Immortalization eliminates a roadblock during cellular reprogramming into iPS cells

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Abstract

The overexpression of defined transcription factors in somatic cells results in their reprogramming into induced pluripotent stem (iPS) cells1,2,3. The extremely low efficiency and slow kinetics of in vitro reprogramming suggest that further rare events are required to generate iPS cells. The nature and identity of these events, however, remain elusive. We noticed that the reprogramming potential of primary murine fibroblasts into iPS cells decreases after serial passaging and the concomitant onset of senescence. Consistent with the notion that loss of replicative potential provides a barrier for reprogramming, here we show that cells with low endogenous p19Arf (encoded by the Ink4a/Arf locus, also known as Cdkn2a locus) protein levels and immortal fibroblasts deficient in components of the Arf–Trp53 pathway yield iPS cell colonies with up to threefold faster kinetics and at a significantly higher efficiency than wild-type cells, endowing almost every somatic cell with the potential to form iPS cells. Notably, the acute genetic ablation of Trp53 (also known as p53) in cellular subpopulations that normally fail to reprogram rescues their ability to produce iPS cells. Our results show that the acquisition of immortality is a crucial and rate-limiting step towards the establishment of a pluripotent state in somatic cells and underscore the similarities between induced pluripotency and tumorigenesis.

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Figure 1: Reprogramming efficiency of fibroblasts is influenced by replicative potential and Arf expression status.
Figure 2: Transcription-factor-induced downregulation of Ink4a/Arf expression in cells undergoing reprogramming.
Figure 3: Cellular immortalization enhances reprogramming potential and kinetics.
Figure 4: Trp53 deficiency rescues reprogramming potential in cells that normally fail to form iPS cells.

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Acknowledgements

We thank M. Roussel and C. Sherr for providing us with Arf–GFP cells, D. C. Bennett and E. Sviderskaya for sharing Melan A cells, and A. Ventura and T. Jacks for tail biopsies of conditional Trp53-mutant mice. We also thank A. Tzatsos and N. Bardeesy for suggestions, for critical reading of the manuscript and for providing Ink4a/Arf-/- MEFs. We are grateful to P. Follett for blastocyst injections and L. Prickett and K. Folz-Donahue for assistance with FACS. J.U. was supported by a postdoctoral fellowship from the Mildred Scheel Foundation, J.M.P. by an ECOR fellowship, and M.S. by a fellowship from the Schering Foundation. J.G.R. was supported by an NIH Skin Disease Research Center Grant. N.M. was supported by a graduated scholarship from the Natural Sciences and Engineering Council of Canada. Support to K.H. came from the NIH Director’s Innovator Award, the Harvard Stem Cell Institute, the Kimmel Foundation and the V Foundation.

Author Contributions J.U., J.M.P. and K.H. conceived the study, interpreted results and wrote the manuscript, J.U. and J.M.P. performed most of the experiments with help from W.K., R.M.W. and A.K. M.S., N.M. and J.G.R. provided essential study material and helped with interpretation of results.

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Correspondence to Konrad Hochedlinger.

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Utikal, J., Polo, J., Stadtfeld, M. et al. Immortalization eliminates a roadblock during cellular reprogramming into iPS cells. Nature 460, 1145–1148 (2009) doi:10.1038/nature08285

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