Reprogramming somatic cells to induced pluripotent stem (iPS) cells has been accomplished by expressing pluripotency factors and oncogenes1,2,3,4,5,6,7,8, but the low frequency and tendency to induce malignant transformation9 compromise the clinical utility of this powerful approach. We address both issues by investigating the mechanisms limiting reprogramming efficiency in somatic cells. Here we show that reprogramming factors can activate the p53 (also known as Trp53 in mice, TP53 in humans) pathway. Reducing signalling to p53 by expressing a mutated version of one of its negative regulators, by deleting or knocking down p53 or its target gene, p21 (also known as Cdkn1a), or by antagonizing reprogramming-induced apoptosis in mouse fibroblasts increases reprogramming efficiency. Notably, decreasing p53 protein levels enabled fibroblasts to give rise to iPS cells capable of generating germline-transmitting chimaeric mice using only Oct4 (also known as Pou5f1) and Sox2. Furthermore, silencing of p53 significantly increased the reprogramming efficiency of human somatic cells. These results provide insights into reprogramming mechanisms and suggest new routes to more efficient reprogramming while minimizing the use of oncogenes.
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We are grateful to the CMRB Histology & Bioimaging and Cell culture Platforms for assistance, S. Boue for microarray analysis, S. Kim for help with maintenance of mouse colonies, Y. Richaud for technical assistance, M. Nagao for preparation of mouse neural stem cells, K. Brennand and F. Gage for preparation of human neural stem cells, I. Verma and A. Consiglio for advice and help with lentiviral transduction, Y. Dayn for chimaeric mouse production, and all members of the Gene Expression Laboratory and CMRB for discussions and M. Serrano for sharing unpublished results. J.S. was partially supported by Astellas Pharma Inc. T.K. was partially supported by Japan Society for the Promotion of Science. Work in the laboratory of G.M.W. was supported by NIH grants (5 R01 CA061449 and CA100845). Work in the laboratory of J.C.I.B. was supported by grants from the NIH, Tercel, Marato, G. Harold and Leila Y. Mathers Charitable Foundation and Fundacion Cellex.
Author Contributions T.K. and J.S. contributed to the experimental work, project planning, data analysis and writing the manuscript and contributed equally to this work. Y.V.W. contributed to the experimental work, data analysis, writing the manuscript and established Mdmx mutant mice. S.M., L.B.M. and A.R. contributed to the experimental work, data analysis and writing the manuscript. G.M.W. and J.C.I.B. contributed to project planning and writing the manuscript, and supervised all the work. G.M.W. and J.C.I.B. are co-contributing corresponding authors.
This file contains Supplementary Tables 1-3, Supplementary References and Supplementary Figures 1-27 with Legends. (PDF 12910 kb)
This file contains microarray analysis on MEF, 2F-p53KD-iPS cell clones (#1 and #6) and mouse ES. (XLS 8680 kb)
In this movie file beating cells were observed in embryoid bodies (d10) derived from 2F-iPS clone #1. (MOV 566 kb)
In this movie file beating cells were observed in embryoid bodies (d14) derived from 2F-iPS clone #3. (MOV 567 kb)
In this movie file beating cells were observed in embryoid bodies (d15) derived from 2F-iPS clone #6. (MOV 593 kb)
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Kawamura, T., Suzuki, J., Wang, Y. et al. Linking the p53 tumour suppressor pathway to somatic cell reprogramming. Nature 460, 1140–1144 (2009). https://doi.org/10.1038/nature08311
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