", "is", "their", "ability", "to", "phagocytose", "foreign", "material", "and", "present", "antigenic", "peptides", "on", "their", "cell", "surface.", "To", "evaluate", "phagocytic", "function,", "fluorescently", "labeled", "E.", "coli", "Bioparticles®", "were", "added", "to", "macrophage", "cultures", "followed", "by", "FACS", "analysis.", "Nontransduced", "(94.6%)", "as", "well", "as", "lentiviral", "vector", "transduced", "(98.7%)", "hES", "cell", "derived", "macrophages", "were", "found", "to", "be", "capable", "of", "phagocytosing", "the", "Bioparticles®", "in", "comparison", "to", "fetal", "liver", "CD34", "cell", "derived", "macrophages", "(95.8%)", "(Fig", "3B).", "These", "values", "are", "representative", "of", "triplicate", "experiments.", "Magi-CXCR4", "cells", "with", "no", "phagocytic", "capacity", "were", "used", "as", "non-phagocytic", "cell", "controls", "and", "similarly", "exposed", "to", "E.", "coli", "Bioparticles®", "(Fig", "3B).", "No", "uptake", "of", "the", "bacteria", "could", "be", "seen.", "Thus,", "uptake", "of", "E.", "coli", "Bioparticles®", "by", "macrophages", "is", "indicative", "of", "active", "ingestion.Macrophages,", "as", "effector", "cells,", "play", "a", "key", "role", "in", "the", "inflammatory", "response.", "Activated", "macrophages", "secrete", "various", "cytokines,", "two", "of", "the", "major", "ones", "being", "IL-1", "and", "TNF-α.", "To", "determine", "if", "hES", "cell", "derived", "macrophages", "have", "such", "a", "capacity,", "cells", "were", "stimulated", "with", "LPS.", "On", "days", "1,", "2,", "and", "3", "post-stimulation,", "culture", "supernatants", "were", "analyzed", "by", "ELISA", "to", "detect", "IL-1", "and", "TNF-α.", "As", "seen", "in", "figure", "4A,", "there", "were", "no", "significant", "differences", "in", "IL-1", "secretion", "between", "the", "three", "sets", "of", "macrophages.", "Similarly,", "nontransduced", "and", "transduced", "hES", "cell", "derived", "macrophages", "were", "also", "capable", "of", "TNF-α", "secretion", "upon", "LPS", "stimulation.", "However,", "levels", "of", "the", "respective", "cytokines", "detected", "were", "slightly", "lower", "than", "those", "from", "fetal", "liver", "CD34", "cell", "derived", "macrophages", "(Fig", "4B).", "The", "values", "of", "cytokine", "secretion", "levels", "represent", "triplicate", "experiments.Figure", "4Cytokine", "IL-1", "and", "TNFα", "secretion", "by", "stimulated", "hES", "cell", "derived", "macrophages:", "Macrophages", "derived", "from", "transduced", "and", "nontransduced", "hES", "and", "fetal", "liver", "CD34", "cells", "were", "stimulated", "with", "5", "μg/ml", "LPS.", "On", "days", "1,", "2,", "and", "3", "post-stimulation,", "supernatants", "were", "collected", "and", "assayed", "by", "ELISA", "for", "(A)", "IL-1", "and", "(B)", "TNFα.", "Experiments", "were", "done", "in", "triplicate.", "hES", "cell", "derived", "macrophages", "support", "productive", "HIV-1", "infectionThe", "above", "data", "have", "shown", "that", "hES", "cell", "derived", "macrophages", "are", "very", "similar", "to", "normal", "human", "macrophages", "based", "on", "phenotypic", "and", "functional", "analysis.", "In", "addition", "to", "being", "important", "cells", "of", "the", "immune", "system,", "macrophages", "are", "among", "the", "major", "target", "cells", "for", "certain", "viral", "infections,", "particularly", "for", "HIV-1.", "We", "wanted", "to", "determine", "if", "hES", "cell", "derived", "macrophages", "were", "susceptible", "to", "HIV-1", "infection", "compared", "to", "standard", "macrophages.", "In", "these", "studies,", "we", "only", "used", "an", "R5-tropic", "strain", "of", "HIV-1", "since", "macrophages", "are", "natural", "targets", "for", "this", "virus.", "Our", "results", "from", "challenge", "studies", "of", "these", "cells", "clearly", "indicated", "the", "capacity", "of", "hES", "cell", "derived", "macrophages", "in", "supporting", "a", "productive", "infection.", "Levels", "of", "virus", "increased", "up", "to", "15", "days", "similar", "to", "non-hES", "derived", "macrophages", "showing", "that", "the", "initial", "viral", "input", "was", "amplified", "in", "productive", "viral", "infection.", "However,", "the", "levels", "of", "viral", "yield", "were", "found", "to", "be", "slightly", "lower", "for", "the", "ES", "cell", "derived", "macrophages.", "In", "the", "case", "of", "GFP-ES", "macrophages,", "there", "was", "a", "decline", "in", "viral", "titer.", "This", "could", "be", "due", "to", "possible", "lower", "numbers", "of", "cells", "present", "in", "the", "initial", "cultures.", "As", "a", "first", "step", "towards", "the", "use", "of", "hES", "cells", "for", "hematopoietic", "stem", "cell", "and", "HIV", "gene", "therapies,", "we", "have", "shown", "here", "that", "phenotypically", "and", "functionally", "normal", "macrophages", "could", "be", "derived", "from", "hES-CD34", "cells.", "Both", "non", "transduced", "and", "lentiviral", "vector", "transduced", "hES", "cells", "were", "found", "to", "be", "capable", "of", "generating", "CD34", "cells", "that", "give", "rise", "to", "macrophages", "which", "could", "support", "productive", "HIV-1", "infection.", "Current", "sources", "of", "CD34", "cells", "consist", "of", "human", "bone", "marrow,", "cytokine", "mobilized", "peripheral", "blood,", "fetal", "liver,", "and", "cord", "blood", "[34].", "However,", "the", "number", "of", "cells", "that", "can", "be", "obtained", "for", "manipulations", "is", "not", "unlimited.", "Therefore,", "deriving", "CD34", "cells", "for", "therapeutic", "and", "investigative", "purposes", "from", "hES", "cells", "with", "unlimited", "growth", "potential", "has", "the", "advantage", "of", "a", "consistent", "and", "uniform", "source.", "The", "ability", "to", "obtain", "phenotypically", "normal", "and", "functionally", "competent", "macrophages", "from", "hES", "cells", "is", "important", "to", "evaluate", "their", "potential", "therapeutic", "utilities", "in", "the", "future.", "Additionally,", "testing", "of", "transgenic", "hES", "cells", "derived", "via", "lentiviral", "vector", "gene", "transduction", "is", "also", "helpful", "to", "determine", "the", "stability", "of", "the", "transgene", "expression", "and", "their", "capacity", "for", "differentiation", "into", "end", "stage", "mature", "cells", "such", "as", "macrophages.", "Based", "on", "these", "considerations,", "both", "non-", "transduced", "and", "lentiviral", "vector", "transduced", "hES", "cells", "were", "evaluated", "for", "their", "capacity", "to", "give", "rise", "to", "CD34", "progenitor", "cells.", "In", "colony", "forming", "assays", "using", "semisolid", "methylcellulose", "medium,", "the", "morphology", "of", "myelomonocytic", "colonies", "derived", "from", "hES", "CD34", "cells", "appeared", "similar", "to", "that", "of", "fetal", "liver", "CD34", "cells.", "When", "subsequently", "cultured", "in", "cytokine", "media", "that", "promotes", "macrophage", "differentiation,", "morphologically", "normal", "macrophages", "were", "obtained", "with", "hES-CD34", "cells", "similar", "to", "that", "of", "fetal", "liver", "CD34", "cells.", "At", "higher", "magnification,", "the", "macrophages", "displayed", "flat", "projecting", "cellular", "borders", "with", "fried", "egg", "appearance", "with", "distinct", "refractory", "lysosomal", "granules", "in", "the", "cytoplasm", "(data", "not", "shown).", "Lentiviral", "vector", "transduced", "hES", "cells", "also", "did", "not", "display", "any", "abnormal", "growth", "or", "differentiation", "characteristics", "as", "compared", "to", "nontransduced", "hES-CD34", "cells", "indicating", "no", "adverse", "effects", "due", "to", "vector", "integration", "and", "expression.", "Transduced", "cells", "gave", "rise", "to", "cystic", "bodies", "with", "similar", "CD34", "cell", "content", "and", "profiles", "upon", "development.", "The", "transduced", "hES-CD34", "cells", "also", "gave", "rise", "to", "apparently", "normal", "macrophages", "that", "expressed", "the", "transgene", "as", "shown", "by", "GFP", "expression.", "These", "results", "are", "consistent", "with", "those", "of", "others", "that", "showed", "normal", "differentiation", "of", "hES", "cells", "to", "other", "cell", "types", "following", "lentiviral", "transduction", "[32].", "A", "requirement", "for", "successful", "cellular", "and", "HIV-1", "gene", "therapy", "is", "that", "mature", "end", "stage", "cells", "derived", "from", "CD34", "progenitor", "cells", "be", "phenotypically", "and", "functionally", "normal", "to", "maintain", "and", "restore", "the", "body's", "immunological", "function.", "Accordingly,", "hES", "cell", "derived", "macrophages", "were", "evaluated", "to", "determine", "if", "they", "met", "these", "criteria.", "Macrophages", "display", "distinct", "cell", "surface", "markers", "upon", "end", "stage", "differentiation.", "To", "determine", "whether", "hES", "cell", "derived", "macrophages", "display", "these", "surface", "markers,", "FACS", "analysis", "was", "performed", "to", "detect", "the", "presence", "of", "CD14,", "HLA-DR", "(MHCII),", "CD4,", "CCR5,", "and", "CXCR4.", "As", "observed", "in", "Fig", "2A,", "both", "nontransduced", "and", "transduced", "hES", "cell", "derived", "macrophages", "expressed", "all", "of", "these", "markers", "with", "some", "differences", "in", "their", "levels", "of", "expression.", "HLA-DR,", "CD4,", "and", "CCR5", "expression", "profiles", "were", "comparable", "between", "all", "cell", "types", "analyzed.", "Even", "though", "all", "cell", "types", "analyzed", "stained", "positive", "for", "CD14,", "relative", "expression", "of", "CD14", "was", "slightly", "lower", "on", "hES", "cell", "derived", "macrophages", "compared", "to", "fetal", "liver", "CD34", "cell", "derived", "macrophages.", "On", "the", "contrary,", "the", "levels", "of", "CXCR4,", "a", "chemokine", "receptor", "involved", "in", "cellular", "homing,", "were", "found", "to", "be", "higher", "on", "hES-CD34", "cell", "derived", "macrophages.", "This", "may", "be", "due", "to", "inherent", "differences", "in", "the", "cell", "types", "and/or", "due", "to", "their", "physiological", "state", "at", "the", "time", "of", "harvest", "[35].", "Additional", "hES", "cell", "lines", "need", "to", "be", "evaluated", "in", "the", "future", "to", "establish", "if", "these", "differences", "are", "consistent.", "A", "major", "functional", "role", "of", "macrophages", "in", "vivo", "is", "their", "ability", "to", "serve", "as", "professional", "antigen", "presenting", "cells.", "During", "this", "process", "macrophages", "present", "antigen", "peptide", "fragments", "complexed", "with", "both", "classes", "of", "MHC", "molecules", "and", "deliver", "a", "costimulatory", "signal", "through", "the", "expression", "of", "B7", "molecules.", "Upon", "stimulation", "with", "LPS,", "hES-CD34", "cell", "derived", "macrophages", "had", "shown", "upregulation", "of", "the", "costimulatory", "molecule", "B7.1", "similar", "to", "cells", "derived", "from", "fetal", "liver.", "Furthermore,", "the", "hES-CD34", "cell", "derived", "macrophages", "also", "showed", "a", "normal", "capacity", "to", "ingest", "foreign", "particles", "in", "phagocytosis", "assays", "using", "E.coli", "Bioparticles®.", "In", "addition", "to", "antigen", "presentation", "and", "phagocytosis,", "macrophages", "also", "play", "a", "critical", "role", "in", "inflammation", "and", "secrete", "cytokines", "in", "response", "to", "external", "stimuli.", "When", "exposed", "to", "LPS,", "the", "hES-CD34", "cell", "derived", "macrophages", "secreted", "two", "important", "cytokines", "IL-1", "and", "TNF-α", "similar", "to", "that", "of", "fetal", "liver", "derived", "cells.", "The", "above", "data", "has", "established", "that", "phenotypically", "and", "functionally", "normal", "macrophages", "could", "be", "derived", "from", "hES-CD34", "cells.", "Macrophages", "in", "addition", "to", "playing", "important", "physiological", "roles", "are", "also", "major", "cell", "targets", "for", "certain", "viral", "infections,", "particularly", "HIV-1.", "Here", "we", "evaluated", "the", "susceptibility", "of", "hES-CD34", "cell", "derived", "macrophages", "to", "be", "productively", "infected", "with", "HIV-1.", "Similar", "to", "that", "of", "fetal", "liver", "CD34", "cell", "derived", "cells,", "the", "hES-CD34", "macrophages", "also", "supported", "HIV-1", "infection", "although", "the", "levels", "of", "viral", "yield", "differed", "somewhat.", "However", "this", "should", "not", "be", "a", "major", "concern", "for", "testing", "anti-HIV", "genes", "in", "these", "cells.", "In", "all", "the", "above", "experiments,", "the", "vector", "transduced", "transgenic", "macrophages", "also", "behaved", "similarly", "to", "that", "of", "nontransduced", "cells", "showing", "that", "they", "were", "also", "physiologically", "normal.", "The", "lack", "of", "vector", "toxicity", "on", "cellular", "maturation", "is", "encouraging", "for", "future", "work", "with", "transduced", "hES-CD34", "cells", "to", "derive", "other", "important", "differentiated", "cells", "like", "T", "cells", "and", "dendritic", "cells", "relevant", "for", "HIV", "studies.", "Although", "there", "are", "numerous", "studies", "on", "hES", "cell", "differentiation", "into", "many", "important", "end", "stage", "mature", "cells,", "systematic", "work", "on", "hES", "cell", "hematopoietic", "differentiation", "and", "thorough", "characterization", "of", "end", "stage", "mature", "cells", "that", "participate", "in", "critical", "immune", "responses", "has", "just", "begun", "[21,27-29].", "Our", "current", "results", "established", "that", "physiologically", "normal", "macrophages", "could", "be", "derived", "from", "hES", "cells", "and", "that", "these", "cells", "have", "the", "potential", "for", "use", "in", "cellular", "and", "gene", "therapies.", "To", "our", "knowledge", "this", "is", "the", "first", "demonstration", "that", "hES", "cell", "derivatives", "can", "be", "used", "for", "infectious", "disease", "research.", "Due", "to", "the", "extensive", "ability", "for", "hES", "cells", "to", "self-renew,", "large", "numbers", "of", "differentiated", "cells", "can", "be", "derived", "so", "that", "infection", "studies", "and", "evaluation", "tests", "can", "be", "carried", "out", "in", "a", "more", "standardized", "way.", "Our", "results", "showing", "that", "both", "normal", "and", "transgenic", "derivative", "macrophages", "support", "HIV-1", "infection", "points", "out", "to", "their", "utility", "for", "testing", "anti-HIV", "constructs", "transduced", "into", "hES-CD34", "cells", "and", "pave", "the", "way", "for", "their", "application", "in", "stem", "cell", "based", "HIV", "gene", "therapy.", "So", "far", "a", "number", "of", "studies", "including", "our", "own", "have", "tested", "many", "gene", "therapeutic", "constructs", "in", "CD34", "cells", "from", "conventional", "sources.", "These", "constructs", "include", "anti-HIV", "ribozymes,", "RNA", "decoys,", "transdominant", "proteins,", "bacterial", "toxins,", "anti-sense", "nucleic", "acids,", "and", "most", "recently", "siRNAs", "[36-50].", "In", "addition,", "a", "number", "of", "cellular", "molecules", "that", "aid", "in", "HIV-1", "infection", "such", "as", "cellular", "receptors", "and", "coreceptors", "CD4,", "CCR5", "and", "CXCR4", "have", "also", "been", "successfully", "tested", "in", "CD34", "cell", "derived", "macrophages", "and", "T", "cells", "[16,18,38].", "Some", "of", "these", "approaches", "have", "progressed", "into", "clinical", "evaluations", "as", "well", "[14,51,52].", "Based", "on", "our", "current", "results,", "many", "of", "these", "novel", "anti-HIV", "constructs", "can", "also", "be", "tested", "in", "hES-CD34", "cells", "for", "their", "potential", "application.", "Although", "there", "are", "advantages", "of", "using", "hES", "cell", "derived", "CD34", "cells", "for", "potential", "cellular", "therapies,", "transplantation", "of", "these", "cells", "constitutes", "an", "allogenic", "source", "with", "immune", "rejection", "as", "a", "major", "issue.", "However,", "a", "recent", "study", "using", "human", "leukocyte", "reconstituted", "mice", "suggested", "that", "hESCs", "and", "their", "derivative", "cell", "types", "were", "less", "prone", "to", "invoking", "an", "allogeneic", "response", "[53].", "Other", "recent", "studies", "demonstrated", "successful", "engraftment", "of", "primary", "and", "secondary", "recipients", "with", "hES", "cell", "derived", "hematopoietic", "cells", "in", "both", "immunodeficient", "mice", "and", "in", "vivo", "fetal", "sheep", "models", "adding", "further", "support", "that", "any", "obstacles", "could", "be", "overcome", "[23,54,55].", "Moreover,", "multiple", "novel", "strategies", "to", "avoid", "immune-mediated", "rejection", "of", "hES", "cell-derived", "cells", "have", "been", "proposed", "[56,57].", "It", "is", "not", "too", "far", "in", "the", "future", "that", "even", "autologous", "hES", "cells", "may", "be", "derived", "from", "specific", "individuals", "for", "deriving", "CD34", "cells", "which", "can", "be", "used", "for", "cell", "replacement", "therapy.", "Phenotypically", "normal", "and", "functionally", "competent", "macrophages", "could", "be", "derived", "from", "hES-CD34", "cells.", "Since", "these", "cells", "are", "susceptible", "to", "HIV-1", "infection,", "they", "provide", "a", "uniform", "source", "of", "macrophages", "for", "viral", "infection", "studies.", "Based", "on", "these", "results,", "it", "is", "also", "now", "feasible", "to", "transduce", "hES-CD34", "cells", "with", "anti-HIV", "genes", "such", "as", "inhibitory", "siRNAs", "and", "test", "their", "antiviral", "efficacy", "in", "down", "stream", "differentiated", "cells", "such", "as", "macrophages", "which", "are", "among", "the", "primary", "cells", "that", "need", "to", "be", "protected", "against", "HIV-1", "infection.", "Thus,", "the", "potential", "utility", "of", "hES", "derived", "CD34", "hematopoietic", "cells", "for", "HIV-1", "gene", "therapy", "can", "be", "evaluated.", "Growth,", "propagation", "and", "lentiviral", "transduction", "of", "hES", "cellsThe", "NIH", "approved", "human", "ES", "H1", "cell", "line", "was", "obtained", "from", "WiCell", "(Madison,", "Wisconsin).", "hES", "cell", "colonies", "were", "cultured", "on", "mouse", "embryonic", "fibroblasts", "(MEF)", "(Chemicon,", "Temecula,", "CA)", "in", "the", "presence", "of", "DMEM-F12", "(Invitrogen,", "Carlsbad,", "CA)", "supplemented", "with", "20%", "KNOCKOUT", "serum", "replacement", "with", "1", "mM", "L-glutamine,", "1%", "Nonessential", "Amino", "Acids,", "0.1", "mM", "β-mercaptoethanol,", "0.5%", "penicillin/streptomycin,", "and", "4", "ng/ml", "human", "basic", "fibroblast", "growth", "factor.", "Culture", "medium", "was", "replaced", "daily", "with", "fresh", "complete", "DMEM-F12.", "Mature", "colonies", "were", "subcultured", "weekly", "by", "digesting", "with", "collagenase", "IV", "as", "previously", "described", "[5].", "A", "VSV-G", "pseudotyped", "lentiviral", "vector", "(SINF-EF1a-GFP)", "containing", "a", "GFP", "reporter", "gene", "(kindly", "supplied", "by", "R.", "Hawley,", "George", "Washington", "University)", "was", "used", "for", "hES", "cell", "transductions", "as", "previously", "described", "(30,", "58).", "Generation", "of", "the", "pseudotyped", "vector", "in", "293T", "cells", "and", "its", "concentration", "by", "ultracentrifugation", "were", "described", "previously", "[30,48].", "For", "vector", "transduction,", "the", "undifferentiated", "hES", "cells", "were", "prepared", "into", "small", "clumps", "of", "50–100", "cells", "with", "enzyme", "digestion", "as", "done", "for", "routine", "passaging", "of", "cells.", "The", "cell", "clumps", "were", "incubated", "with", "the", "vector", "for", "2", "hrs", "in", "the", "presence", "of", "polybrene", "6", "ug/ml.", "A", "secondary", "cycle", "of", "transduction", "was", "done", "by", "adding", "fresh", "vector", "and", "incubating", "for", "another", "2", "hrs.", "The", "general", "vector", "titers", "were", "1", "×", "107", "and", "the", "multiplicity", "of", "infection", "was", "10.", "The", "transduction", "efficiency", "was", "about", "50%.", "The", "transduced", "colonies", "were", "cultured", "on", "MEF", "like", "above.", "Derivation", "and", "purification", "of", "CD34", "cells", "from", "hES", "cellsUndifferentiated", "hES", "cells", "were", "cultured", "on", "S17", "mouse", "bone", "marrow", "stromal", "cell", "monolayers", "to", "derive", "cystic", "bodies", "containing", "CD34+", "hematopoietic", "progenitor", "stem", "cells.", "hES", "cell", "cultures", "were", "treated", "with", "collagenase", "IV(1", "mg/ml)", "for", "10", "minutes", "at", "37°C", "and", "subsequently", "detached", "from", "the", "plate", "by", "gentle", "scraping", "of", "the", "colonies.", "The", "hES", "cell", "clusters", "were", "then", "transferred", "to", "irradiated", "(35", "Gy)", "S17", "cell", "layers", "and", "cultured", "with", "RPMI", "differentiation", "medium", "containing", "15%", "FBS", "(HyClone,", "Logan,", "UT),", "2", "mM", "L-glutamine,", "0.1", "mM", "β-mercaptoethanol,", "1%", "MEM-nonessential", "amino", "acids,", "and", "1%", "penicillin/streptomycin.", "Media", "was", "changed", "every", "2", "to", "3", "days", "during", "14–17", "days", "of", "culture", "on", "S17", "cells", "[20].After", "allowing", "adequate", "time", "for", "differentiation,", "hES", "cystic", "bodies", "were", "harvested", "and", "processed", "into", "a", "single", "cell", "suspension", "by", "collagenase", "IV", "treatment", "followed", "by", "digestion", "with", "trypsin/EDTA", "supplemented", "with", "2%", "chick", "serum", "(Invitrogen,", "Carlsbad,", "CA)", "for", "20", "minutes", "at", "37°C.", "Cells", "were", "washed", "twice", "with", "PBS", "and", "filtered", "through", "a", "70", "uM", "cell", "strainer", "to", "obtain", "a", "single", "cell", "suspension.", "To", "assess", "the", "levels", "of", "CD34", "cells", "in", "the", "bulk", "cell", "suspension,", "cells", "were", "labeled", "with", "PE", "conjugated", "anti-CD34", "antibody", "(BD", "Biosciences,", "San", "Jose,", "CA)", "and", "analyzed", "by", "FACS.", "To", "purify", "the", "CD34", "cells,", "Direct", "CD34", "Progenitor", "Cell", "Isolation", "Kit", "(Miltenyi", "Biotech,", "Auburn,", "CA)", "was", "used", "following", "the", "manufacturer's", "protocol.", "Isolated", "CD34", "hematopoietic", "progenitor", "stem", "cells", "were", "then", "analyzed", "by", "FACS", "as", "mentioned", "above", "to", "determine", "cell", "purity.", "For", "comparative", "experiments,", "human", "CD34", "hematopoietic", "progenitor", "cells", "were", "also", "purified", "from", "fetal", "liver", "tissue", "as", "described", "above.", "Derivation", "of", "macrophages", "from", "hES", "cell", "derived", "and", "human", "fetal", "CD34", "cellsCD34", "cells", "were", "cultured", "initially", "in", "semisolid", "media", "to", "derive", "myelomonocytic", "colonies", "followed", "by", "liquid", "culture", "in", "cytokine", "supplemented", "media", "as", "described", "below.", "Purified", "CD34+", "progenitor", "cells", "(~2.5", "×", "105", "to", "4.0", "×", "105)", "were", "placed", "directly", "into", "Methocult", "semisolid", "medium", "(Stem", "Cell", "Technologies,", "Vancouver,", "BC),", "mixed,", "and", "cultured", "in", "35", "mm", "plates.", "Myeloid", "colonies", "were", "allowed", "to", "develop", "for", "12–15", "days.", "Upon", "differentiation", "and", "proliferation,", "myelomonocytic", "colonies", "were", "harvested", "by", "the", "addition", "of", "5", "ml", "DMEM", "containing", "10%", "FBS,", "10", "ng/ml", "each", "GM-CSF", "and", "M-CSF.", "Cells", "(~106)", "were", "placed", "in", "a", "35", "mm", "well", "and", "allowed", "to", "adhere", "for", "48", "hours.", "At", "two", "and", "four", "days", "post-harvest,", "medium", "was", "replaced", "with", "fresh", "complete", "DMEM", "supplemented", "with", "10", "ng/ml", "GM-CSF", "and", "M-CSF.", "By", "4–5", "days,", "cells", "developed", "into", "mature", "macrophages", "which", "were", "used", "for", "subsequent", "phenotypic", "and", "functional", "characterization.", "Phenotypic", "analysis", "of", "hES", "cell", "derived", "macrophagesTo", "determine", "if", "nontransduced", "and", "lentiviral", "vector", "transduced", "hES", "cell", "derived", "macrophages", "display", "normal", "macrophage", "surface", "markers,", "FACS", "analysis", "was", "performed", "using", "respective", "fluorochrome", "conjugated", "antibodies.", "Fetal", "liver", "derived", "CD34+", "cells", "as", "well", "as", "nontransduced", "and", "transduced", "hES", "cell", "derived", "macrophages", "were", "evaluated", "in", "parallel.", "Cells", "were", "scraped", "from", "their", "wells,", "washed", "two", "times", "with", "PBS,", "and", "stained", "with", "the", "following", "antibodies:", "PE-CD14,", "PE-HLA-DR,", "PECY5-CD4,", "PECY5-CCR5,", "PECY5-CXCR4", "(BD", "Biosciences,", "San", "Jose,", "CA).", "A", "blocking", "step", "was", "first", "performed", "by", "incubating", "the", "cells", "with", "the", "respective", "isotype", "control", "for", "30", "minutes", "at", "4C", "before", "staining", "with", "the", "respective", "cell", "surface", "marker", "antibodies.", "Isotype", "control", "staining", "was", "used", "to", "determine", "background", "levels.", "FACS", "analysis", "was", "performed", "on", "a", "Beckman-Coulter", "EPICS", "®", "XL-MCL", "flow", "cytometer", "with", "data", "analysis", "using", "EXPO32", "ADC", "software", "(Coulter", "Corporation,", "Miami,", "FL).", "A", "minimum", "of", "8,000", "cells", "were", "analyzed", "in", "each", "FACS", "evaluation.", "Functional", "analysis", "of", "hES", "cell", "derived", "macrophagesPhysiological", "roles", "of", "macrophages", "include", "phagocytic", "and", "immune", "related", "functions.", "To", "determine", "if", "hES", "cell", "derived", "macrophages", "were", "functionally", "normal,", "a", "stimulation", "assay", "to", "determine", "upregulation", "of", "the", "costimulatory", "molecule", "B7.1", "was", "performed.", "Activated", "macrophages", "upregulate", "the", "expression", "of", "B7.1", "upon", "activation", "with", "various", "stimuli.", "Accordingly,", "fetal", "liver", "CD34,", "nontransduced", "hES,", "and", "GFP-alone", "transduced", "hES", "cell", "derived", "macrophages", "were", "stimulated", "by", "the", "addition", "of", "LPS", "(5", "ug/ml)", "to", "the", "cell", "culture", "medium.", "Twenty-four", "hours", "post-stimulation,", "cells", "were", "stained", "with", "an", "anti-B7.1", "antibody", "labeled", "with", "PE-Cy5", "(BD", "Biosciences,", "San", "Jose,", "CA)", "and", "analyzed", "by", "FACS.", "To", "assess", "the", "hES", "cell", "derived", "macrophages'", "phagocytic", "function,", "5", "ug/ml", "of", "fluorescently", "labeled", "E.", "coli", "Bioparticles®", "(Invitrogen,", "Carlsbad,", "CA)", "were", "added", "directly", "to", "the", "cell", "culture", "medium.", "Four", "hours", "later,", "macrophages", "were", "washed", "six", "times", "with", "PBS", "and", "fresh", "medium", "with", "10", "ng/ml", "GM-CSF", "and", "M-CSF", "was", "added.", "Twenty-four", "hours", "later,", "cells", "were", "analyzed", "by", "FACS", "for", "the", "presence", "of", "ingested", "Bioparticles®", "which", "can", "be", "detected", "in", "the", "PE", "(FL2)", "channel.", "Lentiviral", "vector", "transduced", "Magi-CXCR4", "cells,", "a", "HeLa", "cell", "derivative", "with", "no", "phagocytic", "capacity,", "were", "used", "as", "non-phagocytic", "cell", "controls", "and", "similarly", "exposed", "to", "E.", "coli", "Bioparticles®Human", "ES", "cell", "derived", "macrophages", "were", "also", "analyzed", "for", "their", "ability", "to", "secrete", "two", "major", "cytokines,", "IL-1", "and", "TNF-α,", "upon", "external", "stimulation.", "Accordingly,", "macrophages", "were", "stimulated", "with", "5", "ug/ml", "of", "LPS", "during", "culture.", "On", "days", "1,", "2,", "and", "3", "post-stimulation,", "cell", "culture", "supernatant", "samples", "were", "collected", "and", "analyzed", "by", "a", "Quantikine®", "ELISA", "kit", "(R&D", "Systems,", "Minneapolis,", "MN).", "Non-stimulated", "supernatants", "were", "also", "analyzed", "for", "basal", "levels", "of", "cytokine", "secretion.", "HIV-1", "infection", "of", "hES", "cell", "derived", "macrophagesTo", "determine", "if", "hES", "cell", "derived", "macrophages", "can", "be", "infected", "with", "HIV-1", "and", "support", "viral", "replication,", "cells", "were", "challenged", "with", "a", "macrophage", "R5-tropic", "BaL-1", "strain", "of", "HIV-1.", "An", "m.o.i.", "of", "0.01", "in", "the", "presence", "of", "4", "ug/ml", "polybrene", "was", "used.", "At", "different", "days", "post-infection,", "culture", "supernatants", "were", "collected", "and", "assayed", "for", "p24", "antigen", "by", "ELISA.", "To", "quantify", "viral", "p24", "levels,", "a", "Coulter-p24", "kit", "(Beckman", "Coulter,", "Fullerton,", "CA)", "was", "used." ]
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"neoorganogenesis", "potential,", "the", "ability", "of", "hESCs,", "and", "the", "more", "differentiated", "myogenic", "ASCs", "to", "contribute", "to", "tissue", "repair", "in", "the", "old", "will", "be", "greatly", "restricted", "due", "to", "the", "conserved", "inhibitory", "influence", "of", "aged", "differentiated", "niches.", "Significantly,", "this", "work", "establishes", "that", "hESC-derived", "factors", "enhance", "the", "regenerative", "potential", "of", "both", "young", "and,", "importantly,", "aged", "muscle", "stem", "cells", "in", "vitro", "and", "in", "vivo;", "thus,", "suggesting", "that", "the", "regenerative", "outcome", "of", "stem", "cell-based", "replacement", "therapies", "will", "be", "determined", "by", "a", "balance", "between", "negative", "influences", "of", "aged", "tissues", "on", "transplanted", "cells", "and", "positive", "effects", "of", "embryonic", "cells", "on", "the", "endogenous", "regenerative", "capacity.", "Comprehensively,", "this", "work", "points", "toward", "novel", "venues", "for", "in", "situ", "restoration", "of", "tissue", "repair", "in", "the", "old", "and", "identifies", "critical", "determinants", "of", "successful", "cell-replacement", "therapies", "for", "aged", "degenerating", "organs.", "Embryonic", "stem", "cells", "(ESCs)", "are", "distinguished", "by", "their", "ability", "to", "self-renew", "and", "to", "differentiate", "into", "any", "other", "cell", "type", "via", "asymmetric", "cell", "divisions,", "in", "which", "one", "daughter", "cell", "maintains", "‘stemness’", "while", "the", "other", "daughter", "cell", "differentiates", "into", "a", "particular", "tissue", "type.", "ESCs,", "including", "those", "of", "human", "origin", "(hESCs),", "are", "derived", "from", "the", "blastocyst", "and", "can", "be", "propagated", "in", "vitro", "(Evans", "&", "Kaufman,", "1981;", "Thomson", "et", "al.,", "1998;", "Wobus", "&", "Boheler,", "2005).", "Their", "tremendous", "potential", "for", "organogenesis", "has", "created", "a", "great", "interest", "in", "using", "hESCs", "for", "replacing", "tissues", "and", "organs", "lost", "to", "disease,", "or", "old", "age", "(reviewed", "in", "Wobus", "&", "Boheler,", "2005).", "As", "such,", "the", "use", "of", "hESCs", "is", "particularly", "important,", "due", "to", "the", "fact", "that", "adult", "organ", "stem", "cells", "are", "often", "limited", "in", "number,", "cell-fate", "plasticity,", "expansion", "capacity,", "telomere", "length,", "and", "lifespan", "(Mayhall", "et", "al.,", "2004).", "The", "general", "goal", "behind", "most", "cell-replacement", "approaches", "is", "to", "expand", "and", "then", "differentiate", "hESCs", "in", "vitro,", "thus", "producing", "a", "cell", "type", "of", "interest,", "such", "as", "neuronal,", "blood,", "endothelial,", "pancreatic,", "bone,", "and", "others.", "These", "differentiated", "cells", "are", "expected", "to", "replace", "their", "dysfunctional", "counterparts", "in", "vivo.", "The", "scope", "of", "disorders", "that", "can", "be", "potentially", "treated", "with", "a", "neoorganogenesis", "approach", "is", "large", "and", "includes", "many", "that", "are", "currently", "incurable,", "such", "as", "muscle", "atrophy,", "diabetes,", "Alzheimer's", "disease,", "Parkinson's", "disease,", "and", "other", "degenerative", "diseases", "that", "often", "accompany", "human", "aging", "(McDonald", "et", "al.,", "1999;", "Liu", "et", "al.,", "2000;", "Hori", "et", "al.,", "2002;", "Kim", "et", "al.,", "2002;", "Blyszczuk", "et", "al.,", "2003).", "While", "many", "studies", "have", "focused", "on", "the", "derivation,", "propagation,", "and", "in", "vitro", "differentiation", "of", "hESCs", "(reviewed", "in", "Hoffman", "&", "Carpenter,", "2005;", "Wobus", "&", "Boheler,", "2005),", "relatively", "few", "have", "examined", "the", "properties", "of", "these", "cells", "and", "their", "more", "differentiated", "progeny", "in", "the", "aged,", "as", "opposed", "to", "the", "young,", "systemic", "and", "local", "organ", "environments.", "Recently", "published", "data", "suggest", "that", "these", "extrinsic", "cues", "become", "altered", "with", "age", "in", "ways", "that", "preclude", "activation", "of", "organ", "stem", "cells", "(such", "as", "satellite", "cells),", "inhibit", "repair-specific", "molecular", "signaling", "(such", "as", "delta-Notch),", "and", "interfere", "with", "productive", "tissue", "repair", "(Conboy", "et", "al.,", "2003,", "2005;", "Janzen", "et", "al.,", "2006;", "Krishnamurthy", "et", "al.,", "2006;", "Molofsky", "et", "al.,", "2006).", "Furthermore,", "at", "least", "two", "lines", "of", "evidence", "suggest", "that", "stem", "cell-based", "tissue-replacement", "therapies", "might", "be", "hindered", "in", "the", "elderly,", "because", "all", "cells", "along", "the", "developmental", "lineage", "(e.g.,", "stem", "cells,", "more", "differentiated", "progenitor", "cells", "or", "even", "tissues", "containing", "a", "pool", "of", "precursors)", "might", "rapidly", "‘age’", "and", "fail", "to", "contribute", "to", "organ", "repair", "when", "introduced", "into", "the", "old", "organism", "in", "vivo.", "First,", "in", "heterochronic", "tissue-transplantation", "studies,", "the", "age", "of", "the", "host", "environment", "determined", "the", "regenerative", "outcome,", "as", "both", "young", "and", "old", "skeletal", "muscle", "explants", "containing", "differentiated", "and", "precursor", "cells", "effectively", "regenerated", "in", "young,", "but", "not", "in", "old", "animals", "(Zacks", "&", "Sheff,", "1982;", "Carlson", "&", "Faulkner,", "1989).", "Second,", "using", "parabiotically", "paired", "young", "and", "old", "mice,", "the", "regenerative", "potential", "of", "muscle", "and", "liver", "was", "shown", "to", "be", "influenced", "by", "the", "age", "of", "the", "systemic", "environment", "(Conboy", "et", "al.,", "2005).", "Thus,", "we", "sought", "to", "determine", "whether", "key", "molecular", "identifiers", "of", "stem", "cell", "properties,", "the", "rate", "of", "cell", "proliferation,", "and", "the", "myogenic", "capacity", "would", "be", "influenced", "by", "the", "age", "of", "extrinsic", "milieu,", "regardless", "of", "whether", "stem", "cells", "are", "embryonic", "or", "the", "more", "differentiated,", "muscle-specific", "satellite", "cells.", "Satellite", "cells", "are", "muscle", "stem", "cells", "situated", "in", "direct", "contact", "with", "myofibers,", "the", "differentiated", "muscle", "cells.", "When", "myofibers", "are", "damaged,", "quiescent", "satellite", "cells", "are", "activated", "to", "proliferate", "and", "then", "differentiate", "into", "fusion-competent", "myoblasts", "that", "continue", "to", "proliferate", "and", "can", "form", "primary", "cultures,", "but", "are", "also", "capable", "of", "producing", "new,", "multinucleated", "myofibers", "or", "myotubes", "in", "vitro", "and", "in", "vivo", "(Morgan", "et", "al.,", "2002;", "Collins", "et", "al.,", "2005;", "Wagers", "&", "Conboy,", "2005).", "Activated", "satellite", "cells", "express", "myogenic", "markers,", "such", "as", "Myf5,", "M-cadherin,", "and", "Paired", "box", "gene", "7", "(Pax7);", "fusion-competent", "myoblasts", "express", "high", "levels", "of", "desmin,", "and", "de", "novo", "generated", "myofibers", "or", "myotubes", "express", "embryonic", "myosin", "heavy", "chain", "(eMyHC)", "and", "continue", "to", "express", "desmin", "(Schultz", "&", "McCormick,", "1994;", "Wagers", "&", "Conboy,", "2005).", "While", "desmin", "can", "be", "also", "present", "in", "smooth", "and", "cardiac", "muscle", "cells,", "the", "isolation", "of", "hind", "limb", "skeletal", "muscle", "with", "subsequent", "purification", "of", "myofibers", "away", "from", "all", "interstitial", "cells,", "as", "well", "as", "purification", "of", "associated", "muscle", "stem", "cells", "results", "in", "primary", "cultures", "that", "are", "uniformly", "of", "skeletal", "muscle", "lineage.", "Every", "desmin+", "cell", "in", "such", "cultures", "is", "a", "fusion-competent", "myoblast,", "and", "is", "able", "to", "produce", "multinucleated", "myotubes", "after", "48", "h", "of", "culture", "in", "differentiation-promoting", "medium", "[Dulbecco's", "modified", "Eagle's", "medium", "(DMEM)", "with", "2%", "horse", "serum].", "Some", "of", "these", "myogenic", "cells", "fuse", "into", "myotubes,", "even", "in", "the", "mitogen-rich", "medium", "[(Opti-MEM", "(Invitrogen,", "Carlsbad,", "CA,", "USA)", "with", "5–10%", "mouse", "serum", "or", "DMEM", "with", "10%", "fetal", "bovine", "serum,", "FBS]", "(Conboy", "&", "Rando,", "2002;", "Conboy", "et", "al.,", "2003;", "and", "see", "below).", "An", "experimental", "system", "was", "developed", "that", "(i)", "provided", "the", "ability", "to", "study", "the", "regenerative", "response", "of", "hESCs", "and", "of", "muscle", "stem", "cells", "in", "various", "heterochronic", "environments", "in", "vitro;", "and", "(ii)", "allowed", "examination", "of", "the", "effects", "of", "hESCs", "on", "muscle", "repair,", "in", "vivo,", "after", "transplantation", "into", "young", "vs.", "old", "hosts.", "This", "model", "allowed", "us", "to", "address", "both", "the", "negative", "effects", "of", "the", "aged", "niche", "on", "key", "stem", "cell", "properties", "and", "the", "positive", "effects", "of", "hESCs", "on", "the", "aged", "muscle-specific", "organ", "progenitor", "cells", "in", "vitro,", "and", "on", "the", "regenerative", "capacity", "of", "old", "muscle", "in", "vivo.", "The", "resulting", "data", "demonstrate", "that", "the", "composition", "of", "conserved", "extrinsic", "cues,", "regulating", "stem", "cell", "responses,", "becomes", "altered", "with", "age", "in", "ways", "that", "inhibit", "both", "hESCs", "and", "adult", "stem", "cell", "regenerative", "potential.", "Specifically,", "molecular", "markers", "of", "stem", "cell", "functionality,", "e.g.", "Oct4", "(in", "hESCs)", "and", "Myf5", "(in", "muscle", "stem", "cells),", "the", "rate", "of", "cell", "proliferation,", "and", "the", "capacity", "for", "myogenic", "differentiation", "are", "all", "dominantly", "inhibited", "by", "the", "aged", "systemic", "milieu,", "and", "by", "the", "old", "differentiated", "muscle", "tissue.", "However,", "while", "satellite", "cells", "are", "unable", "to", "deter", "the", "inhibitory", "affects", "of", "aged", "systemic", "and", "local", "niches,", "hESCs", "are", "capable", "of", "antagonizing", "the", "aged", "environments,", "thereby", "enhancing", "the", "regenerative", "potential", "of", "both", "young", "and", "old", "muscle", "stem", "cells", "in", "vitro", "and", "in", "vivo.", "Thus,", "a", "complex", "interplay", "between", "negative", "regulation", "of", "hESCs", "and", "adult", "muscle", "stem", "cells", "by", "the", "aged", "niche,", "and", "positive", "regulation", "of", "the", "host's", "regenerative", "responses", "by", "hESCs", "will", "likely", "determine", "the", "success", "of", "hESC-based", "cell-replacement", "therapies", "in", "the", "old.", "Regenerative", "responses", "of", "adult", "muscle", "stem", "cells", "and", "hESCs", "are", "dominantly", "inhibited", "by", "the", "aged", "systemic", "milieuPrevious", "work", "established", "that", "the", "upregulation", "of", "repair-specific", "molecular", "signaling", "mechanisms,", "such", "as", "Notch,", "and", "successful", "engagement", "of", "resident", "muscle", "stem", "cells", "in", "tissue", "repair", "are", "largely", "determined", "by", "the", "age", "of", "the", "systemic", "milieu,", "rather", "than", "by", "the", "cell-autonomous", "age", "of", "muscle", "cells,", "or", "by", "the", "differences", "in", "their", "numbers", "(Conboy", "&", "Rando,", "2005;", "Conboy", "et", "al.,", "2005).", "Intriguingly,", "these", "experiments", "also", "hinted", "at", "a", "small", "but", "persistent", "inhibitory", "effect", "of", "the", "aged", "systemic", "milieu", "on", "the", "performance", "of", "young", "stem", "cells.", "Exploring", "this", "further,", "we", "found", "that", "young", "serum", "permits", "satellite", "cells", "to", "be", "myogenic,", "while", "old", "serum", "inhibits", "the", "satellite", "cell", "regenerative", "potential", "not", "only", "alone,", "but", "also", "when", "mixed", "with", "young", "serum,", "suggesting", "a", "dominant", "over-riding", "of", "‘young’", "serum", "factors", "(Fig.", "1).", "Myofiber", "cultures,", "in", "which", "satellite", "cells", "have", "been", "activated", "by", "injury", "in", "vivo,", "were", "established", "from", "young", "(2–3", "months)", "and", "old", "(22–24", "months)", "C57-BL/6", "male", "mice,", "as", "previously", "described", "(Conboy", "&", "Rando,", "2002;", "Conboy", "et", "al.,", "2005).", "As", "previously", "shown,", "this", "method", "is", "well", "suited", "for", "the", "assessment", "of", "satellite", "cell", "regenerative", "myogenic", "capacity", "(Conboy", "&", "Rando,", "2002;", "Wagers", "&", "Conboy,", "2005).", "Isolated", "myofiber", "explants", "with", "associated", "satellite", "cells", "were", "cultured", "overnight", "in", "the", "presence", "of", "young", "or", "old", "serum", "(alone", "at", "5%", "and", "10%,", "and", "mixed", "at", "5%", "young", "+", "5%", "old);", "bromodeoxyuridine", "(BrdU)", "was", "added", "for", "the", "last", "2", "h", "of", "culture", "to", "measure", "the", "rate", "of", "cell", "proliferation.", "The", "effects", "of", "heterochronic", "systemic", "milieu", "on", "myogenic", "potential", "were", "examined", "as", "generation", "of", "proliferating", "myoblasts", "that", "express", "desmin", "and", "Myf5,", "and", "that", "spontaneously", "form", "multinucleated", "nascent", "myotubes.", "As", "shown", "in", "Fig.", "1A", "and", "quantified", "in", "Fig.", "1B,", "the", "age", "of", "sera", "clearly", "determined", "satellite", "cell", "regenerative", "potential", "and", "old", "serum", "strongly", "inhibited", "the", "myogenic", "potential", "of", "young", "satellite", "cells", "either", "when", "present", "alone,", "or", "when", "mixed", "with", "young", "sera.", "Similar", "data", "was", "obtained", "by", "using", "another", "myogenic", "marker,", "Pax7", "(Supplementary", "Fig.", "S1).", "Additionally,", "there", "were", "two", "to", "three", "times", "fewer", "total", "cells", "generated", "in", "the", "presence", "of", "aged", "serum", "(not", "shown).Fig.", "1The", "age", "of", "sera", "determined", "the", "regenerative", "potential", "of", "satellite", "cells.", "(A)", "Young", "satellite", "cells", "were", "cultured", "either", "in", "5%", "or", "10%", "young", "(Young),", "10%", "old", "(Old),", "or", "in", "a", "5%+", "5%", "mouse", "sera", "combination", "(young", "+", "old).", "Cells", "were", "analyzed", "by", "immunofluorescence", "microscopy,", "using", "anti-BrdU", "(red),", "antidesmin", "(green)", "or", "anti-Myf5", "antibodies", "(green,", "small", "panels).", "Similar", "results", "are", "shown", "for", "Pax7", "immunodetection", "(Supplementary", "Fig.", "S1).", "Hoechst", "(blue)", "labeled", "nuclei.", "(B)", "Three", "independent", "experiments", "were", "quantified", "[300", "young", "myofibers", "per", "experiment]", "as", "percentage", "of", "desmin+/Myf5+/BrdU+", "de", "novo", "generated", "cells", "for", "each", "age", "and", "culture", "condition.", "On", "average,", "two", "to", "three", "fewer", "cells", "were", "generated", "when", "cultured", "in", "the", "presence", "of", "old.", "Shown", "are", "identical", "microscope", "fields", "at", "×40", "magnification.", "At", "least", "three", "independent", "experiments", "produced", "similar", "results.", "(*)", "indicates", "P≤", "0.001", "as", "compared", "to", "young", "sera.Importantly,", "it", "was", "not", "simply", "the", "dilution", "of", "young", "serum", "factors", "that", "resulted", "in", "diminished", "myogenic", "capacity", "when", "young", "and", "old", "sera", "were", "mixed,", "because", "young", "sera", "promoted", "robust", "myogenesis", "both", "at", "10%", "and", "5%.", "Thus,", "old", "serum", "factors", "dominantly", "inhibited", "the", "myogenic", "capacity", "of", "young", "satellite", "cells", "even", "in", "the", "presence", "of", "young", "serum.", "This", "observation", "suggests", "that", "satellite", "cells", "of", "young", "mice", "engage", "in", "efficient", "myogenic", "responses,", "in", "part,", "because", "the", "inhibitory", "influence", "of", "old", "circulatory", "milieu", "is", "absent.These", "data", "reveal", "that", "the", "regenerative", "potential", "of", "young", "muscle", "stem", "cells", "is", "determined", "by", "the", "age", "of", "the", "systemic", "milieu,", "prompting", "us", "to", "investigate", "whether", "hESCs", "would", "similarly", "succumb", "to", "inhibitory", "factors", "present", "in", "the", "aged", "circulation.To", "determine", "the", "effects", "of", "aged", "serum", "on", "stem", "cell", "self-renewal/pluripotency,", "we", "analyzed", "hESC", "expression", "of", "Oct4", "and", "studied", "the", "rate", "of", "hESC", "proliferation,", "by", "assessing", "BrdU", "incorporation", "(Fig.", "2)", "and", "Ki67", "expression", "(Supplementary", "Fig.", "S2).", "Specifically,", "these", "determinants", "of", "hESC", "regenerative", "potential", "were", "examined", "in", "the", "presence", "of", "heterochronic", "(young", "vs.", "old)", "mouse", "sera", "added", "to", "typical", "hESC", "medium,", "e.g.,", "MEF-conditioned", "medium", "(MCM).", "Oct4", "is", "expressed", "by", "self-renewing,", "pluripotent", "ESCs", "in", "culture,", "by", "the", "totipotent", "inner", "cell", "mass", "of", "the", "blastocyst", "and", "by", "the", "germ", "cells", "(Nichols", "et", "al.,", "1998;", "Pesce", "et", "al.,", "1999).", "Most", "cells", "in", "control", "cultures", "or", "young", "conditions", "expressed", "high", "levels", "of", "this", "marker", "of", "‘stemness’,", "and", "maintained", "their", "normal", "phenotype", "and", "morphology", "throughout", "the", "various", "co-culture", "experiments", "performed", "in", "this", "study", "(see", "below).Fig.", "2The", "regenerative", "potential", "of", "embryonic", "stem", "cells", "was", "negatively", "affected", "by", "aged", "mouse", "sera.", "(A)", "hESCs", "were", "cultured", "in", "MCM", "with", "10%", "young", "(young)", "or", "old", "(old)", "mouse", "serum,", "or", "in", "three", "control", "media:", "MCM", "without", "mouse", "sera;", "GM", "(myoblast", "medium", "of", "Ham's", "F10", "with", "20%", "FBS)", "and", "DMEM/FBS", "(hESC", "differentiation", "medium", "of", "DMEM", "with", "10%", "FBS).", "BrdU", "was", "added", "for", "the", "last", "2", "h", "of", "culture", "to", "measure", "the", "rate", "of", "cell", "proliferation.", "Immunodetection", "assays", "were", "performed", "for", "BrdU", "(red),", "Oct4", "(red),", "and", "Ki67", "(Supplementary", "Fig.", "S2).", "Hoechst", "(blue)", "labels", "nuclei.", "A", "high", "rate", "of", "hESC", "proliferation", "and", "Oct4", "expression", "is", "displayed", "in", "all", "control", "media", "and", "in", "the", "presence", "of", "young", "mouse", "serum.", "In", "contrast,", "hESC", "proliferation", "and", "Oct4", "expression", "are", "inhibited", "in", "the", "presence", "of", "old", "mouse", "serum,", "either", "alone", "or", "when", "mixed", "with", "young", "serum.", "MCM", "with", "mouse", "sera", "at", "5%", "gave", "results", "similar", "to", "those", "observed", "with", "10%", "young", "mouse", "sera", "or", "in", "control", "media", "(Supplementary", "Fig.", "S3).", "(B)", "Three", "independent", "experiments", "yielded", "similar", "results", "and", "were", "quantified", "as", "percentage", "of", "BrdU+", "and", "Oct4+", "cells", "for", "each", "culture", "condition.", "*", "indicates", "P", "<", "0.001", "as", "compared", "to", "young", "serum.Importantly,", "at", "10%", "aged", "serum", "dramatically", "inhibited", "the", "self-renewal", "and", "proliferative", "potential", "of", "hESCs,", "as", "judged", "by", "highly", "diminished", "Oct4", "expression", "and", "a", "lack", "of", "BrdU", "incorporation.", "Again,", "the", "inhibitory", "factors", "in", "the", "aged", "milieu", "were", "dominant", "over", "the", "young,", "as", "evidenced", "by", "a", "decline", "in", "Oct4", "expression,", "the", "low", "rate", "of", "BrdU", "incorporation,", "and", "Ki67", "expression", "in", "young", "and", "old", "mixed", "environments", "(5%", "young", "+", "5%", "old", "sera", "in", "MCM).", "Similar", "to", "the", "data", "shown", "for", "adult", "stem", "cells", "(ASCs)", "(Fig.", "1),", "it", "was", "not", "simply", "a", "dilution", "of", "young", "serum", "factors", "as", "hESCs", "robustly", "proliferated", "and", "expressed", "high", "levels", "of", "Oct4", "when", "cultured", "with", "5%", "young", "sera", "in", "MCM", "(Supplementary", "Fig.", "S3).", "Quantification", "of", "multiple", "independent", "experiments", "has", "demonstrated", "that", "hESC", "expression", "of", "Oct4", "and", "BrdU", "incorporation", "have", "been", "reduced", "by", "two-", "to", "threefold", "in", "the", "aged", "milieu", "(Fig.", "2B).As", "expected,", "hESCs", "cultured", "in", "control", "media,", "including", "MCM", "alone", "that", "does", "not", "contain", "either", "young", "or", "old", "serum,", "also", "displayed", "a", "high", "rate", "of", "proliferation", "and", "Oct4", "expression", "(Fig.", "2,", "control", "medium).", "Additionally,", "in", "this", "experimental", "set-up", "there", "was", "no", "general", "inhibitory", "effect", "of", "sera", "per", "se", "on", "hESC", "proliferation", "and", "Oct4", "expression,", "as", "10%", "young", "mouse", "sera", "(young)", "and", "10–20%", "of", "FBS", "(growth", "medium", "and", "DMEM/FBS)", "allowed", "for", "a", "high", "rate", "of", "cell", "proliferation", "and", "for", "uniformly", "high", "Oct4", "levels", "(Fig.", "2).When", "instead", "of", "immediate", "exposure", "to", "aged", "mouse", "serum,", "hESCs", "were", "first", "cultured", "overnight", "in", "MCM,", "these", "cells", "were", "no", "longer", "susceptible", "to", "the", "negative", "effects", "of", "old", "systemic", "milieu", "(Fig.", "3),", "suggesting", "that", "hESC-produced", "factors", "established", "an", "embryonic", "microniche", "that", "may", "provide", "temporary", "protection", "from", "the", "aged", "environment.", "It", "appears", "that", "satellite", "cells", "do", "not", "have", "such", "anti-aging", "ability,", "because", "despite", "an", "initial", "activation", "in", "entirely", "young", "environments,", "e.g.,", "after", "muscle", "injury", "to", "young", "muscle,", "isolated", "satellite", "cells", "remain", "susceptible", "to", "inhibition", "by", "the", "old", "mouse", "serum", "(Figs", "1", "and", "4C).", "Similarly,", "culturing", "satellite", "cells", "isolated", "from", "noninjured", "muscle", "in", "growth-promoting", "medium", "for", "1–2", "days", "does", "not", "protect", "against", "the", "inhibitory", "affects", "of", "aged", "systemic", "milieu", "(not", "shown).Fig.", "3Embryonic", "stem", "cells", "produce", "youthful", "microniche", "in", "culture.", "(A)", "As", "opposed", "to", "immediate", "exposure", "to", "old", "mouse", "serum", "after", "passaging", "(10%", "old),", "preculturing", "of", "hESCs", "for", "24", "h", "in", "feeder-free", "conditions,", "e.g.,", "Matrigel™", "+", "MCM,", "prior", "to", "replacing", "MCM", "with", "MCM", "+", "10%", "old", "mouse", "sera,", "resulted", "in", "continuously", "high", "BrdU", "incorporation", "and", "Oct4", "expression", "(embryonic", "microniche", "+", "10%", "old).", "BrdU", "was", "added", "for", "the", "last", "2", "h", "of", "culture", "to", "measure", "the", "rate", "of", "cell", "proliferation.", "Immunodetection", "of", "BrdU", "and", "Oct4", "(both", "in", "red)", "was", "performed", "as", "described", "in", "Experimental", "procedures.", "Hoechst", "(blue)", "labels", "nuclei.", "(B)", "Three", "independent", "experiments", "yielded", "similar", "results", "and", "were", "quantified", "as", "percentage", "of", "BrdU+/Oct4+", "for", "each", "condition.", "*", "indicates", "P", "<", "0.001", "as", "compared", "to", "‘old", "+", "MCM’.Fig.", "4Aged", "muscle", "niche", "inhibits", "the", "regenerative", "potential", "of", "hESCs", "and", "satellite", "cells.", "(A)", "Immunodetection", "of", "a", "mouse-specific", "M-cadherin", "(green)", "or", "desmin", "(red;", "both", "human", "and", "mouse", "proteins", "are", "detected)", "revealed", "that", "hESCs", "underwent", "muscle", "lineage", "differentiation", "when", "co-cultured", "with", "young,", "but", "not", "old", "myofibers.", "The", "myogenic", "progeny", "of", "hESCs", "appears", "M-cadherin−/desmin+", "(white", "arrow", "in", "young),", "as", "opposed", "to", "M-cadherin−/desmin−", "hESCs", "that", "lack", "myogenic", "commitment", "(white", "arrow", "in", "old).", "M-cadherin+/desmin+", "cells", "are", "the", "myogenic", "progeny", "of", "mouse", "satellite", "cells", "(yellow", "arrows).", "To", "assess", "the", "effects", "of", "secreted", "factors", "produced", "by", "young", "vs.", "old", "myofibers", "on", "the", "rate", "of", "hESC", "proliferation,", "transient,", "2", "h", "BrdU", "incorporation", "was", "examined", "in", "hESCs", "cultured", "for", "48", "h", "with", "supernatants", "produced", "by", "heterochronic", "myofiber", "explants", "(See", "Experimental", "procedures", "for", "details).", "As", "compared", "to", "young", "myofiber-derived", "supernatants", "(young", "myofiber", "supernant),", "exposure", "to", "old", "myofiber-derived", "supernatants", "(old", "myofiber", "supernant)", "inhibited", "hESCs", "proliferation,", "as", "judged", "by", "BrdU", "immunodetection", "(red).", "As", "expected,", "the", "rate", "of", "hESCs", "proliferation", "was", "high", "in", "control", "media", "(shown", "in", "Fig.", "2).", "Hoechst", "(blue)", "labels", "nuclei", "in", "all", "experiments.", "Quantification", "of", "desmin+/BrdU+", "hESCs", "in", "direct", "myofiber", "cocultures,", "or", "with", "muscle", "supernatants,", "is", "shown", "in", "(B).", "*", "indicates", "P≤", "0.001", "as", "compared", "to", "young.", "(C)", "Transwell", "co-cultures", "between", "purified", "young", "satellite", "cells", "and", "myofibers", "isolated", "from", "uninjured", "young", "(young", "myofiber)", "and", "old", "(old", "myofiber)", "muscle", "demonstrated", "that", "satellite", "cell", "regenerative", "myogenic", "capacity", "was", "inhibited", "by", "the", "aged", "differentiated", "muscle.", "Myogenic", "potential", "was", "determined", "by", "the", "ability", "of", "satellite", "cells", "to", "generate", "proliferating", "desmin+", "myoblasts", "(immunodetection", "shown", "in", "green)", "and", "by", "rate", "of", "proliferation", "(2", "h", "BrdU", "incorporation;", "immunodetection", "shown", "in", "red).", "(D)", "Satellite", "cell", "regenerative", "potential", "was", "quantified", "as", "percentage", "of", "desmin+/BrdU+", "cells", "for", "transwell", "co-cultures", "with", "young", "or", "old", "uninjured", "myofibers", "(i.e.,", "RM,", "resting", "muscle).", "n", "=", "3;", "*", "indicates", "P≤", "0.05", "as", "compared", "to", "young.Comprehensively,", "these", "data", "establish", "that", "the", "inhibition", "of", "stem", "cell", "regenerative", "potential", "by", "the", "aged", "systemic", "milieu", "is", "conserved", "between", "species", "(mouse", "vs.", "human)", "and", "cell", "types", "(adult", "vs.", "embryonic", "stem", "cells).", "As", "summarized", "in", "Table", "1,", "aged", "mouse", "sera", "similarly", "affected", "the", "expression", "of", "key", "molecular", "identifiers", "of", "both", "embryonic", "and", "adult", "stem", "cells,", "e.g.,", "Oct4", "in", "hESCs", "and", "Myf5", "in", "mouse", "ASCs.", "As", "expected,", "adult", "mouse", "stem", "cells", "did", "not", "express", "Oct4,", "and", "hESCs", "did", "not", "express", "Myf5", "in", "these", "experimental", "conditions", "(not", "shown).", "Moreover,", "aged", "systemic", "milieu", "had", "similar", "inhibitory", "effects", "on", "proliferation", "of", "hESCs", "and", "ASCs,", "suggesting", "that", "not", "only", "the", "regenerative", "capacity,", "but", "also", "the", "presence", "and", "expansion", "of", "stem", "cells", "will", "be", "significantly", "restricted", "in", "aged", "organs.", "Intriguingly,", "prolonged", "culturing", "of", "hESCs", "in", "their", "preferred", "in", "vitro", "conditions", "enables", "generation", "of", "an", "embryonic", "microniche", "that", "antagonizes", "the", "inhibitory", "influences", "of", "aged", "circulatory", "factors.Table", "1Conservation", "of", "stem", "cell", "aging", "in", "the", "systemic", "environmentRate", "of", "proliferation", "ESC/ASC", "(percentage", "of", "BrdU)Call-fate", "identifier", "ESC", "(percentage", "of", "Oct4)/", "ASC", "(percentage", "of", "Myf5)10%", "young59.5", "±", "0.8,", "59.3", "±", "4.099.0", "±", "0.1,", "50.7", "±", "9.510%", "old32.7", "±", "2.1,", "27.3", "±", "3.517.6", "±", "3.2,", "18.1", "±", "5.95%", "young", "+", "5%", "old31.0", "±", "2.6,", "38.0", "±", "2.020.6", "±", "3.5,", "17.1", "±", "4.2Quantified", "results", "from", "Figs", "1,", "2", "are", "summarized", "and", "presented", "as", "mean", "percentages", "from", "experimental", "replicates", "±", "SE.", "Rate", "of", "proliferation", "(BrdU)", "and", "cell-fate", "identifier", "(Oct4", "or", "Myf5)", "are", "shown", "for", "both", "ESCs", "and", "ASCs", "cultured", "in", "heterochronic", "systemic", "conditions", "of", "10%", "young", "(young),", "10%", "old", "(old)", "or", "in", "5%+", "5%", "mouse", "sera", "combination", "(young", "+", "old).", "Results", "for", "5%", "young", "mouse", "sera", "are", "very", "similar", "to", "those", "for", "10%", "young", "mouse", "sera", "and", "are", "shown", "in", "Fig.", "1", "(ASCs)", "and", "Supplementary", "Fig.", "S3", "(hESCs).", "The", "regenerative", "potential", "of", "hESCs", "and", "ASCs", "is", "inhibited", "by", "aged", "differentiated", "muscleAfter", "establishing", "that", "the", "aged", "systemic", "niche", "negatively", "affects", "the", "regenerative", "capacity", "of", "hESCs", "and", "of", "ASCs,", "we", "then", "assessed", "whether", "myogenic", "potential", "and", "the", "rate", "of", "cell", "proliferation", "would", "be", "inhibited", "in", "hESCs", "and", "ASCs", "by", "the", "aged", "local", "muscle", "niche.", "Myofibers", "with", "associated", "satellite", "cells", "were", "isolated", "from", "young", "and", "old", "injured", "muscle,", "and", "were", "directly", "co-cultured", "with", "hESCs", "in", "typical", "hESC", "differentiation", "medium", "(DMEM/FBS).", "Similar", "to", "Fig.", "1,", "the", "myogenic", "potential", "in", "these", "co-cultures", "was", "assayed", "by", "the", "expression", "of", "desmin,", "which", "is", "present", "in", "both", "fusion-competent", "myoblasts", "and", "newly", "formed", "myotubes.", "To", "analyze", "whether", "hESCs,", "mouse", "myogenic", "progenitor", "cells", "or", "both", "could", "express", "desmin", "in", "direct", "co-cultures,", "we", "costained", "these", "cells", "with", "a", "mouse-specific", "antibody", "to", "a", "myogenic", "marker,", "M-cadherin,", "which", "does", "not", "react", "with", "human", "protein,", "and", "a", "desmin-specific", "antibody", "that", "recognizes", "both", "mouse", "and", "human", "proteins.", "As", "shown", "in", "Fig.", "4A,", "hESCs", "underwent", "myogenic", "differentiation", "in", "co-cultures", "with", "young", "myofibers", "(M-cadherin−/desmin+", "mononucleated", "cells,", "white", "arrow", "in", "young).", "These", "myogenic", "progeny", "of", "hESCs", "in", "co-cultures", "with", "young", "myofibers", "could", "be", "of", "skeletal,", "smooth", "or", "cardiac", "muscle", "lineages", "(Debus", "et", "al.,", "1983;", "Fischman", "&", "Danto,", "1985;", "Schultz", "&", "McCormick,", "1994).", "As", "expected,", "the", "young", "mouse", "muscle", "progenitor", "cells", "(M-cadherin+/desmin+)", "were", "more", "advanced", "in", "their", "degree", "of", "myogenic", "differentiation,", "which", "was", "of", "skeletal", "muscle", "lineage,", "as", "judged", "by", "the", "formation", "of", "large,", "multinucleated", "de", "novo", "myotubes", "(yellow", "arrow", "in", "young).", "In", "addition", "to", "the", "myogenically", "differentiated", "human", "cells,", "co-cultures", "with", "young", "myofiber", "explants", "also", "contained", "some", "small", "undifferentiated", "hESC", "colonies,", "as", "determined", "by", "immunoreactivity", "to", "a", "human-specific", "antibody", "to", "the", "nuclear", "mitotic", "apparatus", "protein,", "NuMA", "and", "Oct4", "expression", "(Supplementary", "Fig.", "S4).In", "contrast,", "when", "co-cultured", "with", "the", "aged", "mouse", "myofibers,", "only", "mouse", "cells", "appeared", "desmin+", "(Fig.", "4A,", "yellow", "arrow", "in", "old).", "These", "aged", "myogenic", "cells", "were", "of", "skeletal", "muscle", "lineage,", "based", "on", "spontaneous", "generation", "of", "multinucleated", "myotubes", "(see", "Fig.", "5B)", "and", "based", "on", "induced", "differentiation", "into", "myotubes", "in", "DMEM", "+", "2%", "horse", "serum", "(not", "shown).", "Importantly,", "the", "myogenic", "differentiation", "of", "hESCs", "failed", "in", "the", "aged", "co-cultures", "(Fig.", "4A,", "white", "arrow", "in", "old).", "Furthermore,", "colonies", "of", "hESCs", "in", "co-cultures", "with", "aged", "myofibers", "typically", "differentiated", "into", "cells", "with", "fibroblast", "morphology,", "which", "lacked", "Oct4", "expression", "(not", "shown).", "Spontaneous", "production", "of", "desmin+", "myogenic", "cells", "in", "control", "hESC", "cultures", "without", "myofibers,", "or", "with", "young/old", "mouse", "sera", "was", "less", "than", "0.1%", "(not", "shown).Fig.", "5In", "vitro", "co-culture", "with", "hESCs", "enhanced", "myogenesis", "of", "mouse", "cells.", "(A)", "1", "×", "105", "hESCs", "or", "control", "hMSCs", "were", "co-cultured", "with", "5", "×", "106", "primary", "mouse", "myoblasts.", "hESCs", "expressing", "Oct4", "(immunodetection", "shown", "in", "red)", "dramatically", "enhanced", "myotube", "formation", "of", "co-cultured", "mouse", "myoblasts", "(immunodetection", "of", "eMyHC", "is", "shown", "in", "green),", "as", "compared", "to", "co-cultures", "between", "mouse", "myoblasts", "and", "human", "mesenchymal", "stem", "cells", "(Mb", "+", "hMSCs)", "or", "myoblasts", "alone", "(Mb", "alone).", "Experiments", "were", "carried", "out", "in", "myoblast", "differentiation", "medium.", "Hoechst", "(blue)", "labels", "nuclei", "throughout", "this", "figure.", "(B)", "1", "×", "105", "hESCs", "or", "control", "hMSCs", "were", "co-cultured", "with", "young", "or", "old", "myofiber-associated", "satellite", "cells,", "as", "described", "in", "Experimental", "procedures.", "Co-culture", "with", "hESCs", "(myofiber", "+", "hESC),", "but", "not", "hMSCs", "(myofiber", "+", "hMSC)", "or", "control", "medium", "(DMEM/FBS),", "greatly", "enhanced", "the", "myogenic", "potential", "of", "both", "young", "and", "old", "myofiber-associated", "satellite", "cells,", "based", "on", "immunodetection", "of", "percentage", "of", "desmin+", "de", "novo", "generated", "myoblasts", "and", "multinucleated", "myotubes.", "These", "experiments", "were", "carried", "out", "in", "GM.", "Shown", "are", "myogenic", "responses", "of", "mouse", "cells", "only,", "judged", "by", "lack", "of", "immunoreactivity", "to", "human-specific/hESC-specific", "antigens,", "such", "as", "NuMA", "and", "Oct4;", "and", "presence", "of", "mouse-specific", "immunoreactivity,", "e.g.,", "M-cadherin", "(not", "shown).", "Both", "young", "and", "old", "myofiber", "associated", "satellite", "cells", "exhibited", "considerable", "myogenic", "improvement", "over", "control", "conditions.", "n", "=", "3.In", "concert", "with", "the", "conservation", "of", "inhibitory", "affects", "of", "aged", "systemic", "niche,", "the", "negative", "influence", "of", "local", "muscle", "niche", "was", "also", "found", "to", "be", "conserved", "in", "its", "inhibition", "of", "hESC", "and", "ASC", "regenerative", "responses.", "Specifically,", "the", "myogenic", "capacity", "(generation", "of", "desmin+", "myoblasts)", "was", "inhibited", "in", "young", "satellite", "cells", "co-cultured", "in", "a", "transwell", "system", "with", "aged", "myofibers", "(Fig.", "4B).", "In", "addition,", "hESC", "and", "ASC", "proliferation", "(BrdU", "incorporation)", "was", "also", "inhibited", "by", "aged", "differentiated", "muscle", "(Fig.", "4A,C).", "These", "data", "suggest", "that", "not", "only", "systemic", "but", "also", "local", "organ", "niches", "would", "inhibit", "key", "stem", "cell", "properties,", "e.g.,", "myogenic", "capacity", "and", "the", "rate", "of", "proliferation", "in", "the", "aged", "organism.", "The", "conserved", "inhibitory", "influences", "of", "the", "differentiated", "muscle", "niche", "on", "hESC", "and", "ASC", "regenerative", "responses", "are", "summarized", "in", "Table", "2.Table", "2Conservation", "of", "stem", "cell", "aging", "in", "the", "local", "organ", "nicheRate", "of", "proliferation", "ESC/ASC", "(percentage", "of", "BrdU)Myogenic", "differentiation", "ESC/ASC", "(percentage", "of", "desmin)Young", "myofiber60.2", "±", "2.5,", "40.5", "±", "2.67.4", "±", "0.9,", "47.6", "±", "5.0Old", "myofiber30.1", "±", "4.3,", "21.5", "±", "4.11.3", "±", "0.7,", "19.7", "±", "4.7Quantified", "results", "from", "Fig.", "4", "are", "summarized", "and", "presented", "as", "mean", "percentages", "from", "experimental", "replicates", "±", "SE.", "Rate", "of", "proliferation", "(BrdU)", "and", "myogenic", "differentiation", "(desmin)", "are", "shown", "for", "both", "ESCs", "and", "ASCs,", "in", "the", "presence", "of", "young", "vs.", "old", "differentiated", "muscle", "environments", "(young", "myofiber", "or", "old", "myofiber).", "hESCs", "indirectly", "enhance", "and", "rejuvenate", "the", "regeneration", "of", "skeletal", "muscleWhile", "hESC", "properties", "were", "inhibited", "by", "aged", "differentiated", "muscle,", "the", "myogenic", "potential", "of", "aged", "satellite", "cells", "seemed", "to", "be", "enhanced", "by", "co-cultures", "with", "hESCs", "(Fig.", "4A).", "Therefore,", "we", "further", "explored", "the", "enhancing", "and", "rejuvenating", "effects", "of", "hESCs", "on", "myogenic", "potential", "in", "vitro", "and", "in", "vivo,", "using", "human", "mesenchymal", "stem", "cells", "(hMSCs)", "as", "a", "negative", "control.", "First,", "we", "examined", "the", "effects", "of", "hESCs", "on", "myotube", "generation", "by", "co-culture", "with", "primary", "myoblasts", "freshly", "derived", "from", "activated-by-injury", "satellite", "cells", "(Conboy", "et", "al.,", "2003).", "As", "shown", "in", "Fig.", "5A", "(Mb", "+", "hESC),", "primary", "myoblasts", "underwent", "very", "rapid", "and", "robust", "nascent", "myotube", "formation,", "when", "co-cultured", "with", "hESCs", "for", "48", "h", "in", "myoblast", "differentiation", "medium.", "Namely,", "remarkably", "large", "fused", "myotubes", "containing", "approximately", "50–70", "nuclei", "formed", "around", "hESCs", "colonies", "(Fig.", "5A).", "In", "contrast,", "when", "co-cultured", "with", "hMSCs,", "myotube", "formation", "was", "no", "greater", "than", "in", "myoblast", "cultures", "alone", "(Fig.", "5A,", "Mb", "+", "hMSC", "and", "Mb", "alone).", "Encouraged", "by", "these", "data,", "we", "analyzed", "the", "myogenic", "potential", "of", "young", "and", "old", "satellite", "cells", "co-cultured", "with", "hESCs", "for", "48", "h.", "As", "shown", "in", "Fig.", "5B,", "hESCs", "conferred", "a", "much-enhanced", "myogenic", "capacity", "on", "both", "young", "and,", "importantly,", "old", "myofiber-associated", "satellite", "cells", "(rapid", "formation", "of", "desmin+", "myogenic", "cells,", "many", "of", "which", "formed", "de", "novo", "multinucleated", "myotubes).", "Control", "co-cultures", "of", "these", "satellite", "cells", "with", "hMSCs", "displayed", "no", "enhanced", "myogenicity.", "In", "summary,", "while", "the", "myogenic", "potential", "(production", "of", "desmin+", "fusion-competent", "cells)", "was", "more", "pronounced", "in", "young", "vs.", "old", "myofiber-associated", "satellite", "cells", "under", "all", "experimental", "conditions,", "a", "finding", "that", "is", "consistent", "with", "previous", "data", "(Conboy", "et", "al.,", "2003),", "a", "clear", "increase", "in", "myogenic", "potential", "of", "old", "satellite", "cells", "was", "noted", "in", "co-cultures", "with", "hESCs,", "as", "compared", "to", "control", "cultures", "devoid", "of", "hESCs", "(Fig.", "4A,B).Interestingly,", "in", "addition", "to", "the", "rejuvenating", "effects", "of", "direct", "co-cultures", "shown", "in", "Fig.", "5,", "soluble", "factors", "present", "in", "hESC-conditioned", "culture", "supernatants", "were", "also", "able", "to", "enhance", "myogenesis", "of", "aged", "satellite", "cells", "(Supplementary", "Fig.", "S5).", "Thus,", "in", "agreement", "with", "the", "notion", "that", "an", "established", "embryonic", "microniche", "antagonizes", "the", "inhibitory", "effects", "of", "the", "aged", "environment", "on", "stem", "cell", "responses", "(Fig.", "3),", "the", "hESC-produced", "factors", "enhanced", "myogenic", "capacity", "of", "even", "old", "mouse", "satellite", "cells.Establishing", "that", "hESC-produced", "factors", "enhance", "adult", "myogenesis", "and", "rejuvenate", "the", "regenerative", "capacity", "of", "even", "aged", "satellite", "cells", "in", "vitro", "prompted", "us", "to", "examine", "whether", "the", "regeneration", "of", "old", "injured", "muscle", "will", "be", "improved", "by", "hESC", "transplantation", "in", "vivo.", "Additionally,", "based", "on", "the", "data", "shown", "above,", "we", "speculated", "that", "even", "if", "the", "host's", "repair", "capacity", "is", "improved,", "hESCs", "themselves", "will", "not", "be", "efficiently", "maintained", "or", "expanded", "in", "the", "context", "of", "old", "systemic", "and", "local", "organ", "environments,", "and", "will", "not", "directly", "contribute", "to", "the", "repair", "of", "aged", "skeletal", "muscle.", "To", "test", "these", "hypotheses,", "we", "injected", "5", "×", "105", "hESCs", "or", "control", "hMSCs", "into", "the", "tibialis", "anterior", "(TA)", "and", "gastrocnemius", "muscles", "of", "young", "and", "old", "mice", "at", "24", "h", "after", "cardiotoxin-induced", "injury,", "when", "activation/proliferation", "of", "endogenous", "satellite", "cells", "normally", "begins", "(Conboy", "et", "al.,", "2003,", "2005;", "Wagers", "&", "Conboy,", "2005).", "To", "avoid", "immune", "response", "against", "hESC", "antigens,", "mice", "were", "immunosuppressed", "using", "FK506", "(Ito", "&", "Tanaka,", "1997;", "Dumont,", "2000).", "Muscle", "was", "isolated", "5", "days", "post-injury,", "when", "nascent", "differentiated", "myofibers", "normally", "replace", "the", "damaged", "tissue", "(Conboy", "et", "al.,", "2003),", "and", "10", "µm", "cryosections", "were", "analyzed", "for", "the", "success", "in", "tissue", "repair", "using", "hematoxylin", "and", "eosin", "(H&E)", "histochemistry", "and", "eMyHC", "immunodetection.", "H&E", "analysis", "reveals", "newly", "formed", "myofibers,", "based", "on", "their", "smaller", "size", "and", "centrally", "located", "nuclei.", "Additionally,", "de", "novo", "myofibers", "in", "the", "damaged", "area", "appear", "positive", "for", "eMyHC,", "while", "undamaged", "myofibers", "remain", "negative.", "As", "shown", "in", "Fig.", "6A", "and", "quantified", "in", "6B,", "injection", "of", "hESCs", "significantly", "enhanced", "regeneration", "of", "skeletal", "muscle.", "Remarkably,", "this", "positive", "embryonic", "effect", "was", "especially", "pronounced", "in", "old", "tissue.Fig.", "6Skeletal", "muscle", "regeneration", "following", "hESC", "transplantation", "is", "a", "balance", "between", "the", "inhibitory", "influence", "of", "aged", "niches", "and", "the", "rejuvenating", "effects", "of", "hESCs.", "Young", "and", "old", "tibialis", "anterior", "and", "gastrocnemius", "muscles", "were", "injured", "by", "cardiotoxin", "injection.", "hESCs", "or", "hMSCs", "were", "transplanted", "at", "the", "site", "of", "injury", "and", "were", "analyzed", "by", "cryosectioning", "at", "Day", "5", "after", "injury", "(as", "described", "in", "Experimental", "procedures).", "(A)", "Newly", "regenerated", "myofibers", "were", "detected", "using", "eMyHC-specific", "antibody", "(green)", "and", "staining", "with", "H&E.", "In", "H&E", "staining,", "newly", "regenerated", "areas", "contain", "smaller,", "immature", "myofibers", "with", "centrally", "located", "nuclei.", "Uninjured", "myofibers", "are", "much", "larger,", "by", "comparison,", "with", "peripherally", "restricted", "nuclei.", "Poorly", "regenerated", "areas", "lack", "new", "myofibers", "and", "contain", "areas", "of", "fibrosis", "and", "inflammation.", "eMyHC", "immunodetection", "is", "specific", "for", "regenerating", "areas", "of", "muscle", "only.", "Both", "assays", "showed", "dramatic", "enhancement", "of", "muscle", "regeneration", "in", "‘old", "+", "hESC’", "vs.", "‘old", "+", "hMSC’.", "Regeneration", "improvement", "was", "also", "seen", "in", "young", "+", "hESC,", "as", "compared", "to", "young", "+", "hMSC.", "(B)", "Quantification", "of", "muscle", "regeneration", "was", "performed", "by", "analyzing", "the", "density", "of", "newly", "formed", "myofibers", "per", "mm2", "of", "injury", "site,", "which", "is", "the", "volume", "that", "typically", "covers", "the", "whole", "injured", "area.", "Multiple,", "10", "µm", "H&E", "sections", "were", "examined", "through", "the", "entire", "volume", "of", "injury", "in", "multiple,", "independently", "injured", "muscles.", "n", "=", "20;", "*", "indicates", "P", "<", "0.001", "(‘old", "+", "hMSC’", "compared", "to", "young", "+", "hMSC", "and", "‘old", "+", "hMSC’", "compared", "to", "‘old", "+", "hESC’.", "(C)", "H&E", "and", "immunofluoresence", "staining", "for", "Oct4,", "and", "a", "human-specific", "antibody", "to", "NuMA,", "revealed", "the", "failure", "of", "hESCs", "to", "expand", "or", "persist", "in", "old,", "but", "the", "presence", "of", "hESCs", "in", "young", "muscle", "at", "5", "days", "post-transplantation.", "Hoechst", "(blue)", "labels", "nuclei.Importantly,", "such", "enhanced", "and", "rejuvenated", "muscle", "repair", "stems", "from", "an", "indirect", "induction,", "as", "hESCs", "themselves", "(or", "control", "hMSCs)", "did", "not", "physically", "contribute", "to", "the", "mouse", "myofibers,", "as", "judged", "by", "near", "absence", "(less", "than", "0.1%)", "of", "human-specific", "NuMA+", "nuclei", "in", "de", "novo", "desmin+", "myofibers,", "analyzed", "through", "multiple", "injury", "sites.", "An", "example", "of", "one", "regenerated", "myofiber", "from", "young", "muscle", "injected", "with", "hESCs,", "with", "NuMA+", "nucleus", "in", "a", "field", "of", "NuMA−/desmin+", "mouse", "myofibers,", "is", "shown", "in", "Supplementary", "Fig.", "S6.", "No", "such", "NuMA+/desmin+", "myofibers", "were", "detected", "in", "aged", "regenerated", "muscle", "(not", "shown).In", "agreement", "with", "the", "in", "vitro", "data,", "establishing", "that", "aged", "systemic", "and", "local", "niches", "inhibit", "hESC", "proliferation", "and", "Oct4", "expression", "(Figs", "2", "and", "4", "and", "Supplementary", "Fig.", "S2),", "hESCs", "failed", "to", "expand", "or", "even", "persist", "in", "old", "muscle,", "as", "judged", "by", "the", "absence", "of", "NuMA+/Oct4+", "hESC-derived", "cells", "in", "the", "aged", "tissue.", "In", "contrast,", "colonies", "of", "Numa+/Oct4+", "hESC-derived", "cells", "that", "did", "not", "undergo", "myogenic", "differentiation", "were", "easily", "detected", "in", "young", "regenerating", "muscle", "(Fig.", "6C).", "This", "finding", "validates", "several", "technical", "aspects", "of", "these", "experiments,", "and", "confirms", "the", "contrasting", "effects", "of", "young", "and", "old", "systemic", "and", "local", "organ", "niches", "on", "hESC", "self-renewal.These", "data", "further", "confirm", "and", "extrapolate", "our", "findings", "and", "demonstrate", "that", "when", "exposed", "to", "both", "aged", "systemic", "and", "local", "organ", "niches,", "hESCs", "fail", "to", "persist", "and", "do", "not", "contribute", "to", "tissue", "repair", "directly.", "At", "the", "same", "time,", "these", "embryonic", "cells", "indirectly", "but", "significantly", "improve", "the", "repair", "of", "aged", "injured", "muscle", "in", "vivo.", "The", "data", "presented", "here", "establish", "for", "the", "first", "time", "that", "both", "the", "local", "environment", "of", "old", "differentiated", "organ,", "e.g.,", "skeletal", "muscle", "and", "the", "systemic", "milieu", "dramatically", "affect", "the", "regenerative", "potential", "of", "both", "hESCs", "and", "mouse", "post-natal", "myogenic", "progenitor", "cells.", "Not", "only", "are", "the", "factors", "promoting", "myogenic", "differentiation", "and", "proliferation", "of", "hESCs", "likely", "to", "become", "depleted", "with", "age,", "but", "the", "aged", "systemic", "and", "local", "organ", "niches", "are", "likely", "to", "contain", "dominant", "inhibitors", "of", "ASC", "and", "hESC", "regenerative", "potential", "(Figs", "1,", "2,", "and", "4,", "summarized", "in", "Tables", "1", "and", "2).", "Importantly,", "the", "similar", "inhibitory", "effects", "of", "old", "mouse", "serum", "and", "old", "myofibers", "on", "satellite", "cell", "(Figs", "1", "and", "4C)", "and", "hESC", "(Figs", "2", "and", "4A)", "proliferation", "and", "regenerative", "capacity", "suggest", "the", "conservation", "of", "elements", "in", "age-specific", "extrinsic", "regulatory", "mechanisms", "between", "evolutionarily", "distinct", "species", "and", "stem", "cell", "types.", "Additionally,", "a", "similarity", "in", "the", "inhibitory", "properties", "between", "systemic", "and", "local", "organ", "niches", "is", "also", "of", "interest", "and", "may", "indicate", "that", "molecules", "produced", "by", "old", "tissues", "have", "circulatory/endocrine", "activity;", "and/or", "that", "age-specific", "systemic", "inhibitory", "components", "become", "deposited", "in", "the", "old", "tissues.", "Humans", "display", "broad", "genetic", "polymorphisms", "and", "behavioral", "variations,", "which", "makes", "the", "identification", "of", "age-specific", "molecular", "changes", "complicated.", "In", "contrast,", "laboratory", "mice", "are", "genetically", "and", "environmentally", "controlled.", "Establishing", "that", "age-specific", "signals,", "regulating", "stem", "cell", "responses,", "are", "evolutionarily", "conserved", "and", "soluble", "enables", "the", "formation", "of", "rational", "approaches", "for", "the", "identification", "and", "characterization", "of", "the", "inhibitors", "involved,", "and", "for", "revealing", "the", "precise", "timing", "of", "their", "first", "appearance", "in", "serum", "and", "differentiated", "tissues", "with", "advancing", "age.", "Significantly,", "these", "experiments", "have", "also", "revealed", "that", "not", "only", "are", "hESCs", "able", "to", "protect", "themselves", "against", "the", "negative", "influences", "of", "aged", "mouse", "sera", "(Fig.", "3),", "but", "these", "cells", "also", "produce", "factors", "that", "dramatically", "enhance", "the", "myogenic", "capacity", "of", "primary", "myoblasts", "and", "young", "and", "old", "satellite", "cells", "(Fig.", "5),", "and", "also", "significantly", "improve", "repair", "of", "young", "and", "old", "injured", "muscle", "in", "vivo", "(Fig.", "6).", "Identification", "of", "these", "embryonic", "factors", "would", "allow", "us", "to", "potentially", "enrich", "the", "arsenal", "of", "therapeutic", "tools", "for", "combating", "age-specific", "degenerative", "disorders.", "The", "interactions", "between", "hESCs", "and", "heterochronic", "differentiated", "niches,", "initially", "identified", "in", "vitro,", "have", "been", "confirmed", "by", "in", "vivo", "experiments.", "Namely,", "while", "the", "regenerative", "capacity,", "or", "presence,", "of", "hESCs", "is", "greatly", "restricted", "in", "aged,", "as", "compared", "to", "young", "skeletal", "muscle", "(where", "transplanted", "cells", "experience", "both", "old", "systemic", "and", "local", "environments),", "embryonic", "cells", "indirectly", "enhance", "and", "rejuvenate", "muscle", "repair", "when", "introduced", "at", "the", "time", "of", "muscle", "stem", "cell", "activation", "in", "the", "host,", "e.g.,", "at", "Day", "1", "after", "the", "injury", "(Fig.", "6).", "It", "remains", "to", "be", "determined", "whether", "the", "percentage", "of", "hESCs", "direct", "contribution", "to", "desmin+", "myofibers", "in", "young", "muscle", "will", "be", "increased", "by", "transplanting", "these", "cells", "at", "a", "different", "time-point", "after", "muscle", "injury,", "e.g.,", "at", "Days", "3–5", "(as", "in", "co-cultures", "with", "myofibers", "pre-injured", "for", "3", "days,", "Fig.", "4A).", "In", "any", "case,", "the", "virtual", "lack", "of", "hESC", "and", "hMSC", "direct", "contribution", "to", "the", "newly", "regenerated", "skeletal", "muscle,", "when", "small", "numbers", "of", "these", "cells", "were", "injected", "into", "injured", "tissue,", "is", "completely", "consistent", "with", "the", "body", "of", "previous", "data", "demonstrating", "that", "myofiber-associated", "satellite", "cells", "conduct", "rapid", "and", "robust", "muscle", "repair", "and", "greatly", "outnumber", "injected", "human", "cells", "(Collins", "et", "al.,", "2005;", "Wagers", "&", "Conboy,", "2005);", "that", "compared", "to", "muscle-specific", "satellite", "cells,", "the", "myogenic", "differentiation", "of", "hESCs", "in", "vitro", "remains", "very", "small", "(Fig.", "5,", "Table", "2),", "and", "that", "control", "hMSCs", "are", "not", "normally", "myogenic", "unless", "these", "cells", "overexpress", "exogenous", "constitutively", "active", "domain", "of", "Notch", "(Dezawa", "et", "al.,", "2005).", "Intriguingly,", "the", "failure", "of", "hESCs", "to", "strive", "in", "old", "skeletal", "muscle", "might", "represent", "a", "therapeutically", "desirable", "outcome.", "For", "example,", "while", "in", "young", "tissue", "hESC", "derivatives", "putatively", "would", "go", "on", "to", "produce", "teratomas,", "it", "is", "unlikely", "that", "teratoma", "formation", "would", "occur", "after", "hESC", "transplantation", "into", "aged", "skeletal", "muscle.", "Thus,", "the", "indirect", "beneficial", "effects", "of", "hESCs", "on", "tissue", "repair", "are", "unlikely", "to", "be", "compromised", "by", "the", "oncogenic", "properties", "of", "these", "embryonic", "cells", "in", "the", "context", "of", "old", "skeletal", "muscle.", "Comprehensively,", "the", "results", "of", "this", "work", "increase", "our", "understanding", "of", "aging", "as", "a", "process,", "reveal", "evolutionary", "conserved", "age-specific", "interactions", "between", "stem", "cells", "and", "their", "differentiated", "niches,", "and", "suggest", "novel", "therapeutic", "approaches", "for", "improving", "the", "regenerative", "responses", "of", "endogenous", "or", "transplanted", "stem", "cells", "in", "old", "individuals.", "Animal", "strainsYoung", "(2–3", "months),", "C57-BL/6", "male", "mice", "were", "obtained", "from", "pathogen-free", "breeding", "colonies", "at", "Jackson", "Laboratories", "(Bar", "Harbor,", "ME,", "USA).", "Aged", "22–24", "months", "C57-BL/6", "male", "mice", "were", "obtained", "from", "the", "National", "Institute", "on", "Aging", "(NIH).", "Animals", "were", "maintained", "in", "the", "North-West", "Animal", "Facility", "of", "the", "University", "of", "California,", "Berkeley,", "CA,", "USA,", "and", "handled", "in", "accordance", "with", "the", "Administrative", "Panel", "on", "Laboratory", "Animal", "Care", "at", "UC", "Berkeley.", "Muscle", "injury", "and", "isolationMyofiber", "cultures,", "in", "which", "satellite", "cells", "were", "activated", "by", "in", "vivo", "injury,", "were", "set", "up", "as", "previously", "described", "(Conboy", "&", "Rando,", "2002;", "Conboy", "et", "al.,", "2005).", "Briefly,", "mice", "were", "injured", "by", "direct", "injection", "with", "5", "ng", "cardiotoxin", "(CTX-1)", "(Sigma,", "St", "Louis,", "MO,", "USA)", "into", "the", "tibialis", "anterior", "and", "gastrocnemius", "muscles", "using", "a", "28-gauge", "needle.", "After", "1–5", "days", "post-injection,", "injured", "or", "uninjured", "muscle", "tissue", "was", "dissected", "out.", "Once", "isolated,", "whole", "muscle", "was", "prepared", "for", "cryosectioning", "(see", "below)", "or", "myofiber", "fragments", "were", "obtained", "from", "hind", "limb", "muscles", "by", "enzymatic", "digestion", "(see", "below),", "trituration,", "and", "multiple", "sedimentation", "and", "washing", "procedures.", "Additionally,", "blood", "was", "collected", "from", "mice", "for", "the", "isolation", "of", "sera.", "Briefly,", "blood", "cells", "were", "coagulated", "at", "37", "°C", "for", "15’", "and", "then", "were", "centrifuged", "repeatedly", "at", "5900", "g,", "4", "°C", "in", "a", "microfuge", "for", "3’", "to", "isolate", "sera.", "Mixtures", "of", "young", "and", "old", "sera", "were", "made", "1", ":", "1.", "For", "example,", "in", "5%+", "5%", "conditions,", "50", "µL", "of", "young", "and", "50", "µL", "old", "serum", "were", "added", "to", "900", "µL", "of", "culture", "medium", "(Opti-MEM", "or", "MCM,", "see", "co-culture", "procedures", "below).", "Myofiber", "explant", "culturesExplant", "and", "primary", "cell", "cultures", "were", "generated", "from", "C57-BL/6", "mice,", "as", "previously", "described", "(Conboy", "&", "Rando,", "2002;", "Conboy", "et", "al.,", "2003).", "Dissected", "gastrocnemius", "and", "tibialis", "anterior", "muscles", "underwent", "enzymatic", "digestion", "at", "37", "°C", "in", "DMEM", "(Invitrogen)/Pen-Strep", "(Invitrogen)/0.2%", "Collagenase", "Type", "IIA", "(Sigma)", "solution.", "Isolated", "fibers", "were", "resuspended", "in", "GM", "(Ham's", "F10", "nutrient", "mixture", "(Mediatech,", "Inc.,", "Herndon,", "VA,", "USA),", "20%", "FBS", "(Mediatech),", "5", "ng", "mL−1", "bFGF", "(Chemicon,", "Temecula,", "CA,", "USA)", "and", "1%", "Pen-Strep,", "and", "cultured", "on", "ECM-coated", "(BD", "Biosciences,", "San", "Jose,", "CA,", "USA)", "plates", "(diluted", "1", ":", "500", "in", "PBS).", "Cultures", "of", "primary", "myoblasts", "were", "derived", "from", "isolated", "fibers,", "through", "repeated", "passaging,", "and", "were", "maintained", "in", "GM.", "Myoblast", "differentiation", "medium", "[DMEM,", "supplemented", "with", "2%", "horse", "serum", "(Mediatech)]", "was", "used", "to", "promote", "rapid", "formation", "of", "myotubes", "from", "cultured", "myoblasts", "(Morgan", "&", "Partridge,", "2003).", "Human", "embryonic", "and", "mesenchymal", "stem", "cell", "cultureThe", "federally", "approved", "hESC", "line,", "H7", "(NIH", "no.", "WA07,", "obtained", "from", "WiCell", "Research", "Institue,", "Madison,", "WI,", "USA),", "was", "used", "in", "accordance", "with", "the", "UC", "Berkeley", "and", "UC", "San", "Francisco", "Committee", "on", "Human", "Research", "guidelines,", "and", "in", "accordance", "with", "NIH", "guidelines.", "To", "propagate", "hESCs,", "routine", "culturing", "and", "maintenance", "was", "performed", "using", "standard", "in", "vitro", "conditions", "for", "both", "feeder-dependent", "and", "feeder-free", "cultures", "(Geron", "Corporation,", "2002).", "Briefly,", "hESCs", "grown", "on", "MEFs", "were", "cultured", "in", "standard", "hESC", "medium", "[Knockout™", "DMEM,", "20%", "KSR,", "1%", "NEAA,", "1", "mm", "l-glutamine", "(Invitrogen),", "0.1", "mmβ-mercaptoethanol", "(Sigma)]", "and", "were", "supplemented", "with", "4", "ng", "mL−1", "hbFGF", "(Invitrogen).", "Feeder-free", "hESC", "cultures", "were", "maintained", "in", "MEF-conditioned", "hESC", "medium", "(MCM),", "4", "ng", "mL−1", "hbFGF.", "Differentiation", "medium", "for", "hESCs", "(DMEM/FBS)", "was", "made", "by", "replacing", "KSR", "with", "20%", "FBS", "(Hyclone,", "Logan,", "UH,", "USA).", "hMSCs", "were", "maintained", "in", "mesenchymal", "stem", "cell", "GM,", "MSC-GM™", "and", "were", "cultured", "according", "to", "supplier", "recommendations", "(Cambrex", "Walkersville,", "MD,", "USA).", "hESCs", "and", "hMSCs", "were", "typically", "seeded", "onto", "chambered", "slides", "coated", "with", "a", "3%", "GFR", "Matrigel™", "(BD", "Biosciences)", "substrate", "in", "PBS.", "Cells", "were", "typically", "incubated", "for", "48", "h", "at", "37", "°C,", "5%", "CO2,", "under", "the", "various", "experimental", "conditions", "employed,", "then", "were", "fixed", "with", "70%", "EtOH/PBS", "at", "4", "°C.", "hESCs", "and", "hMSCs", "were", "analyzed", "24–48", "h", "after", "experimental", "treatments,", "during", "which", "no", "apoptosis-related", "differences", "in", "cell", "numbers", "were", "observed.", "Heterochronic", "co-culture", "systemsHeterochronic", "systemic", "cultures", "were", "established", "by", "culturing", "myofiber", "explants", "(in", "GM)", "or", "hESCs", "(in", "MCM)", "in", "the", "presence", "of", "young,", "old", "or", "young", "+", "old", "sera", "for", "48", "h", "(Figs", "1", "and", "2", "and", "Supplementary", "Figs", "S1–3).", "In", "such", "cultures,", "hESCs", "were", "passaged", "immediately", "prior", "to", "sera", "exposure.", "In", "contrast,", "preculturing", "of", "hESCs", "for", "24", "h", "in", "MCM,", "prior", "to", "replacing", "MCM", "with", "MCM", "+", "10%", "old", "mouse", "sera", "was", "done", "for", "embryonic", "microniche", "experiments", "(Fig.", "3).", "For", "heterochronic", "local", "organ", "niche", "cultures,", "hESCs", "were", "co-cultured", "directly", "with", "myofiber", "explants", "for", "48", "h", "in", "GM,", "or", "were", "cultured", "in", "the", "presence", "of", "supernatants", "derived", "from", "cultured", "myofiber", "explants", "for", "48", "h", "(Figs", "4A", "and", "5).", "Specifically,", "1", "×", "105", "hESCs", "or", "control", "hMSCs", "were", "co-cultured", "with", "identical", "volume,", "e.g.,", "100", "µL,", "of", "young", "or", "old", "myofiber", "fragments", "with", "their", "associated", "satellite", "cells", "(Fig.", "5).", "In", "experiments", "shown", "in", "Supplementary", "Fig.", "S5,", "culture-conditioned", "supernatant", "produced", "by", "hESCs", "grown", "in", "MCM", "was", "used", "as", "a", "medium", "in", "which", "1", "×", "105", "of", "myofiber-associated", "young", "or", "old", "satellite", "cells", "were", "cultured", "for", "48", "h.", "In", "direct", "co-cultures,", "mouse", "vs.", "human", "cells", "were", "distinguished", "by", "immunodetection", "with", "human-specific/hESC-specific", "and", "mouse-specific", "antibodies", "(Supplementary", "Fig.", "S4", "and", "see", "below).", "To", "prepare", "muscle", "supernatants,", "explants", "were", "cultured", "for", "24", "h", "in", "GM", "and", "cellular", "debris", "was", "removed", "from", "conditioned", "media", "by", "multiple", "rounds", "of", "centrifugation.", "The", "absence", "of", "cells", "was", "confirmed", "by", "microscopic", "examination.", "To", "mimic", "the", "local", "organ", "niche", "for", "satellite", "cell", "assays", "(Fig.", "4B),", "1.0", "µm", "transwell", "(Corning,", "NY,", "USA)", "co-cultures", "of", "uninjured", "explants", "with", "activated", "satellite", "cells", "were", "established.", "Activated-by-injury", "(24", "h", "post-injury)", "satellite", "cells", "were", "seeded", "onto", "ECM-coated", "12-well", "plates", "in", "Opti-MEM", "(Invitrogen)", "and", "5%", "FBS.", "Transwells", "were", "placed", "over", "satellite", "cells", "and", "contained", "isolated", "myofiber", "explants", "from", "uninjured", "young", "or", "old", "muscle", "(i.e.,", "resting", "muscle).", "Satellite", "cells", "were", "cultured", "for", "72–96", "h", "in", "the", "presence", "of", "myofiber", "explants", "and", "were", "fixed", "for", "immunodetection,", "as", "described", "above.", "Cell", "transplantationhESCs", "were", "grown", "on", "MEFs", "and", "expanded", "in", "6-well", "plates.", "Cells", "were", "treated", "with", "1", "mg", "mL−1", "Collagenase", "Type", "IV", "(Invitrogen)", "for", "5–10", "min,", "were", "washed", "and", "then", "incubated", "with", "0.5", "mg", "mL−1", "Dispase", "(Invitrogen)", "to", "lift", "only", "human", "cell", "colonies.", "Isolated", "hESCs", "were", "washed", "several", "times", "and", "resuspended", "in", "100", "µL", "hESC", "medium.", "Similarly,", "hMSCs", "were", "expanded", "in", "6-well", "plates,", "lifted", "with", "Trypsin/EDTA", "(Invitrogen),", "washed", "and", "resuspended", "in", "100", "µL", "hESC", "medium.", "Approximately", "5", "×", "105", "hESCs", "or", "hMSCs", "were", "injected", "into", "24", "h", "post-injured", "gastrocnemius", "and", "tibialis", "anterior", "muscles", "of", "young", "and", "old", "mice,", "using", "a", "21-gauge", "needle.", "Immunosuppression", "of", "animals", "was", "achieved", "by", "intraperitoneal", "injection", "of", "1", "mg", "kg−1", "FK506", "(Sigma)", "at", "48", "h", "prior", "to", "cell", "transplantation,", "and", "on", "each", "day", "following", "transplantation.", "Immunodetection", "and", "histological", "analysisTo", "assay", "the", "affects", "of", "heterochronic", "local", "and", "systemic", "environments", "on", "stem", "cell", "regenerative", "potential,", "hESC,", "hMSC,", "and", "myofiber-derived", "precursor", "cell", "cultures", "were", "fixed", "with", "70%", "EtOH/PBS", "at", "4", "°C,", "and", "were", "analyzed", "by", "indirect", "immunofluorescence.", "Combinations", "of", "antibodies", "were", "used", "to", "co-stain", "cultures", "and", "histosections,", "in", "order", "to", "determine", "the", "percentages", "of", "cells", "that", "proliferated", "or", "differentiated", "and", "to", "distinguish", "hESCs", "from", "mouse", "cells.", "Antibodies", "to", "the", "myogenic", "transcription", "factors,", "Myf5/Pax7,", "the", "intermediate", "filament", "protein,", "desmin,", "and", "the", "marker", "of", "newly", "formed", "myotubes,", "eMyHC,", "were", "used", "to", "reveal", "commitment", "to", "myogenic", "differentiation.", "Cell", "commitment", "to", "this", "differentiation", "program", "was", "assessed", "by", "the", "efficiency", "of", "myotube", "formation,", "estimated", "by", "the", "number", "of", "nuclei", "per", "myotube.", "Ki67,", "a", "cell", "cycle", "related", "nuclear", "protein", "consistently", "absent", "in", "quiescent", "cells,", "was", "used", "as", "a", "marker", "for", "proliferation.", "Whereas", "Ki67", "appears", "in", "all", "active", "phases", "of", "the", "cell", "cycle,", "BrdU", "staining", "allowed", "exclusive", "detection", "of", "cells", "in", "S-phase,", "thereby", "enabling", "accurate", "quantification", "of", "DNA", "synthesis.", "In", "select", "cultures,", "10", "µm", "BrdU", "was", "added", "for", "2", "h", "prior", "to", "fixation.", "BrdU-specific", "immunostaining", "required", "nuclear", "permeabilization", "with", "treatment", "of", "4N", "HCl.", "hESCs", "were", "distinguished", "from", "mouse", "cells", "by", "using", "a", "species-specific", "antibody", "to", "the", "cell-surface", "marker", "M-cadherin", "for", "murine", "and", "the", "nuclear", "marker", "NuMA", "for", "human", "cells.", "Antibodies", "to", "Oct4", "were", "used", "as", "a", "marker", "of", "hESC", "self-renewal/pluripotency.", "Following", "permeabilization", "in", "PBS,", "+1%", "FBS,", "+0.25%", "Triton", "X-100,", "cells", "were", "incubated", "with", "primary", "antibodies", "(concentration", "determined", "as", "per", "manufacturer's", "recommendations)", "for", "1", "h", "at", "room", "temperature", "in", "PBS,", "+1%", "FBS,", "washed", "several", "times,", "and", "then", "incubated", "with", "fluorophore-conjugated,", "species-specific", "secondary", "antibodies", "(diluted", "1", ":", "500", "in", "PBS", "+", "1%", "FBS)", "for", "1", "h", "at", "room", "temperature.", "For", "histological", "analysis,", "dissected", "muscle", "was", "treated", "in", "a", "25%", "sucrose/PBS", "solution,", "frozen", "in", "OCT", "compound", "(Tissue", "Tek)", "and", "cryosectioned", "at", "10", "µm.", "Immunostaining", "was", "performed", "in", "the", "manner", "described", "above,", "or", "H&E", "staining", "of", "cryosections", "was", "performed.", "Nuclei", "were", "visualized", "by", "Hoechst", "staining", "for", "all", "immunostains.", "Samples", "were", "analyzed", "at", "room", "temperature", "by", "using", "a", "Zeiss", "Axioscope", "40", "fluorescent", "microscope,", "and", "imaged", "with", "an", "Axiocan", "MRc", "camera", "and", "AxioVision", "software.", "All", "images", "depict", "identical", "microscope", "fields", "at", "×20", "magnification,", "unless", "otherwise", "noted.", "ReagentsAntibodies", "to", "Oct4", "(ab18976),", "BrdU", "(BU1/75", "(ICR1),", "and", "Ki67", "(ab15580)", "were", "purchased", "from", "Abcam", "(Cambridge,", "MA,", "USA).", "Antibody", "to", "M-cadherin", "(clone", "12G4)", "was", "acquired", "from", "Upstate", "Biotechnology", "(Lake", "Placid,", "NY,", "USA),", "and", "NuMA", "antibody", "(Catalog", "number", "NA09L)", "from", "EMD", "Biosciences", "(San", "Diego,", "CA,", "USA).", "Antibody", "to", "developmental", "eMyHC", "(clone", "RNMy2/9D2)", "was", "acquired", "from", "Vector", "Laboratories", "(Burlingame,", "CA,", "USA).", "Myf5", "(GTX77876)", "and", "Pax7", "(GTX77888)", "antibodies", "were", "obtained", "from", "GeneTex", "(San", "Antonio,", "TX,", "USA).", "Desmin", "antibodies", "(clone", "DE-U-10", "and", "Catalog", "number", "D8281),", "BrdU", "labeling", "reagent", "and", "FK506", "(Catalog", "number", "F4679)", "were", "obtained", "from", "Sigma.", "Fluorophore-conjugated", "secondary", "antibodies", "(Alexa", "Fluor)", "were", "obtained", "from", "Molecular", "Probes", "(Eugene,", "OR,", "USA).", "Statistical", "analysesA", "minimum", "of", "three", "replicates", "were", "undertaken", "for", "each", "experimental", "condition.", "Quantified", "data", "are", "presented", "as", "means", "±", "SE.", "Significance", "testing", "was", "performed", "using", "one-way", "analysis", "of", "variance", "(anova)", "to", "compare", "data", "from", "different", "experimental", "groups.", "P", "values", "of", "<", "0.05", "were", "considered", "as", "statistically", "significant." ]
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9, 9, 9, 9, 9, 9, 8, 9, 9, 9, 9, 9, 9, 9, 9, 8, 12, 12, 12, 9, 9, 9, 9, 9, 9, 9, 9, 9, 0, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 8, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 2, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 8, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 8, 12, 12, 9, 9, 8, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 8, 9, 9, 9, 8, 12, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 14, 10, 9, 9, 9, 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"they", "were", "maintained.", "hESCs", "could", "be", "readily", "distinguished", "from", "embryoid", "bodies", "(EB)", "differentiated", "from", "them", "and", "the", "karyotypically", "abnormal", "hESC", "line", "BG01V.", "The", "embryonal", "carcinoma", "(EC)", "line", "NTera2", "is", "a", "useful", "model", "for", "evaluating", "characteristics", "of", "hESCs.", "Expression", "of", "subsets", "of", "individual", "genes", "was", "validated", "by", "comparing", "with", "published", "databases,", "MPSS", "(Massively", "Parallel", "Signature", "Sequencing)", "libraries,", "and", "parallel", "analysis", "by", "microarray", "and", "RT-PCR.Conclusionwe", "show", "that", "Illumina's", "bead", "array", "platform", "is", "a", "reliable,", "reproducible", "and", "robust", "method", "for", "developing", "base", "global", "profiles", "of", "cells", "and", "identifying", "similarities", "and", "differences", "in", "large", "number", "of", "samples.", "Embryonic", "stem", "cells", "(ESCs),", "derived", "from", "the", "inner", "cell", "mass", "of", "pre-implantation", "embryos,", "have", "been", "recognized", "as", "the", "most", "pluripotent", "stem", "cell", "population.", "Human", "ES", "cells", "(hESCs)", "can", "be", "maintained", "and", "propagated", "on", "mouse", "or", "human", "fibroblast", "feeders", "for", "extended", "periods", "in", "media", "containing", "basic", "fibroblast", "growth", "factor", "(bFGF)", "[1-4]", "while", "retaining", "the", "ability", "to", "differentiate", "into", "ectoderm,", "endoderm", "and", "mesoderm", "as", "well", "as", "trophoectoderm", "and", "germ", "cells.", "Gene", "expression", "in", "hESC", "has", "been", "investigated", "by", "a", "variety", "of", "techniques", "including", "massively", "parallel", "signature", "sequencing", "(MPSS),", "serial", "analysis", "of", "gene", "expression", "(SAGE),", "expressed", "sequence", "tag", "(EST)", "scan,", "large", "scale", "microarrays,", "focused", "cDNA", "microarrays,", "and", "immunocytochemistry", "[5-7].", "Markers", "for", "hESCs", "that", "may", "also", "contribute", "to", "the", "\"stemness\"", "phenotype", "have", "been", "established", "and", "markers", "that", "distinguish", "ESCs", "from", "embryoid", "bodies", "(EB)", "have", "been", "developed.", "Novel", "stage-specific", "genes", "that", "distinguish", "between", "hESCs", "and", "EBs", "have", "been", "identified", "and", "allelic", "differences", "between", "ESC", "have", "begun", "to", "be", "recognized", "[8-10].", "As", "the", "potential", "of", "hESCs", "and", "their", "derivatives", "for", "regenerative", "medicine", "is", "being", "evaluated,", "it", "has", "become", "clear", "that", "the", "overall", "state", "of", "the", "cells,", "degree", "of", "contamination", "and", "comparisons", "of", "the", "more", "than", "a", "hundred", "different", "newly", "derived", "lines", "will", "need", "to", "be", "performed.", "It", "will", "be", "necessary", "to", "develop", "methods", "to", "monitor", "and", "assess", "hESC", "and", "their", "derivatives", "on", "a", "routine", "basis.", "Since", "differentiated", "cells", "are", "often", "scattered", "within", "or", "at", "the", "edge", "of", "colonies", "[11]", "and", "the", "differentiation", "is", "so", "subtle", "that", "morphological", "characteristics", "and", "even", "immunohistochemistry", "are", "insufficient", "to", "detect", "it,", "larger", "scale", "methods", "of", "analysis", "need", "to", "be", "developed.", "Our", "strategy", "was", "to", "compare", "a", "variety", "of", "different", "hESC", "lines", "that", "were", "derived", "and", "expanded", "by", "three", "different", "institutions", "(WiCell", "Research", "Institute,", "BresaGen,", "Inc.,", "and", "Technion-Israel", "Institute", "of", "Technology),", "and", "cultured", "in", "two", "separate", "laboratories", "(Burnham", "Institute", "and", "NIA)", "to", "a", "baseline", "set", "of", "data", "against", "which", "cell", "samples", "can", "be", "compared.", "By", "using", "cells", "grown", "in", "different", "conditions", "we", "expected", "to", "be", "able", "to", "identify", "core", "commonalities", "and", "by", "comparing", "feeders", "and", "embryoid", "bodies", "(EB)", "with", "hESC", "identify", "measures", "of", "contamination", "and", "early", "markers", "of", "differentiation.", "Further,", "by", "comparing", "embryonal", "carcinoma", "cell", "(EC)", "and", "karyotypically", "variant", "lines", "with", "hESC,", "we", "would", "be", "able", "to", "directly", "assess", "their", "utility", "as", "surrogates", "(for", "quality", "control", "purposes)", "for", "hESC.", "We", "employed", "a", "pre-commercial", "prototype", "of", "the", "Illumina", "HumanRef-8", "BeadChip", "[12],", "a", "genome-scale", "bead", "based", "array", "technology", "that", "combines", "the", "sensitivity", "and", "low", "cost", "of", "a", "focused", "array", "with", "the", "coverage", "of", "a", "large", "scale", "array,", "while", "requiring", "much", "smaller", "sample", "sizes", "than", "MPSS,", "EST", "scan", "or", "SAGE.", "We", "show", "that", "the", "Illumina", "bead", "based", "array", "correctly", "identified", "blinded", "duplicates", "as", "the", "closest", "related", "samples", "and", "readily", "distinguished", "between", "hESC", "lines,", "as", "well", "as", "between", "ESCs", "and", "EBs", "derived", "from", "them.", "This", "array", "allowed", "us", "to", "estimate", "the", "degree", "of", "feeder", "contamination", "present", "in", "the", "cultures.", "Similarities", "and", "differences", "between", "EC", "line", "NTera2", "and", "hESC", "lines", "could", "be", "determined", "and", "verified,", "and", "the", "database", "comparisons", "allowed", "us", "to", "identify", "core", "self-renewal", "pathways", "that", "regulate", "hESC", "propagation.", "Multiple", "hESC", "lines", "can", "be", "assessed", "by", "Illumina", "bead", "arrayForty-eight", "samples", "were", "selected", "from", "multiple", "laboratories", "and", "gene", "expression", "profiles", "were", "examined", "using", "a", "bead", "array", "containing", "24,131", "transcripts", "derived", "from", "the", "Human", "RefSeq", "database", "that", "included", "full", "length", "and", "splice", "variants.", "Each", "gene", "was", "represented", "by", "sequences", "containing", "an", "average", "of", "thirty", "beads", "to", "provide", "an", "internal", "measure", "of", "reliability.", "Samples", "included", "7", "hESC", "lines", "BG01,", "BG02,", "BG03,", "I6,", "H1,", "H7", "and", "H9,", "EBs", "that", "were", "differentiated", "from", "hESCs", "of", "the", "three", "BG", "lines,", "human", "fibroblast", "feeder", "HS27", "(ATCC),", "hESC-derived", "fibroblasts,", "karyotypically", "abnormal", "hESC", "line", "BG01", "Variant", "(BG01V)", "[13]", "and", "EC", "line", "NTera2", "[14].", "Samples", "were", "blinded", "and", "biological", "and", "technical", "repeats", "were", "examined", "at", "the", "same", "time.", "A", "single", "slide", "contained", "eight", "replicates", "and", "six", "such", "slides", "were", "used", "for", "the", "present", "set", "of", "samples.", "Results", "were", "normalized", "to", "average", "following", "Illumina", "Beadstudio", "manual", "and", "the", "quality", "of", "each", "sample", "was", "assessed", "by", "immunocytochemitsry", "and", "RT-PCR", "prior", "to", "subjecting", "them", "for", "analysis", "(data", "not", "shown).", "Results", "from", "the", "entire", "sample", "set", "are", "available", "for", "download", "as", "an", "excel", "spreadsheet", "(Additional", "file", "1)", "and", "a", "CD", "of", "the", "results", "is", "available", "upon", "request.", "The", "total", "number", "of", "genes", "identified", "as", "expressed", "at", ">0.99", "confidence", "is", "summarized", "in", "Table", "1.", "Intensity", "results", "are", "reported", "in", "arbitrary", "units", "and", "ranged", "from", "10", "to", "20,000", "(a", "two", "thousand", "fold", "range).", "Although", "the", "sensitivity", "of", "the", "array", "has", "been", "reported", "to", "be", "high,", "in", "the", "present", "report", "we", "have", "restricted", "our", "analysis", "to", "expression", "of", "at", "least", "100", "units", "in", "any", "one", "sample.", "Using", "this", "cutoff,", "on", "average", "cells", "expressed", "approximately", "8,000", "transcripts", "(Table", "1,", "2),", "a", "number", "similar", "to", "the", "number", "detected", "by", "SAGE,", "MPSS", "and", "EST", "analysis", "[5-7,10,15,16].", "As", "with", "other", "analysis,", "genes", "with", "the", "highest", "abundance", "were", "housekeeping", "genes,", "ribosomal", "genes", "and", "structural", "genes", "(Table", "2", "and", "Additional", "file", "1).", "These", "genes", "were", "similar", "in", "most", "samples", "though", "relative", "levels", "varied.Table", "1Correlation", "coefficients", "of", "paired", "samples", "in", "this", "bead", "array", "In", "order", "to", "test", "the", "reproducibility", "and", "reliability", "of", "the", "bead", "array,", "duplicate", "samples", "of", "hESC", "lines", "H9,", "I6,", "and", "EC", "line", "NTera2", "and", "human", "fibroblast", "feeders", "(HS27)", "were", "run", "at", "the", "same", "time", "and", "correlation", "coefficients", "(R2)", "of", "duplicates", "were", "generated", "using", "the", "entire", "data", "of", "all", "genes", "with", "expression", "level", ">0", "(§),", "or", "genes", "with", "detection", "confidence", ">0.99", "(*),", "or", "genes", "with", "detection", "confidence", ">0.99", "and", "expression", "level", ">", "100", "arbitrary", "units", "(#).", "Note", "that", "the", "correlation", "coefficients", "are", "in", "the", "range", "of", "0.9382–0.9761", "and", "the", "number", "of", "genes", "was", "in", "the", "range", "of", "10,000–14,000.Duplicate", "SamplesNo.", "of", "all", "genes", "(expr.>0)§R2of", "all", "genes", "(expr.", ">0)No.", "of", "genes", "(>0.99)*R2", "of", "genes", "(>0.99)*No.", "(>0.99,", "level>100)#H918,8990.868113,6720.97087,408I619,1390.866312,5700.97616,826NTera219,1620.874114,0360.93827,147Feeder18,1570.872410,6060.97517,021Table", "2Distribution", "of", "genes", "with", "expression", "levels", "<50", "and", ">50–10,000", "as", "detected", "by", "Illumina", "bead", "array", "in", "8", "hESC", "populations", "All", "human", "ESC", "samples", "were", "hybridized", "in", "one", "experiment", "and", "the", "relative", "detection", "levels", "of", "genes", "were", "binned", "to", "obtain", "a", "global", "overview", "of", "transcription,", "approximately", "8,", "000", "genes", "(~50%)", "were", "greater", "than", "100", "arbitrary", "units.", "The", "numbers", "are", "similar", "to", "results", "obtained", "by", "other", "large", "scale", "analysis", "such", "as", "MPSS.Abundance", "(relative", "detection", "levels)H9H9", "on", "human", "feedersI6BG01BG02BG03BG01VPooled", "(H1,", "H7,", "H9)No.%No.%No.%No.%No.%No.%No.%No.%<502,90921.26,06738.43,55925.34,52830.74,25630.54,70632.24194.35,80334.4>5010,81778.89,74761.610,48474.710,23269.39,69469.59,91567.89,43895.711,08565.6>1008,12659.27,53947.77,40952.87,70352.27,49653.77,43050.87,23073.38,21748.7>5003,07722.42,94718.62,62618.73,06520.82,95021.12,85219.52,85128.92,94117.4>10001,62911.91,62510.31,49010.61,63811.11,58911.41,51710.41,56615.91,5549.2>50002481.82571.62561.82561.72631.92621.82752.82511.5>10000900.7940.61120.8920.61000.71070.71011.0940.6Total", "No.", "of", "genes", "detected", "at", ">0.99", "confidence13,72615,81414,04314,76013,95014,6219,85716,888One", "of", "the", "advantages", "of", "the", "Illumina", "arrays", "is", "the", "ability", "of", "running", "multiple", "samples", "simultaneously", "thus", "allowing", "multiple", "pairwise", "comparisons", "to", "be", "performed", "readily.", "To", "show", "the", "similarity", "of", "relative", "gene", "expression", "between", "samples,", "we", "have", "used", "Illumina", "Beadstudio", "and", "clustering", "software", "packages", "Pcluster", "[17]", "and", "TreeView", "[18]", "to", "generate", "a", "heat-map", "(Figure", "1)", "and", "a", "dendrogram", "(Figure", "2).", "Based", "on", "their", "properties,", "we", "classified", "some", "of", "our", "samples", "into", "four", "groups,", "(A)", "undifferentiated", "hESCs", "(including", "a", "sample", "from", "karyotypically", "abnormal", "variant,", "designated", "as", "\"ES\",", "n", "=", "11);", "(B)", "differentiated", "ES", "cells", "and", "EBs", "(designated", "as", "\"EB\",", "n", "=", "6);", "(C)", "hESC", "derived", "neural", "cells", "(designated", "as", "\"NS\",", "n", "=", "3);", "and", "(D)", "hESC", "derived", "mesenchyme", "and", "human", "fibroblast", "feeder", "cells", "(designated", "as", "\"FB\",", "n", "=", "5)", "and", "these", "groups", "were", "shown", "in", "the", "heat-map.", "Comparing", "the", "overall", "pattern", "of", "expression,", "we", "made", "several", "important", "observations:", "1)", "Duplicates", "clustered", "close", "to", "each", "other", "and", "were", "more", "related", "to", "each", "other", "than", "to", "any", "other", "sample;", "2)", "ESCs", "appeared", "more", "similar", "to", "each", "other", "than", "to", "EBs;", "3)", "NTera2", "cells", "appeared", "more", "similar", "to", "ESCs", "while", "differentiated", "NTera2", "and", "EBs", "can", "be", "readily", "distinguished", "from", "their", "parent", "populations", "(Figure", "2);", "4)", "BG01V", "appeared", "similar", "to", "undifferentiated", "BG01", "cells;", "5)", "In", "general", "ESC", "lines", "grown", "in", "one", "laboratory", "appeared", "more", "similar", "than", "samples", "grown", "in", "other", "laboratories,", "suggesting", "that", "culture", "conditions", "affected", "gene", "expression", "but", "that", "this", "effect", "was", "much", "smaller", "than", "the", "effect", "of", "differentiation.Figure", "1Unsupervised", "two-way", "hierarchical", "cluster", "analysis", "of", "differentially", "expressed", "genes", "illustrated", "in", "a", "heat-map.", "Each", "row", "represents", "the", "relative", "levels", "of", "expression", "of", "a", "single", "gene.", "Each", "column", "represents", "a", "sample.", "The", "samples", "include", "four", "groups", "of", "cells,", "ES", "designates", "11", "samples", "of", "hESCs,", "EB", "contains", "6", "samples", "of", "differentiated", "ESCs", "and", "EBs,", "NS", "consists", "of", "3", "hESC", "derived", "neural", "cells", "and", "FB", "is", "a", "collection", "of", "hESC", "derived", "mesenchyme", "and", "fibroblasts.", "High", "expressions", "relative", "to", "mean", "are", "colored", "red.", "Low", "expressions", "are", "colored", "green.", "Black", "represents", "no", "significant", "change", "in", "expression", "level", "between", "mean", "and", "sample.", "Samples", "cluster", "closer", "within", "their", "own", "group", "than", "samples", "from", "other", "groups.Figure", "2Dendrogram", "of", "unsupervised", "one-way", "hierarchical", "clustering", "analysis", "of", "relative", "expression", "of", "genes", "in", "selected", "samples.", "The", "clustering", "analysis", "was", "based", "on", "the", "average", "linkage", "and", "Euclidean", "distances", "as", "the", "similarity", "metric", "using", "differentially", "expressed", "genes", "identified", "by", "ANOVA", "(p", "<", "0.05).", "hESCs", "clustered", "together", "and", "BG", "lines", "cultured", "in", "the", "same", "laboratory", "shared", "the", "largest", "similarities.", "EBs", "were", "separated", "from", "hESCs", "from", "which", "they", "were", "derived.", "EC", "line", "NTera2", "and", "feeder", "cells", "can", "be", "distinguished", "from", "hESCs", "respectively.The", "global", "analysis", "suggested", "that", "the", "bead", "arrays", "used", "were", "sufficiently", "sensitive", "such", "that", "individual", "subsets", "of", "genes", "could", "be", "analyzed,", "different", "populations", "of", "cells", "could", "be", "readily", "distinguished", "and", "that", "a", "subset", "of", "candidate", "genes", "could", "be", "sufficient", "to", "distinguish", "between", "groups", "of", "cells.", "The", "comparison", "across", "multiple", "samples", "will", "allow", "a", "set", "of", "core", "stem", "cell", "markers", "to", "be", "identified.", "In", "subsequent", "sections", "we", "have", "performed", "such", "analysis.", "Readers", "are", "urged", "to", "analyze", "the", "expression", "of", "desired", "genes", "directly", "as", "it", "is", "impossible", "to", "test", "every", "gene", "given", "the", "large", "body", "of", "data", "generated.", "Comparison", "between", "MPSS", "and", "Illumina", "bead", "array", "resultsWe", "have", "previously", "used", "EST", "scan", "and", "MPSS", "to", "analyze", "pooled", "samples", "of", "ESCs", "and", "EBs", "from", "three", "different", "WiCell", "lines", "(H1,", "H7", "and", "H9)", "[5].", "Comparison", "between", "the", "two", "methodologies", "indicated", "that", "while", "there", "is", "good", "concordance", "for", "genes", "expressed", "at", "high", "levels,", "this", "does", "not", "hold", "for", "genes", "expressed", "at", "lower", "levels.", "As", "a", "test", "of", "the", "quality", "of", "the", "data", "generated", "in", "these", "experiments", "and", "to", "evaluate", "whether", "comparisons", "can", "be", "made", "across", "different", "methodologies,", "we", "re-ran", "the", "identical", "samples", "on", "the", "bead", "array", "platform.", "The", "complete", "comparison", "of", "gene", "expression", "is", "shown", "in", "Additional", "file", "2", "and", "is", "summarized", "in", "Tables", "3", "and", "Table", "4.", "Overall,", "concordance", "in", "Illumina", "array", "was", "better", "than", "that", "evident", "between", "EST", "scan", "and", "MPSS", "datasets", "[9],", "but", "clearly", "showed", "much", "wider", "differences", "than", "that", "seen", "with", "running", "duplicates", "in", "the", "same", "assay", "format.", "Nevertheless,", "this", "comparison", "provides", "an", "independent", "verification", "of", "the", "data", "and", "suggests", "that", "if", "a", "sample", "is", "detected", "in", "more", "than", "one", "large-scale", "analysis,", "the", "reliability", "of", "the", "gene", "expression", "detection", "is", "high,", "which", "also", "reduces", "the", "number", "of", "individual", "genes", "needed", "to", "be", "verified.", "Caution", "should", "be", "observed", "in", "comparing", "different", "samples", "run", "on", "different", "platforms,", "especially", "when", "there", "has", "not", "been", "rigorous", "bioinformatic", "matching", "of", "the", "source", "sequences", "used", "to", "identify", "genes", "in", "the", "platforms.", "Often", "genes", "called", "by", "the", "same", "symbol", "originate", "from", "different", "database", "records,", "which", "may", "originate", "from", "different", "splice", "variants", "or", "contain", "sequence", "differences", "due", "to", "polymorphisms", "or", "outright", "error", "[19].Table", "3Expression", "of", "hESC", "specific", "markers", "in", "pooled", "hESC", "sample", "as", "detected", "by", "Illumina", "bead", "array", "The", "expression", "of", "previously", "identified", "hESC", "markers", "was", "examined", "in", "all", "hESC", "samples", "(the", "values", "displayed", "represent", "the", "expression", "level", "of", "pooled", "H1,", "H7", "and", "H9).", "Most", "of", "the", "genes", "were", "also", "identified", "using", "Illumina", "bead", "array", "in", "all", "8", "hESC", "populations", "in", "this", "study", "(1*),", "the", "gene", "CER1", "was", "detected", "in", "all", "except", "one", "duplicate", "of", "H9", "(2*),", "Nanog", "was", "not", "detected", "in", "all", "populations", "(3*)", "and", "Sox2,", "Lin41,", "NR6A1", "and", "FoxD3", "were", "not", "detected", "in", "the", "array", "although", "they", "were", "present", "in", "the", "chips", "for", "hybridization", "(4*).AccessionSymbolPooled", "ES", "(H1,", "7,", "9)CommentsNM_003641.1IFITM188441*NM_020997.2LEFTB6579.61*NM_024674.3LIN283944.11*NM_175849.1DNMT3B33911*NM_003212.1TDGF131691*NM_001769.2CD92930.81*NM_000165.2GJA12404.41*NM_021195.2CLDN62247.21*NM_004360.2CDH11972.91*NM_021127.1PMAIP11601.51*NM_032805.1ZNF2061504.51*NM_003577.1UTF11444.11*XM_050625.2SFRP21353.91*NM_006548.3IMP-212061*NM_152312.2GYLTL1B1066.61*NM_015973.2GAL1043.31*NM_003240.2EBAF944.11*NM_054023.2SCGB3A2890.51*NM_020990.2CKMT1742.41*NM_033668.1ITGB1694.71*NM_003744.3NUMB618.31*NM_007015.1LECT1597.41*NM_021912.2GABRB3482.71*NM_006729.2DIAPH2467.11*NM_000222.1KIT188.91*NM_005454.1CER1151.52*NM_024865.1NANOG56.93*NM_002701.1POU5F1694.41*NM_003106SOX2ND4*NM_006458.2LIN41ND4*NM_001489.3NR6A1ND4*NM_012183FOXD3ND4*Table", "4Comparison", "of", "MPSS", "and", "Illumina", "bead", "array", "results", "The", "samples", "were", "analyzed", "by", "MPSS", "and", "bead", "array.", "The", "number", "of", "genes", "detected", "by", "each", "method", "and", "the", "degree", "of", "overlap", "is", "summarized.", "Note", "much", "higher", "degree", "of", "overlap", "when", "the", "top", "2000", "hits", "were", "compared.", "*:", "Most", "of", "the", "genes", "detected", "by", "MPSS", "were", "novel", "genes", "not", "included", "in", "the", "bead", "array.ESNo.%Common", "in", "both", "(Top", "2000", "hits)1,62281.1Common", "in", "both", "(All", "hits)5,07146.0By", "bead", "array", "only3,46231.4By", "MPSS", "only", "*2,50422.7Total11,037100EBNo.%Common", "in", "both", "(All", "hits)5,16843.1By", "bead", "array", "only4,13134.4By", "MPSS", "only", "*2,69422.5Total11,993100", "Human", "feeders", "and", "hESCs", "can", "be", "readily", "distinguished", "and", "contamination", "can", "be", "readily", "assessedFor", "all", "samples,", "we", "conducted", "an", "unsupervised", "one-way", "hierarchical", "clustering", "analysis.", "The", "clustering", "analysis", "was", "based", "on", "the", "average", "linkage", "and", "Euclidean", "distances", "as", "the", "similarity", "metric", "using", "differentially", "expressed", "genes", "identified", "by", "ANOVA", "(P", "<", "0.05).", "The", "analysis", "revealed", "the", "underlying", "features", "and", "variation", "patterns", "of", "gene", "expression", "in", "each", "cell", "types.", "Figure", "2", "shows", "results", "of", "the", "cluster", "analysis", "of", "relative", "gene", "expression", "in", "selected", "samples.", "As", "one", "of", "our", "purposes", "of", "this", "study", "was", "to", "distinguish", "between", "human", "fibroblast", "feeders", "cells", "and", "hESCs", "and", "hEBs,", "wishing", "to", "readily", "detect", "feeder", "contamination", "in", "hESCs,", "we", "included", "one", "of", "the", "human", "feeder", "cells", "HS27", "(ATCC)", "in", "this", "study.", "We", "have", "been", "using", "HS27", "as", "feeder", "cells", "for", "H9", "hESCs", "for", "more", "than", "two", "years", "and", "all", "hESCs", "grown", "on", "HS27", "had", "normal", "karyotype,", "expressed", "all", "undifferentiated", "markers,", "and", "made", "teratomas", "with", "all", "germ", "layers", "(data", "not", "show).", "The", "global", "pairwise", "comparison", "clearly", "showed", "that", "human", "feeders", "were", "far", "more", "dissimilar", "to", "hESCs", "than", "hESCs", "grown", "in", "different", "laboratories,", "hESCs", "compared", "to", "their", "differentiated", "EBs", "that", "contained", "mesodermal", "tissue,", "and", "hESCs", "compared", "to", "the", "karyotypically", "variant", "hESC", "line", "BG01V.", "Pairwise", "comparisons", "of", "human", "feeders", "with", "hESCs", "resulted", "in", "a", "correlation", "coefficient", "of", "0.66,", "which", "was", "less", "than", "the", "correlation", "coefficient", "of", "0.71–0.74", "observed", "between", "hESCs", "and", "their", "corresponding", "EBs.", "The", "large", "difference", "between", "human", "feeders", "and", "hESCs", "suggested", "that", "it", "would", "be", "possible", "to", "identify", "markers", "that", "were", "robust", "and", "reliable", "in", "distinguishing", "the", "two", "populations,", "and", "these", "markers", "would", "be", "sufficiently", "sensitive", "in", "detecting", "contamination", "of", "feeders.", "We", "examined", "the", "data", "to", "develop", "a", "list", "of", "genes", "that", "had", "high", "levels", "of", "expression", "in", "human", "feeder", "cells", "maintained", "in", "hESC", "medium", "but", "whose", "expression", "was", "low", "or", "absent", "in", "either", "ESCs", "or", "EBs.", "The", "absence", "of", "expression", "in", "EBs", "was", "used", "as", "a", "control", "for", "spontaneous", "differentiation", "of", "ESC", "colonies", "(including", "mesodermal", "differentiation)", "which", "may", "occur", "and", "the", "markers", "selected", "should", "be", "able", "to", "distinguish", "between", "these", "two", "events.", "A", "complete", "list", "of", "genes", "expressed", "at", "least", "ten-fold", "higher", "in", "human", "feeders", "is", "provided", "in", "Figure", "3.", "Quantitative", "RT-PCR", "(qPCR)", "was", "used", "to", "verify", "the", "fold", "change", "of", "the", "expression", "of", "4", "genes,", "including", "THBS1,", "MMP3,", "TNFRSF11B", "and", "KRTHA4", "(Figure", "3C).", "Further", "confirmation", "can", "also", "be", "done", "using", "immunocytochemistry,", "as", "antibodies", "against", "these", "genes", "are", "commercially", "available.Figure", "3Human", "fibroblast", "feeder", "cells", "can", "be", "distinguished", "from", "hESCs", "and", "EBs.", "Bead", "array", "identified", "lists", "of", "genes", "that", "were", "uniquely", "expressed", "in", "human", "fibroblast", "feeders", "as", "compared", "to", "hESCs", "(A)", "and", "hEBs", "(B).", "The", "four", "genes", "whose", "expression", "was", "confirmed", "by", "qPCR", "(C)", "were", "in", "bold.", "In", "the", "graph", "(C),", "gene", "expression", "of", "each", "gene", "in", "feeder", "cells", "was", "designated", "as", "1", "fold", "and", "the", "bars", "represented", "fold", "decrease", "for", "each", "gene.Thus", "this", "comparison", "allowed", "us", "to", "distinguish", "between", "hESCs", "and", "human", "feeders", "and", "identify", "candidate", "markers", "that", "could", "detect", "feeder", "cell", "contamination", "should", "human", "feeders", "be", "used", "in", "the", "propagation", "of", "hESCs.", "hESCs", "and", "EBs", "can", "be", "distinguished", "from", "each", "otherIllumina", "bead", "array", "analysis", "confirmed", "that", "hESCs", "could", "be", "readily", "distinguished", "from", "EBs", "by", "global", "analysis.", "This", "raised", "the", "possibility", "that", "specific", "subsets", "of", "markers", "could", "be", "identified.", "We", "and", "others", "have", "used", "MPSS", "and", "EST", "scan", "and", "generated", "array", "data", "to", "make", "lists", "of", "hESC-specific", "genes", "[5,9,10,20].", "As", "discussed", "above,", "most", "hESC", "markers", "identified", "by", "MPSS", "have", "been", "detected", "in", "the", "present", "bead", "array", "analysis", "(Table", "3),", "confirming", "the", "utility", "of", "these", "previously", "identified", "markers", "for", "use", "in", "assessing", "undifferentiated", "status", "of", "hESCs.", "In", "addition,", "we", "have", "generated", "a", "list", "of", "genes", "differentially", "expressed", "at", "higher", "level", "in", "EBs", "than", "in", "hESCs,", "a", "subset", "of", "which", "is", "shown", "in", "Table", "5.", "These", "markers", "were", "common", "to", "all", "EB", "samples", "tested", "and", "included", "genes", "known", "to", "be", "expressed", "in", "ectoderm,", "endoderm", "and", "mesoderm.", "The", "entire", "set", "of", "differentially", "expressed", "genes", "is", "provided", "in", "Additional", "file", "3.", "Thus,", "the", "bead", "array", "format,", "which", "allows", "multiple", "pairwise", "comparisons,", "can", "be", "used", "to", "identify", "genes", "that", "are", "expressed", "by", "all", "differentiating", "EB", "samples", "in", "the", "present", "study.", "Our", "data", "suggested", "that", "a", "core", "set", "of", "limited", "markers", "might", "be", "sufficient", "to", "monitor", "the", "process", "of", "differentiation.", "By", "suitable", "selection", "of", "different", "germ", "cell", "layer", "specific", "markers", "one", "may", "also", "assess", "the", "overall", "quality", "of", "differentiation", "toward", "germ", "cells.Table", "5Genes", "which", "are", "differentially", "expressed", "at", "higher", "levels", "in", "EBs", "than", "in", "hESCsSymbolAll", "EBAll", "ESEB/ESRELN11120.52224.0SST1394.25.5253.5SLC40A11210.19.1133.0IGF24896.938.4127.5SLN2017.817118.7DCN7588.176.299.6ANXA84048.941.996.6AQP11665.420.581.2APOB1256.415.879.5AHSG1414.320.868.0NID21476.731.447.0FGB221052.442.2LUM8439.4205.441.1MGP2772.469.839.7THBD1206.434.435.1SERPINA11255.643.728.7HAND112294437.428.1HBE11106.542.626.0TTR7661.2347.122.1HBG21601.185.618.7COL2A11636.291.817.8KIAA09771370.87817.6AFP8941552.316.2COL3A11355796714.0IGFBP38446.3603.414.0PAX61577.8118.213.3APOA19398.1709.813.2FRZB3523.7315.711.2SPON21548.7159.49.7CEBPD1100.2122.79.0DLK13355.8374.89.0RDC11589.7192.18.3BMP41851.5227.38.1PITX21057.9131.28.1ACTA25045629.88.0GAS11215.61547.9AGTRL11053.8135.17.8COL5A15876.4765.47.7CDKN1C3134.1412.97.6CXCL142220.5312.67.1DOK41011.1145.27.0ARHGDIB1635.3246.56.6FLRT21879.5314.36.0MSX12771.8499.75.5", "Smaller", "but", "distinct", "differences", "among", "undifferentiated", "hESC", "linesOur", "cluster", "analysis", "indicated", "that", "BG01,", "BG02", "and", "BG03", "cell", "lines", "were", "overall", "more", "similar", "to", "each", "other", "than", "to", "other", "lines", "(Figure", "1", "and", "2),", "but", "nevertheless", "showed", "additional", "differences", "than", "technical", "or", "biological", "repeats", "of", "the", "same", "sample.", "This", "raised", "the", "possibility", "that", "this", "microarray", "strategy", "may", "be", "sufficiently", "sensitive", "to", "identify", "relatively", "cell", "type", "specific", "candidate", "genes", "that", "could", "be", "used", "to", "distinguish", "one", "hESC", "population", "from", "another", "or", "to", "identify", "differences", "that", "were", "due", "to", "varied", "isolation", "and", "growth", "conditions.", "As", "a", "test", "we", "looked", "for", "differences", "between", "BG01,", "BG02", "and", "BG03,", "which", "were", "grown", "in", "the", "same", "laboratory", "under", "the", "same", "conditions.", "Lists", "of", "candidate", "genes", "are", "shown", "in", "Figure", "4A,", "C", "and", "4E", "and", "the", "comparison", "of", "these", "three", "lines", "are", "shown", "in", "scatter", "plots", "in", "Figure", "4B,", "D", "and", "4F.Figure", "4BG", "lines", "show", "small", "but", "distinct", "differences", "as", "assessed", "by", "bead", "array.", "These", "three", "hESC", "lines", "share", "high", "similarities", "as", "shown", "by", "the", "scatterplots", "of", "BG01", "vs", "BG02", "(B),", "BG01", "vs", "BG03", "(D)", "and", "BG02", "vs", "BG03", "(F).", "Comparisons", "of", "all", "three", "lines", "were", "made", "and", "lists", "of", "selected", "genes", "that", "were", "specifically", "expressed", "in", "BG01", "(A),", "BG02", "(C)", "and", "BG03", "(F)", "are", "shown.", "Correlation", "coefficients", "(R2)", "were", "generated", "using", "all", "genes", "with", "expression", "level", ">0", "(black", "and", "blue", "dots),", "or", "all", "genes", "with", "detection", "confidence", ">0.99", "(blue", "dots).", "Genes", "outside", "the", "two", "thin", "red", "lines", "were", "detected", "at", ">2.5-", "fold", "difference.We", "reasoned", "as", "well", "that", "such", "a", "global", "comparison", "should", "allow", "us", "to", "distinguish", "between", "male", "and", "female", "lines", "if", "genes", "present", "on", "the", "Y", "chromosome", "were", "expressed", "at", "high", "levels", "in", "the", "undifferentiated", "state", "and", "were", "detected", "by", "the", "bead", "array.", "Several", "such", "candidate", "genes", "were", "identified.", "The", "most", "robust", "were", "RPS4Y,", "RPS4Y2,", "and", "EIF1AY", "(Figure", "5).", "To", "confirm", "that", "these", "were", "useful", "markers,", "we", "designed", "RT-PCR", "primers", "and", "tested", "their", "expression", "in", "a", "male", "(BG01)", "and", "a", "female", "(BG03)", "line", "(Figure", "5B).", "We", "noted", "that", "several", "of", "these", "continued", "to", "be", "expressed", "at", "high", "levels", "as", "ESCs", "differentiated", "to", "form", "EBs", "and", "upon", "further", "differentiation", "(data", "not", "shown),", "suggesting", "that", "these", "markers", "might", "be", "used", "in", "adult", "stem", "cell", "and", "germ", "cell", "populations", "as", "well.Figure", "5Male", "and", "female", "hESC", "lines", "can", "be", "distinguished", "by", "genes", "identified", "by", "bead", "array.", "Five", "potential", "genes", "RPS4Y,", "RPS4Y2,", "EIF1AY,", "VCY,", "and", "AMELY", "are", "located", "in", "the", "Y", "chromosome.", "By", "comparing", "the", "expression", "level", "of", "these", "genes", "in", "all", "hESC", "lines,", "we", "have", "found", "that", "3", "out", "of", "5", "were", "specifically", "expressed", "in", "male", "hESC", "lines", "I6,", "BG01", "and", "BG02", "(A)", "and", "this", "was", "verified", "by", "RT-PCR", "in", "male", "line", "BG01", "and", "female", "line", "BG03", "(B).", "G3PDH", "was", "used", "as", "an", "internal", "control.", "*:", "represents", "the", "gene", "expression", "level", "is", "detected", "at", "<0.99", "confidence.In", "summary,", "our", "data", "suggest", "that", "the", "bead", "array", "format", "is", "sufficiently", "sensitive", "and", "global", "that", "it", "can", "distinguish", "one", "cell", "line", "from", "another", "even", "if", "those", "two", "cell", "lines", "are", "grown", "in", "the", "same", "laboratory", "under", "virtually", "identical", "conditions.", "Bead", "array", "can", "also", "be", "used", "to", "distinguish", "between", "male", "and", "female", "lines.", "Comparison", "of", "diploid", "pluripotent", "cells", "with", "NTera2", "and", "BG01", "variantOur", "previous", "results", "have", "suggested", "that", "EC", "lines", "share", "many", "of", "the", "properties", "of", "hESCs", "and", "can", "be", "used", "as", "a", "useful", "model", "for", "initial", "testing", "of", "biological", "questions", "[21].", "More", "recently", "we", "have", "identified", "BG01V", "as", "a", "karyotypically", "abnormal", "variant", "that", "behaves", "much", "like", "its", "normal", "counterpart", "BG01,", "but", "is", "not", "subject", "to", "the", "same", "constraints", "of", "use", "as", "karyotypically", "normal", "hESCs", "[13].", "Given", "the", "sensitivity", "of", "the", "bead", "array", "analysis,", "we", "tested", "its", "ability", "to", "detect", "the", "overall", "similarities", "and", "differences", "between", "NTera2", "and", "a", "pooled", "ESC", "sample", "or", "between", "the", "karyotypically", "abnormal", "BG01V", "and", "its", "normal", "parent", "line", "(Figure", "6).Figure", "6Diploid", "pluripotent", "EC", "cell", "line", "NTera2", "and", "karyotypically", "abnormal", "hESC", "line", "BG01V", "can", "be", "distinguished", "from", "normal", "hESCs", "using", "Illumina", "array.", "Comparison", "of", "NTera2", "and", "pooled", "hESC", "sample", "resulted", "a", "correlation", "coefficient", "of", "0.8997.", "Two", "lists", "of", "genes,", "which", "were", "specifically", "expressed", "in", "NTera2", "(C)", "or", "in", "hESCs", "(E)", "were", "identified.", "Likewise,", "while", "sharing", "similarities", "with", "BG01", "(B,", "correlation", "coefficient=", "0.9043),", "BG01V", "was", "different", "from", "BG01", "in", "expression", "for", "many", "genes,", "particularly", "genes", "from", "the", "TGFβ", "pathway", "(D,", "F).", "Black", "dots", "represent", "genes", "that", "were", "detected", "at", ">0", "expression", "level,", "blue", "dots", "represent", "genes", "that", "were", "detected", "both", "at", ">", "0", "expression", "level", "and", "at", ">0.99", "confidence.", "Genes", "plotted", "outside", "the", "two", "thin", "red", "lines", "were", "detected", "at", ">2.5-", "fold", "difference.Our", "results", "showed", "that,", "while", "NTera2", "shared", "a", "high", "similarity", "with", "hESCs", "[21],", "it", "did", "have", "important", "differences", "with", "hESC", "lines.", "Examining", "these", "differences", "(summarized", "in", "Figure", "6C", "and", "6E),", "we", "noted", "that", "some", "reflected", "the", "origin", "of", "the", "tumor", "cells", "from", "which", "this", "line", "was", "derived", "[14].", "Several", "germ", "cell", "markers", "such", "as", "GAGE2,", "GAGE7", "and", "GAGE8", "were", "highly", "expressed", "in", "NTera2", "but", "were", "absent", "(or", "present", "at", "low", "levels)", "in", "any", "of", "the", "hESC", "lines", "examined", "(See", "Figure", "6C", "and", "Additional", "file", "1.", "Note", "that", "the", "GAGE", "genes", "are", "highly", "similar", "in", "sequence,", "making", "it", "difficult", "to", "distinguish", "one", "family", "member", "from", "another", "through", "hybridization;", "thus,", "while", "all", "of", "these", "GAGE", "gene", "probes", "gave", "positive", "signal,", "it", "is", "difficult", "to", "say", "if", "the", "signal", "came", "from", "the", "specific", "gene", "itself", "or", "from", "cross-hybridization", "from", "one", "of", "the", "other", "family", "members).", "None", "of", "these", "were", "present", "in", "BG01V,", "indicating", "that", "the", "karyotypically", "abnormal", "variant", "is", "not", "the", "equivalent", "of", "a", "teratocarcinoma", "line", "such", "as", "NTera2.", "In", "addition", "to", "the", "expression", "of", "germ", "cell", "markers,", "we", "noticed", "a", "significant", "difference", "in", "the", "expression", "of", "genes", "in", "the", "TGFβ", "pathway,", "such", "as", "GDF3", "(Figure", "6C),", "TGFBI,", "CDKN1A,", "IGFBP7,", "IGFBP3,", "NODAL,", "CER1", "and", "BMP2", "(Figure", "6E).", "This", "is", "consistent", "with", "the", "postulated", "role", "of", "this", "pathway", "in", "germ", "cell", "differentiation", "[22,23]", "and", "suggests", "that", "TGFβ", "pathway", "cannot", "be", "reliably", "tested", "using", "NTera2", "as", "a", "model", "for", "hESC.The", "BG01V", "showed", "clear", "differences", "from", "its", "normal", "counterpart", "and", "some", "major", "changes", "are", "summarized", "in", "Figure", "6D", "and", "6F.", "Early", "markers", "of", "differentiation", "appeared", "to", "be", "present", "at", "higher", "levels", "in", "BG01V", "as", "compared", "to", "any", "of", "the", "hESC", "lines", "examined,", "although", "hESC", "specific", "genes", "continued", "to", "be", "expressed", "at", "high", "levels", "(see", "Additional", "file", "4).", "In", "particular,", "the", "Wnt", "pathway", "and", "the", "TGFβ", "signaling", "pathway", "(Figure", "6D),", "both", "of", "which", "involved", "in", "the", "early", "process", "of", "differentiation", "[24,25],", "appeared", "to", "be", "activated", "(Additional", "file", "4),", "suggesting", "that", "the", "role", "of", "growth", "factors", "and", "signaling", "in", "these", "early", "events", "cannot", "be", "readily", "studied", "in", "this", "cell", "line.In", "summary,", "the", "analysis", "highlighted", "the", "utility", "of", "the", "potential", "reference", "standards", "NTera2", "and", "BG01V,", "demonstrated", "their", "general", "similarity", "and", "provided", "detail", "on", "potential", "caveats", "to", "their", "application.", "Global", "arrays", "provide", "a", "snapshot", "of", "the", "state", "of", "the", "cells", "and", "identify", "core", "self-renewal", "pathwaysWe", "have", "utilized", "a", "small", "fraction", "of", "the", "data", "to", "demonstrate", "the", "overall", "utility", "of", "this", "approach", "and", "its", "sensitivity", "in", "identifying", "small", "differences", "in", "cell", "populations.", "An", "additional", "potential", "application", "of", "such", "an", "analysis", "is", "the", "ability", "to", "examine", "the", "general", "state", "of", "a", "particular", "signaling", "pathway", "and", "determine", "whether", "it", "is", "active.", "By", "comparing", "across", "many", "samples,", "a", "procedure", "previously", "expensive", "and", "difficult", "in", "terms", "of", "the", "RNA", "and", "replicate", "requirement,", "one", "can", "rapidly", "identify", "key", "regulatory", "pathways.To", "test", "whether", "we", "could", "use", "such", "multiple", "pairwise", "comparisons", "to", "elucidate", "the", "major", "regulatory", "pathways", "that", "may", "be", "required", "for", "hESC", "self-renewal,", "we", "examined", "several", "metabolic", "pathways.", "The", "results", "of", "the", "analysis", "of", "the", "insulin/insulin-like", "growth", "factor", "(IGF)", "signaling", "pathway", "are", "shown", "in", "Figure", "7.", "Using", "the", "same", "4", "groups", "of", "samples", "as", "in", "Figure", "1,", "we", "conducted", "PAM", "(Prediction", "Analysis", "of", "Microarray)", "[26],", "in", "search", "for", "biomarkers", "used", "in", "diagnostic", "identification", "of", "these", "four", "groups,", "ES,", "EB,", "NS,", "and", "FB.", "In", "PAM,", "a", "list", "of", "significant", "IGF", "pathway", "genes", "whose", "expression", "characterizes", "each", "diagnostic", "class", "was", "obtained.", "The", "average", "gene", "expression", "level", "in", "each", "class", "was", "divided", "by", "the", "within-class", "standard", "deviation.", "The", "nearest", "centroid", "classification", "computed", "took", "the", "gene", "expression", "profile", "from", "a", "new", "sample", "and", "compared", "it", "to", "each", "of", "these", "class", "centroids.", "For", "cross-validation", "of", "prediction", "results,", "multiple", "classification", "processes", "were", "performed", "on", "two", "data", "sets", "randomly", "constructed", "each", "time", "from", "the", "entire", "gene", "expression", "dataset.", "The", "first", "dataset,", "consisting", "of", "70%", "of", "the", "total", "data,", "was", "used", "as", "the", "training", "dataset,", "and", "the", "other", "dataset,", "containing", "the", "remaining", "30%", "of", "data,", "was", "used", "for", "the", "data", "prediction", "and", "verification", "process.", "The", "final", "biomarkers", "were", "determined", "in", "such", "a", "way", "that", "the", "misclassification", "error", "rate", "was", "minimal.", "The", "resulting", "graph", "(Figure", "7)", "showed", "the", "shrunken", "class", "centroids", "for", "genes", "that", "had", "at", "least", "one", "nonzero", "difference", "in", "each", "diagnostic", "class.", "The", "genes", "with", "nonzero", "components", "in", "each", "class", "were", "almost", "mutually", "exclusive", "and", "represented", "candidate", "biomarkers", "for", "the", "diagnosis", "of", "each", "class.", "All", "data", "analyses", "were", "performed", "using", "the", "bioconductor", "package", "[17].Figure", "7Identification", "of", "diagnostic", "markers", "by", "PAM.", "The", "shrunken", "class", "centroids", "for", "genes", "which", "have", "at", "least", "one", "nonzero", "difference", "are", "shown.", "The", "genes", "with", "nonzero", "components", "in", "each", "class", "were", "almost", "mutually", "exclusive", "and", "were", "the", "candidate", "molecular", "markers", "for", "the", "diagnosis", "of", "the", "four", "groups", "of", "cell", "populations,", "including,", "(from", "left", "to", "right)", "hESC", "derived", "mesenchyme", "and", "human", "fibroblast", "feeder", "cells", "(\"FB\",", "n", "=", "5),", "undifferentiated", "hESCs", "(\"ES\",", "n", "=", "11),", "hESC", "derived", "neural", "cells", "(\"NS\",", "n", "=", "3),", "and", "differentiated", "ES", "cells", "and", "EB,", "(\"EB\",", "n", "=", "6).", "The", "identified", "biomarkers", "can", "be", "used", "to", "distinguish", "the", "four", "groups", "of", "cell", "populations.", "Undifferentiated", "hESCs", "have", "been", "analyzed", "by", "EST", "scan,", "MPSS,", "SAGE", "and", "microarray", "[5,10,16].", "The", "goal", "of", "these", "experiments", "including", "our", "own", "is", "to", "develop", "a", "low", "cost", "reliable", "method", "to", "assess", "multiple", "samples", "to", "generate", "a", "global", "database", "of", "markers", "and", "to", "provide", "a", "method", "of", "identifying", "core", "measures", "of", "similarities", "and", "differences", "across", "multiple", "laboratories.", "We", "and", "others", "have", "proposed", "three", "alternative", "methods", "of", "assessment:", "Quantitative", "RT-PCR", "[9,20],", "focused", "arrays", "[27]", "or", "a", "large", "scale", "array", "with", "bioinformatics", "tools", "being", "utilized", "to", "focus", "on", "appropriate", "subsets", "of", "genes", "[5,7,15,16,28].", "Each", "of", "these", "methods", "has", "its", "advantages", "and", "disadvantages.", "The", "present", "results", "suggest", "that", "the", "global", "Illumina", "bead", "array", "retains", "the", "advantages", "of", "low", "cost", "per", "sample", "associated", "with", "focused", "arrays", "yet", "still", "has", "the", "strength", "of", "the", "global", "attributes", "of", "MPSS", "or", "EST", "scan", "while", "requiring", "much", "less", "RNA", "and", "turnaround", "time.", "To", "test", "this", "array", "format", "we", "examined", "samples", "from", "a", "variety", "of", "laboratories", "in", "a", "blinded", "fashion", "to", "determine", "whether", "the", "array", "was", "sufficiently", "sensitive", "and", "rapid", "for", "routine", "assessment.", "Duplicates", "using", "100", "ng", "of", "RNA", "were", "run", "and", "results", "obtained", "forty-eight", "hours", "later.", "The", "resolution", "was", "sufficient", "that", "ESC", "samples", "could", "be", "distinguished", "from", "one", "another", "and", "a", "variant", "karyotypically", "abnormal", "subclone", "could", "be", "distinguished", "from", "the", "parent", "population", "(correlation", "coefficient", "=", "0.9043).", "Aliquots", "of", "the", "pooled", "ES", "and", "pooled", "EB", "samples,", "which", "we", "had", "prepared", "for", "MPSS,", "were", "included", "in", "this", "run", "to", "compare", "these", "two", "methods", "directly.", "The", "current", "analysis", "confirms", "that", "comparison", "across", "platforms", "is", "difficult", "and", "that", "only", "positive", "results", "can", "be", "treated", "with", "any", "reliability.", "The", "absence", "of", "expression", "cannot", "be", "readily", "interpreted.", "In", "particular,", "genes", "expressed", "at", "low", "levels", "(greater", "than", "70%", "of", "all", "genes", "detected)", "should", "not", "be", "assessed", "in", "cross", "platform", "comparisons.", "The", "limited", "concordance", "at", "low", "levels", "raises", "a", "question", "as", "to", "how", "many", "genes", "are", "actually", "expressed", "by", "any", "one", "cell", "line", "and", "whether", "the", "cutoff", "of", "3", "tpm", "used", "for", "MPSS", "or", "100", "intensity", "units", "for", "bead", "arrays", "is", "a", "reasonable", "cutoff.", "We", "used", "100", "units", "for", "our", "analysis", "and", "we", "would", "suggest", "that", "readers", "exercise", "similar", "caution.", "Nevertheless", "even", "at", "this", "higher", "cutoff", "the", "arrays", "were", "remarkably", "sensitive", "and", "allowed", "us", "to", "readily", "distinguish", "between", "samples", "including", "cells", "grown", "in", "the", "same", "laboratory.", "The", "basis", "of", "the", "sensitivity", "could", "be", "attributed", "to", "a", "limited", "set", "of", "genes", "and", "those", "genes", "could", "be", "identified", "for", "future", "use.", "For", "example", "BG01V,", "while", "much", "more", "similar", "to", "BG01", "than", "to", "any", "other", "cell", "type,", "could", "still", "be", "distinguished", "from", "a", "biological", "replicate", "of", "BG01", "by", "the", "expression", "of", "a", "particular", "subset", "of", "differentiation", "markers", "(Figure", "6).", "EC", "cells", "such", "as", "NTera2", "could", "be", "distinguished", "from", "hESCs", "by", "the", "expression", "of", "germ", "cell", "markers", "and", "the", "presence", "of", "a", "partially", "inactivated", "TGFβ", "(BMP)", "signaling", "pathway", "(Figure", "6).", "Distinguishing", "ESCs", "from", "EBs", "was", "relatively", "straightforward.", "We", "have", "confirmed", "the", "utility", "of", "previously", "identified", "markers", "for", "use", "in", "this", "platform", "as", "well", "as", "identified", "an", "additional", "set", "of", "markers", "that", "can", "serve", "as", "biomarkers", "to", "distinguish", "between", "the", "hESC", "and", "EB", "states.", "A", "subset", "of", "these", "markers", "have", "been", "used", "to", "develop", "a", "qPCR", "assay", "that", "shows", "such", "a", "high", "sensitivity", "that", "changes", "in", "cell", "behavior", "can", "be", "detected", "after", "as", "little", "as", "twenty-four", "hours", "and", "the", "development", "of", "EBs", "can", "be", "reliably", "staged", "[10,20].", "During", "the", "identification", "of", "ES", "and", "EB", "specific", "markers,", "we", "have", "noticed", "that", "some", "known", "hESC", "markers,", "such", "as", "Nanog,", "was", "not", "detected", "in", "all", "populations", "of", "hESCs", "that", "were", "included", "in", "this", "analysis.", "Several", "ESC-specific", "gene,", "including", "Lin41,", "Sox2", "and", "FoxD3,", "were", "not", "detected", "in", "the", "array", "either", "(Table", "3).", "We", "believe", "that", "the", "problem", "with", "Lin", "41,", "Sox2", "and", "FoxD3", "is", "a", "technical", "one", "as", "we", "were", "able", "to", "confirm", "expression", "using", "alternate", "methods.", "We", "are", "in", "progress", "of", "redesigning", "appropriate", "probes", "for", "these", "genes.", "In", "the", "case", "of", "the", "gene", "Nanog,", "there", "are", "several", "pseudo", "genes", "in", "the", "genome", "for", "Nanog", "and", "it", "has", "been", "a", "major", "technical", "challenge", "designing", "primers", "or", "probes", "that", "are", "specific", "and", "sensitive.", "We", "believe", "that", "a", "partial", "explanation", "for", "the", "variability", "in", "Nanog", "expression", "is", "due", "to", "the", "lack", "of", "sensitivity", "to", "this", "gene.", "However,", "immunocytochemistry", "while", "not", "strictly", "quantitative", "shows", "similar", "variability", "when", "used", "to", "assess", "Nanog", "expression", "in", "different", "cell", "lines", "[9,27,28].", "This", "large", "comparison", "between", "samples", "allowed", "us", "to", "identify", "markers", "that", "distinguish", "human", "feeder", "cells", "from", "hESC.", "While", "we", "have", "listed", "19", "potential", "markers", "(Figure", "3)", "and", "identified", "several", "hundred", "potential", "markers", "as", "shown", "in", "Additional", "file", "5,", "we", "suggest", "that", "as", "few", "as", "3–4", "genes", "may", "be", "sufficient.", "Previously", "we", "found", "that", "as", "few", "as", "four", "were", "satisfactory", "to", "distinguish", "between", "hESCs", "and", "hEBs,", "which", "are", "two", "much", "more", "closely", "related", "samples", "[9].", "In", "this", "study", "we", "have", "confirmed", "by", "qPCR", "the", "differential", "expression", "of", "four", "genes,", "THBS1,", "MMP3,", "TNFRSF11B", "and", "KRTH4,", "to", "separate", "human", "fibroblast", "feeders", "and", "hESCs", "(Figure", "3).", "Several", "markers", "such", "as", "MMP3", "and", "TNFRSF11B", "have", "commercially", "available", "antibodies", "(R&D", "systems)", "that", "may", "be", "used", "to", "further", "confirm", "contamination", "of", "feeder", "cells", "by", "immunocytochemistry.", "Efforts", "to", "identify", "other", "useful", "antibodies", "based", "on", "these", "results", "continue", "[29].", "While", "we", "have", "focused", "on", "the", "immediate", "utility", "of", "the", "Illumina", "array", "platform,", "it", "is", "important", "to", "remember", "that", "this", "array", "provides", "a", "global", "snapshot", "of", "cell", "state", "and", "the", "data", "obtained", "can", "be", "readily", "compared", "in", "order", "to", "determine", "key", "signaling", "pathways.", "The", "ability", "to", "compare", "multiple", "samples", "in", "one", "run", "enhances", "data", "selectivity", "and", "reliability.", "To", "make", "such", "analysis", "more", "readily", "available,", "we", "utilized", "several", "software", "tools", "including", "the", "software", "package", "available", "through", "Illumina.", "The", "BeadStudio", "software", "provided", "with", "the", "BeadLab", "and", "BeadStudio", "genetic", "analysis", "systems", "for", "use", "with", "the", "bead", "array", "datasets", "provides", "a", "useful", "set", "of", "analytical", "and", "presentation", "tools", "that", "allow", "straightforward", "comparisons,", "which", "are", "sufficient", "for", "average", "users.", "For", "detailed", "analysis", "we", "recommend", "using", "more", "specific", "commercial", "tools", "or", "software", "packages", "developed", "by", "NCBI.", "In", "summary,", "the", "Illumina", "bead", "array", "has", "several", "key", "strengths", "including", "high", "throughput,", "low", "cost", "and", "high", "sensitivity.", "By", "using", "this", "array,", "we", "can", "readily", "detect", "contaminating", "feeders", "and", "spontaneous", "differentiation,", "differentiate", "male", "and", "female", "lines", "and", "distinguish", "between", "one", "undifferentiated", "population", "and", "another.", "Such", "a", "global", "analysis", "allows", "us", "to", "assess", "context", "dependent", "signaling", "and", "identify", "biomarkers", "of", "particular", "states", "of", "cells.", "Our", "future", "efforts", "will", "focus", "on", "data", "mining", "and", "developing", "better", "cross", "platform", "comparison", "tools", "and", "generating", "focused", "high", "throughput", "arrays", "for", "quality", "control", "in", "clinical", "and", "research", "settings.", "hESC", "cultureThe", "hESC", "lines", "H1,", "H7", "and", "H9", "(WiCell,", "Madison,", "WI)", "were", "cultured", "on", "feeder", "layers", "derived", "from", "mitotically", "inactivated", "HS27", "human", "fibroblast", "cells", "(HS27,", "ATCC),", "or", "mouse", "embryonic", "fibroblsts", "or", "under", "feeder-free", "conditions", "on", "Matrigel", "(BD,", "Franklin", "Lakes,", "NJ)", "coated", "plates", "for", "at", "least", "10", "passages.", "Culture", "medium", "for", "all", "cultures", "was", "composed", "of", "DMEM/F12-Glutamax", "1:1,", "20%", "Knockout", "Serum", "Replacement,", "2", "mM", "nonessential", "amino", "acids,", "100", "μM", "beta-mercaptoethanol,", "50", "μg/ml", "Pen-Strep", "(all", "from", "Invitrogen,", "Carlsbad,", "CA),", "and", "4", "ng/ml", "human", "recombinant", "basic", "fibroblast", "growth", "factor", "(bFGF/FGF2;", "PeproTech", "Inc.,", "Rocky", "Hill,", "NJ.)", "Feeder-free", "cultures", "were", "prepared", "for", "gene", "expression", "analysis", "by", "manually", "harvesting", "individual", "colonies", "with", "uniform", "typical", "undifferentiated", "ESC", "morphology.BG01", "(46,", "XY),", "BG02", "(46,", "XY),", "BG03", "(46,", "XX),", "I6", "(46,", "XY)", "and", "BG01V", "(BG01", "karyotypic", "variant:", "49,", "XXY,", "+12,", "+17):", "Cells", "were", "maintained", "for", "3", "(BG01V),", "7", "(BG02),", "8", "(BG01),", "or", "21", "(BG03)", "passages", "under", "feeder-free", "condition", "on", "fibronectin-coated", "plates", "in", "medium", "that", "had", "been", "conditioned", "by", "mouse", "embryonic", "fibroblasts", "for", "24", "hours.", "Culture", "medium", "was", "DMEM/F12,", "1:1", "supplemented", "with", "20%", "Knockout", "Serum", "Replacement,", "2", "mM", "non-essential", "amino", "acids,", "2", "mM", "L-glutamine,", "50", "μg/ml", "Pen-Strep,", "100", "μM", "beta-mercaptoethanol,", "and", "4", "ng/ml", "of", "bFGF.Different", "hESC", "lines", "were", "grown", "in", "slightly", "different", "culture", "conditions", "as", "described", "above.", "H", "lines", "were", "grown", "on", "Matrigel", "coated", "dishes,", "while", "BG", "lines", "on", "fibronectin", "treated", "dishes.", "These", "coating", "substrata", "supported", "the", "growth", "of", "hESCs", "similarly,", "as", "evaluated", "by", "colony", "morphology,", "immunocytochemistry", "and", "proliferation", "rate", "(data", "not", "shown).Embryoid", "bodies", "(EBs)", "were", "prepared", "from", "BG", "lines", "as", "described", "in", "[5].", "Cells", "were", "aggregated", "and", "cultured", "on", "non-adherent", "substrata", "for", "fourteen", "days.Other", "cellsNTera2", "cells", "were", "purchased", "from", "ATCC", "and", "cultured", "in", "parallel", "with", "hESCssamples", "using", "protocols", "described", "previously", "[21].", "HS27", "embryonic", "human", "newborn", "foreskin", "cells", "(ATCC", "CRL-1634)", "were", "grown", "in", "DMEM", "with", "10%FBS.All", "samples", "included", "in", "this", "study", "can", "be", "found", "in", "Additional", "file", "6.Bead", "array", "gene", "expression", "analysisRNA", "was", "isolated", "from", "cultured", "cells", "using", "the", "Qiagen", "RNEasy", "kit", "(Qiagen,", "Inc,", "Valencia,", "CA).", "Sample", "amplification", "was", "performed", "using", "100", "ng", "of", "total", "RNA", "as", "input", "material", "by", "the", "method", "of", "Van", "Gelder", "et", "al", "[30].", "Amplified", "RNA", "synthesized", "from", "limited", "quantities", "of", "heterogenous", "cDNA", "[30]", "was", "performed", "using", "the", "Illumina", "RNA", "Amplification", "kit", "(Ambion,", "Inc.,", "Austin,", "TX)", "following", "the", "Manufacturer", "instructions.", "Labeling", "was", "achieved", "by", "use", "of", "the", "incorporation", "of", "biotin-16-UTP", "(Perkin", "Elmer", "Life", "and", "Analytical", "Sciences,", "Boston,", "MA)", "present", "at", "a", "ratio", "of", "1:1", "with", "unlabeled", "UTP.", "Labeled,", "amplified", "material", "(700", "ng", "per", "array)", "was", "hybridized", "to", "a", "pilot", "version", "of", "the", "Illumina", "HumanRef-8", "BeadChip", "according", "to", "the", "Manufacturer's", "instructions", "(Illumina,", "Inc.,", "San", "Diego,", "CA).", "Amersham", "fluorolink", "streptavidin-Cy3", "(GE", "Healthcare", "Bio-Sciences,", "Little", "Chalfont,", "UK)", "following", "the", "BeadChip", "manual.", "Arrays", "were", "scanned", "with", "an", "Illumina", "Bead", "array", "Reader", "confocal", "scanner", "according", "to", "the", "Manufacturer's", "instructions.", "Array", "data", "processing", "and", "analysis", "was", "performed", "using", "Illumina", "BeadStudio", "software.Identification", "of", "differentially", "expressed", "genes", "and", "clustering", "analysisDifferentially", "expressed", "genes", "between", "ES", "and", "EB", "were", "identified", "by", "ANOVA", "at", "p", "value", "0.05", "using", "bioconductor", "[17].", "Unsupervised", "hierarchical", "clustering", "analysis", "and", "principal", "component", "analysis", "(PCA)", "were", "conducted", "using", "software", "Pcluster", "[31]", "and", "TreeView", "[18].Identification", "of", "diagnostic", "markersPAM", "(prediction", "analysis", "of", "microarray)", "was", "employed", "for", "the", "identification", "of", "diagnostic", "markers", "from", "insulin", 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[ "BackgroundHuman", "stem", "cells", "are", "viewed", "as", "a", "possible", "source", "of", "neurons", "for", "a", "cell-based", "therapy", "of", "neurodegenerative", "disorders,", "such", "as", "Parkinson's", "disease.", "Several", "protocols", "that", "generate", "different", "types", "of", "neurons", "from", "human", "stem", "cells", "(hSCs)", "have", "been", "developed.", "Nevertheless,", "the", "cellular", "mechanisms", "that", "underlie", "the", "development", "of", "neurons", "in", "vitro", "as", "they", "are", "subjected", "to", "the", "specific", "differentiation", "protocols", "are", "often", "poorly", "understood.ResultsWe", "have", "designed", "a", "focused", "DNA", "(oligonucleotide-based)", "large-scale", "microarray", "platform", "(named", "\"NeuroStem", "Chip\")", "and", "used", "it", "to", "study", "gene", "expression", "patterns", "in", "hSCs", "as", "they", "differentiate", "into", "neurons.", "We", "have", "selected", "genes", "that", "are", "relevant", "to", "cells", "(i)", "being", "stem", "cells,", "(ii)", "becoming", "neurons,", "and", "(iii)", "being", "neurons.", "The", "NeuroStem", "Chip", "has", "over", "1,300", "pre-selected", "gene", "targets", "and", "multiple", "controls", "spotted", "in", "quadruplicates", "(~46,000", "spots", "total).", "In", "this", "study,", "we", "present", "the", "NeuroStem", "Chip", "in", "detail", "and", "describe", "the", "special", "advantages", "it", "offers", "to", "the", "fields", "of", "experimental", "neurology", "and", "stem", "cell", "biology.", "To", "illustrate", "the", "utility", "of", "NeuroStem", "Chip", "platform,", "we", "have", "characterized", "an", "undifferentiated", "population", "of", "pluripotent", "human", "embryonic", "stem", "cells", "(hESCs,", "cell", "line", "SA02).", "In", "addition,", "we", "have", "performed", "a", "comparative", "gene", "expression", "analysis", "of", "those", "cells", "versus", "a", "heterogeneous", "population", "of", "hESC-derived", "cells", "committed", "towards", "neuronal/dopaminergic", "differentiation", "pathway", "by", "co-culturing", "with", "PA6", "stromal", "cells", "for", "16", "days", "and", "containing", "a", "few", "tyrosine", "hydroxylase-positive", "dopaminergic", "neurons.ConclusionWe", "characterized", "the", "gene", "expression", "profiles", "of", "undifferentiated", "and", "dopaminergic", "lineage-committed", "hESC-derived", "cells", "using", "a", "highly", "focused", "custom", "microarray", "platform", "(NeuroStem", "Chip)", "that", "can", "become", "an", "important", "research", "tool", "in", "human", "stem", "cell", "biology.", "We", "propose", "that", "the", "areas", "of", "application", "for", "NeuroStem", "microarray", "platform", "could", "be", "the", "following:", "(i)", "characterization", "of", "the", "expression", "of", "established,", "pre-selected", "gene", "targets", "in", "hSC", "lines,", "including", "newly", "derived", "ones,", "(ii)", "longitudinal", "quality", "control", "for", "maintained", "hSC", "populations,", "(iii)", "following", "gene", "expression", "changes", "during", "differentiation", "under", "defined", "cell", "culture", "conditions,", "and", "(iv)", "confirming", "the", "success", "of", "differentiation", "into", "specific", "neuronal", "subtypes.", "Modern", "DNA", "microarrays", "permit", "a", "comprehensive", "analysis", "of", "quantitative", "and", "qualitative", "changes", "in", "RNA", "transcript", "abundance,", "outlining", "the", "cross-sections", "of", "gene", "expression", "and", "alterations", "of", "these", "in", "response", "to", "genetic", "or", "environmental", "stimuli.", "Genome-scale", "microarrays", "(cDNA-", "or", "oligonucleotide-based)", "are", "most", "valuable", "when", "screening", "populations", "of", "cells", "for", "the", "novel", "genes", "reflecting", "potential", "diagnostic", "and", "prognostic", "markers", "or", "for", "an", "identification", "of", "novel", "therapeutic", "targets.", "On", "the", "other", "hand,", "custom", "microarray", "platforms", "that", "focus", "on", "specific", "pre-selected", "subset", "of", "genes", "relevant", "to", "a", "particular", "field", "of", "investigation", "can", "be", "less", "costly", "and", "more", "suitable", "for", "detection", "of", "smaller", "gene", "expression", "changes.", "Microarray", "technology", "has", "added", "important", "information", "on", "both", "normal", "development", "and", "pathological", "changes", "in", "neurons.", "This", "is", "well", "illustrated", "by", "multiple", "studies", "on", "substantia", "nigra", "dopaminergic", "neurons,", "which", "degenerate", "in", "Parkinson's", "disease", "(PD)", "[1-5].", "The", "shortcomings", "of", "pharmacological", "therapies", "in", "PD", "have", "stimulated", "a", "search", "for", "alternative", "treatment", "strategies.", "In", "successful", "cases,", "transplants", "of", "human", "embryonic", "mesencephalic", "dopaminergic", "neurons", "can", "both", "restore", "dopaminergic", "neurotransmission", "and", "provide", "some", "symptomatic", "relief", "[6-8].", "A", "wider", "application", "of", "neural", "transplantation", "in", "PD", "is,", "however,", "currently", "not", "feasible", "due", "to", "the", "unpredictable", "and", "variable", "outcome,", "the", "risks", "of", "unwanted", "side-effects", "(dyskinesias)", "[9,10]", "and", "ethical", "and", "practical", "problems", "associated", "with", "using", "donor", "cells", "obtained", "from", "aborted", "embryos", "and", "fetuses", "[11,12].", "Human", "embryonic", "stem", "cells", "(hESCs)", "are", "considered", "a", "promising", "future", "source", "of", "cells", "for", "cell", "replacement", "therapy", "in", "PD", "and", "other", "neurological", "conditions", "[13].", "They", "could", "constitute", "a", "virtually", "infinite", "source", "of", "self-renewing", "cells", "that", "can", "be", "persuaded", "to", "differentiate", "into", "specific", "types", "of", "neural", "cells,", "including", "dopaminergic", "neurons", "[14-16].", "The", "molecular", "mechanisms", "that", "govern", "development", "of", "cultured", "hESCs", "into", "specific", "types", "of", "neural", "cells", "are", "not", "fully", "understood.", "To", "promote", "our", "understanding", "of", "such", "mechanisms,", "it", "would", "be", "valuable", "to", "have", "tools", "that", "readily", "and", "reproducibly", "can", "help", "to", "characterize", "the", "cells", "as", "they", "differentiate", "from", "pluripotent", "stem", "cells", "into", "post-mitotic", "neurons.", "This", "important", "issue", "was", "addressed", "in", "earlier", "studies", "by", "Luo", "et", "al.", "and", "Yang", "et", "al.,", "who", "designed", "small-to-moderate", "scale", "custom", "microarray", "platforms", "(281", "and", "755", "gene", "targets,", "respectively)", "[17,18].", "In", "addition", "SuperArray", "Bioscience", "Corporation", "(Frederick,", "MD,", "USA)", "have", "manufactured", "a", "range", "of", "small-scale", "arrays", "(263", "gene", "targets", "for", "human", "array;", "[19]).", "We", "sought", "to", "create", "an", "improved", "and", "updated", "microarray", "platform", "for", "hESC/neuronal", "differentiation-oriented", "gene", "expression", "studies.", "Therefore,", "we", "generated", "a", "specialized", "large-scale", "DNA", "microarray", "platform", "(the", "\"NeuroStem", "Chip\")", "that", "has", "over", "1,300", "pre-selected", "gene", "targets", "and", "multiple", "controls", "spotted", "in", "quadruplicates", "(~46,000", "spots", "total).", "Here", "we", "introduce", "the", "platform", "and", "the", "advantages", "it", "can", "offers", "to", "neuroscientists", "and", "stem", "cell", "biologists:", "particularly,", "in", "the", "niche", "of", "gene", "expression-oriented", "characterization", "of", "the", "samples", "using", "an", "assay", "of", "pre-selected,", "already", "established", "gene", "targets.", "In", "the", "current", "study,", "we", "use", "the", "NeuroStem", "Chip", "to", "characterize", "an", "undifferentiated", "population", "of", "pluripotent", "hESCs", "(cell", "line", "SA02,", "Cellartis", "AB,", "Göteborg,", "Sweden)", "and", "compare", "the", "gene", "expression", "in", "those", "cells", "with", "that", "of", "a", "hESC-derived", "cell", "population", "rich", "in", "neurons,", "including", "tyrosine", "hydroxylase-positive", "dopaminergic", "neurons.", "Stem", "cells", "have", "unique", "biological", "characteristics,", "but", "only", "a", "limited", "number", "of", "genes", "are", "currently", "recognized", "as", "established", "stem", "cell", "markers.", "Examples", "include", "POU", "domain,", "class", "5,", "transcription", "factor", "1", "(Oct3/4),", "signal", "transducer", "and", "activator", "of", "transcription", "3", "(Stat3),", "teratocarcinoma-derived", "growth", "factor", "(Tdgf1),", "Enk-pending", "(Nanog),", "undifferentiated", "embryonic", "cell", "transcription", "factor", "1", "(Utf1)", "and", "DNA", "methyltransferase", "3B", "(Dnmt3b)", "[20].", "At", "the", "same", "time,", "hundreds", "of", "genes", "are", "suggested", "as", "candidate", "markers", "for", "\"stemness\",", "but", "their", "coupling", "to", "the", "undifferentiated", "stem", "cell", "state", "is", "not", "yet", "fully", "verified", "[21].", "The", "concept", "of", "\"stemness\"", "(term", "introduced", "in", "1986", "by", "Grossman", "&", "Levine)", "is", "defined", "as", "\"core", "stem", "cell", "properties", "that", "underlie", "self-renewal", "and", "the", "ability", "to", "generate", "differentiated", "progeny\"", "[22].", "Considering", "the", "complexity", "of", "the", "processes", "involved,", "stemness", "can", "hardly", "be", "ensured", "by", "co-operation", "of", "just", "a", "few", "genes.", "Nevertheless,", "three", "stemness", "genes", "(namely,", "Oct3/4,", "Stat3", "and", "Nanog)", "are", "considered", "\"master\"-genes", "that", "control", "the", "self-renewing", "process", "[23,24].", "Various", "types", "of", "stem", "cells,", "such", "as", "hematopoietic,", "mesenchymal", "and", "neural", "(HSCs,", "MSCs", "and", "NSCs,", "respectively),", "embryonic", "germ", "and", "embryonic", "carcinoma", "cells", "(EGCs", "and", "ECCs,", "respectively)", "are", "all", "characterized", "by", "variations", "in", "gene", "expression", "profiles,", "and", "only", "a", "few", "gene", "markers", "are", "associated", "with", "all", "these", "cell", "types", "[25,26].", "We", "have", "aimed", "to", "embrace", "the", "most", "comprehensive", "set", "of", "those", "genes", "into", "a", "solitary", "array,", "the", "NeuroStem", "Chip.", "Thereby,", "it", "is", "possible", "to", "employ", "it", "to", "monitor", "the", "relative", "expression", "levels", "of", "numerous", "known", "and", "candidate", "stemness", "genes", "in", "a", "single", "experiment.", "Similar", "to", "the", "genetic", "bases", "underlying", "stemness,", "cell", "differentiation", "is", "associated", "with", "altered", "expression", "levels", "of", "certain", "recognized", "or", "candidate", "genes", "[25].", "We", "therefore", "incorporated", "gene", "markers", "of", "development", "and", "differentiation", "in", "general,", "and", "that", "of", "neuronal", "and", "dopaminergic", "differentiation", "in", "particular,", "into", "the", "NeuroStem", "Chip.", "Examples", "include", "markers", "for", "the", "processes", "of", "neuronal", "maturation,", "axonal", "branching,", "neural/neuronal", "survival,", "etc.", "Finally,", "we", "ensured", "that", "known", "markers", "for", "specific", "types", "of", "neurons,", "allowing", "identification", "of", "individual", "cell", "types,", "were", "present", "on", "the", "chip.", "We", "paid", "special", "attention", "to", "genes", "associated", "with", "the", "differentiation", "and", "maturation", "of", "dopaminergic", "neurons.", "In", "many", "published", "studies,", "the", "expression", "of", "only", "a", "single", "(tyrosine", "hydroxylase,", "TH)", "or", "2–3", "markers", "for", "dopaminergic", "neurons", "(e.g.", "amino", "acid", "decarboxylase", "(AADC),", "dopamine", "transporter", "(DAT),", "vesicular", "monoamine", "transporter", "2", "(VMAT2))", "have", "been", "used", "to", "indicate", "dopaminergic", "identity", "of", "neurons.", "In", "contrast,", "the", "NeuroStem", "Chip", "includes", "oligonucleotide", "probes", "for", "88", "genes", "related", "to", "dopaminergic", "neurons,", "thus", "being", "more", "comprehensive", "in", "this", "sense,", "compared", "to", "other", "existing", "microarray", "platforms,", "including", "focused", "ones", "[17,18].", "Those", "entries", "encompass", "recognized", "and", "candidate", "markers", "for", "dopaminergic", "neurons", "(mature", "and", "early)", "and", "progenitors,", "as", "well", "as", "markers", "for", "the", "maturation", "and", "differentiation", "of", "the", "latter", "(Table", "1).", "Table", "2", "represents", "conditional", "functional", "breakdown", "of", "genes", "targeted", "by", "the", "NeuroStem", "microarray", "platform.", "A", "number", "of", "important", "gene", "groups", "that", "are", "included", "in", "the", "chip", "are", "not", "mentioned", "in", "Table", "2.", "Among", "these,", "entries", "related", "to", "Dickkopf", "gene", "family,", "galanin-,", "melatonin-,", "vasoactive", "intestinal", "peptide", "(VIP)-,", "cAMP", "response", "element-binding", "protein", "(CREB)-", "and", "B", "cell", "leukemia", "2", "(Bcl2)", "oncogene-related", "are", "present.", "Many", "of", "them", "play", "potentially", "important,", "yet", "undefined,", "roles", "in", "the", "biology", "of", "stem", "cells.", "Additionally,", "we", "included", "some", "genes", "implicated", "in", "disease", "mechanisms", "of", "neurodegenerative", "disorders", "(most", "importantly,", "Parkinson's", "disease", "and", "Alzheimer's", "disease)", "in", "the", "chip.", "Furthermore,", "we", "incorporated", "a", "number", "of", "markers", "for", "distinct", "differentiation", "pathways", "(e.g.", "hematopoietic", "and", "pancreatic)", "and", "cell", "types", "(e.g.", "cancer", "subtypes", "and", "a", "range", "of", "normal", "cell", "types)", "to", "serve", "as", "essential", "controls.", "Taken", "together,", "we", "believe", "that", "in", "its", "present", "form", "NeuroStem", "Chip", "represents", "currently", "most", "comprehensive", "gene", "expression", "platform", "for", "studies", "on", "stem", "cells,", "neural/neuronal", "differentiation,", "human", "neurodegeneration", "and", "neuronal", "survival,", "both", "in", "vivo", "and", "in", "vitro.", "The", "complete", "layout", "of", "NeuroStem", "Chip", "will", "be", "disclosed", "to", "the", "academic", "community,", "upon", "request.", "The", "microarray", "format", "we", "selected", "relies", "on", "long", "oligonucleotide", "molecules", "(69–71", "nucleotides)", "printed", "over", "a", "solid", "surface.", "We", "spotted", "the", "synthesized", "oligonucleotides", "(Operon", "Biotechnologies)", "with", "a", "constant", "concentration", "across", "the", "slides,", "and", "evaluated", "the", "quality", "and", "consistency", "of", "spotting", "in", "a", "series", "of", "control", "experiments.", "We", "then", "illustrated", "the", "utility", "and", "technical", "reliability", "of", "the", "NeuroStem", "Chip", "by", "characterizing", "the", "gene", "expression", "profile", "of", "commonly", "utilized", "hESC", "line", "SA02", "(Sahlgrenska", "2;", "[27]),", "including", "(i)", "undifferentiated", "cells", "and", "(ii)", "cells", "committed", "towards", "neuronal/dopaminergic", "differentiation", "pathway.", "For", "the", "first", "of", "these,", "we", "used", "total", "RNA", "sample", "purified", "from", "hESC", "colonies", "that", "exhibited", "morphology", "consistent", "with", "cell", "proliferation", "and", "the", "absence", "of", "spontaneous", "differentiation", "(Figure", "1A).", "We", "also", "evaluated", "the", "expression", "of", "the", "cell", "cycle", "marker", "Ki67", "and", "the", "pluripotency", "marker", "OCT3/4", "in", "the", "sample", "by", "immunocytochemistry", "(Figure", "1B–E).", "Co-culturing", "of", "ESCs", "with", "murine", "stromal", "cells", "(including", "PA6", "cell", "line)", "rapidly", "generates", "dopaminergic", "neurons", "from", "ESCs", "by", "an", "unexplained", "mechanism", "termed", "stromal", "cell-derived", "inducing", "activity", "(SDIA;", "[28,29]).", "We", "therefore", "committed", "hESCs", "toward", "the", "neuronal/dopaminergic", "differentiation", "pathway", "by", "co-culturing", "with", "PA6", "cells", "for", "16", "days,", "resulting", "in", "appearance", "of", "cells", "positive", "for", "early", "and", "late", "neuronal", "markers,", "including", "nestin,", "β-III-tubulin,", "and", "TH,", "the", "established", "marker", "of", "dopaminergic", "neurons", "(Figure", "2).", "To", "verify", "the", "expression", "of", "some", "key", "stem", "cell-", "and", "neural", "phenotype-associated", "genes", "we", "performed", "RT-PCR", "comparing", "RNA", "samples", "from", "the", "undifferentiated", "hESCs", "with", "hESCs", "of", "the", "same", "line", "differentiated", "toward", "neuronal/dopaminergic", "pathway,", "as", "described", "above.", "The", "expression", "profile", "outlined", "by", "RT-PCR", "confirmed", "the", "identity", "of", "the", "sample", "used", "(Figure", "3).", "After", "performing", "RNA", "integrity", "tests,", "we", "incorporated", "fluorescent", "labels", "to", "the", "amplified", "RNA", "samples", "from", "hESCs", "(Cyanine", "3-CTP", "(Cy3)", "and", "Cyanine", "5-CTP", "(Cy5)),", "hESC-derived", "cells", "containing", "TH-positive", "neurons", "(Cy3", "and", "Cy5)", "and", "human", "universal", "reference", "RNA", "(Cy5),", "and", "hybridized", "aliquots", "with", "NeuroStem", "microarray", "slides", "using", "the", "following", "conditions:", "hESC", "vs.", "reference,", "Cy3", ":", "Cy5", "=", "(i)", "20:10", "pmol,", "and", "(ii)", "10:5", "pmol;", "and", "hESC", "vs.", "hESC-derived", "cells,", "Cy3", ":", "Cy5", "=", "(iii)", "30:20", "pmol,", "respectively.", "Universal", "reference", "RNA", "has", "been", "previously", "established", "as", "a", "standard", "reference", "material", "for", "microarray", "experiments,", "proving", "an", "ability", "to", "effectively", "hybridize", "to", "a", "large", "fraction", "of", "microarray", "spots", "[30].", "We", "performed", "two-color", "hybridizations", "(e.g.", "for", "the", "experiment", "vs.", "reference)", "following", "an", "established", "protocol", "[31],", "and", "included", "dye-flip", "technical", "replicates", "in", "the", "analysis", "(Figure", "4).", "Using", "the", "online", "software", "program", "BASE", "[32]", "we", "sequentially", "filtered", "the", "data", "by", "background", "subtraction,", "negative", "flagging,", "negative", "intensities", "and", "for", "inconsistent", "data", "amongst", "replicates", "[33].", "Figure", "5A", "shows", "a", "comparison", "of", "the", "spot", "intensities", "prior", "to", "normalization", "(M", "versus", "A", "plot),", "with", "the", "Log2", "of", "the", "expression", "ratio", "between", "Cy3/Cy5", "being", "plotted", "as", "a", "function", "of", "the", "log10", "of", "the", "mean", "of", "the", "total", "expression", "intensities", "for", "Cy3", "and", "Cy5", "channels.", "The", "deviation", "of", "the", "line", "from", "zero", "revealed", "a", "need", "for", "normalization,", "so", "prior", "to", "data", "analyses", "we", "normalized", "signals", "using", "a", "locally", "weighted", "scatterplot-smoothing", "regression", "(LOWESS)", "algorithm", "(Figure", "5A–B;", "fitted", "line)", "implemented", "in", "BASE.", "Since", "the", "reproducibility", "of", "two-color", "microarray", "gene", "expression", "data", "is", "critically", "important,", "we", "calculated", "Pearson", "correlation", "coefficients", "of", "the", "reporters", "present", "in", "the", "filtered", "database", "comparing", "the", "average", "expression", "ratios", "(7005", "for", "hESCs", "vs.", "universal", "reference;", "6947", "for", "undifferentiated", "vs.", "neuronal/dopaminergic", "lineage-committed", "hESCs).", "Results", "obtained", "revealed", "that", "data", "were", "consistent", "across", "technical", "replicates", "(dye-swap", "and", "amount", "of", "loaded", "material),", "showing", "general", "high", "reproducibility:", "e.g.,", "correlation", "coefficients", "were", "greater", "than", "0.96", "for", "technical", "replicates", "and", "0.78", "for", "dye-swapping", "samples", "in", "hESCs", "vs.", "universal", "reference", "hybridizations", "(Table", "3).", "To", "detect", "genes", "with", "high", "expression", "levels", "in", "hESC", "samples,", "we", "filtered", "data", "for", "intensity", "values", ">100", "in", "the", "hESC", "sample", "and", "performed", "clustering", "analysis", "using", "the", "TIGR", "MultiExperiment", "Viewer", "(MEV;", "[34]).", "To", "visualize", "variations", "of", "spot/reporter", "per", "technical", "replicate,", "hierarchical", "clustering", "was", "performed", "by", "K-means", "classifier", "based", "on", "the", "linear-correlation-based", "distance", "(Pearson,", "centred)", "method.", "The", "optimal", "number", "of", "clusters", "was", "determined", "empirically", "to", "produce", "the", "most", "balanced", "ratio", "of", "entries", "to", "cluster", "of", "highly", "expressed", "genes.", "A", "cluster", "of", "101", "genes", "up-regulated", "in", "the", "hESC", "sample", "[see", "Additional", "file", "1],", "was", "plotted", "in", "a", "centroid", "graph", "(Figure", "5C);", "the", "variation", "across", "technical", "replicates", "was", "low.", "We", "merged", "technical", "replicates", "to", "generate", "a", "list", "of", "the", "most", "up-regulated", "genes", "expressed", "in", "the", "hESC", "sample", "compared", "to", "the", "universal", "reference", "RNA", "(Table", "4).", "Standard", "error", "of", "the", "mean", "expressed", "as", "percentage", "was", "calculated", "for", "the", "4", "technical", "replicates,", "and", "was", "6.7%", "for", "the", "top", "25", "genes", "up-regulated", "in", "hESC", "samples,", "compared", "to", "universal", "reference", "RNA.", "We", "performed", "the", "analysis", "of", "microarray", "data,", "as", "described", "in", "the", "Methods,", "and", "spot", "error", "values", "were", "generally", "in", "the", "lower", "range,", "indicating", "high", "stringency", "of", "the", "signals", "and", "low", "variance.", "As", "seen", "in", "Table", "4", "and", "Table", "5,", "the", "NeuroStem", "Chip", "identified", "numerous", "genes", "associated", "with", "stem", "cells.", "In", "particular,", "homeo", "box", "expressed", "in", "ES", "cells", "1", "(Hesx1)", "gene", "was", "identified", "as", "the", "most", "up-regulated", "in", "the", "ES", "cell", "preparation,", "compared", "to", "universal", "reference", "RNA.", "Highly", "expressed", "in", "pluripotent", "ESCs,", "Hesx1", "expression", "is", "down-regulated", "upon", "embryonic", "stem", "cell", "differentiation", "[35,36],", "as", "also", "clearly", "seen", "in", "differentiation", "experiment", "of", "our", "own", "(Table", "4).", "Similarly,", "Gremlin", "1", "homolog,", "cysteine", "knot", "superfamily", "gene", "(Grem1,", "also", "known", "as", "Cktsf1b1", "and", "Dand2)", "is", "a", "recognized", "factor", "of", "cell-fate", "determination", "of", "ESCs", "[37].", "Many", "more", "genes", "highly", "up-regulated", "in", "the", "hESC", "sample", "in", "comparison", "with", "universal", "reference", "RNA", "are", "associated", "with", "stem", "cells:", "further", "examples", "include", "Gap", "junction", "protein", "α1", "(Gja1)", "and", "Zic", "family", "member", "3", "heterotaxy", "1", "(Zic3)", "(Table", "4)", "[20].", "The", "expression", "of", "fibroblast", "growth", "factor", "receptor", "2", "(Fgfr2)", "is", "of", "particular", "interest.", "Basic", "fibroblast", "growth", "factor", "(FGF2,", "bFGF)", "supports", "hESC", "proliferation", "and", "their", "ability", "to", "maintain", "undifferentiated", "phenotype", "when", "cultured", "in", "vitro", "[38,39].", "Moreover,", "in", "some", "hESC", "lines", "a", "very", "high", "concentration", "of", "FGF2", "could", "substitute", "for", "the", "need", "of", "feeder", "cells", "[40].", "At", "the", "same", "time,", "genes", "listed", "in", "Table", "4", "represent", "the", "most", "highly", "up-regulated", "entries", "in", "a", "relatively", "limited", "group", "of", "genes", "(Figure", "5C).", "Many", "other", "genes", "involved", "in", "maintenance", "of", "ESC", "phenotype", "(i.e.", "established", "or", "candidate", "markers", "of", "stem", "cells)", "have", "lower", "levels", "of", "expression", "(Table", "5).", "Examples", "include", "undifferentiated", "embryonic", "cell", "transcription", "factor", "1", "(Utf1),", "DNA", "methyltransferase", "3B", "(Dnmt3b),", "developmental", "pluripotency", "associated", "4", "(Dppa4,", "a", "newly", "established", "pluripotency", "marker", "[41])", "and", "numerous", "candidate", "markers", "of", "\"stemness\":", "e.g.", "genes", "for", "KIAA1573", "protein,", "forkhead", "box", "O1A", "(Foxo1a),", "high-mobility", "group", "box", "1", "(Hmgb1),", "C-terminal", "binding", "protein", "2", "(Ctbp2)", "and", "left-right", "determination", "factor", "1", "(Lefty1),", "as", "well", "as", "others.", "For", "numerous", "established", "or", "candidate", "markers", "of", "stem", "cells", "the", "expression", "levels", "were", "not", "considerably", "higher", "(Log2", "ratio", "<", "1)", "in", "the", "hESC", "sample", "compared", "to", "the", "universal", "reference", "RNA.", "For", "example,", "the", "expression", "of", "Nanog,", "DNA", "(cytosine-5-)-methyltransferase", "3α", "(Dnmt3a),", "MutS", "homolog", "2,", "colon", "cancer,", "nonpolyposis", "type", "1", "(E.", "coli)", "(Msh2),", "Thy-1", "cell", "surface", "antigen", "(Thy1),", "high-mobility", "group", "box", "2", "(Hmgb2),", "transcription", "factor", "3", "(Tcf3),", "Nanos", "homolog", "1", "(Nanos1),", "MyoD", "family", "inhibitor", "(Mdfi),", "Calumenin", "(Calu)", "and", "soluble", "thymidine", "kinase", "1", "(Tk1)", "was", "detected", "in", "hES", "SA02", "cells", "with", "Log2", "ratio", "value", "<", "1.", "Expression", "levels", "of", "those", "genes", "range", "from", "being", "inconsiderably", "higher", "to", "nearly", "equal", "to", "that", "in", "universal", "reference", "RNA", "sample.", "We", "believe", "that", "those", "findings", "could", "be", "explained", "by", "cellular", "composition", "of", "human", "universal", "reference", "RNA", "sample", "[42],", "which", "includes", "pooled", "RNA", "samples", "from", "proliferating", "cells", "(e.g.,", "skin", "and", "testis", "cell", "lines).", "Thus,", "the", "relative", "difference", "between", "gene", "expression", "of", "certain", "markers", "of", "stem", "cells", "in", "undifferentiated", "hESCs", "and", "universal", "reference", "RNA", "is", "naturally", "decreased.", "Taken", "together,", "the", "gene", "expression", "signature", "of", "hES", "SA02", "cell", "line", "profiled", "by", "NeuroStem", "Chip", "is", "indeed", "characteristic", "for", "pluripotent", "stem", "cells,", "providing", "proof-of-concept.", "Notably,", "comparison", "of", "expression", "profiles", "of", "undifferentiated", "hESCs", "and", "hESC-derived", "cells", "committed", "toward", "dopaminergic", "differentiation", "pathway", "by", "co-culturing", "with", "SDIA", "for", "16", "days", "have", "revealed", "that", "many", "of", "the", "stem", "cell", "marker", "genes", "mentioned", "above", "were", "down-regulated", "in", "differentiation", "(Table", "5).", "Expectedly,", "Hesx1,", "Grem1,", "Dnmt3b,", "Utf1", "and", "Nanog", "could", "be", "listed", "among", "these.", "At", "the", "same", "time,", "numerous", "other", "genes,", "including", "Pitx2,", "Dlk1", "and", "Msx1", "were", "up-regulated", "in", "the", "latter", "sample", "([see", "Additional", "file", "2],", "Figure", "3).", "Table", "1", "lists", "24", "dopaminergic", "system-related", "entries", "(e.g.,", "Ptx3,", "Th,", "Lhx1)", "with", "gene", "expression", "up-regulated", "by", "Day", "16", "of", "hESC", "differentiation", "protocol;", "few", "more", "genes", "have", "demonstrated", "less", "prominent", "up-regulation", "(Log2", "ratio", "values", "in", "the", "range", "of", "0.7/0.97–1.0).", "The", "gene", "expression", "profiles", "generated", "are", "therefore", "consistent", "with", "the", "results", "of", "earlier", "studies", "utilizing", "hSC-derived", "samples", "with", "similar", "characteristics", "[43,44].", "Diversity", "of", "NeuroStem", "Chip", "entries", "responsive", "to", "hESC", "commitment", "toward", "neuronal/dopaminergic", "differentiation", "pathway", "clearly", "illustrates", "the", "complexity", "of", "that", "pathway.", "The", "cell", "population", "obtained", "after", "16", "day", "exposure", "to", "SDIA", "is", "highly", "heterogeneous.", "Only", "around", "0.2%", "of", "the", "cells", "are", "TH-positive", "cells", "(Figure", "2).", "This", "heterogeneity,", "with", "an", "apparent", "presence", "of", "residual", "pluripotent", "cells", "explains", "the", "presence", "of", "stem", "cell", "marker", "genes,", "including", "homeobox", "transcription", "factor", "Nanog,", "as", "revealed", "by", "RT-PCR", "data", "(Figure", "3).", "It", "would", "be", "therefore", "impossible", "to", "apply", "the", "platform", "to", "identify", "novel", "genes", "associated", "with", "the", "process", "of", "differentiation;", "for", "that", "application,", "the", "genome-scale", "microarray", "platforms", "(e.g.,", "Affymetrix)", "are", "clearly", "superior.", "Nevertheless,", "being", "based", "upon", "a", "moderate", "assay", "of", "pre-selected", "specific", "gene", "targets,", "the", "comparative", "analysis", "of", "microarray", "data", "derived", "from", "undifferentiated", "and", "dopaminergic", "differentiate", "pathway-committed", "hESCs", "provides", "a", "valuable", "cross-cut", "of", "complex", "relationship", "between", "factors", "driving", "or", "indicative", "to", "neuronal/dopaminergic", "differentiation", "[see", "Additional", "file", "2].", "RT-PCR", "analyses", "have", "validated", "the", "overall", "reliability", "of", "NeuroStem", "microarray", "platform:", "all", "of", "the", "entries", "detected", "in", "the", "hybridization", "experiments", "have", "demonstrated", "similar", "trends", "when", "analyzed", "by", "RT-PCR", "means", "(Figure", "3).", "Those", "entries", "include", "Sox2,", "En1", "and", "Nanog", "(ratio", "of", "differentiated/undifferentiated", "hESC", "sample", "normalized", "spot", "intensity", "<", "0.75,", "down-regulated),", "Gadph,", "Aldh1a1,", "Sdha,", "Tubb", "and", "Nestin", "(ratio", ".1.0,", "unchanged),", "Actb,", "Th,", "Msx1", "and", "Pitx2", "(ratio", ">1.25,", "up-regulated).", "Some", "of", "the", "housekeeping", "genes", "(Gapdh,", "Sdha,", "Tubb,", "Actb)", "have", "somewhat", "different", "expression", "in", "undifferentiated", "vs.", "differentiated", "cells,", "consistent", "with", "previous", "reports", "on", "certain", "established", "housekeeping", "genes", "(including", "Gapdh)", "being", "variable", "in", "human", "samples", "[45].", "Importantly,", "all", "the", "observed", "gene", "expression", "trends", "were", "similar", "in", "both", "microarray", "and", "RT-PCR.", "Our", "experiment", "therefore", "confirms", "that", "the", "NeuroStem", "Chip", "microarray", "platform", "can", "still", "identify", "gene", "expression", "changes", "related", "to", "early", "stages", "of", "differentiation", "of", "hESC", "into", "dopaminergic", "neurons.", "Recent", "technological", "advances", "have", "led", "to", "DNA", "microarrays", "which", "contain", "over", "hundred", "thousand", "of", "spots", "of", "DNA", "material,", "reaching", "a", "truly", "genomic", "scale.", "Highly", "specialized", "DNA", "microarrays", "of", "smaller", "scale", "(e.g.", "the", "NeuroStem", "Chip)", "still", "have", "an", "important", "role", "in", "the", "directed", "studies", "in", "particular", "fields.", "Since", "they", "are", "significantly", "less", "expensive,", "compared", "to", "many", "recognized", "large-scale", "platforms", "(e.g.", "Affymetrix", "Human", "Genome", "platforms),", "they", "have", "a", "clear", "advantage", "in", "routine", "work", "involving", "samples", "from,", "e.g.,", "multiple", "cell", "culture", "conditions.", "While", "there", "is", "a", "risk", "that", "one", "will", "miss", "out", "on", "changes", "in", "genes", "previously", "not", "believed", "to", "be", "relevant", "to", "neural", "differentiation,", "the", "restricted", "number", "of", "genes", "in", "the", "NeuroStem", "Chip", "also", "simplifies", "analysis", "and", "adds", "power.", "NeuroStem", "Chip", "is", "comparable", "to", "other", "stem", "cell-related", "focused", "microarray", "platforms", "in", "regards", "to", "manufacturing", "costs", "and", "technical", "simplicity", "of", "the", "recommended", "hybridization", "protocols.", "At", "the", "same", "time,", "it", "currently", "implies", "an", "advantage", "in", "both", "the", "scale", "and", "the", "spectrum", "of", "pre-selected,", "specific", "gene", "targets", "assayed.", "Some", "suggested", "areas", "of", "application", "for", "NeuroStem", "microarray", "platform", "could", "be", "the", "following:", "(i)", "characterization", "of", "the", "expression", "of", "established,", "pre-selected", "gene", "targets", "in", "human", "stem", "cell", "(hSC)", "lines,", "including", "newly", "derived", "ones,", "(ii)", "longitudinal", "quality", "control", "for", "maintained", "hSC", "populations,", "(iii)", "following", "gene", "expression", "changes", "during", "differentiation", "under", "defined", "cell", "culture", "conditions,", "and", "(iv)", "confirming", "the", "success", "of", "differentiation", "into", "specific", "neuronal", "subtypes.", "In", "addition,", "the", "NeuroStem", "Chip", "can", "be", "used", "to", "characterize", "gene", "changes", "in", "intracerebral", "grafts", "of", "human", "cells,", "even", "when", "they", "are", "transplanted", "into", "experimental", "animals.", "We", "specifically", "wish", "to", "stress", "that", "we", "are", "about", "to", "make", "the", "NeuroStem", "Chip", "available", "at", "a", "non-profit", "cost", "to", "the", "research", "community.", "We", "believe", "it", "has", "the", "potential", "to", "become", "an", "important", "screening", "tool", "in", "the", "expanding", "field", "of", "hSC", "studies", "in", "application", "to", "neurological/neurodegenerative", "disorders.", "Human", "embryonic", "stem", "cell", "(hESC)", "culturesUndifferentiated", "hESCs", "of", "SA02", "(Sahlgrenska", "2)", "line", "(Cellartis", "AB,", "Göteborg,", "Sweden;", "see", "NIH", "Human", "Embryonic", "Stem", "Cell", "Registry", "at", "[46])", "were", "maintained", "over", "a", "monolayer", "of", "human", "\"feeder", "cells\"", "(hFCs;", "human", "foreskin", "fibroblasts,", "ATCC;", "cell", "line", "CCD-1112Sk).", "Feeder", "cells", "were", "grown", "in", "hFC", "medium", "(Iscove's", "modified", "Dulbecco's", "medium", "(IMDM)", "supplemented", "with", "10%", "heat-inactivated", "FCS", "(Stem", "Cell", "Technologies,", "USA)", "and", "0.5%", "Penicillin/Streptomycin", "mix)", "for", "11", "passages.", "One", "day", "prior", "to", "hESC", "plating,", "hFC", "medium", "was", "washed", "away", "from", "the", "hFCs,", "the", "latter", "were", "resuspended", "in", "a", "hESC", "proliferation", "medium", "(VitroHES", "media", "(Vitrolife", "AB,", "Sweden)", "supplemented", "with", "4", "ng/ml", "human", "recombinant", "basic", "FGF", "(hrbFGF,", "Biosource", "International,", "USA)", "and", "plated", "in", "a", "central", "ring", "of", "gelatinized", "in", "vitro", "fertilization", "(IVF)", "dishes", "with", "a", "cell", "density", "of", "120,000", "cells/dish.", "The", "outer", "rings", "of", "the", "IVF", "dishes", "were", "filled", "with", "Dulbecco's", "modified", "Eagle", "medium", "(DMEM)", "supplemented", "with", "0.5%", "Penicillin/Streptomycin", "mix.", "One", "half", "of", "the", "culture", "medium", "was", "replaced", "every", "other", "day.", "The", "cells", "were", "maintained", "at", "37°C,", "5%", "CO2,", "95%", "humidity", "settings.", "Every", "6", "days,", "fragments", "of", "the", "hESC", "colonies", "(around", "10–14", "colonies", "per", "dish,", "measuring", "around", "0.015", "×", "0.015", "mm)", "that", "had", "an", "unaltered", "morphology", "(indicating", "lack", "of", "spontaneous", "differentiation)", "were", "mechanically", "cut", "from", "dishes", "using", "stem", "cell", "knives/transfer", "pipettes", "(SweMed", "Lab", "International", "AB,", "Sweden)", "and", "then", "plated", "on", "fresh", "hFCs.", "Commitment", "of", "hESCs", "towards", "neuronal/dopaminergic", "differentiation", "pathwayCo-culturing", "with", "the", "PA6", "stromal", "cell", "line", "(MC3T3-G2/Pa6,", "from", "RIKEN", "Cell", "Bank", "Japan", "(RCB", "1127),", "derived", "from", "newborn", "mouse", "calvaria", "rapidly", "generates", "high", "numbers", "of", "DA", "neurons", "from", "mouse", "and", "monkey", "ESCs", "by", "an", "unknown", "mechanism", "named", "stromal-derived", "inducing", "activity", "(SDIA;", "[28,29]).", "For", "differentiation", "experiments,", "PA6", "cells", "were", "plated", "on", "gelatine-coated", "T25", "flasks", "at", "16", "×", "103", "cells/cm2", "(400,000", "cells/flask)", "density", "2", "days", "prior", "to", "introducing", "hESCs", "into", "the", "co-culture", "and", "cultured", "at", "PA6", "culturing", "media", "(containing", "minimum", "essential", "medium", "alpha", "(α-MEM)", "supplemented", "with", "10%", "FCS", "and", "0.5%", "Penicillin/Streptomycin).", "Alternatively,", "PA6", "cells", "were", "plated", "over", "Type", "I", "collagen-coated", "glass", "cover-slips", "placed", "in", "wells", "of", "4-well-plates", "(50,000", "cells/well,", "for", "immunocytochemical", "(ICC)", "analysis).", "Three", "hours", "prior", "to", "initiation", "of", "co-culture,", "PA6", "cells", "were", "rinsed", "3", "times", "with", "PBS", "and", "media", "was", "replaced", "with", "co-culture", "media", "(Glasgow's", "modified", "Eagle's", "media", "(G-MEM)", "supplemented", "with", "8%", "knock-out", "serum", "replacement", "(KSR),", "2", "mM", "glutamine,", "0.1", "mM", "non-essential", "amino-acids", "(NEAA),", "1", "mM", "pyruvate,", "0.1", "mM", "β-mercaptoethanol", "(βME)", "and", "4", "ng/μl", "bFGF).", "Fragments", "of", "hESC", "colonies", "(80–90", "per", "flask;", "4–5", "per", "well", "of", "4-well-plate)", "presenting", "undifferentiated", "morphology", "were", "manually", "passaged", "onto", "the", "confluent", "PA6", "monolayer", "and", "cell", "co-cultures", "were", "maintained", "at", "37°C,", "5%", "CO2,", "95%", "humidity", "settings.", "One", "half", "of", "the", "co-culture", "medium", "was", "replaced", "every", "other", "day", "for", "first", "10", "days,", "and", "daily", "onwards.", "Characterization", "of", "hESCs", "and", "hESC-derived", "cells", "by", "immunocytochemistry", "(ICC)", "and", "RT-PCRIVF", "dishes", "with", "hESCs", "grown", "atop", "hFCs", "and", "4-well", "plate", "dishes", "with", "hESCs", "growing", "atop", "PA6", "cells", "were", "fixed", "with", "4%", "paraformaldehyde", "(PFA)", "for", "15", "minutes", "at", "the", "day", "of", "passage/harvest", "(Day", "6", "of", "hESC/hFC", "co-culturing)", "and", "Day", "16", "of", "co-culturing", "with", "PA6", "cells,", "respectively.", "Cells", "were", "pre-incubated", "with", "blocking", "solution", "containing", "PBS,", "0.5%", "Triton", "X-100", "and", "5%", "of", "donkey", "serum.", "They", "were", "then", "incubated", "with", "primary", "antibodies", "in", "blocking", "solution", "overnight", "at", "room", "temperature.", "After", "three", "washes", "with", "PBS,", "cells", "were", "incubated", "with", "the", "donkey", "anti-rabbit", "IgG", "conjugated", "with", "FITC", "or", "anti-mouse", "Cy3", "(1:200,", "Jackson", "ImmunoResearch", "Laboratories).", "Cells", "were", "then", "washed", "once", "with", "PBS,", "incubated", "with", "1:1000", "DAPI", "in", "PBS", "for", "10", "minutes,", "followed", "by", "another", "wash", "with", "PBS.", "Coverslips", "were", "mounted", "onto", "glass", "slides", "with", "PVA", "mounting", "medium", "containing", "anti-fading", "reagent", "DABCO.", "The", "following", "primary", "antibodies", "were", "used:", "mouse", "anti-Oct3/4", "(1:500,", "Santa", "Cruz", "Biotechnology", "Inc.);", "rabbit", "anti-Ki67", "(1:200,", "Novocastra", "Ltd.);", "rabbit", "anti-TH", "(1:500,", "Chemicon).", "Immunostained", "cell", "cultures", "were", "visualized", "using", "a", "Zeiss", "fluorescent", "microscope", "attached", "to", "a", "Nikon", "digital", "camera.Using", "RT-PCR,", "all", "RNA", "samples", "used", "in", "this", "study", "were", "tested", "negative", "for", "the", "presence", "of", "gDNA", "(data", "not", "shown).", "The", "intron-spanning", "primers", "for", "RT-PCR", "were", "selected", "from", "published", "works", "or", "designed", "using", "Oligo", "4.0", "software", "(Molecular", "Biology", "Insight)", "or", "Clone", "Manager", "Suite", "7.1", "(Sci", "Ed", "Software,", "NC,", "USA)", "and", "ordered", "from", "TAG", "Copenhagen", "A/S,", "Denmark,", "as", "the", "following:", "Sox2,", "SRY-box", "2:", "5'-TAC", "CTC", "TTC", "CTC", "CC", "CTC", "CA-3',", "5'-ACT", "CTC", "CTC", "TTT", "TGC", "ACC", "CC-3';", "En1,", "Engrailed", "1:", "5'-AAG", "GGA", "CGA", "AAC", "TGC", "GAA", "CTC", "C-3',", "5'-GAC", "ACG", "AAA", "GGA", "AAC", "ACA", "CAC", "TCT", "CG-3'", "[47];", "Nanog:", "5'-TGC", "TTA", "TTC", "AGG", "ACA", "GCC", "T-3',", "5'-TCT", "GGT", "CTT", "CTG", "TTT", "CTT", "GAC", "T-3'", "[48];", "Gapdh,", "glyceraldehydes-3-phosphate", "dehydrogenase:", "5'-GGA", "AGG", "TGA", "AGG", "TCG", "GAG", "TCA", "A-3',", "5'-GAT", "CTC", "GCT", "CCT", "GGA", "AGA", "TGG", "T-3';", "Aldh1A1,", "aldehyde", "dehydrogenase", "1", "family,", "member", "A1:", "5'-GGG", "CAG", "CCA", "TTT", "CTT", "CTC", "AC-3',", "5'-CTT", "CTT", "AGC", "CCG", "CTC", "AAC", "AC-3'", "[49];", "Sdha,", "succinate", "dehydrogenase:", "5'-TGG", "GAA", "CAA", "GAG", "GGC", "ATC", "TG-3',", "5'-CCA", "CCA", "CTG", "CAT", "CAA", "ATT", "CAT", "G-3'", "[50];", "Tubb,", "β-tubulin:", "5'-CTC", "ACA", "AGT", "ACG", "TGC", "CTC", "GAG-3',", "5'-GCA", "CGA", "CGC", "TGA", "AGG", "TGT", "TCA-3';", "Nestin:", "5'-AGA", "GGG", "GAA", "TTC", "CTG", "CT", "GAG-3',", "5'-CTG", "AGG", "ACC", "AGG", "ACT", "CTC", "TA-3'", "[47];", "Actb,", "β-actin:", "5'-CAT", "CGA", "GCA", "CGG", "CAT", "CGT", "CA-3',", "5'-TAG", "CAC", "AGC", "CTG", "GAT", "AGC", "AAC-3'", "[51];", "Th,", "Tyrosine", "hydroxylase:", "5'-CGA", "GCT", "GTG", "AAG", "GTG", "TTT", "G-3',", "5'-TTG", "GTG", "ACC", "AGG", "TGA", "TGA", "C-3';", "Msx1,", "homolog", "of", "Drosophila", "muscle", "segment", "homolog", "1:", "5'-CTC", "AAG", "CTG", "CCA", "GAA", "GAT", "GC-3',", "5'-TCC", "AGC", "TCT", "GCC", "TCT", "TGT", "AG-3';", "Pitx2,", "paired-like", "homeodomain", "transcription", "factor", "2:", "5'-ACC", "TTA", "CGG", "AAG", "CCC", "GAG", "TC-3',", "5'-TGG", "ATA", "GGG", "AGG", "CGG", "ATG", "TA-3'", "[49].", "cDNA", "was", "synthesized", "from", "1", "mg", "of", "total", "RNA", "using", "SuperScript", "II", "(Invitrogen),", "and", "RT-PCR", "amplifications", "were", "performed", "using", "the", "MiniOpticon", "system", "(Bio-Rad)", "with", "REDTaq", "Polymerase", "(Sigma-Aldrich)", "essentially", "as", "described", "by", "the", "manufacturer.", "Following", "initial", "denaturation", "for", "5", "min", "at", "95°C,", "DNA", "amplifications", "were", "performed", "for", "35", "(En1,", "Nanog,", "Aldh1a1),", "33", "(Sox2,", "Nestin,", "Th,", "Msx1),", "32", "(Tubb,", "Pitx2),", "27", "(Sdha),", "25", "(Actb)", "or", "22", "(Gapdh)", "cycles", "of", "1", "min", "at", "95°C,", "1", "min", "at", "55°C", "(En1,", "Pitx2),", "57°C", "(Sox2,", "Nanog,", "Sdha,", "Nestin),", "58°C", "(Tubb),", "58.5°C", "(Aldh1a1,", "Th)", "or", "59°C", "(Gapdh,", "Actb,", "Msx1),", "and", "1", "min", "at", "72°C.", "The", "final", "extension", "was", "5", "min", "at", "72°C.", "Twenty", "μl", "volumes", "of", "RT-PCR", "products", "were", "analyzed", "by", "electrophoresis", "at", "1%", "agarose", "gels", "and", "visualized", "by", "ethidium", "bromide", "staining.RNA", "purification", "and", "fluorescent", "dye", "incorporationFor", "RNA", "purification", "of", "undifferentiated", "hESCs,", "the", "latter", "were", "mechanically", "separated", "from", "hFCs,", "collected", "in", "a", "500", "μl", "volume", "of", "VitroHES", "media,", "rinsed", "in", "PBS", "buffer", "and", "spun", "down", "at", "300", "rcf", "for", "5", "min.", "hESC-derived", "cells", "grown", "atop", "PA6", "cells", "were", "harvested", "using", "a", "papain", "dissociation", "kit", "(Worthington", "Biochemical", "Corporation),", "rinsed", "in", "PBS", "buffer", "and", "spun", "down", "as", "described", "above.", "The", "resulting", "cell", "pellets", "were", "resuspended", "in", "RLT", "buffer", "(Qiagen,", "USA),", "passed", "through", "the", "shredder", "column", "(Qiagen)", "and", "stored", "at", "-80°C", "until", "the", "RNA", "sample", "was", "purified", "following", "the", "RNeasy", "Micro", "Kit", "(Qiagen)", "protocol", "(without", "carrier", "RNA);", "with", "DNase", "I", "(Quiagen)", "treatment", "incorporated", "to", "the", "latter.", "RNA", "integrity", "was", "tested", "using", "both", "ND-1000", "specrophotometer", "(NanoDrop,", "USA)", "and", "RNA", "Nano", "LabChip/2100", "Bioanalyzer", "system", "(Agilent", "Technologies,", "USA).Fluorescent", "label", "(24", "nmol", "of", "the", "Cyanine", "3-CTP", "(Cy3);", "PerkinElmer,", "USA)", "was", "incorporated", "to", "350–500", "ng", "of", "total", "RNA", "amplified", "using", "Low", "RNA", "Input", "Fluorescent", "Linear", "Amplification", "Kit", "(Agilent", "Technologies),", "generally", "following", "the", "kit", "manufacturer's", "protocol.", "Similarly,", "24", "nmols", "of", "the", "Cyanine", "5-CTP", "(Cy5;", "PerkinElmer)", "fluorescent", "label", "were", "incorporated", "to", "400", "ng", "sample", "of", "Human", "Universal", "Reference", "RNA", "(Stratagene,", "USA);", "in", "addition,", "dye-swap", "replicate", "amplification", "were", "performed.", "Amplified", "fluorescent", "cRNA", "samples", "were", "purified", "using", "RNeasy", "mini-columns", "(Quiagen),", "and", "fluorescence", "of", "the", "eluted", "products", "was", "measured", "using", "ND-1000", "specrophotometer", "(NanoDrop).", "Microarray", "technologyLong", "oligonucleotide", "probes", "(69–71", "nucleotides)", "matching", "gene", "targets", "of", "interest", "were", "selected", "from", "Operon", "V2", "and", "V3", "human", "AROS", "sets", "(Operon", "Biotechnologies", "Inc.,", "USA).", "Arrays", "were", "produced", "by", "the", "SweGene", "DNA", "Microarray", "Resource", "Centre,", "Department", "of", "Oncology", "at", "Lund", "University", "(Sweden)", "using", "a", "MicroGrid", "II", "600R", "arrayer", "fitted", "with", "MicroSpot", "10", "K", "pins", "(Harvard", "BioRobotics,", "USA).", "Printing", "was", "performed", "in", "a", "temperature-", "(18–20°C)", "and", "humidity-", "(44–49%", "RH)", "controlled", "area", "on", "Corning", "UltraGAPS", "aminosilane", "slides", "(Corning", "Inc.,", "USA)", "with", "140", "μm", "spot-to-spot", "centerdistance", "and", "90–110", "μm", "average", "spot", "size.", "Following", "printing,", "arrays", "were", "dried", "for", "48", "hours", "andstored", "in", "a", "dessicator", "until", "used.", "Microarray", "slides", "were", "UV-cross-linked", "(800", "mJ/cm2),", "pre-hybridizedwith", "fluorescently", "labeled", "samples", "using", "the", "Pronto!", "Universal", "Microarray", "Hybridization", "Kit", "(Corning)", "and", "subsequently", "hybridized", "with", "test", "(Cy3-labeled)/reference", "(Cy5-labeled)", "RNA", "samples", "(or", "in", "reverse", "dye-labeling", "order)", "at", "42°C", "for", "17", "h", "using", "a", "MAUI", "hybridization", "station", "(BioMicro", "Systems", "Inc.,", "USA)", "and", "the", "Pronto!", "Universal", "Microarray", "Hybridization", "Kit,", "generally", "following", "manufacturer's", "instructions,", "with", "several", "minor", "adaptations", "[31].", "Data", "acquisition", "and", "statistical", "analysisImmediately", "following", "the", "washing", "steps,", "the", "fluorescence", "intensities", "were", "measured", "using", "a", "confocal", "laser", "scanner", "(G2505B,", "Agilent", "Technologies).", "After", "image", "formatting", "by", "Tiff", "Image", "Channel", "Splitter", "Utility", "(Agilent", "Technologies)", "and", "grid", "annotation,", "a", "complete", "set", "of", "spots", "was", "visually", "inspected", "for", "each", "slide.", "Using", "GenePix", "Pro", "(Molecular", "Devices", "Corp.", "USA)", "flags", "for", "artifactual", "spots", "were", "annotated", "for", "each", "spot.", "Median", "pixel", "intensity", "minus", "the", "median", "local", "background", "for", "both", "dyes", "was", "used", "to", "obtain", "a", "test", "over", "reference", "intensity", "ratio.", "Data", "normalization", "was", "performed", "per", "array", "subgrid", "using", "LOWESS", "curve", "fitting", "with", "a", "smoothing", "factor", "of", "0.33", "[52,53].", "All", "normalizations,", "filtering,", "merging", "of", "technical", "replicates", "and", "analyses", "were", "performed", "in", "the", "BioArray", "Software", "Environment", "database", "[32].", "To", "visualize", "sample-dependent", "variation", "of", "spot", "intensities,", "data", "was", "uploaded", "to", "the", "TIGR", "MultiExperiment", "Viewer", "(MEV;", "[34])." ]
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[ "BackgroundHuman", "embryonic", "stem", "cells", "provide", "access", "to", "the", "earliest", "stages", "of", "human", "development", "and", "may", "serve", "as", "a", "source", "of", "specialized", "cells", "for", "regenerative", "medicine.", "Thus,", "it", "becomes", "crucial", "to", "develop", "protocols", "for", "the", "directed", "differentiation", "of", "embryonic", "stem", "cells", "into", "tissue-restricted", "precursors.Methods", "and", "FindingsHere,", "we", "present", "culture", "conditions", "for", "the", "derivation", "of", "unlimited", "numbers", "of", "pure", "mesenchymal", "precursors", "from", "human", "embryonic", "stem", "cells", "and", "demonstrate", "multilineage", "differentiation", "into", "fat,", "cartilage,", "bone,", "and", "skeletal", "muscle", "cells.ConclusionOur", "findings", "will", "help", "to", "elucidate", "the", "mechanism", "of", "mesoderm", "specification", "during", "embryonic", "stem", "cell", "differentiation", "and", "provide", "a", "platform", "to", "efficiently", "generate", "specialized", "human", "mesenchymal", "cell", "types", "for", "future", "clinical", "applications.Lorenz", "Studer", "and", "colleagues", "describe", "the", "use", "of", "embryonic", "stem", "cells", "to", "derive", "mesenchymal", "precursors", "and", "then", "fat,", "cartilage,", "bone,", "and", "skeletal", "muscle", "cells.", "Embryonic", "stem", "(ES)", "cells", "are", "pluripotent", "cells", "derived", "from", "the", "inner", "cell", "mass", "of", "the", "blastocyst", "that", "can", "be", "maintained", "in", "culture", "for", "an", "extended", "period", "of", "time", "without", "losing", "differentiation", "potential.", "The", "successful", "isolation", "of", "human", "ES", "cells", "(hESCs)", "has", "raised", "the", "hope", "that", "these", "cells", "may", "provide", "a", "universal", "tissue", "source", "to", "treat", "many", "human", "diseases.", "However,", "directed", "differentiation", "of", "hESCs", "into", "specific", "tissue", "types", "poses", "a", "formidable", "challenge.", "Protocols", "are", "currently", "available", "for", "only", "a", "few", "cell", "types,", "mostly", "of", "neural", "identity", "[1–3],", "and", "differentiation", "into", "many", "of", "the", "cell", "types", "derived", "from", "the", "paraxial", "mesoderm", "has", "not", "been", "reported,", "with", "the", "exception", "of", "a", "recent", "study", "indicating", "osteoblastic", "differentiation", "[4].", "Mesenchymal", "stem", "cells", "(MSCs)", "have", "been", "isolated", "from", "the", "adult", "bone", "marrow", "[5],", "adipose", "tissue", "[6],", "and", "dermis", "and", "other", "connective", "tissues", "[7].", "Harvesting", "MSCs", "from", "any", "of", "these", "sources", "requires", "invasive", "procedures", "and", "the", "availability", "of", "a", "suitable", "donor.", "The", "number", "of", "MSCs", "that", "can", "be", "obtained", "from", "a", "single", "donor", "is", "limited,", "and", "the", "capacity", "of", "these", "cells", "for", "long-term", "proliferation", "is", "rather", "poor.", "In", "contrast,", "hESCs", "could", "provide", "an", "unlimited", "number", "of", "specialized", "cells.", "In", "this", "study,", "we", "present", "techniques", "for", "the", "generation", "and", "purification", "of", "mesenchymal", "precursors", "from", "hESCs", "and", "their", "directed", "differentiation", "in", "vitro", "into", "various", "mesenchymal", "derivatives,", "including", "skeletal", "myoblasts.", "Our", "isolation", "method", "for", "mesenchymal", "precursors", "is", "the", "first", "example,", "to", "our", "knowledge,", "of", "efficiently", "deriving", "structures", "of", "the", "paraxial", "mesoderm", "from", "ES", "cells,", "and", "further", "highlights", "the", "potential", "of", "hESCs", "for", "basic", "biology", "and", "regenerative", "medicine.", "Cell", "Culture", "and", "FACSUndifferentiated", "hESCs,", "H1", "(WA-01,", "XY,", "passages", "40–65)", "and", "H9", "(WA-09,", "XX,", "passages", "35–45),", "were", "cultured", "on", "mitotically", "inactivated", "mouse", "embryonic", "fibroblasts", "(Specialty", "Media,", "Phillipsburg,", "New", "Jersey,", "United", "States)", "and", "maintained", "under", "growth", "conditions", "and", "passaging", "techniques", "described", "previously", "[3].", "OP9", "cells", "were", "maintained", "in", "alpha", "MEM", "medium", "containing", "20%", "fetal", "bovine", "serum", "(FBS)", "and", "2", "mM", "L-glutamine.", "Mesenchymal", "differentiation", "was", "induced", "by", "plating", "10", "×", "103", "to", "25", "×", "103", "cells/cm2", "on", "a", "monolayer", "of", "OP9", "cells", "in", "the", "presence", "of", "20%", "heat-inactivated", "FBS", "in", "alpha", "MEM", "medium.", "Flow-activated", "cell", "sorting", "(FACS)", "(CD73-PE;", "PharMingen,", "San", "Diego,", "California,", "United", "States)", "was", "performed", "on", "a", "MoFlo", "(Cytomation,", "Fort", "Collins,", "Colorado,", "United", "States).", "All", "human", "ES", "cell–derived", "mesenchymal", "precursor", "cell", "(hESMPC)", "lines", "in", "this", "study", "are", "of", "polyclonal", "origin.", "Primary", "human", "bone", "marrow–derived", "MSCs", "and", "primary", "human", "foreskin", "fibroblasts", "(both", "from", "Poietics,", "Cambrex,", "East", "Rutherford,", "New", "Jersey,", "United", "States)", "were", "grown", "in", "alpha", "MEM", "medium", "containing", "10%", "FBS", "and", "2", "mM", "L-glutamine.", "Adipocytic", "DifferentiationhESMPCs", "are", "grown", "to", "confluence", "followed", "by", "exposure", "to", "1", "mM", "dexamethasone,", "10", "μg/ml", "insulin,", "and", "0.5", "mM", "isobutylxanthine", "(all", "from", "Sigma,", "St.", "Louis,", "Missouri,", "United", "States)", "in", "alpha", "MEM", "medium", "containing", "10%", "FBS", "for", "2–4", "wk.", "Data", "were", "confirmed", "in", "hESMPC-H1.1,", "-H1.2,", "-H1.3,", "and", "-H9.1", "(hESMPC-H1.4", "was", "not", "tested).", "Chondrocytic", "DifferentiationDifferentiation", "of", "hESMPCs", "was", "induced", "in", "pellet", "culture", "[5]", "by", "exposure", "to", "10", "ng/ml", "TGF-β3", "(R", "&", "D", "Systems,", "Minneapolis,", "Minnesota,", "United", "States)", "and", "200", "μM", "ascorbic", "acid", "(Sigma)", "in", "alpha", "MEM", "medium", "containing", "10%", "FBS", "for", "3–4", "wk.", "Data", "were", "confirmed", "in", "hESMPC-H1.1,", "-H1.3,", "and", "-H9.1", "(hESMPC-H1.2", "and", "-H1.4", "were", "not", "tested).", "Osteogenic", "DifferentiationhESMPCs", "were", "plated", "at", "low", "density", "(1", "×", "103", "to", "2.5", "×", "103", "cells/cm2)", "on", "tissue-culture-treated", "dishes", "in", "the", "presence", "of", "10", "mM", "β-glycerol", "phosphate", "(Sigma),", "0.1", "μM", "dexamethasone,", "and", "200", "μM", "ascorbic", "acid", "in", "alpha", "MEM", "medium", "containing", "10%", "FBS", "for", "3–4", "wk.", "Data", "were", "confirmed", "in", "hESMPC-H1.1,", "-H1.3,", "and", "-H9.1", "(hESMPC-H1.2", "and", "-H1.4", "were", "not", "tested).", "Myogenic", "DifferentiationConfluent", "hESMPCs", "were", "maintained", "for", "2–3", "wk", "in", "alpha", "MEM", "medium", "with", "20%", "heat-inactivated", "FBS.", "More", "rapid", "induction", "was", "observed", "in", "the", "presence", "of", "medium", "conditioned", "for", "24", "h", "by", "differentiated", "C2C12", "cells.", "Coculture", "of", "hESMPCs", "and", "C2C12", "cells", "was", "carried", "out", "in", "alpha", "MEM", "with", "3%", "horse", "serum", "and", "1%", "FBS", "[8].", "Data", "were", "confirmed", "in", "hESMPC-H1.3,", "-H1.4,", "and", "-H9.1", "(hESMPC-H1.1", "and", "-H1.2", "were", "not", "tested).", "CytochemistryImmunocytochemistry", "for", "all", "surface", "markers", "was", "performed", "on", "live", "cells.", "Monoclonal", "antibodies", "VCAM,", "STRO-1,", "ICAM-1(CD54),", "CD105,", "CD29,", "and", "MF20", "were", "from", "Developmental", "Studies", "Hybridoma", "Bank", "(University", "of", "Iowa,", "Iowa", "City,", "Iowa,", "United", "States);", "CD73,", "CD44,", "and", "ALCAM(CD166)", "were", "from", "BD", "Biosciences", "Pharmingen", "(San", "Diego,", "California,", "United", "States).", "All", "other", "immunocytochemical", "analyses", "were", "performed", "after", "fixation", "in", "4%", "paraformaldehyde", "and", "0.15%", "picric", "acid,", "followed", "by", "permeabilization", "in", "0.3%", "Triton", "X100.", "Polyclonal", "antibodies", "used", "were", "MyoD", "(Santa", "Cruz", "Biotechnology,", "Santa", "Cruz,", "California,", "United", "States)", "and", "nestin", "(gift", "from", "R.", "McKay);", "monoclonal", "antibodies", "were", "vimentin,", "alpha", "smooth", "muscle", "actin,", "fast-switch", "myosin,", "pan-cytokeratin", "(all", "from", "Sigma),", "and", "human", "nuclear", "antigen", "(Chemicon,", "Temecula,", "California,", "United", "States).Alkaline", "phosphatase", "reaction", "was", "performed", "using", "a", "commercially", "available", "kit", "(Kit-86;", "Sigma)", "and", "the", "mineral", "was", "stained", "with", "silver", "nitrate", "according", "to", "the", "von", "Kossa", "method.", "Fat", "granules", "were", "visualized", "by", "Oil", "Red", "O", "staining", "solution", "(Sigma).", "Alcian", "Blue", "(Sigma)", "was", "used", "to", "detect", "extracellular", "matrix", "proteoglycans", "in", "chondrogenic", "cultures.", "Gene-Expression", "AnalysesRT-PCR", "analysisTotal", "RNA", "was", "extracted", "by", "using", "the", "RNeasy", "kit", "and", "DNase", "I", "treatment", "(Qiagen,", "Valencia,", "California,", "United", "States).", "Total", "RNA", "(2", "μg", "each)", "was", "reverse", "transcribed", "(SuperScript;", "Invitrogen,", "Carlsbad,", "California,", "United", "States).", "PCR", "conditions", "were", "optimized", "and", "linear", "amplification", "range", "was", "determined", "for", "each", "primer", "by", "varying", "annealing", "temperature", "and", "cycle", "number.", "PCR", "products", "were", "identified", "by", "size,", "and", "identity", "was", "confirmed", "by", "DNA", "sequencing.", "Primer", "sequences,", "cycle", "numbers,", "and", "annealing", "temperatures", "are", "provided", "in", "Table", "S1.Affymetrix", "analysisTotal", "RNA", "(5", "μg)", "from", "primary", "MSCs,", "from", "hESMPC-H9.1,", "hESMPC-H1.2,", "and", "three", "samples", "of", "undifferentiated", "hESCs", "(H1;", "passages", "42–46),", "were", "processed", "by", "the", "Memorial", "Sloan-Kettering", "Cancer", "Center", "Genomics", "Core", "Facility", "and", "hybridized", "on", "Affymetrix", "(Santa", "Clara,", "California,", "United", "States)", "U133A", "human", "oligonucleotide", "arrays.", "Data", "were", "analyzed", "using", "MAS5.0", "(Affymetrix)", "software.", "Transcripts", "selectively", "expressed", "in", "each", "of", "the", "mesenchymal", "cell", "populations", "(MSC,", "hESMPC-H9.1,", "and", "hESMPC-H1.2)", "were", "defined", "as", "those", "called", "“increased”", "by", "the", "MAS5.0", "algorithm", "in", "each", "of", "three", "comparisons", "with", "independent", "samples", "of", "undifferentiated", "hESCs.", "A", "Venn", "diagram", "was", "generated", "to", "visualize", "overlap", "in", "gene", "expression.", "Further", "statistical", "analyses", "were", "performed", "as", "described", "below.", "Mesenchymal", "differentiation", "of", "hESCs", "(lines", "H1", "[WA-01]", "and", "H9", "[WA-09])", "[9]", "was", "induced", "by", "plating", "undifferentiated", "hESCs", "on", "a", "monolayer", "of", "murine", "OP9", "stromal", "cells", "[10],", "in", "the", "presence", "of", "20%", "heat-inactivated", "FBS", "in", "alpha", "MEM", "medium.", "OP9", "cells", "have", "been", "previously", "shown", "to", "induce", "blood", "cell", "differentiation", "from", "mouse", "ES", "cells", "[11].", "After", "40", "d", "of", "coculture,", "cells", "were", "harvested", "and", "sorted", "by", "FACS", "for", "CD73,", "a", "surface", "marker", "expressed", "in", "adult", "MSCs", "[5]", "(Figure", "1A).", "An", "average", "of", "5%", "CD73+", "cells", "was", "obtained", "from", "the", "mixed", "culture", "of", "OP9", "and", "differentiated", "hESC", "progeny.", "CD73+", "cells", "were", "replated", "in", "the", "absence", "of", "stromal", "feeders", "on", "tissue", "culture", "plates", "and", "expanded", "in", "alpha", "MEM", "medium", "with", "20%", "FBS", "for", "7–14", "d.", "We", "next", "established", "the", "membrane", "antigen", "profile", "of", "the", "resulting", "population", "of", "flat", "spindle-like", "cells.", "The", "H1-", "and", "H9-derived", "CD73+", "cells", "expressed", "a", "comprehensive", "set", "of", "markers", "that", "are", "considered", "to", "define", "adult", "MSCs,", "including", "CD105(SH2),", "STRO-1,", "VCAM", "(CD106),", "CD29(integrin", "β1),", "CD44,", "ICAM", "-1(CD54),", "ALCAM(CD166),", "vimentin,", "and", "alpha", "smooth", "muscle", "actin", "(Figure", "1B", "and", "1C).", "The", "cells", "were", "negative", "for", "hematopoietic", "markers", "such", "as", "CD34,", "CD45,", "and", "CD14.", "They", "were", "also", "negative", "for", "neuroectodermal,", "epithelial,", "and", "muscle", "cell", "markers", "including", "nestin,", "pancytokeratin,", "and", "desmin", "(data", "not", "shown).", "The", "human", "identity", "of", "these", "presumed", "mesenchymal", "cells", "(termed", "hESMPC-H1.1,", "-H1.2,", "-H1.3,", "-H1.4,", "and", "-H9.1)", "was", "confirmed", "for", "all", "experiments", "by", "immunocytochemistry", "for", "human", "nuclear", "antigen", "to", "rule", "out", "the", "possibility", "of", "contamination", "with", "OP9", "cells", "(Figure", "S1).", "To", "further", "characterize", "hESMPCs,", "we", "performed", "genome-wide", "expression", "analysis", "using", "oligonucleotide", "arrays", "(Affymetrix", "U133A).", "The", "expression", "profiles", "of", "hESMPC-H1.2", "and", "hESMPC-H9.1", "were", "compared", "with", "that", "of", "human", "primary", "adult", "MSCs.", "Housekeeping", "genes", "for", "each", "of", "the", "mesenchymal", "cell", "populations", "were", "eliminated", "by", "subtracting", "those", "transcripts", "also", "expressed", "in", "at", "least", "one", "of", "three", "independent", "samples", "of", "undifferentiated", "hESCs.", "Based", "on", "this", "analysis,", "1,280", "transcripts", "were", "selectively", "expressed", "in", "hESMPC-H1.2,", "932", "transcripts", "in", "hESMPC-H9.1,", "and", "1,218", "transcripts", "in", "primary", "adult", "MSCs.", "A", "remarkable", "overlap", "of", "579", "transcripts", "shared", "among", "the", "three", "mesenchymal", "populations", "was", "observed", "(Figure", "1D).", "Using", "the", "genes", "that", "were", "selected", "in", "the", "initial", "filter,", "we", "performed", "a", "statistical", "analysis", "on", "the", "expression", "levels", "to", "determine", "whether", "the", "genes", "were", "expressed", "significantly", "differently", "in", "the", "two", "cell", "types.", "We", "used", "a", "Bayesian", "extension", "to", "the", "standard", "t-test", "[12]", "to", "assess", "this", "difference.", "Of", "the", "579", "genes,", "412", "of", "them", "were", "significantly", "different,", "at", "a", "false", "discovery", "rate", "cutoff", "of", "0.05.", "The", "relative", "fold", "changes", "were", "also", "extremely", "large", "in", "many", "of", "the", "cases.", "We", "also", "looked", "at", "the", "variance", "of", "the", "expression", "levels", "within", "the", "cell", "types.", "For", "the", "MSCs,", "94%", "had", "a", "coefficient", "of", "variation", "less", "than", "20%", "for", "the", "expression", "(log", "transformed);", "for", "the", "ES-derived", "cells,", "72%", "had", "a", "coefficient", "of", "variation", "less", "than", "20%.", "Numerous", "known", "MSC", "markers", "were", "included", "in", "the", "list", "of", "412", "genes,", "such", "as", "the", "mesenchymal", "stem", "cell", "protein", "DSC54", "(13.9-fold", "increase,", "p", "<", "0.001),", "neuropilin", "1", "(30.4-fold", "increase,", "p", "<", "0.001),", "hepatocyte", "growth", "factor", "(48.1-fold", "increase,", "p", "<", "0.001),", "forkhead", "box", "D1", "(14.8-fold", "increase,", "p", "<", "0.001),", "and", "notch", "homolog", "2", "(2.9-fold", "increase,", "p", "<", "0.001)", ".", "Table", "S2", "lists", "the", "p-values", "from", "the", "test,", "the", "mean", "and", "standard", "deviation", "of", "the", "expression", "levels,", "and", "the", "relative", "fold", "change", "of", "all", "412", "genes", "between", "the", "two", "types.", "Known", "markers", "of", "MSCs,", "such", "as", "mesenchymal", "stem", "cell", "protein", "DSC54,", "were", "all", "included", "within", "the", "579", "shared", "transcripts.", "These", "findings", "support", "the", "immunocytochemical", "data", "and", "suggest", "that", "hESMPCs", "and", "primary", "MSCs", "are", "highly", "related.", "MSCs", "are", "characterized", "functionally", "by", "their", "ability", "to", "differentiate", "into", "mesenchymal", "tissues,", "such", "as", "fat,", "cartilage,", "and", "bone.", "Therefore,", "we", "tested", "whether", "hESMPCs", "have", "the", "same", "potential", "(Figure", "2).", "Adipocytic", "differentiation", "of", "hESMPCs", "was", "induced", "under", "conditions", "described", "previously", "for", "primary", "adult", "MSCs", "[5].", "Appearance", "of", "cells", "harboring", "fat", "granules", "was", "observed", "after", "10–14", "d", "in", "culture.", "After", "3", "wk", "of", "induction,", "more", "than", "70%", "of", "the", "cells", "displayed", "Oil", "Red", "O+", "fat", "granules,", "and", "PPARγ,", "a", "marker", "of", "adipocytic", "differentiation,", "was", "detected", "by", "RT-PCR.", "(Figure", "2A).", "Chondrocytic", "differentiation", "was", "achieved", "using", "the", "pellet", "culture", "system", "[5].", "After", "28", "d", "in", "culture,", "more", "than", "50%", "of", "all", "cells", "exhibited", "robust", "staining", "for", "Alcian", "Blue,", "a", "marker", "specific", "for", "extracellular", "matrix", "proteoglycans.", "Chondrocytic", "differentiation", "was", "confirmed", "by", "the", "gene", "expression", "of", "collagen", "II", "and", "aggrecan,", "two", "components", "of", "extracellular", "matrix", "selectively", "expressed", "by", "chondrocytes,", "using", "RT-PCR", "(Figure", "2B).", "Osteogenic", "differentiation", "was", "induced", "in", "the", "presence", "of", "β-glycerolphosphate", "[5].", "Osteogenesis", "was", "demonstrated", "by", "specific", "staining", "for", "calcium", "deposition", "in", "the", "matrix", "(von", "Kossa,", "Figure", "2C;", "or", "Alizarin", "Red,", "Figure", "S2A)", "and", "increased", "expression", "of", "bone-specific", "alkaline", "phosphatase", "and", "bone", "sialoprotein", "at", "day", "28", "of", "treatment", "(Figures", "2C", "and", "S2B).", "At", "day", "28,", "Alizarin", "Red", "staining", "was", "detected", "in", "approximately", "70%", "of", "all", "cells.", "Throughout", "these", "studies,", "human", "adult", "MSCs", "and", "foreskin", "fibroblasts", "were", "used", "as", "positive", "and", "negative", "controls,", "respectively.", "In", "addition", "to", "adipocytic,", "chondrocytic,", "and", "osteogenic", "differentiation,", "reports", "suggested", "that", "adult", "MSCs", "can", "form", "skeletal", "muscle", "[13].", "Although", "generation", "of", "skeletal", "muscle", "cells", "from", "adult", "MSCs", "remains", "controversial,", "we", "tested", "whether", "hESMPCs", "exhibit", "this", "potential.", "Under", "the", "conditions", "previously", "described", "[13],", "hESMPC-H1.1", "and", "-H9.1", "did", "not", "yield", "significant", "numbers", "of", "MyoD+", "cells", "after", "15–20", "d", "in", "culture.", "However,", "when", "confluent", "cells", "were", "maintained", "in", "culture", "in", "the", "presence", "or", "absence", "of", "5-AzaC", "without", "passage", "for", "more", "than", "21", "d,", "expression", "of", "specific", "skeletal", "muscle", "markers", "such", "as", "MyoD", "and", "fast-switch", "myosin", "was", "observed", "(Figure", "3A).", "More", "rapid", "myogenic", "differentiation", "was", "obtained", "in", "the", "presence", "of", "24-h-conditioned", "medium", "from", "the", "murine", "myoblastic", "cell", "line", "C2C12", "previously", "induced", "to", "form", "myotubes", "[14].", "Direct", "coculture", "of", "hESMPCs", "with", "C2C12", "cells", "led", "to", "the", "formation", "of", "hESMPC-derived", "myotubes,", "as", "visualized", "by", "expression", "of", "human", "nuclear", "antigen", "(Figure", "3B),", "similar", "to", "those", "formed", "by", "host", "C2C12", "cells.", "After", "1", "wk", "of", "coculture,", "myotubes", "composed", "of", "human", "nuclei", "accounted", "for", "more", "than", "10%", "of", "the", "total", "number", "of", "human", "cells", "present,", "and", "each", "human", "myotube", "was", "composed", "of", "up", "to", "ten", "human", "nuclei.", "Human", "cell", "contribution", "to", "myotubes", "in", "coculture", "was", "confirmed", "by", "expression", "of", "human", "muscle-specific", "transcripts", "such", "as", "MyoD,", "myosin", "heavy", "chain", "IIa,", "and", "myogenin", "(data", "not", "shown).", "These", "data", "demonstrate", "that", "hESMPCs", "can", "give", "rise", "to", "mesenchymal", "derivatives", "typically", "obtained", "from", "primary", "adult", "MSCs,", "as", "well", "as", "to", "cells", "expressing", "markers", "of", "skeletal", "muscle.", "One", "concern", "for", "the", "clinical", "application", "of", "hESC-derived", "progeny", "in", "regenerative", "medicine", "is", "the", "risk", "of", "teratoma", "formation", "due", "to", "the", "presence", "of", "residual", "undifferentiated", "ES", "cells", "among", "the", "differentiated", "progeny.", "We", "did", "not", "detect", "markers", "of", "undifferentiated", "hESCs,", "such", "as", "Nanog", "[15]", "or", "Oct-4", "[16],", "in", "any", "of", "the", "hESMPCs", "by", "RT-PCR", "(see", "Figure", "2D)", "and", "immunocytochemistry", "(data", "not", "shown),", "suggesting", "the", "lack", "of", "any", "undifferentiated", "ES", "cells", "in", "hESMPC", "cultures.", "However,", "future", "in", "vivo", "studies", "are", "required", "to", "rule", "out", "the", "potential", "of", "these", "cells", "for", "teratoma", "formation.", "Previous", "studies", "have", "demonstrated", "the", "derivation", "of", "neural", "cells", "[1–3],", "hematopoietic", "[17]", "and", "endothelial", "lineages", "[18],", "and", "cardiomyocytes", "[19]", "from", "hESCs.", "This", "study", "presents", "the", "induction", "of", "paraxial", "mesoderm", "with", "the", "generation", "of", "multipotent", "mesenchymal", "precursors.", "We", "calculate", "that", "under", "these", "conditions", "a", "single", "undifferentiated", "hESC", "yields", "an", "average", "of", "one", "CD73+", "cell", "at", "day", "40", "of", "differentiation,", "suggesting", "a", "balance", "between", "cell", "proliferation", "and", "cell", "selection.", "There", "were", "no", "obvious", "differences", "in", "marker", "and", "gene-expression", "profile", "or", "in", "differentiation", "behavior", "among", "the", "five", "hESMPC", "lines", "generated.", "However,", "some", "of", "the", "lines", "(e.g.,", "hESMPC9.1)", "exhibited", "a", "tendency", "of", "spontaneous", "osteogenic", "differentiation", "after", "long-term", "propagation.", "Directed", "differentiation", "of", "hESCs", "into", "somatic", "stem-cell-like", "precursors", "represents", "a", "substantial", "advancement", "in", "harnessing", "the", "developmental", "potential", "of", "hESCs.", "The", "high", "purity,", "unlimited", "availability,", "and", "multipotentiality", "of", "hESMPCs", "will", "provide", "the", "basis", "for", "future", "therapeutic", "efforts", "using", "these", "cells", "in", "preclinical", "animal", "models", "of", "disease.", "Such", "in", "vivo", "studies", "will", "also", "be", "required", "to", "properly", "assess", "the", "safety", "profile", "of", "these", "cells.", "Furthermore,", "our", "system", "also", "offers", "a", "novel", "platform", "to", "study", "basic", "mechanisms", "of", "mesodermal", "induction", "and", "differentiation", "during", "early", "human", "development." ]
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[ "Mapping", "sites", "within", "the", "genome", "that", "are", "hypersensitive", "to", "digestion", "with", "DNaseI", "is", "an", "important", "method", "for", "identifying", "DNA", "elements", "that", "regulate", "transcription.", "The", "standard", "approach", "to", "locating", "these", "DNaseI-hypersensitive", "sites", "(DHSs)", "has", "been", "to", "use", "Southern", "blotting", "techniques,", "although", "we,", "and", "others,", "have", "recently", "published", "alternative", "methods", "using", "a", "range", "of", "technologies", "including", "high-throughput", "sequencing", "and", "genomic", "array", "tiling", "paths.", "In", "this", "article,", "we", "describe", "a", "novel", "protocol", "to", "use", "real-time", "PCR", "to", "map", "DHS.", "Advantages", "of", "the", "technique", "reported", "here", "include", "the", "small", "cell", "numbers", "required", "for", "each", "analysis,", "rapid,", "relatively", "low-cost", "experiments", "with", "minimal", "need", "for", "specialist", "equipment.", "Presented", "examples", "include", "comparative", "DHS", "mapping", "of", "known", "TAL1/SCL", "regulatory", "elements", "between", "human", "embryonic", "stem", "cells", "and", "K562", "cells.", "Mapping", "the", "location", "of", "DNaseI-hypersensitive", "sites", "(DHSs)", "remains", "central", "to", "developing", "our", "understanding", "of", "transcriptional", "regulation.", "Elements", "with", "a", "range", "of", "transcriptional", "regulatory", "functions", "have", "been", "identified", "initially", "as", "DHSs.", "These", "include", "transcriptional", "enhancers", "(1,2)", "and", "repressors", "(3,4)", "as", "well", "as", "chromatin", "insulators", "and", "barrier", "elements", "(5,6).", "A", "number", "of", "techniques", "have", "been", "published", "recently", "that", "permit", "the", "mapping", "of", "DHSs", "without", "the", "need", "for", "Southern", "blotting", "(7–13).", "These", "include", "high-throughput", "sequencing", "of", "cloned", "DNA", "libraries", "derived", "from", "DNaseI-digested", "chromatin", "(8,9),", "and", "a", "number", "of", "different", "approaches", "that", "use", "genomic", "tiling", "path", "arrays", "to", "map", "DHSs", "(7,11,13).", "While", "these", "approaches", "have", "the", "advantage", "of", "covering", "large", "genomic", "regions", "with", "a", "limited", "number", "of", "experiments,", "they", "are", "inherently", "costly", "and", "less", "applicable", "to", "the", "rapid", "DHS", "mapping", "of", "specific", "genomic", "sites", "in", "a", "range", "of", "cell", "types.", "An", "alternative", "approach", "that", "does", "permit", "the", "targeted", "semi-quantitative", "DHS", "mapping", "of", "specific", "loci", "has", "used", "quantitative", "real-time", "PCR", "to", "map", "sites", "from", "digested", "DNA", "(10).", "This", "technique", "depends", "on", "the", "quantification", "of", "relative", "loss", "of", "PCR", "signal", "observed", "when", "PCR", "primers", "amplify", "across", "regions", "of", "digested", "DNA", "compared", "with", "amplification", "of", "undigested", "DNA.", "This", "approach", "has", "been", "reported", "as", "showing", "good", "sensitivity,", "but", "a", "limitation", "is", "the", "large", "number", "of", "PCR", "reactions", "required", "to", "quantify", "the", "calculated", "loss", "of", "signal", "with", "significant", "certainty.", "In", "this", "article,", "we", "present", "an", "alternative", "method", "to", "identify", "DHS", "using", "real-time", "PCR", "by", "adapting", "the", "protocol", "we", "have", "used", "to", "map", "DHS", "using", "genomic", "tiling", "path", "arrays", "(11).", "We", "have", "previously", "demonstrated", "the", "high", "sensitivity", "and", "specificity", "of", "the", "basic", "protocol", "with", "regard", "to", "identifying", "DHS", "across", "large", "genomic", "regions", "(11).", "Here,", "we", "detail", "the", "laboratory", "protocol", "that", "permits", "the", "rapid", "comparative", "mapping", "of", "known", "and", "candidate", "DHS", "between", "different", "cell", "types", "using", "real-time", "PCR.", "As", "examples,", "we", "include", "comparative", "DHS", "mapping", "of", "regulatory", "elements", "located", "across", "the", "extended", "TAL1", "(T-cell", "acute", "lymphocytic", "leukaemia-1,", "also", "known", "as", "SCL", "(stem", "cell", "leukaemia))", "locus", "in", "human", "embryonic", "stem", "(hES)", "cells", "and", "the", "leukaemia", "cell", "line", "K562.", "Generating", "a", "library", "of", "DHSsThe", "basic", "protocol", "is", "outlined", "in", "Figure", "1.", "Nuclei", "are", "extracted", "from", "living", "cells,", "then", "digested", "on", "ice", "for", "1", "h", "with", "a", "range", "of", "DNaseI", "concentrations,", "as", "detailed", "in", "the", "‘Materials", "and", "methods’", "sections.", "Following", "RNaseA", "and", "proteinaseK", "treatment,", "the", "DNA", "is", "extracted", "and", "run", "on", "a", "1%", "agarose", "gel", "to", "check", "for", "the", "size", "of", "digested", "DNA.", "The", "gel", "in", "Figure", "1A", "shows", "the", "DNA", "from", "mouse", "thymocyte", "nuclei", "digested", "with", "0,", "40", "and", "120", "units", "of", "DNaseI.", "Maximal", "enrichment", "at", "DHS", "is", "usually", "observed", "with", "samples", "that", "are", "not", "over-digested.", "In", "the", "experiment", "shown,", "maximal", "enrichment", "at", "a", "known", "control", "DHS", "was", "seen", "with", "40", "units", "DNaseI", "(real-time", "PCR", "quantification", "shown", "in", "Figure", "2B).", "The", "DNA", "is", "then", "blunt-ended", "using", "T4", "polymerase", "(Figure", "1B)", "and", "ligated", "with", "an", "asymmetric", "double-stranded", "linker.", "After", "extraction,", "DNA", "is", "amplified", "using", "a", "biotinylated", "linker-specific", "primer", "and", "35", "thermal", "polymerase", "cycles,", "as", "detailed", "in", "‘Materials", "and", "methods’", "section.", "As", "the", "linker", "will", "ligate", "to", "both", "ends", "of", "digested", "DNA,", "the", "amplification", "will", "represent", "a", "mix", "of", "primer", "extension", "and", "PCR,", "depending", "on", "the", "length", "of", "DNA", "amplified.", "As", "has", "been", "previously", "reasoned", "(13),", "one", "double", "strand", "of", "DNA", "in", "a", "region", "of", "DNaseI", "hypersensitivity", "is", "more", "likely", "to", "be", "digested", "twice", "within", "a", "short", "distance", "than", "non-hypersensitive", "DNA.", "This", "will", "lead", "to", "the", "preferential", "amplification", "of", "DNA", "from", "regions", "of", "DHS.", "The", "amplified", "DNA", "is", "then", "extracted", "using", "para-magnetic", "streptavidin", "beads,", "which", "provides", "a", "DNA", "library", "representative", "of", "whole-genome", "DHS.", "Agarose", "gel", "electrophoresis", "of", "the", "DNA", "recovered", "from", "the", "beads", "confirms", "that", "the", "vast", "majority", "of", "these", "products", "are", "between", "300", "and", "500", "base", "pairs", "in", "length", "(Figure", "1B).", "Real-time", "PCR", "quantification", "of", "DNA", "within", "the", "DHS", "libraryFigure", "2", "documents", "the", "quantification", "of", "specific", "DNA", "sites", "from", "different", "mouse-cell-derived", "DNA", "libraries.", "Figure", "2A", "shows", "the", "quantification", "of", "material", "from", "two", "primer", "sets,", "which", "are", "within", "(primer", "pair", "A)", "and", "3′", "(primer", "pair", "B)", "to", "the", "mouse", "Stil", "promoter.", "The", "left-hand", "panels", "of", "Figure", "2A", "show", "the", "Sybr-green", "real-time", "fluorescence", "profiles", "using", "serial", "5-fold", "dilutions", "of", "quantified", "mouse", "genomic", "DNA", "standards", "with", "primers", "A", "and", "B.", "A", "calculated", "standard", "curve", "then", "permits", "the", "quantification", "of", "DNA", "from", "this", "sequence", "in", "library", "samples.", "The", "right-hand", "panels", "of", "Figure", "2A", "show", "the", "quantification", "of", "samples", "from", "0", "and", "40", "units", "DNaseI-treated", "material", "using", "primers", "A", "and", "B.", "While", "there", "is", "no", "difference", "in", "amplification", "between", "the", "samples", "using", "primer", "pair", "B,", "with", "primer", "pair", "A,", "the", "40", "units", "DNaseI", "sample", "amplifies", "∼5.5", "PCR", "cycles", "before", "the", "0", "units", "sample.", "This", "equates", "to", "greater", "than", "40-fold", "enrichment", "at", "the", "Stil", "promoter", "compared", "with", "no", "observed", "enrichment", "downstream", "of", "the", "promoter.", "Figure", "2B", "shows", "the", "quantifiable", "differences", "in", "enrichment", "at", "the", "porphobilinogen", "deaminase", "(Hmbs)", "promoter", "between", "primary", "mouse", "thymocytes", "and", "the", "mouse", "T-cell", "line", "BW5147.", "This", "representative", "primary", "mouse", "cell", "experiment", "was", "performed", "with", "the", "DNA", "shown", "in", "Figure", "1A,", "which", "confirms", "that", "with", "these", "cells,", "maximal", "enrichment", "is", "observed", "from", "DNA", "that", "is", "not", "over-digested.", "The", "comparison", "of", "the", "thymocytes", "with", "the", "BW5147", "cells", "illustrates", "another", "common", "finding", "that,", "in", "our", "experience", "of", "primary", "cell", "experiments,", "maximal", "enrichment", "is", "often", "obtained", "using", "lower", "amounts", "of", "DNaseI", "compared", "with", "cell", "lines.", "Comparison", "of", "real-time", "PCR", "DHS", "with", "Southern", "blottingAs", "the", "protocol", "generates", "template", "by", "primer", "extending", "away", "from", "a", "DNaseI-digestion", "site,", "as", "well", "as", "PCR", "amplification", "between", "DNaseI-digestion", "sites,", "there", "is", "the", "potential", "for", "the", "genomic", "size", "of", "the", "real-time", "DHS", "to", "be", "larger", "than", "sites", "revealed", "by", "Southern", "blotting.", "There", "is", "also", "potential", "for", "signal", "to", "be", "lost", "at", "the", "most", "‘open’", "stretch", "of", "DNA,", "due", "to", "over-digestion", "with", "DNaseI.", "These", "issues", "are", "addressed", "in", "Figure", "3.", "The", "upper", "panel", "shows", "a", "Southern", "blot", "of", "the", "human", "STIL", "(SCL/TAL1", "interrupting", "locus)", "promoter", "in", "K562", "cells.", "The", "BglII", "restricted", "fragment", "is", "probed", "from", "the", "3′", "end,", "as", "shown", "in", "the", "upper", "panel.", "This", "reveals", "a", "central", "DHS", "∼500", "bp", "wide,", "with", "a", "suggestion", "of", "weaker", "hypersensitivity", "for", "a", "few", "hundred", "base", "pairs", "5′", "and", "3′", "to", "the", "central", "region.", "The", "lower", "panel", "shows", "real-time", "PCR", "data", "from", "K562", "material", "using", "10", "primer", "sets,", "each", "generating", "an", "amplicon", "∼120", "bp", "long.", "This", "permits", "the", "quantification", "of", "enrichments", "over", "1200", "bp,", "centred", "around", "the", "STIL", "transcription", "start", "site.", "The", "lower", "panel", "represents", "the", "mean", "±", "SD", "enrichment", "from", "three", "independent", "biological", "replicates", "from", "K562", "cells.", "The", "black", "bar", "denotes", "the", "location", "of", "the", "‘core’", "hypersensitive", "site", "as", "defined", "by", "Southern", "blotting.", "There", "is", "good", "correlation", "between", "the", "location", "of", "the", "DHS", "between", "the", "two", "techniques.", "The", "5′", "extension", "of", "enrichment", "seen", "in", "the", "lower", "panel", "appears", "to", "reflect", "the", "weaker", "5′", "extension", "seen", "in", "the", "Southern", "blot", "in", "the", "upper", "panel.", "A", "dip", "in", "enrichment", "is", "observed", "in", "the", "lower", "panel", "over", "the", "previously", "mapped", "transcription-factor-binding", "sites", "(14),", "which", "may", "represent", "over-digestion", "of", "the", "most", "accessible", "core", "region", "of", "the", "DHS.", "Quantification", "of", "DHS", "across", "the", "TAL1", "locus", "in", "hES", "and", "K562", "cellsAs", "examples", "of", "relative", "enrichments", "at", "different", "regulatory", "elements,", "we", "present", "a", "comparison", "of", "enrichments", "across", "the", "extended", "TAL1", "locus", "from", "human", "embryonic", "stem", "(hES)", "cells", "and", "K562", "cells", "in", "Figure", "4B.", "Relative", "quantification", "of", "mRNA", "expression", "using", "real-time", "PCR", "shows", "that", "both", "of", "these", "cell", "types", "express", "STIL,", "but", "only", "K562", "cells", "express", "TAL1", "(Figure", "4A).", "TAL1", "expression", "is", "critical", "to", "the", "establishment", "of", "haematopoiesis", "in", "a", "developing", "embryo", "(15).", "Post", "embryogenesis,", "TAL1", "expression", "is", "maintained", "in", "the", "non-lymphoid", "haematopoietic", "system,", "although", "in", "addition,", "expression", "is", "observed", "in", "a", "range", "of", "non-haematopoietic", "tissues,", "including", "endothelium", "and", "brain", "(16–18).", "Over", "the", "past", "10", "years,", "we", "and", "others", "have", "dissected", "the", "regulatory", "elements", "that", "direct", "the", "expression", "of", "TAL1", "to", "different", "tissues", "(11,19–31).", "These", "include", "the", "3′", "haematopoietic", "stem", "cell", "enhancer", "(+19)", "(24,30),", "the", "5′", "endothelial-haematopoietic", "enhancer", "(−4)", "(31),", "the", "3′", "erythroid", "enhancer", "(+40", "in", "mouse,", "+50", "in", "human)", "(28),", "and", "a", "number", "of", "neuronal", "elements", "(25,26).", "The", "location", "of", "the", "STIL", "promoter", "has", "been", "previously", "mapped", "(14),", "although", "no", "other", "STIL", "regulatory", "elements", "have", "yet", "been", "identified.", "Figure", "4B", "shows", "the", "mean", "±", "SD", "ratio", "(DNaseI-treated", "enrichment/DNaseI-untreated", "enrichment)", "of", "quantitative", "enrichments", "from", "two", "independent", "hES", "cell", "biological", "replicates", "and", "two", "independent", "K562", "biological", "replicates,", "using", "primer", "sets", "from", "different", "locations", "as", "indicated", "in", "the", "figure.", "Primers", "indicated", "in", "red", "correspond", "to", "known", "regulatory", "elements.", "The", "hES", "cell", "cultures", "were", "maintained", "as", "detailed", "in", "methods.", "Both", "cell", "types", "show", "significant", "enrichment", "at", "the", "STIL", "promoter.", "Four", "control", "regions", "were", "selected:", "−16", "kb,", "upstream", "of", "TAL1;", "+5", "and", "+22", "kb,", "within", "the", "TAL1", "locus;", "+70", "kb,", "downstream", "of", "TAL1.", "These", "sequences", "were", "chosen", "as", "control", "regions", "as", "they", "show", "limited", "homology", "between", "a", "number", "of", "species", "(26)", "and", "were", "considered", "highly", "unlikely", "to", "represent", "regulatory", "elements.", "All", "four", "regions", "showed", "no", "enrichments", "between", "DNaseI-treated", "and", "-untreated", "samples", "in", "either", "the", "hES", "cells", "or", "K562", "cells.", "The", "most", "striking", "differences", "between", "hES", "cells", "and", "K562", "cells", "were", "found", "at", "the", "TAL1", "regulatory", "elements.", "With", "K562", "cells,", "there", "is", "prominent", "enrichment", "at", "the", "TAL1", "promoter", "1a,", "and", "the", "−4,", "+19", "and", "+50", "enhancers.", "These", "cells", "express", "high", "levels", "of", "TAL1", "transcripts,", "and", "were", "originally", "derived", "from", "a", "patient", "with", "blastic", "transformation", "of", "chronic", "myeloid", "leukaemia.", "K562", "cells", "are", "relatively", "undifferentiated,", "although", "they", "have", "a", "partial", "erythroid", "phenotype.", "This", "potentially", "explains", "the", "marked", "enrichment", "seen", "at", "the", "+19", "stem", "cell", "enhancer", "and", "the", "+50", "erythroid", "enhancer.", "This", "is", "similar", "to", "data", "obtained", "from", "primary", "mouse", "erythroid", "cells", "(day", "14.5", "foetal", "liver),", "which", "show", "the", "highest", "enrichments", "at", "the", "mouse", "+40", "enhancer", "(homologous", "to", "the", "human", "+50)", "(data", "not", "shown).", "In", "contrast,", "the", "hES", "cells", "show", "markedly", "reduced", "enrichment", "at", "the", "TAL1", "regulatory", "elements", "when", "compared", "with", "K562", "cells.", "Although", "there", "is", "enrichment", "at", "the", "+50", "enhancer,", "there", "is", "minimal", "enrichment", "at", "the", "−4,", "promoter", "1A", "and", "+19", "elements.", "Reduced/absent", "accessibility", "of", "regulatory", "elements", "to", "DNaseI", "digestion", "is", "consistent", "with", "the", "lack", "of", "expression", "of", "TAL1", "in", "hES", "cells", "(Figure", "4A).", "As", "ES", "cells", "differentiate", "to", "form", "embryoid", "bodies,", "expression", "of", "TAL1", "is", "rapidly", "switched", "on,", "appearing", "from", "day", "3", "of", "mouse", "ES", "cell", "differentiation", "(32).", "The", "relative", "accessibility", "of", "the", "+50", "enhancer", "may", "reflect", "a", "poised", "chromatin", "state", "in", "hES", "cells,", "which", "permits", "a", "rapid", "response", "to", "the", "changing", "transcription", "factor", "environment", "that", "accompanies", "differentiation.", "The", "development", "of", "techniques", "that", "permit", "the", "rapid", "comparative", "mapping", "of", "DHS", "between", "different", "cell", "types", "will", "greatly", "facilitate", "the", "study", "of", "transcriptional", "regulation", "in", "both", "normal", "and", "diseased", "cells.", "Recently", "published", "high-throughput", "techniques", "that", "map", "DHS", "sites", "using", "high-throughput", "sequencing", "(8,9)", "and", "genomic", "array", "tiling", "paths", "(7,11,13)", "have", "clear", "advantages", "of", "scale", "over", "more", "targeted", "approaches.", "Major", "disadvantages", "of", "these", "approaches", "include", "cost", "and", "lack", "of", "focus.", "This", "makes", "them", "less", "suitable", "for", "many", "laboratories", "that", "want", "to", "assess", "the", "chromatin", "accessibility", "of", "a", "number", "of", "defined", "or", "presumed", "regulatory", "elements", "in", "a", "range", "of", "cell", "types.", "A", "real-time-PCR-based", "approach", "to", "DHS", "mapping", "has,", "therefore,", "a", "number", "of", "potential", "advantages", "for", "researchers", "interested", "in", "specific", "regulatory", "questions", "at", "defined", "loci.", "Real-time", "PCR", "is", "relatively", "inexpensive", "compared", "with", "the", "large-scale", "techniques,", "and", "permits", "a", "rapid,", "focused", "DHS", "analysis", "of", "defined", "regions", "of", "DNA", "from", "multiple", "cell", "types.", "It", "also", "provides", "flexibility,", "as", "any", "genomic", "region", "can", "be", "analysed", "from", "the", "DNA", "library", "derived", "from", "the", "DNaseI-treated", "samples", "by", "designing", "further", "real-time", "PCR", "primer", "sets.", "We", "have", "previously", "shown", "that", "our", "basic", "technique", "for", "amplifying", "a", "library", "of", "DHS", "generates", "a", "template", "representative", "of", "known", "DHS", "with", "excellent", "sensitivity", "and", "specificity", "(11).", "Although", "the", "experiments", "presented", "in", "this", "paper", "were", "each", "performed", "using", "5", "million", "cells/digestion", "condition,", "we", "have", "obtained", "reproducible", "data", "using", "5-fold", "fewer", "cells", "as", "starting", "material.", "We", "feel", "our", "technique", "can", "deliver", "acceptable", "specificity", "and", "sensitivity", "for", "DHS", "mapping", "with", "small", "numbers", "of", "cells,", "and", "will", "therefore", "be", "of", "use", "to", "those", "researchers", "working", "with", "limited", "numbers", "of", "primary", "cells.", "An", "alternative", "approach", "using", "real-time", "PCR", "to", "define", "DHS", "has", "been", "previously", "published", "(10).", "The", "two", "approaches", "differ", "in", "that", "Dorscher", "et", "al.", "quantify", "the", "DHS", "through", "the", "loss", "of", "PCR", "signal", "obtained", "from", "DHS", "when", "DNA", "is", "digested,", "whereas", "our", "approach", "uses", "real-time", "PCR", "to", "quantify", "a", "gain", "of", "signal", "observed", "from", "DHS.", "Dorscher", "et", "al.", "report", "excellent", "sensitivity", "using", "their", "approach", "to", "map", "DHS.", "However,", "the", "technique", "depends", "on", "large", "numbers", "of", "comparative", "quantitative", "real-time", "PCR", "reactions", "across", "a", "region", "in", "both", "digested", "and", "undigested", "material,", "in", "order", "to", "quantify", "the", "loss", "of", "enrichment.", "One", "advantage", "of", "our", "technique", "published", "here", "is", "that", "data", "can", "be", "obtained", "using", "far", "fewer", "quantitative", "PCR", "reactions.", "The", "technique", "is", "highly", "reproducible,", "with", "relatively", "little", "variation", "in", "quantifiable", "enrichments", "observed", "between", "different", "biological", "replicates.", "Moreover,", "we", "demonstrate", "tissue", "specificity,", "with", "variable", "enrichment", "at", "known", "regulatory", "elements", "between", "different", "cell", "types.", "The", "technique", "published", "here", "permits", "the", "rapid", "comparative", "analysis", "of", "DHS", "between", "different", "cell", "types", "from", "relatively", "small", "numbers", "of", "cells.", "It", "will", "have", "potential", "use", "for", "researchers", "across", "a", "broad", "spectrum", "of", "biology", "for", "the", "study", "of", "transcriptional", "regulation", "in", "both", "healthy", "and", "diseased", "tissues.", "Cell", "cultureCell", "lines", "were", "maintained", "in", "culture", "as", "previously", "described", "(11).", "Care", "was", "taken", "to", "ensure", "maximum", "viability", "of", "cells", "when", "taken", "for", "experiments.", "The", "primary", "thymocytes", "used", "for", "Figure", "2", "were", "prepared", "by", "gentle", "physical", "disassociation", "of", "a", "whole", "thymus", "gland", "into", "PBS", "supplemented", "with", "2%", "FCS.", "Cells", "were", "filtered", "to", "ensure", "that", "a", "single-cell", "suspension", "was", "taken", "forward", "for", "nuclei", "isolation.", "Human", "embryonic", "stem", "cells", "(hES)", "were", "grown", "in", "chemically", "defined", "media", "in", "the", "presence", "of", "Activin", "and", "FGF2,", "as", "detailed", "previously", "(33).", "In", "these", "conditions,", "hES", "cells", "remain", "homogenously", "undifferentiated.", "Nuclei", "preparationUp", "to", "3", "×", "107", "cells", "were", "washed", "in", "ice-cold", "PBS", "and", "resuspended", "in", "2", "ml", "of", "ice-cold", "cell", "lysis", "buffer", "[300", "mM", "sucrose,", "10", "mM", "Tris", "pH", "7.4,", "15", "mM", "NaCl,", "5", "mM", "MgCl,", "0.1", "mM", "EGTA,", "60", "mM", "KCl,", "0.2%", "NP-40,", "0.5", "mM", "DTT,", "0.5", "μM", "spermidine,", "1×", "protease", "inhibitor", "(complete,", "Roche)].", "After", "5", "min,", "the", "lysed", "cells", "were", "spun", "at", "500", "g", "for", "5", "min", "at", "4°C", "with", "brakes", "off.", "After", "careful", "removal", "of", "supernatant,", "the", "nuclei", "were", "gently", "resuspended", "in", "200", "μl", "of", "ice-cold", "reaction", "buffer", "(20", "μl", "10×", "DNaseI", "buffer,", "4", "μl", "glycerol,", "176", "μl", "water)", "using", "pipette", "tips", "with", "cut", "off", "ends.", "The", "nuclei", "were", "spun", "again", "at", "500", "g", "for", "5", "min", "at", "4°C", "and,", "following", "supernatant", "removal,", "were", "resuspended", "in", "30", "μl", "of", "reaction", "buffer", "per", "DNaseI", "condition.", "For", "example,", "if", "2", "×", "107", "cells", "were", "taken", "to", "look", "at", "four", "different", "conditions", "(e.g.", "0,", "20,", "60,", "120", "units", "DNaseI),", "the", "nuclei", "were", "resuspended", "in", "120", "μl", "of", "reaction", "buffer.", "Separate", "30", "μl", "aliquots", "were", "then", "taken", "and", "gently", "mixed", "with", "70", "μl", "of", "DNaseI", "mix", "(see", "Table", "1)", "on", "ice.", "This", "made", "a", "final", "digestion", "volume", "of", "100", "μl", "for", "each", "sample,", "which", "was", "left", "to", "incubate", "for", "1", "h", "on", "ice", "in", "the", "cold", "room.", "After", "1", "h,", "700", "μl", "of", "nuclei", "lysis", "buffer", "(100", "mM", "tris", "HCL", "pH", "8,", "5", "mM", "EDTA", "pH", "8,", "200", "mM", "NaCl,", "0.2%", "SDS)", "was", "added", "to", "each", "sample", "with", "50", "μg", "proteinase", "K.", "Following", "gentle", "mixing", "with", "inversion,", "the", "lysed", "samples", "were", "incubated", "at", "55°C", "for", "1", "h.", "RNaseA", "(10", "μg", "(Ambion))", "was", "then", "added", "to", "each", "sample", "and", "further", "incubated", "at", "37°C", "for", "30", "min.", "DNA", "was", "then", "extracted", "using", "standard", "phenol–chloroform", "techniques.", "Care", "was", "taken", "to", "use", "cut-off", "tips", "and", "very", "gentle", "pipetting", "to", "reduce", "non-specific", "DNA", "sheering.", "Following", "precipitation,", "DNA", "was", "resuspended", "in", "200", "μl", "of", "0.1", "TE", "and", "quantified", "using", "spectophotometry.", "Samples", "(1", "μg)", "were", "analysed", "using", "gel-electrophoresis,", "as", "shown", "in", "Figure", "1A.", "DNA", "library", "preparationFollowing", "quantification,", "7.5", "μg", "of", "DNA", "was", "taken", "for", "each", "sample", "condition.", "This", "was", "blunt-ended", "using", "T4", "polymerase", "(10", "μl", "10×", "buffer,", "0.5", "μl", "25", "mM", "dNTP,", "3", "μl", "BSA,", "1", "μl", "T4", "polymerase", "(3", "U/μl", "NE", "Biolabs),", "85.5", "μl", "of", "water/DNA", "in", "0.1", "TE).", "The", "samples", "were", "mixed", "gently", "on", "ice,", "then", "put", "at", "12°C", "for", "16", "min.", "The", "reaction", "was", "stopped", "with", "excess", "EDTA", "(4", "μl", "0.5", "M)", "followed", "by", "75°C", "for", "10", "min.", "The", "DNA", "was", "further", "extracted", "using", "phenol–chloroform", "and", "precipitated", "with", "ethanol.", "Glycogen", "carrier", "(5", "μg)", "was", "used", "at", "this", "stage,", "and", "the", "pellet", "resuspended", "in", "80", "μl", "water.The", "blunt-ended", "DNA", "was", "then", "ligated", "to", "the", "LP21–25", "linker.", "A", "stock", "of", "linker", "was", "prepared", "by", "mixing", "80", "μl", "LP21", "(100", "μM:", "GAATTCAGATCTCCCGGGTCA)", "with", "80", "μl", "LP25", "(100", "μM:", "GCGGTGACCCGGGAGATCTG", "AATTC)", "and", "240", "μl", "water.", "The", "mix", "was", "then", "placed", "on", "a", "95°C", "hot", "block", "and", "the", "power", "supply", "turned", "off.", "When", "the", "block", "had", "cooled", "to", "room", "temperature,", "the", "LP21–25", "linker", "was", "aliquoted", "and", "frozen.", "The", "80", "μl", "DNA", "sample", "was", "split", "into", "two", "samples", "for", "ligation", "(5", "μl", "10×", "buffer,", "3", "μl", "LP21–25", "linker,", "3", "μl", "ligase", "(NE", "biolabs", "400", "U/μl", "and", "39", "μl", "DNA).", "This", "was", "mixed", "well", "with", "a", "pipette", "and", "left", "at", "16°C", "overnight.Following", "ligation,", "the", "DNA", "was", "precipitated", "with", "ethanol,", "and", "resuspended", "in", "42", "μl", "water.", "The", "sample", "was", "amplified", "using", "Vent", "exo-polymerase", "and", "a", "biotinylated", "LP25", "primer.", "The", "mix", "(5", "μl", "ThermoPol", "10×", "buffer,", "0.5", "μl", "B-LP25", "(200", "μM),", "2", "μl", "Vent", "exo-(NE", "Biolabs", "2", "U/μl),", "1", "μl", "dNTP", "(25", "mM)", "and", "41.5", "μl", "DNA)", "was", "amplified", "with", "the", "following", "thermal", "cycle:", "(95°C", "3", "min,", "95°C", "30", "s,", "61°C", "30", "s,", "72°C", "30", "s)", "×", "35", "cycles.Following", "amplification,", "the", "biotinylated", "products", "were", "extracted", "using", "Dynal", "streptavidin", "beads.", "For", "each", "sample,", "30", "μl", "of", "beads", "(Dyabeads", "M-270;", "Dynal", "biotech)", "were", "washed", "twice", "in", "2×", "binding", "buffer", "(10", "mM", "Tris", "HCL", "pH7.5,", "1", "mM", "EDTA,", "2", "M", "NaCl)", "using", "a", "magnet,", "and", "then", "resuspended", "in", "50", "μl", "of", "2×", "binding", "buffer/sample.", "Each", "post", "amplification", "sample", "(50", "μl)", "was", "then", "mixed", "with", "50", "μl", "of", "resuspended", "beads", "and", "incubated", "at", "room", "temperature", "on", "a", "rotator", "for", "1", "h.", "After", "binding,", "the", "samples", "were", "washed", "twice", "with", "2×", "binding", "buffer,", "then", "once", "with", "1×", "TE.", "After", "the", "last", "wash,", "each", "sample", "was", "resuspended", "in", "30", "μl", "0.1", "TE.", "The", "paired", "samples", "that", "were", "split", "before", "ligation", "were", "then", "pooled,", "making", "a", "60", "μl", "final", "aliquot.", "This", "was", "heated", "at", "95°C", "for", "10", "min", "to", "free", "the", "DNA", "from", "the", "beads,", "and", "then", "stored", "at", "4°C.", "Real-time", "PCR", "quantificationWe", "have", "used", "Stratagene", "Brilliant", "SYBR", "Green", "QPCR", "Master", "Mix", "as", "our", "standard", "kit", "for", "quantification", "of", "genomic", "controls", "and", "samples.", "We", "have", "used", "a", "range", "of", "primer", "sets.", "The", "HMBS", "primers", "are:", "human—Forward;", "ATGCTGCCTATTTCAAGGTTGT,", "Reverse;", "GAATT", "GGAACATTGCGACAGT,", "and", "mouse—Forward;", "CGGAGTCATGTCCGGTAAC,", "Reverse;", "CGACCAA", "TAGACGACGAGAA.", "Primers", "from", "the", "TAL1", "locus", "(human", "and", "mouse)", "are", "available", "on", "request.Amplification", "conditions", "and", "PCR", "mixes", "were", "as", "recommended", "by", "Stratagene.", "Genomic", "standard", "curves", "were", "calculated", "for", "each", "primer", "using", "serial", "5-fold", "dilutions", "from", "50", "ng", "genomic", "DNA/PCR", "reaction.", "For", "each", "sample", "PCR,", "a", "45", "μl", "stock", "was", "made", "using", "1.3", "μl", "of", "sample", "DNA", "+", "43.7", "μl", "water.", "This", "stock", "was", "used", "as", "5", "μl", "per", "real-time", "PCR", "reaction,", "and", "following", "amplification,", "quantified", "relative", "to", "the", "genomic", "DNA", "standard", "curve,", "for", "each", "primer", "set.", "The", "quantifications", "shown", "in", "Figure", "3", "represent", "the", "absolute", "quantification", "of", "DNA", "at", "each", "primer", "set", "for", "the", "5", "μl", "PCR", "sample.The", "mRNA", "expression", "levels", "of", "TAL1", "and", "STIL", "in", "Figure", "4A", "were", "normalised", "relative", "to", "β-actin", "and", "plotted", "relative", "to", "mRNA", "expression", "from", "normal", "human", "donor", "CD34", "+", "cells,", "as", "described", "previously", "(11)." ]
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