OTAR3088/Cellfinder-cleaned_CoNNL2 · Datasets at Hugging Face

tokens sequencelengths 2.46k 11.4k	ner_tags sequencelengths 2.46k 11.4k
[ "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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[ "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." ]	[ 2, 12, 12, 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, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 0, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 14, 9, 9, 14, 9, 2, 9, 9, 9, 9, 2, 12, 12, 12, 12, 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, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 5, 15, 9, 5, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 5, 9, 15, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 5, 9, 9, 9, 9, 9, 9, 9, 9, 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, 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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, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 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"(...TRUNCATED)	[9,12,12,12,9,9,9,9,9,9,9,14,12,9,9,9,9,9,9,9,9,9,14,12,9,9,9,9,8,9,9,9,9,9,9,9,9,9,9,9,9,9,9,2,12,1(...TRUNCATED)
["BackgroundUsing","antibodies","to","specific","protein","antigens","is","the","method","of","choic(...TRUNCATED)	[9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9(...TRUNCATED)
["BackgroundMany","novel","studies","and","therapies","are","possible","with","the","use","of","huma(...TRUNCATED)	[9,9,9,9,9,9,9,9,9,9,9,2,12,12,12,9,9,9,9,9,8,9,9,8,9,9,5,12,12,12,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9(...TRUNCATED)
["This","work","uncovers","novel","mechanisms","of","aging","within","stem","cell","niches","that","(...TRUNCATED)	[9,9,9,9,9,9,9,9,14,10,10,9,9,9,9,9,2,9,2,9,9,9,14,9,8,12,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,(...TRUNCATED)
["BackgroundIn","order","to","compare","the","gene","expression","profiles","of","human","embryonic"(...TRUNCATED)	[9,9,9,9,9,9,9,9,9,2,12,12,12,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,2,12,12,12,9,9,9,9,9,9,9,9,9(...TRUNCATED)
["BackgroundHuman","stem","cells","are","viewed","as","a","possible","source","of","neurons","for","(...TRUNCATED)	[9,12,12,9,9,9,9,9,9,9,8,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,8,9,2,12,12,9,9,9,9,9,9,9,9,9,9,9,9,9,8(...TRUNCATED)
["BackgroundHuman","embryonic","stem","cells","provide","access","to","the","earliest","stages","of"(...TRUNCATED)	[9,12,12,12,9,9,9,9,9,9,9,2,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,14,12,12,9,14,9,9,9,9,(...TRUNCATED)
["Mapping","sites","within","the","genome","that","are","hypersensitive","to","digestion","with","DN(...TRUNCATED)	[9,9,9,9,9,9,9,9,9,9,9,5,9,9,9,9,9,9,0,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9(...TRUNCATED)

[ "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", "Biosciences", "610798),", "CtBP1", "(1:1000,", "BD", "Biosciences", "612042),", "CtBP2", "(1:1000,", "BD", "Biosciences", "612044),", "GS-28", "(1:2000,", "BD", "Biosciences", "611184),", "HDJ-2", "(1:100,", "BD", "Biosciences", "611872),", "L-Caldesmon", "(1:2000,", "BD", "Biosciences", "610660),", "Rabaptin-5", "(1:500,", "BD", "Biosciences", "611080),", "phospho-p130", "Cas", "(Tyr165)", "(1:50,", "Cell", "Signaling", "Technology", "4015),", "phospho-Ras-GAP", "(pY460)", "(1:250,", "BD", "Biosciences", "612736),", "Ras-GAP", "(1:250,", "BD", "Biosciences", "610043),", "Shc-C", "(1:1000,", "BD", "Biosciences", "610642),", "Oct-4", "(Santa", "Cruz", "biotechnology,", "1:200", "SC-8628),", "TNIK", "(1:100,", "BD", "Biosciences,", "612250),", "p130", "Cas", "(1:100,", "BD", "Biosciences,", "610272),", "ERBB2", "(1:100,", "Lab", "Vision,", "9G6.10),", "ERBB3", "(1:100,", "R&D", "Systems,", "MAB348),", "ZO1", "(1:100,", "Invitrogen,", "61–7300),", "or", "Occludin", "(1:100,", "Invitrogen,", "71–1500).", "Hoechst", "(Invitrogen)", "or", "DAPI", "(Sigma)", "were", "used", "to", "identify", "nuclei,", "and", "Triton", "X-100", "was", "omitted", "when", "staining", "for", "extracellular", "antigens", "(ZO1,", "occludin,", "ERBB2/3).", "Images", "were", "captured", "on", "an", "Olympus", "or", "Nikon", "fluorescence", "microscope.", "lllumina", "data", "and", "comparison", "to", "proteomic", "databaseExpression", "levels", "of", "proteins", "detected", "by", "the", "PowerBlot", "assay", "were", "compared", "to", "our", "previous", "published", "database", "of", "multiple", "hESC", "lines", "examined", "using", "the", "Illumina", "bead", "array", "platform", "(Liu", "et", "al.,", "2006).", "Averaged", "transcript", "expression", "signals", "from", "the", "BG01,", "BG02", "and", "BG03", "cell", "lines", "were", "converted", "to", "a", "+/-", "format,", "based", "on", "the", "following", "criteria:", "A", "mean", "transcript", "detection", "level", "of", ">", "5,000", "was", "designated", "as", "++++;", "1,000–5,000", "as", "+++;", "100–1,000", "as", "++;", "30–100", "as", "+;", "and", "signals", "<", "30", "was", "represented", "as", "-.", "In", "parallel,", "the", "protein", "expression", "levels", "were", "converted", "to", "a", "+/-", "format", "based", "on", "these", "criteria:", "i.u.", ">", "10,000", "as", "++++;", "5,000–10,000", "as", "+++;", "1,000–5,000", "as", "++;", "100–1,000", "as", "+.", "In", "addition,", "genes", "were", "categorized", "into", "the", "same", "functional/signaling", "pathways", "as", "per", "the", "western", "blot", "database." ]

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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, 5, 15, 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, 5, 15, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 5, 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, 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, 5, 15, 15, 9, 9, 9, 9, 8, 9, 9, 14, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 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, 8, 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, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 2, 12, 12, 9, 9, 9, 9, 9, 9, 7, 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, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 7, 12, 9, 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, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 5, 9, 9, 9, 5, 9, 9, 9, 9, 5, 9, 9, 9, 9, 9, 5, 9, 9, 9, 9, 9, 9, 5, 15, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 0, 9, 9, 9, 9, 9, 9, 9, 9, 0, 9, 7, 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, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 5, 9, 9, 5, 15, 9, 9, 5, 9, 9, 5, 9, 9, 5, 9, 9, 5, 9, 9, 5, 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, 5, 9, 9, 5, 9, 9, 5, 15, 9, 9, 5, 9, 9, 5, 9, 9, 5, 9, 9, 9, 5, 9, 9, 9, 9, 9, 9, 9, 9, 5, 9, 9, 5, 9, 9, 5, 9, 9, 5, 9, 9, 5, 9, 9, 5, 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, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 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, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 5, 15, 9, 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, 9, 9, 9, 9, 9, 9, 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, 2, 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, 2, 9, 9, 9, 2, 9, 9, 9, 9, 2, 9, 9, 9, 9, 9, 9, 2, 15, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 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, 5, 9, 9, 9, 9, 5, 9, 9, 9, 9, 5, 9, 9, 9, 9, 5, 9, 9, 9, 9, 5, 9, 9, 9, 9, 5, 9, 9, 9, 9, 5, 9, 9, 9, 9, 5, 15, 9, 9, 9, 9, 9, 9, 5, 9, 9, 9, 9, 9, 5, 9, 9, 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", 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9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 5, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 7, 9, 9, 5, 9, 9, 5, 9, 9, 9, 5, 9, 9, 5, 9, 9, 9, 5, 9, 9, 5, 9, 9, 0, 9, 9, 9, 9, 0, 9, 9, 9, 9, 9, 9, 9, 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, 0, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 0, 9, 9, 9, 9, 5, 9, 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, 0, 13, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 0, 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, 0, 9, 9, 9, 5, 15, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 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, 2, 9, 2, 9, 9, 2, 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, 2, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 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, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 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, 2, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 14, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 14, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 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, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 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"(...TRUNCATED)

[9,12,12,12,9,9,9,9,9,9,9,14,12,9,9,9,9,9,9,9,9,9,14,12,9,9,9,9,8,9,9,9,9,9,9,9,9,9,9,9,9,9,9,2,12,1(...TRUNCATED)

["BackgroundUsing","antibodies","to","specific","protein","antigens","is","the","method","of","choic(...TRUNCATED)

[9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9(...TRUNCATED)

["BackgroundMany","novel","studies","and","therapies","are","possible","with","the","use","of","huma(...TRUNCATED)

[9,9,9,9,9,9,9,9,9,9,9,2,12,12,12,9,9,9,9,9,8,9,9,8,9,9,5,12,12,12,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9(...TRUNCATED)

["This","work","uncovers","novel","mechanisms","of","aging","within","stem","cell","niches","that","(...TRUNCATED)

[9,9,9,9,9,9,9,9,14,10,10,9,9,9,9,9,2,9,2,9,9,9,14,9,8,12,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,(...TRUNCATED)

["BackgroundIn","order","to","compare","the","gene","expression","profiles","of","human","embryonic"(...TRUNCATED)

[9,9,9,9,9,9,9,9,9,2,12,12,12,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,2,12,12,12,9,9,9,9,9,9,9,9,9(...TRUNCATED)

["BackgroundHuman","stem","cells","are","viewed","as","a","possible","source","of","neurons","for","(...TRUNCATED)

[9,12,12,9,9,9,9,9,9,9,8,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,8,9,2,12,12,9,9,9,9,9,9,9,9,9,9,9,9,9,8(...TRUNCATED)

["BackgroundHuman","embryonic","stem","cells","provide","access","to","the","earliest","stages","of"(...TRUNCATED)

[9,12,12,12,9,9,9,9,9,9,9,2,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,14,12,12,9,14,9,9,9,9,(...TRUNCATED)

["Mapping","sites","within","the","genome","that","are","hypersensitive","to","digestion","with","DN(...TRUNCATED)

[9,9,9,9,9,9,9,9,9,9,9,5,9,9,9,9,9,9,0,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9(...TRUNCATED)

Datasets:

OTAR3088
/

Cellfinder-cleaned_CoNNL2

Collection including OTAR3088/Cellfinder-cleaned_CoNNL2

Datasets archive