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Genome wide mRNA sequencing of induced pluripotent stem cell (iPSC) diffrentiated neural stem cells (NSCs).

ABSTRACT: The NSC differentiation of six validated iPSC lines were induced using commercially available PSC neural induction medium and following manufacturer method with minor modifications (Gibco). An extensive characterization of generated NSCs on day 14 (at passage P1) was performed using immunocytochemistry (ICC) analysis of the NSC specific markers and by genome wide mRNA sequencing. The iPSC differentiated NSCs expressed NSC specific markers Nestin, PAX6, SOX1 and SOX2 across all six samples. The average correlation coefficient at 95% CI between 6 NSC samples, calculated based on all expressed mRNAs, was 0.97 ± 0.008 suggesting a uniform differentiation of generated NSC lines. The generated NSC showed high expression of neuroepithelial genes/transcription factors (NES, SOX2, SOX1, PAX6, NOTCH1, MSI1,and CHD2) and genes/transcription factors of dorsal tube neuroepithelium (PAX3, GDF7, SOX9 and SNAI2) but lacked the expression of ventral tube neuroepithelial markers (NKX2-2, FOXA2 and SHH) suggesting a dorsal tube neuroepithelial transcriptomic and functional profile of the generated NSCs. A differential gene expression analysis of iPSC’s and NSC’s expressed transcription identified 4,033 mRNAs that were significantly differentially expressed (DE) between iPSCs and differentiated NSCs. The 2,006 DE mRNA were significantly upregulated in differentiated NSCs and were highly enriched in developmental functional categories such as embryonic development (572 mRNAs; FDR adjusted p-value range: 1.92x10-53 – 3.26x10-9), organism development (749 mRNAs; FDR adjusted p-value range: 1.92x10-53 – 4.30x10-9), nervous system development and function (629 mRNAs; FDR adjusted p-value range: 3.23x10-48 – 4.15x10-9), tissue development (655 mRNAs; FDR adjusted p-value range: 3.63x10-40 – 4.32x10-9) and organ development (406 mRNAs; FDR adjusted p-value range: 8.74x10-40 – 2.37x10-9).

ORGANISM(S): Homo sapiens

PROVIDER: GSE156617 | GEO | 2020/08/22

REPOSITORIES: GEO

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Project description:It remains controversial whether the routes from differentiated cells to iPSCs are related to the reverse order of normal developmental processes or independent of them. Here, we generated iPSCs from mouse astrocytes by three (Oct3/4, Klf4 and Sox2 (OKS)), two (OK), or four (OKS plus c-Myc) factors. Sox1, a neural stem cell (NSC)-specific transcription factor, is transiently upregulated during reprogramming and Sox1-positive cells become iPSCs. The upregulation of Sox1 is essential for OK-induced reprogramming. Genome-wide analysis revealed that the gene expression profile of Sox1-expressing intermediate-state cells resembles that of NSCs. Furthermore, the intermediate-state cells are able to generate neurospheres, which can differentiate into both neurons and glial cells. Remarkably, during MEF reprogramming, neither Sox1 upregulation nor an increase in neurogenic potential occurs. Thus, astrocytes are reprogrammed through an NSC-like state, suggesting that reprogramming partially follows the retrograde pathway of normal developmental processes. To investigate the gene expression profile of intermediate-state cells during astrocyte reprogramming, we performed genome-wide gene expression analysis in five samples; starting astrocytes, intermediate-state cells expressing Sox1-GFP, NSCs, iPSCs established from astrocytes, and iPSCs established from MEFs (iPS-MEF-Ng-20D-17) that had previously been reported (Okita, K. et al. Nature 448: 313-317 (2007)). Two (NSCs, iPSCs from astrocytes and MEFs) or three (astrocytes, intermediate-state cells) biological replicates were prepared for microarray samples. Total RNA was extracted with an RNeasy kit (Qiagen). cDNA synthesis and transcriptional amplification were performed using 50-100 ng of total RNA with the GeneChip WT PLUS Reagent Kit (Affymetrix). Fragmented and biotin-labeled cDNA targets were hybridized to GeneChip Mouse Gene 1.0 ST arrays (Affymetrix) according to the manufacturerâ??s protocol. Hybridized arrays were scanned using an Affymetrix GeneChip Scanner.

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Genome wide mRNA sequencing of induced pluripotent stem cell (iPSC) diffrentiated neural stem cells (NSCs).

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