OTAR3088/AL_Test_data · Datasets at Hugging Face

sentence stringlengths 0 960	entities listlengths 0 23	data_source stringclasses 3 values
At any of these two stages, pan-neuronal differentiation can be achieved after three to four weeks.	[ { "end": 40, "label": "Tissue", "start": 28, "text": null } ]	CellFinder
hESC line HUES6 was cultured on MEF feeders as previously described (http://www.mcb.harvard.edu/melton/hues/) or on GFR matrigel coated plates.	[ { "end": 15, "label": "CellLine", "start": 10, "text": null }, { "end": 43, "label": "CellType", "start": 36, "text": null } ]	CellFinder
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.	[]	CellFinder
To differentiate neuroepithelial precursors, colonies were removed by treatment with collagenase IV (Sigma) and washed three times in growth media.	[ { "end": 43, "label": "CellType", "start": 17, "text": null }, { "end": 43, "label": "CellType", "start": 33, "text": null }, { "end": 32, "label": "Tissue", "start": 17, "text": null } ]	CellFinder
The pieces of colonies were resuspended in HUES growth media without FGF2 in an uncoated bacterial Petri dish to form EBs.	[ { "end": 47, "label": "CellType", "start": 43, "text": null }, { "end": 121, "label": "Tissue", "start": 118, "text": null } ]	CellFinder
After one week, EBs were plated on polyornathine/laminin coated plates in DMEM/F12 supplemented with N2 and FGF2.	[ { "end": 19, "label": "Tissue", "start": 16, "text": null } ]	CellFinder
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.	[ { "end": 18, "label": "Tissue", "start": 0, "text": null } ]	CellFinder
Cells could be grown as a monolayer for up to at least ten passages.	[]	CellFinder
Cells were Sox1 and nestin positive and readily differentiated into neurons upon withdrawal of FGF-2.	[]	CellFinder
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].	[ { "end": 97, "label": "CellType", "start": 88, "text": null }, { "end": 51, "label": "CellLine", "start": 44, "text": null }, { "end": 38, "label": "CellType", "start": 0, "text": null }, { "end": 28, "label": "Tissue", "start": 6, "text": null }, { "end": 38, "label": "CellType", "start": 29, "text": null }, { "end": 95, "label": "CellType", "start": 88, "text": null }, { "end": 38, "label": "CellType", "start": 6, "text": null } ]	CellFinder
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.	[]	CellFinder
Cultures were fed weekly and passaged at ∼two to three weeks using collagenases (Roche).	[]	CellFinder
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).	[ { "end": 142, "label": "CellLine", "start": 134, "text": null }, { "end": 129, "label": "CellLine", "start": 123, "text": null }, { "end": 253, "label": "CellType", "start": 246, "text": null }, { "end": 255, "label": "CellType", "start": 246, "text": null }, { "end": 139, "label": "CellLine", "start": 134, "text": null } ]	CellFinder
RNA preparation and array hybridization.	[]	CellFinder
Total RNA from cells was processed as follows.	[]	CellFinder
Cells were lysed in 1 mL of RNA-bee (Teltest).	[]	CellFinder
The RNA was isolated by chloroform extraction of the aqueous phase, followed by isopropanol precipitation as per the manufacturer's instructions.	[]	CellFinder
The precipitated RNA was washed in 75% ethanol and eluted with DEPC-treated water.	[]	CellFinder
Five ug of RNA was treated with RQ1 DNAase (Promega) according to the manufacturer's instructions.	[]	CellFinder
One ug of total RNA for each sample was processed using the Affymetrix GeneChip Whole Transcript Sense Target Labeling Assay (Affymetrix).	[]	CellFinder
Ribosomal RNA was reduced with the RiboMinus Kit (Invitrogen).	[]	CellFinder
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.	[]	CellFinder
Hybridization cocktails containing ∼5 ug of fragmented and labeled DNA target were prepared and applied to GeneChip Human Exon 1.0 ST arrays.	[]	CellFinder
Hybridization was performed for 16 hours using the Fluidics 450 station.	[]	CellFinder
Arrays were scanned using the Affymetrix 3000 7G scanner and GeneChip Operating Software version 1.4 to produce .CEL intensity files.	[]	CellFinder
Detection of AS by RT-PCR.cDNAs were generated from total RNA with Superscript III reverse transcriptase (Invitrogen).	[]	CellFinder
PCR reactions were performed with primer pairs designed for AS targets (annealing at 58 °C and amplification for 30 or 35 cycles).	[]	CellFinder
PCR products were resolved on either 1.5% or 3% agarose gel in TBE.	[]	CellFinder
The Ethidium Bromide-stained gels were scanned with Typhoon 8600 scanner (Molecular Dynamics) for quantification.	[]	CellFinder
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.	[]	CellFinder
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.	[]	CellFinder
The true (false) positive rate was computed as TP (FP) divided by the total number of REAP[+] exons in the experimentally validated set.	[]	CellFinder
The true (false) negative rate was computed as the TN (FN) divided by the total number of REAP[−] exons in the experimentally validated set.	[]	CellFinder
Sensitivity was computed as TP/(TP+FN) and specificity was computed as TN/(FP+TN).	[]	CellFinder
Sequence databases.	[]	CellFinder
Genome sequences of human (hg17), dog (canFam1), rat (rn3), and mouse (mm5) were obtained from UCSC, as were the whole-genome MULTIZ alignments [80].	[]	CellFinder
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.	[]	CellFinder
Known genes that were mapped to different isoform clusters were discarded.	[]	CellFinder
All mRNAs aligned to hg17 that were greater than 300 bases long were clustered together with the known isoforms.	[]	CellFinder
Genes containing less than three exons were removed from further consideration.	[]	CellFinder
A total of 2.7 million spliced ESTs were mapped onto the 17,478 high-quality genes to infer AS.	[]	CellFinder
Exons with canonical splice signals (GT-AG, AT-AC, GC-AG) were retained, resulting in a total of 213,736 exons.	[]	CellFinder
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.	[]	CellFinder
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.	[]	CellFinder
These probesets were used to derive gene and exon-level signal estimates from the CEL files.	[]	CellFinder
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.	[ { "end": 22, "label": "Tissue", "start": 13, "text": null }, { "end": 129, "label": "Tissue", "start": 119, "text": null } ]	CellFinder
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.	[]	CellFinder
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.	[]	CellFinder
Exon array analysis.	[]	CellFinder
The Affymetrix Power Tools (APT) suite of programs was obtained from http://www.affymetrix.com/support/developer/powertools/index.affx.	[]	CellFinder
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.	[]	CellFinder
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).	[]	CellFinder
Briefly, DABG compared the signal for each probe to a background distribution of signals from anti-genomic probes with the same GC content.	[]	CellFinder
The DABG algorithm generated a p-value representing the probability that the signal intensity of a given probe was part of the background distribution.	[]	CellFinder
We considered a probeset with a DABG p-value lower than 0.05 as detected above background.	[]	CellFinder
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.	[ { "end": 470, "label": "CellType", "start": 465, "text": null }, { "end": 450, "label": "CellType", "start": 443, "text": null }, { "end": 452, "label": "CellType", "start": 443, "text": null }, { "end": 85, "label": "CellType", "start": 78, "text": null }, { "end": 115, "label": "CellType", "start": 108, "text": null }, { "end": 239, "label": "CellType", "start": 232, "text": null }, { "end": 279, "label": "CellType", "start": 272, "text": null }, { "end": 188, "label": "CellType", "start": 182, "text": null }, { "end": 156, "label": "CellType", "start": 149, "text": null }, { "end": 22, "label": "CellType", "start": 15, "text": null }, { "end": 139, "label": "CellType", "start": 132, "text": null }, { "end": 69, "label": "CellType", "start": 62, "text": null }, { "end": 40, "label": "CellType", "start": 33, "text": null } ]	CellFinder
Multiple hypothesis testing was corrected by controlling for the false discovery rate (Benjamini-Hochberg).	[]	CellFinder
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.	[]	CellFinder
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.	[]	CellFinder
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.	[]	CellFinder
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)).	[]	CellFinder
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.	[]	CellFinder
Following Belsley et al. [81], we defined the leverage hi of the ith point as hi = n−1 + (xi − μx)2 / sx2(n − 1).	[]	CellFinder
Here we considered a point to have high leverage if hi > 3p/n.	[]	CellFinder
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.	[]	CellFinder
We considered a point to have high influence if \|covi − 1\| > 3p/n.	[]	CellFinder
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.	[]	CellFinder
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.	[]	CellFinder
We considered a point to be an “outlier” if p < 0.01.	[]	CellFinder
Identification of motifs.	[]	CellFinder
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.	[]	CellFinder
All elements had to be greater than 5.	[]	CellFinder
To correct for multiple hypothesis testing, p-values were multiplied by the total number of comparisons.	[]	CellFinder
	[]	CellFinder
BackgroundHuman embryonic stem cells (hESCs) offer a virtually unlimited source of neural cells for structural repair in neurological disorders, such as stroke.	[ { "end": 36, "label": "CellType", "start": 10, "text": null }, { "end": 95, "label": "CellType", "start": 83, "text": null }, { "end": 43, "label": "CellType", "start": 38, "text": null }, { "end": 89, "label": "Tissue", "start": 83, "text": null }, { "end": 36, "label": "CellType", "start": 26, "text": null }, { "end": 25, "label": "Tissue", "start": 16, "text": null } ]	CellFinder
Neural cells can be derived from hESCs either by direct enrichment, or by isolating specific growth factor-responsive and expandable populations of human neural stem cells (hNSCs).	[ { "end": 38, "label": "CellType", "start": 33, "text": null }, { "end": 160, "label": "Tissue", "start": 154, "text": null }, { "end": 171, "label": "CellType", "start": 161, "text": null }, { "end": 171, "label": "CellType", "start": 148, "text": null }, { "end": 169, "label": "CellType", "start": 154, "text": null }, { "end": 12, "label": "CellType", "start": 0, "text": null }, { "end": 6, "label": "Tissue", "start": 0, "text": null }, { "end": 178, "label": "CellType", "start": 173, "text": null } ]	CellFinder
Studies have indicated that the direct enrichment method generates a heterogeneous population of cells that may contain residual undifferentiated stem cells that could lead to tumor formation in vivo.	[ { "end": 156, "label": "CellType", "start": 146, "text": null } ]	CellFinder
Methods/Principal FindingsWe isolated an expandable and homogenous population of hNSCs (named SD56) from hESCs using a defined media supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and leukemia inhibitory growth factor (LIF).	[ { "end": 86, "label": "CellType", "start": 81, "text": null }, { "end": 110, "label": "CellType", "start": 105, "text": null }, { "end": 98, "label": "CellLine", "start": 94, "text": null } ]	CellFinder
These hNSCs grew as an adherent monolayer culture.	[ { "end": 11, "label": "CellType", "start": 6, "text": null } ]	CellFinder
They were fully neuralized and uniformly expressed molecular features of NSCs, including nestin, vimentin and radial glial markers.	[ { "end": 121, "label": "CellType", "start": 110, "text": null }, { "end": 122, "label": "CellType", "start": 117, "text": null }, { "end": 77, "label": "CellType", "start": 73, "text": null } ]	CellFinder
These hNSCs did not express the pluripotency markers Oct4 or Nanog, nor did they express markers for the mesoderm or endoderm lineages.	[ { "end": 125, "label": "Tissue", "start": 117, "text": null }, { "end": 113, "label": "Tissue", "start": 105, "text": null }, { "end": 11, "label": "CellType", "start": 6, "text": null } ]	CellFinder
The self-renewal property of the hNSCs was characterized by a predominant symmetrical mode of cell division.	[ { "end": 38, "label": "CellType", "start": 33, "text": null } ]	CellFinder
The SD56 hNSCs differentiated into neurons, astrocytes and oligodendrocytes throughout multiple passages in vitro, as well as after transplantation.	[ { "end": 42, "label": "CellType", "start": 35, "text": null }, { "end": 75, "label": "CellType", "start": 59, "text": null }, { "end": 8, "label": "CellLine", "start": 4, "text": null }, { "end": 14, "label": "CellType", "start": 9, "text": null }, { "end": 14, "label": "CellType", "start": 4, "text": null }, { "end": 54, "label": "CellType", "start": 44, "text": null } ]	CellFinder
Together, these criteria confirm the definitive NSC identity of the SD56 cell line.	[ { "end": 72, "label": "CellLine", "start": 68, "text": null } ]	CellFinder
Importantly, they exhibited no chromosome abnormalities and did not form tumors after implantation into rat ischemic brains and into naïve nude rat brains and flanks.	[ { "end": 165, "label": "Tissue", "start": 159, "text": null }, { "end": 154, "label": "Tissue", "start": 148, "text": null } ]	CellFinder
Furthermore, hNSCs isolated under these conditions migrated toward the ischemia-injured adult brain parenchyma and improved the independent use of the stroke-impaired forelimb two months post-transplantation.	[ { "end": 110, "label": "Tissue", "start": 94, "text": null }, { "end": 110, "label": "Tissue", "start": 100, "text": null }, { "end": 18, "label": "CellType", "start": 13, "text": null } ]	CellFinder
Conclusions/SignificanceThe SD56 human neural stem cells derived under the reported conditions are stable, do not form tumors in vivo and enable functional recovery after stroke.	[ { "end": 55, "label": "CellType", "start": 39, "text": null }, { "end": 56, "label": "CellType", "start": 46, "text": null }, { "end": 32, "label": "CellLine", "start": 28, "text": null }, { "end": 45, "label": "Tissue", "start": 39, "text": null }, { "end": 125, "label": "Tissue", "start": 119, "text": null }, { "end": 56, "label": "CellType", "start": 28, "text": null } ]	CellFinder
These properties indicate that this hNSC line may offer a renewable, homogenous source of neural cells that will be valuable for basic and translational research.	[ { "end": 102, "label": "CellType", "start": 90, "text": null }, { "end": 96, "label": "Tissue", "start": 90, "text": null } ]	CellFinder
To date there have been no effective treatments for improving residual structural and functional deficits resulting from stroke.	[]	CellFinder
Current therapeutic approaches, such as the use of thrombolytics, benefit only 1 to 4% of patients [1].	[]	CellFinder
Consequently, the majority of stroke patients experience progression of ischemia associated with debilitating neurological deficits.	[]	CellFinder
Recent evidence has suggested that the transplantation of cells derived from cord blood, bone marrow or brain tissue (fetal and adult) enhances sensorimotor function in experimental models of stroke [2], [3].	[ { "end": 87, "label": "Tissue", "start": 77, "text": null }, { "end": 87, "label": "Tissue", "start": 82, "text": null }, { "end": 87, "label": "Tissue", "start": 82, "text": null }, { "end": 81, "label": "Tissue", "start": 77, "text": null }, { "end": 87, "label": "Tissue", "start": 77, "text": null }, { "end": 93, "label": "Tissue", "start": 89, "text": null }, { "end": 100, "label": "Tissue", "start": 89, "text": null }, { "end": 123, "label": "Tissue", "start": 118, "text": null } ]	CellFinder
However, the normal human-derived somatic stem cells used in these studies have a limited capacity to differentiate into the diverse neural cell types optimal for structural and physiological tissue repair and are not amenable for large-scale cell production.	[ { "end": 52, "label": "CellType", "start": 42, "text": null }, { "end": 52, "label": "CellType", "start": 20, "text": null }, { "end": 139, "label": "Tissue", "start": 133, "text": null }, { "end": 52, "label": "CellType", "start": 34, "text": null } ]	CellFinder
Unlike other sources of stem cells, hESC lines possess a nearly unlimited self-renewal capacity and the developmental potential to differentiate into virtually any cell type of the organism.	[]	CellFinder
As such, they constitute an ideal source of cells for regenerative medicine.	[]	CellFinder
The successful derivation of hESC lines from the inner cell mass of preimplantation embryos and their long-term maintenance in vitro over multiple passages has been demonstrated [4] and standardized.	[ { "end": 64, "label": "Tissue", "start": 49, "text": null }, { "end": 91, "label": "Tissue", "start": 84, "text": null } ]	CellFinder
Differentiation and enrichment processes that direct hESCs towards a neural lineage have also been achieved.	[ { "end": 83, "label": "Tissue", "start": 69, "text": null }, { "end": 58, "label": "CellType", "start": 53, "text": null } ]	CellFinder
To promote neuralization, ESCs were cultured in a defined media supplemented with morphogens or growth factors [5], [6], [7] or cultured under conditions that promote “rosettes”, structures morphologically similar to the developing neural tube [8], [9].	[ { "end": 30, "label": "CellType", "start": 26, "text": null }, { "end": 176, "label": "Tissue", "start": 168, "text": null }, { "end": 243, "label": "Tissue", "start": 232, "text": null } ]	CellFinder
This neuralization process has proven invaluable in understanding the specification of hESC-derived neural tissue [10], [11], [12].	[ { "end": 106, "label": "Tissue", "start": 100, "text": null }, { "end": 113, "label": "Tissue", "start": 100, "text": null } ]	CellFinder
However, the enriched neural progeny derived displayed overgrowth and limited migration after grafting into normal newborn mice [13] and lesioned adult rat striatum [12], [14], [15], [16].	[ { "end": 36, "label": "CellType", "start": 22, "text": null }, { "end": 164, "label": "Tissue", "start": 152, "text": null }, { "end": 164, "label": "Tissue", "start": 156, "text": null }, { "end": 28, "label": "Tissue", "start": 22, "text": null } ]	CellFinder

At any of these two stages, pan-neuronal differentiation can be achieved after three to four weeks.

[ { "end": 40, "label": "Tissue", "start": 28, "text": null } ]

CellFinder

hESC line HUES6 was cultured on MEF feeders as previously described (http://www.mcb.harvard.edu/melton/hues/) or on GFR matrigel coated plates.

[ { "end": 15, "label": "CellLine", "start": 10, "text": null }, { "end": 43, "label": "CellType", "start": 36, "text": null } ]

CellFinder

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.

[]

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To differentiate neuroepithelial precursors, colonies were removed by treatment with collagenase IV (Sigma) and washed three times in growth media.

[ { "end": 43, "label": "CellType", "start": 17, "text": null }, { "end": 43, "label": "CellType", "start": 33, "text": null }, { "end": 32, "label": "Tissue", "start": 17, "text": null } ]

CellFinder

The pieces of colonies were resuspended in HUES growth media without FGF2 in an uncoated bacterial Petri dish to form EBs.

[ { "end": 47, "label": "CellType", "start": 43, "text": null }, { "end": 121, "label": "Tissue", "start": 118, "text": null } ]

CellFinder

After one week, EBs were plated on polyornathine/laminin coated plates in DMEM/F12 supplemented with N2 and FGF2.

[ { "end": 19, "label": "Tissue", "start": 16, "text": null } ]

CellFinder

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.

[ { "end": 18, "label": "Tissue", "start": 0, "text": null } ]

CellFinder

Cells could be grown as a monolayer for up to at least ten passages.

[]

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Cells were Sox1 and nestin positive and readily differentiated into neurons upon withdrawal of FGF-2.

[]

CellFinder

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].

[ { "end": 97, "label": "CellType", "start": 88, "text": null }, { "end": 51, "label": "CellLine", "start": 44, "text": null }, { "end": 38, "label": "CellType", "start": 0, "text": null }, { "end": 28, "label": "Tissue", "start": 6, "text": null }, { "end": 38, "label": "CellType", "start": 29, "text": null }, { "end": 95, "label": "CellType", "start": 88, "text": null }, { "end": 38, "label": "CellType", "start": 6, "text": null } ]

CellFinder

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.

[]

CellFinder

Cultures were fed weekly and passaged at ∼two to three weeks using collagenases (Roche).

[]

CellFinder

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).

[ { "end": 142, "label": "CellLine", "start": 134, "text": null }, { "end": 129, "label": "CellLine", "start": 123, "text": null }, { "end": 253, "label": "CellType", "start": 246, "text": null }, { "end": 255, "label": "CellType", "start": 246, "text": null }, { "end": 139, "label": "CellLine", "start": 134, "text": null } ]

CellFinder

RNA preparation and array hybridization.

[]

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Total RNA from cells was processed as follows.

[]

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Cells were lysed in 1 mL of RNA-bee (Teltest).

[]

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The RNA was isolated by chloroform extraction of the aqueous phase, followed by isopropanol precipitation as per the manufacturer's instructions.

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The precipitated RNA was washed in 75% ethanol and eluted with DEPC-treated water.

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Five ug of RNA was treated with RQ1 DNAase (Promega) according to the manufacturer's instructions.

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One ug of total RNA for each sample was processed using the Affymetrix GeneChip Whole Transcript Sense Target Labeling Assay (Affymetrix).

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Ribosomal RNA was reduced with the RiboMinus Kit (Invitrogen).

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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.

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Hybridization cocktails containing ∼5 ug of fragmented and labeled DNA target were prepared and applied to GeneChip Human Exon 1.0 ST arrays.

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Hybridization was performed for 16 hours using the Fluidics 450 station.

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Arrays were scanned using the Affymetrix 3000 7G scanner and GeneChip Operating Software version 1.4 to produce .CEL intensity files.

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Detection of AS by RT-PCR.cDNAs were generated from total RNA with Superscript III reverse transcriptase (Invitrogen).

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PCR reactions were performed with primer pairs designed for AS targets (annealing at 58 °C and amplification for 30 or 35 cycles).

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PCR products were resolved on either 1.5% or 3% agarose gel in TBE.

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The Ethidium Bromide-stained gels were scanned with Typhoon 8600 scanner (Molecular Dynamics) for quantification.

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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.

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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.

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The true (false) positive rate was computed as TP (FP) divided by the total number of REAP[+] exons in the experimentally validated set.

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The true (false) negative rate was computed as the TN (FN) divided by the total number of REAP[−] exons in the experimentally validated set.

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Sensitivity was computed as TP/(TP+FN) and specificity was computed as TN/(FP+TN).

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Sequence databases.

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Genome sequences of human (hg17), dog (canFam1), rat (rn3), and mouse (mm5) were obtained from UCSC, as were the whole-genome MULTIZ alignments [80].

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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.

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Known genes that were mapped to different isoform clusters were discarded.

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All mRNAs aligned to hg17 that were greater than 300 bases long were clustered together with the known isoforms.

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Genes containing less than three exons were removed from further consideration.

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A total of 2.7 million spliced ESTs were mapped onto the 17,478 high-quality genes to infer AS.

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Exons with canonical splice signals (GT-AG, AT-AC, GC-AG) were retained, resulting in a total of 213,736 exons.

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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.

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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.

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These probesets were used to derive gene and exon-level signal estimates from the CEL files.

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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.

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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.

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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.

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Exon array analysis.

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The Affymetrix Power Tools (APT) suite of programs was obtained from http://www.affymetrix.com/support/developer/powertools/index.affx.

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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.

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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).

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Briefly, DABG compared the signal for each probe to a background distribution of signals from anti-genomic probes with the same GC content.

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The DABG algorithm generated a p-value representing the probability that the signal intensity of a given probe was part of the background distribution.

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We considered a probeset with a DABG p-value lower than 0.05 as detected above background.

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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.

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Multiple hypothesis testing was corrected by controlling for the false discovery rate (Benjamini-Hochberg).

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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.

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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.

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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.

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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)).

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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.

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Following Belsley et al. [81], we defined the leverage hi of the ith point as hi = n−1 + (xi − μx)2 / sx2(n − 1).

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Here we considered a point to have high leverage if hi > 3p/n.

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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.

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We considered a point to have high influence if |covi − 1| > 3p/n.

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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.

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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.

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We considered a point to be an “outlier” if p < 0.01.

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Identification of motifs.

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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.

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All elements had to be greater than 5.

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To correct for multiple hypothesis testing, p-values were multiplied by the total number of comparisons.

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BackgroundHuman embryonic stem cells (hESCs) offer a virtually unlimited source of neural cells for structural repair in neurological disorders, such as stroke.

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Neural cells can be derived from hESCs either by direct enrichment, or by isolating specific growth factor-responsive and expandable populations of human neural stem cells (hNSCs).

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Studies have indicated that the direct enrichment method generates a heterogeneous population of cells that may contain residual undifferentiated stem cells that could lead to tumor formation in vivo.

[ { "end": 156, "label": "CellType", "start": 146, "text": null } ]

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Methods/Principal FindingsWe isolated an expandable and homogenous population of hNSCs (named SD56) from hESCs using a defined media supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and leukemia inhibitory growth factor (LIF).

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These hNSCs grew as an adherent monolayer culture.

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They were fully neuralized and uniformly expressed molecular features of NSCs, including nestin, vimentin and radial glial markers.

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These hNSCs did not express the pluripotency markers Oct4 or Nanog, nor did they express markers for the mesoderm or endoderm lineages.

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The self-renewal property of the hNSCs was characterized by a predominant symmetrical mode of cell division.

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The SD56 hNSCs differentiated into neurons, astrocytes and oligodendrocytes throughout multiple passages in vitro, as well as after transplantation.

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Together, these criteria confirm the definitive NSC identity of the SD56 cell line.

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Importantly, they exhibited no chromosome abnormalities and did not form tumors after implantation into rat ischemic brains and into naïve nude rat brains and flanks.

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Furthermore, hNSCs isolated under these conditions migrated toward the ischemia-injured adult brain parenchyma and improved the independent use of the stroke-impaired forelimb two months post-transplantation.

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Conclusions/SignificanceThe SD56 human neural stem cells derived under the reported conditions are stable, do not form tumors in vivo and enable functional recovery after stroke.

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These properties indicate that this hNSC line may offer a renewable, homogenous source of neural cells that will be valuable for basic and translational research.

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To date there have been no effective treatments for improving residual structural and functional deficits resulting from stroke.

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Current therapeutic approaches, such as the use of thrombolytics, benefit only 1 to 4% of patients [1].

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Consequently, the majority of stroke patients experience progression of ischemia associated with debilitating neurological deficits.

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Recent evidence has suggested that the transplantation of cells derived from cord blood, bone marrow or brain tissue (fetal and adult) enhances sensorimotor function in experimental models of stroke [2], [3].

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However, the normal human-derived somatic stem cells used in these studies have a limited capacity to differentiate into the diverse neural cell types optimal for structural and physiological tissue repair and are not amenable for large-scale cell production.

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Unlike other sources of stem cells, hESC lines possess a nearly unlimited self-renewal capacity and the developmental potential to differentiate into virtually any cell type of the organism.

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As such, they constitute an ideal source of cells for regenerative medicine.

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The successful derivation of hESC lines from the inner cell mass of preimplantation embryos and their long-term maintenance in vitro over multiple passages has been demonstrated [4] and standardized.

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Differentiation and enrichment processes that direct hESCs towards a neural lineage have also been achieved.

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To promote neuralization, ESCs were cultured in a defined media supplemented with morphogens or growth factors [5], [6], [7] or cultured under conditions that promote “rosettes”, structures morphologically similar to the developing neural tube [8], [9].

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This neuralization process has proven invaluable in understanding the specification of hESC-derived neural tissue [10], [11], [12].

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However, the enriched neural progeny derived displayed overgrowth and limited migration after grafting into normal newborn mice [13] and lesioned adult rat striatum [12], [14], [15], [16].

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