Abstract
Direct reprogramming of somatic cells into induced pluripotent stem (iPS) cells can be achieved by overexpression of Oct4, Sox2, Klf4 and c-Myc transcription factors, but only a minority of donor somatic cells can be reprogrammed to pluripotency. Here we demonstrate that reprogramming by these transcription factors is a continuous stochastic process where almost all mouse donor cells eventually give rise to iPS cells on continued growth and transcription factor expression. Additional inhibition of the p53/p21 pathway or overexpression of Lin28 increased the cell division rate and resulted in an accelerated kinetics of iPS cell formation that was directly proportional to the increase in cell proliferation. In contrast, Nanog overexpression accelerated reprogramming in a predominantly cell-division-rate-independent manner. Quantitative analyses define distinct cell-division-rate-dependent and -independent modes for accelerating the stochastic course of reprogramming, and suggest that the number of cell divisions is a key parameter driving epigenetic reprogramming to pluripotency.
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Acknowledgements
We would like to thank N. Maheshri, K. Wood, Y. Bugnanim, B. Carey, J. Cassady and F. Soldner for commenting on the manuscript; J. Dausmann, S. Markoulaki, P. Schorderet and M. Pawlak for conducting blastocysts injections; and members of the Jaenisch laboratory for discussions. R.J. is supported by grants from the NIH (RO1-HDO45022, R37-CA084198, RO1-CA087869). J.H. is supported by a postdoctoral fellowship from Helen Hay Whitney Foundation. K.S. is supported by the Society in Science: The Brano-Weiss fellowship. J.v.Z. is supported by the Human Frontiers Science Program. A.v.O. is supported by a National Institutes of Health (NIH) Director’s Pioneer award and an award from the National Cancer Institute (U54CA143874).
Author Contributions J.H., K.S. and R.J. conceived the ideas for this study and designed the experiments. J.H. conducted the reprogramming experiments and data collection. M.P.C. and C.J.L. assisted in construct preparation, transgene induction and blastocyst injections. K.S., B.P., J.v.Z, J.H., and A.v.O. performed statistical and numerical analyses, and also constructed the stochastic models. J.H., K.S. and R.J. wrote the manuscript with contributions from all other authors.
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R.J. is an advisor to Stemgent and a cofounder of Fate Therapeutics.
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Hanna, J., Saha, K., Pando, B. et al. Direct cell reprogramming is a stochastic process amenable to acceleration . Nature 462, 595–601 (2009). https://doi.org/10.1038/nature08592
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DOI: https://doi.org/10.1038/nature08592
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