Abstract
It remains controversial whether the routes from somatic cells to induced pluripotent stem cells (iPSCs) are related to the reverse order of normal developmental processes. Specifically, it remains unaddressed whether or not the differentiated cells become iPSCs through their original tissue stem cell-like state. Previous studies analyzing the reprogramming process mostly used fibroblasts; however, the stem cell characteristics of fibroblasts made it difficult to address this. Here, we generated iPSCs from mouse astrocytes, a type of glial cells, by three (OCT3/4, KLF4, and SOX2), two (OCT3/4 and KLF4), or four (OCT3/4, KLF4, and SOX2 plus c-MYC) factors. Sox1, a neural stem cell (NSC)-specific transcription factor, is transiently up-regulated during reprogramming, and Sox1-positive cells become iPSCs. The up-regulation of Sox1 is essential for OCT3/4- and KLF4-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 fibroblast reprogramming, neither Sox1 up-regulation nor an increase in neurogenic potential occurs. Our results thus demonstrate that astrocytes are reprogrammed through an NSC-like state.
Highlights
Somatic cells can be reprogrammed into a pluripotent state through ectopic expression of defined transcription factors, such as Oct3/4, Klf4, Sox2, and c-Myc (OKSM)4 [1]
Generation of induced pluripotent stem cells (iPSCs) from Mouse Astrocytes by Three (OKS) or Two (OK) Factors—Previous reports have demonstrated that mouse and human astrocytes can be reprogrammed to iPSCs by four factors (OKSM) [22, 23]
The number of alkaline phosphatase (AP)-positive colonies was smaller compared with the OKS method, iPSC colonies were generated with the OCT3/4 and KLF4 (OK) method (Fig. 1D)
Summary
Somatic cells can be reprogrammed into a pluripotent state through ectopic expression of defined transcription factors, such as Oct3/4, Klf4, Sox2, and c-Myc (OKSM)4 [1]. In control samples, which were infected with a retrovirus encoding mCherry and cultured in ESC medium for 18 days, cells remained Sox1-GFP-negative (Fig. 3E). Pluripotency marker genes, which are up-regulated in the late stage of MEF reprogramming (Utf1 and Dnmt3l), were not expressed at all in the Sox1-positive cells during astrocyte reprogramming.
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