Abstract
The introduction of four transcription factors Oct4, Klf4, Sox2 and c-Myc by viral transduction can induce reprogramming of somatic cells into induced pluripotent stem cells (iPSCs), but the use of iPSCs is hindered by the use of viral delivery systems. Chemical-induced reprogramming offers a novel approach to generating iPSCs without any viral vector-based genetic modification. Previous reports showed that several small molecules could replace some of the reprogramming factors although at least two transcription factors, Oct4 and Klf4, are still required to generate iPSCs from mouse embryonic fibroblasts. Here, we identify a specific chemical combination, which is sufficient to permit reprogramming from mouse embryonic and adult fibroblasts in the presence of a single transcription factor, Oct4, within 20 days, replacing Sox2, Klf4 and c-Myc. The iPSCs generated using this treatment resembled mouse embryonic stem cells in terms of global gene expression profile, epigenetic status and pluripotency both in vitro and in vivo. We also found that 8 days of Oct4 induction was sufficient to enable Oct4-induced reprogramming in the presence of the small molecules, which suggests that reprogramming was initiated within the first 8 days and was independent of continuous exogenous Oct4 expression. These discoveries will aid in the future generation of iPSCs without genetic modification, as well as elucidating the molecular mechanisms that underlie the reprogramming process.
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Acknowledgements
We thank JY Guan, JQ Ye, X Li, GF Meng, T Zhao, TT Xiang, JC Wang, HS Liu, X Zhang and X Sui (Peking University) for technical assistance and Dr Hochedlinger (Harvard University) for providing the doxycycline (Dox)-inducible lentiviral vectors. This research was supported by a Ministry of Science and Technology grant (2006AA02A113), Beijing Nature Science Foundation Grant (5100002), Science and Technology Plan of Beijing Municipal Government (D07050701350705), National Basic Research Program for China (973 Program 2009CB941101, 2010CB945204), National Natural Science Foundation of China (90919031 and 30421004), the Chinese Science and Technology Key Project (2009zx10004-403), a Bill and Melinda Gates Foundation Grant (37871) and a 111 Project to HD.
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( Supplementary information is linked to the online version of the paper on the Cell Research website.)
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Supplementary information, Figure S1
Small molecules used at different concentrations in Oct4-induced reprogramming. (PDF 485 kb)
Supplementary information, Figure S2
iPSCs induced from adult mouse fibroblasts by Oct4 and small molecules. (PDF 5438 kb)
Supplementary information, Figure S3
Endogenous expression of reprogramming factors in oct4-induced reprogramming. (PDF 505 kb)
Supplementary information, Table S1
Primers for RT-PCR and genome PCR test. (PDF 56 kb)
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Li, Y., Zhang, Q., Yin, X. et al. Generation of iPSCs from mouse fibroblasts with a single gene, Oct4, and small molecules. Cell Res 21, 196–204 (2011). https://doi.org/10.1038/cr.2010.142
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DOI: https://doi.org/10.1038/cr.2010.142
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