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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.
|
[
{
"end": 332,
"label": "CellType",
"start": 329,
"text": null
},
{
"end": 316,
"label": "CellType",
"start": 311,
"text": null
}
] |
CellFinder
|
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.
|
[
{
"end": 110,
"label": "CellType",
"start": 107,
"text": null
},
{
"end": 94,
"label": "CellType",
"start": 89,
"text": null
}
] |
CellFinder
|
At a defined absolute-valued cutoff, genes were divided into three categories: “enriched in hESCs,” “enriched in NP,” or “unchanged” (Figure 8A).
|
[
{
"end": 97,
"label": "CellType",
"start": 92,
"text": null
}
] |
CellFinder
|
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).
|
[] |
CellFinder
|
However, the fraction of REAP[+] exons decreased significantly in “enriched in hESCs” and “enriched in NPs” categories.
|
[
{
"end": 106,
"label": "CellType",
"start": 103,
"text": null
},
{
"end": 84,
"label": "CellType",
"start": 79,
"text": null
}
] |
CellFinder
|
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).
|
[] |
CellFinder
|
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[+].
|
[] |
CellFinder
|
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[+].
|
[
{
"end": 110,
"label": "CellType",
"start": 107,
"text": null
},
{
"end": 102,
"label": "CellType",
"start": 97,
"text": null
}
] |
CellFinder
|
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.
|
[
{
"end": 211,
"label": "CellType",
"start": 206,
"text": null
},
{
"end": 219,
"label": "CellType",
"start": 216,
"text": null
}
] |
CellFinder
|
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.
|
[] |
CellFinder
|
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.
|
[] |
CellFinder
|
Similar representation as in (B).
|
[] |
CellFinder
|
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.
|
[] |
CellFinder
|
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.
|
[] |
CellFinder
|
In addition, we wanted to identify both common and cell type–specific ISREs.
|
[] |
CellFinder
|
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.
|
[
{
"end": 95,
"label": "CellType",
"start": 92,
"text": null
},
{
"end": 116,
"label": "CellType",
"start": 111,
"text": null
},
{
"end": 211,
"label": "CellType",
"start": 208,
"text": null
},
{
"end": 192,
"label": "CellType",
"start": 187,
"text": null
}
] |
CellFinder
|
Regions of 400 base pairs flanking the exons were targeted for search.
|
[] |
CellFinder
|
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.
|
[] |
CellFinder
|
We were not able to identify 5-mers that were statistically significantly different.
|
[] |
CellFinder
|
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].
|
[
{
"end": 73,
"label": "Tissue",
"start": 64,
"text": null
}
] |
CellFinder
|
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).
|
[] |
CellFinder
|
We enumerated k-mers that were perfectly conserved across all four genomes in the upstream (and downstream) intronic regions.
|
[] |
CellFinder
|
Each conserved k-mer was attributed a χ score representing its enrichment in a set of exons relative to another set of exons.
|
[] |
CellFinder
|
The higher the score, the more frequent the conserved k-mer was in the first set relative to the second set.
|
[] |
CellFinder
|
As a negative control, the associations between REAP scores and exons were shuffled.
|
[] |
CellFinder
|
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).
|
[] |
CellFinder
|
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.
|
[] |
CellFinder
|
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.
|
[] |
CellFinder
|
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).
|
[] |
CellFinder
|
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).
|
[] |
CellFinder
|
It is known that the heterogeneous ribonucleoprotein C (hnRNP C) binding site obtained by SELEX is five “U”s [67].
|
[] |
CellFinder
|
GU-rich sequences in flanking intronic regions were shown to bind to splicing factor ETR-3 to regulate AS [68].
|
[] |
CellFinder
|
CU-rich sequences were shown to bind the splicing factor PTB [69].
|
[] |
CellFinder
|
Of the 5-mers enriched upstream of REAP[+]ES exons, we observed CUAAC, which resembled the splicing branch-signal.
|
[] |
CellFinder
|
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].
|
[
{
"end": 189,
"label": "Tissue",
"start": 180,
"text": null
}
] |
CellFinder
|
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).
|
[
{
"end": 369,
"label": "Tissue",
"start": 360,
"text": null
},
{
"end": 163,
"label": "Tissue",
"start": 155,
"text": null
},
{
"end": 29,
"label": "Tissue",
"start": 20,
"text": null
}
] |
CellFinder
|
Red and green horizontal bars represent conserved intronic elements and nonconserved elements, respectively.
|
[] |
CellFinder
|
Internal exons were divided into REAP[+]NP, REAP[+]ES, and REAP[−] exons.
|
[] |
CellFinder
|
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).
|
[] |
CellFinder
|
The sign represented the direction of change, i.e., positive if enriched in introns flanking REAP[+] versus REAP[−] exon.
|
[] |
CellFinder
|
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.
|
[] |
CellFinder
|
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.
|
[] |
CellFinder
|
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.
|
[] |
CellFinder
|
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).
|
[] |
CellFinder
|
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.
|
[] |
CellFinder
|
G-rich motifs had previously been shown to be part of a bipartite signal that silences AS exons [75].
|
[] |
CellFinder
|
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.
|
[] |
CellFinder
|
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.
|
[
{
"end": 200,
"label": "CellType",
"start": 197,
"text": null
},
{
"end": 192,
"label": "CellType",
"start": 187,
"text": null
}
] |
CellFinder
|
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.
|
[
{
"end": 244,
"label": "Tissue",
"start": 239,
"text": null
},
{
"end": 237,
"label": "Tissue",
"start": 229,
"text": null
},
{
"end": 111,
"label": "CellType",
"start": 106,
"text": null
},
{
"end": 264,
"label": "Tissue",
"start": 250,
"text": null
},
{
"end": 20,
"label": "CellType",
"start": 16,
"text": null
},
{
"end": 64,
"label": "Tissue",
"start": 43,
"text": null
},
{
"end": 52,
"label": "Tissue",
"start": 43,
"text": null
},
{
"end": 227,
"label": "Tissue",
"start": 222,
"text": null
}
] |
CellFinder
|
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.
|
[
{
"end": 160,
"label": "CellType",
"start": 155,
"text": null
}
] |
CellFinder
|
To understand these events, protocols are still being developed to differentiate ESCs into a variety of lineages.
|
[
{
"end": 85,
"label": "CellType",
"start": 81,
"text": null
}
] |
CellFinder
|
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).
|
[
{
"end": 211,
"label": "Tissue",
"start": 199,
"text": null
},
{
"end": 180,
"label": "Tissue",
"start": 172,
"text": null
},
{
"end": 291,
"label": "Tissue",
"start": 280,
"text": null
},
{
"end": 219,
"label": "CellType",
"start": 199,
"text": null
},
{
"end": 299,
"label": "CellType",
"start": 280,
"text": null
},
{
"end": 61,
"label": "CellType",
"start": 56,
"text": null
},
{
"end": 121,
"label": "CellType",
"start": 118,
"text": null
}
] |
CellFinder
|
While many microarray studies have explored molecular differences between hESCs and derived NPs, most, if not all, have focused on transcriptional changes.
|
[
{
"end": 79,
"label": "CellType",
"start": 74,
"text": null
},
{
"end": 95,
"label": "CellType",
"start": 92,
"text": null
}
] |
CellFinder
|
These studies have largely ignored intermediate RNA processing events prior to and during translation.
|
[] |
CellFinder
|
In recent years, AS has gained momentum as being important in development, apoptosis, and cancer.
|
[] |
CellFinder
|
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.
|
[
{
"end": 156,
"label": "Tissue",
"start": 152,
"text": null
},
{
"end": 159,
"label": "CellType",
"start": 152,
"text": null
},
{
"end": 161,
"label": "CellType",
"start": 152,
"text": null
},
{
"end": 124,
"label": "CellType",
"start": 119,
"text": null
},
{
"end": 143,
"label": "CellType",
"start": 140,
"text": null
}
] |
CellFinder
|
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.
|
[] |
CellFinder
|
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.
|
[
{
"end": 239,
"label": "CellType",
"start": 236,
"text": null
}
] |
CellFinder
|
In addition, REAP[+] exons were also enriched for ACEScan-predicted evolutionarily conserved exons [55].
|
[] |
CellFinder
|
As ACEScan utilized a different set of information from REAP, the agreement between both algorithms served to further validate the predicted alternative exons.
|
[] |
CellFinder
|
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.
|
[
{
"end": 32,
"label": "CellType",
"start": 28,
"text": null
},
{
"end": 32,
"label": "CellType",
"start": 22,
"text": null
}
] |
CellFinder
|
Our finding that only a minority of AS events was common between various hESC to NP comparisons is intriguing.
|
[] |
CellFinder
|
A possible explanation is that the cell lines were not only genetically different, but were also exposed to different isolation and culture conditions.
|
[] |
CellFinder
|
In addition, the different differentiation protocols established as optimal for generating Nestin and Sox1 positive neural precursors may lead to vastly different molecular changes.
|
[
{
"end": 133,
"label": "CellType",
"start": 123,
"text": null
},
{
"end": 122,
"label": "Tissue",
"start": 116,
"text": null
},
{
"end": 133,
"label": "CellType",
"start": 116,
"text": null
}
] |
CellFinder
|
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.
|
[] |
CellFinder
|
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].
|
[] |
CellFinder
|
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].
|
[] |
CellFinder
|
We postulated that AS events may serve also to bias the differentiation spectrum of the cells, an important avenue for future work.
|
[] |
CellFinder
|
Experimental validation of REAP[+] exons suggested a high specificity at the expense of relatively moderate sensitivity.
|
[] |
CellFinder
|
We believe that the high FP rates may arise from cross-hybridization effects that remained unaccounted for.
|
[] |
CellFinder
|
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.
|
[
{
"end": 278,
"label": "CellType",
"start": 275,
"text": null
}
] |
CellFinder
|
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.
|
[] |
CellFinder
|
In addition, we validated nine novel AS events that distinguish hESCs and NPs.
|
[
{
"end": 77,
"label": "CellType",
"start": 74,
"text": null
},
{
"end": 69,
"label": "CellType",
"start": 64,
"text": null
}
] |
CellFinder
|
Consistent with our computational results, we observed that the AS patterns in hCNS-SCns were not always similar to those of the derived NPs.
|
[
{
"end": 140,
"label": "CellType",
"start": 137,
"text": null
},
{
"end": 83,
"label": "Tissue",
"start": 79,
"text": null
},
{
"end": 86,
"label": "CellType",
"start": 79,
"text": null
},
{
"end": 88,
"label": "CellType",
"start": 79,
"text": null
}
] |
CellFinder
|
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.
|
[
{
"end": 214,
"label": "CellType",
"start": 211,
"text": null
},
{
"end": 228,
"label": "CellType",
"start": 219,
"text": null
},
{
"end": 223,
"label": "Tissue",
"start": 219,
"text": null
},
{
"end": 226,
"label": "CellType",
"start": 219,
"text": null
}
] |
CellFinder
|
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.
|
[
{
"end": 147,
"label": "CellType",
"start": 142,
"text": null
},
{
"end": 205,
"label": "CellType",
"start": 200,
"text": null
},
{
"end": 221,
"label": "CellType",
"start": 218,
"text": null
}
] |
CellFinder
|
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].
|
[] |
CellFinder
|
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.
|
[
{
"end": 308,
"label": "Tissue",
"start": 300,
"text": null
},
{
"end": 164,
"label": "Tissue",
"start": 142,
"text": null
},
{
"end": 112,
"label": "Tissue",
"start": 103,
"text": null
}
] |
CellFinder
|
In agreement, GO analysis suggested that genes containing REAP[+] exons were enriched in serine/threonine kinase activity, of which SLK is a family member.
|
[] |
CellFinder
|
Future work will be required to study the impact of AS in these genes in hESCs and NPs.
|
[
{
"end": 86,
"label": "CellType",
"start": 83,
"text": null
},
{
"end": 78,
"label": "CellType",
"start": 73,
"text": null
}
] |
CellFinder
|
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.
|
[] |
CellFinder
|
REAP[+] exons were underrepresented in genes that were differentially transcriptionally regulated in hESCs and NPs.
|
[
{
"end": 114,
"label": "CellType",
"start": 111,
"text": null
},
{
"end": 106,
"label": "CellType",
"start": 101,
"text": null
}
] |
CellFinder
|
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.
|
[] |
CellFinder
|
Finally, we identified potential cis-regulatory intronic elements conserved and enriched proximal to the REAP[+] exons.
|
[] |
CellFinder
|
In particular, the FOX1/2 binding site, GCUAG, was conserved and enriched in the flanking introns of a subset of REAP[+] exons.
|
[] |
CellFinder
|
Further studies will be required to explore the importance of FOX1 family members in early neuronal differentiation.
|
[
{
"end": 99,
"label": "Tissue",
"start": 91,
"text": null
}
] |
CellFinder
|
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.
|
[
{
"end": 179,
"label": "CellType",
"start": 171,
"text": null
}
] |
CellFinder
|
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.
|
[
{
"end": 120,
"label": "Tissue",
"start": 114,
"text": null
},
{
"end": 166,
"label": "Tissue",
"start": 158,
"text": null
}
] |
CellFinder
|
Maintenance and differentiation of hESCs and hCNS-SCns.hESC line Cy203 (Cythera) was cultured as previously described [12].
|
[
{
"end": 55,
"label": "CellType",
"start": 46,
"text": null
},
{
"end": 41,
"label": "CellType",
"start": 36,
"text": null
},
{
"end": 71,
"label": "CellLine",
"start": 66,
"text": null
},
{
"end": 53,
"label": "CellType",
"start": 46,
"text": null
},
{
"end": 50,
"label": "Tissue",
"start": 46,
"text": null
}
] |
CellFinder
|
To differentiate into neuroepithelial precursor cells, colonies were manually isolated from mouse embryonic fibroblasts (MEFs) and cut in small pieces.
|
[
{
"end": 119,
"label": "CellType",
"start": 92,
"text": null
},
{
"end": 119,
"label": "CellType",
"start": 108,
"text": null
},
{
"end": 125,
"label": "CellType",
"start": 121,
"text": null
},
{
"end": 53,
"label": "CellType",
"start": 38,
"text": null
},
{
"end": 53,
"label": "CellType",
"start": 22,
"text": null
},
{
"end": 37,
"label": "Tissue",
"start": 22,
"text": null
},
{
"end": 107,
"label": "Tissue",
"start": 98,
"text": null
}
] |
CellFinder
|
These pieces were transferred to a T75 flask with hESCs differentiation media (same hESC medium but 10% KSR and no FGF-2).
|
[
{
"end": 55,
"label": "CellType",
"start": 50,
"text": null
}
] |
CellFinder
|
Medium was changed the next day by transferring the floating hESC aggregates to a new flask.
|
[] |
CellFinder
|
After culturing for a week, the hESC cell aggregates formed mature embroid bodies (EBs; ∼10 um round clusters with dark centers).
|
[
{
"end": 86,
"label": "Tissue",
"start": 83,
"text": null
},
{
"end": 81,
"label": "Tissue",
"start": 67,
"text": null
},
{
"end": 74,
"label": "Tissue",
"start": 67,
"text": null
}
] |
CellFinder
|
EBs were plated on a coated 10-cm dish in hESC differentiation media.
|
[
{
"end": 3,
"label": "Tissue",
"start": 0,
"text": null
}
] |
CellFinder
|
The next day, the medium was changed to DMEM/F12 supplemented with ITS and fibronectin.
|
[] |
CellFinder
|
Medium was changed every other day for a week or until the cells formed rosette-like columnar structures that were isolated manually.
|
[
{
"end": 104,
"label": "Tissue",
"start": 72,
"text": null
}
] |
CellFinder
|
These structures were then transferred to coated dishes in neural induction medium (DMEM/F12 supplemented with N2 and FGF-2) for a week.
|
[
{
"end": 65,
"label": "Tissue",
"start": 59,
"text": null
}
] |
CellFinder
|
Elongated single cells were separated from leftover aggregates using non-enzymatic dissociation.
|
[] |
CellFinder
|
After one to two passages, the cells formed a monolayer of homogeneous NPs (negative for Sox1 immunostaining).
|
[
{
"end": 74,
"label": "CellType",
"start": 71,
"text": null
}
] |
CellFinder
|
Upon confluence, cells will form neurospheres that can also be isolated from the neuroepithelial precursor cells (positive for Sox1 immunostaining).
|
[
{
"end": 112,
"label": "CellType",
"start": 81,
"text": null
},
{
"end": 45,
"label": "Tissue",
"start": 33,
"text": null
},
{
"end": 96,
"label": "Tissue",
"start": 81,
"text": null
}
] |
CellFinder
|
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