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Single-cell RNA transcriptome of induced noradrenergic (iNA) neurons from astrocytes and mouse embryonic fibroblasts

ABSTRACT: We reported the generation of iNA neurons from astrocytes and MEFs by direct cell reprogramming. To compare the transcriptome of iNA neurons induced from this two kinds of starter cells, we prepared the single-cell cDNA library of iNA neurons induced from both starter cells in vitro for 28 days by 10x Genomics. Our results show that the single-cell RNA transcriptome of iNA neurons induced from astrocytes and MEFs is similar.

ORGANISM(S): Mus musculus

PROVIDER: GSE132184 | GEO | 2019/09/01

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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Single-cell RNA transcriptome of induced noradrenergic (iNA) neurons from astrocytes and mouse embryonic fibroblasts

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