Linking the p53 tumour suppressor pathway to somatic cell reprogramming

Reprogramming somatic cells to induced pluripotent stem (iPS) cells has been accomplished by expressing pluripotency factors and oncogenes, but the low frequency and tendency to induce malignant transformation 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. The p53 pathway reduces cancer initiation by inducing apoptosis or cell cycle arrest in response to a variety of stress signals, including overexpressed oncogenes such as c-Myc. Klf4 can either activate or antagonize p53, depending on the cell type used and expression level. Consequently, reprogramming efficiency is probably reduced through oncogene-mediated activation of the p53 pathway. This is consistent with previous results showing that germ cells can be spontaneously reprogrammed in the absence of p53 (ref. 11), and a combination of p53 short interfering RNA (shRNA) and Utf1 expression increased iPS cell formation12 We first determined whether the reprogramming factors, individually or in combination, activate the p53 pathway in mouse embryo fibroblasts (MEFs). Relative to the green fluorescent protein (GFP)retroviral control, c-Myc considerably increased p53 abundance and activity, manifested by increased expression of the cyclin-dependent kinase inhibitor p21 (Fig. 1a). This was achieved by induction of p19, an antagonist of Mdm2, the E3-ubiquitin ligase principally responsible for p53 degradation. Increased p21 protein levels were also observed in MEFs infected with Klf4 alone, with Oct4 and Sox2 (two factors) or with Oct, Sox2 and Klf4 (three factors) (Fig. 1a). Because introducing reprogramming factors increased c-H2ax (also known as c-H2afx) foci (Supplementary Fig. 1), we infer that the expression of reprogramming factors may induce p53 activity by DNA damage. We also compared p53 and p21 expression in a variety of mouse and human cell lines previously used for iPS cell production (Supplementary Fig. 2). Interestingly, keratinocytes, which have higher reprogramming efficiency, display lower p53 and p21 protein levels than other cell types. Moreover, p21 induction in keratinocytes is lower than in fibroblasts after infection with the three factors (Supplementary Fig. 3). Together, these data indicate that the p53 pathway is one determinant of reprogramming efficiency. We therefore tested the effects of reducing p53 signalling by determining reprogramming efficiencies in cells in which p53 function was reduced by shRNA or ablated by homologous recombination. Most cellswere infectedwith shRNA(Supplementary Fig. 4), p53messenger RNA and protein levels were reduced by 60–80% (Fig. 1c and Supplementary Fig. 5), and iPS cell colony formation was increased by 2–4-fold using two different shRNAs (Fig. 1b, c). This probably underestimates p53 suppressive capacity, because functional p53 protein clearly remained present, as indicated by the ability of the p53 activating agent Nutlin-3a (ref. 14) to dose-dependently reduce iPS cell formation inMEFs treated with themost effective p53 shRNA (Fig. 1d). In contrast, reprogramming efficiency was increased by at least tenfold in p53-null MEFs, and this was not reduced by Nutlin-3a (Supplementary Table 1 and Fig. 1e, g). p53 heterozygous MEFs also exhibited higher three-factor-reprogramming efficiency than wild-type MEFs. Although culture stress can induce cellular senescence and activate p53, which would reduce reprogramming, less than 1% of the cells of all p53 genotypes stained with the senescence marker b-galactosidase (Supplementary Fig. 6). Because we did not detect loss of heterozygosity of the p53 gene in iPS cell colonies derived from p53MEFs (Supplementary Fig. 7), the data suggest a p53 dosage-sensitivity to reprogramming (Fig. 1e). We were concerned that because p53-null MEFs are genetically unstable, increased reprogramming efficiency might result from expression of the three factors in variant cells.However, we found that re-expressing p53 protein in the p53-null MEFs markedly reduced reprogramming efficiency (Fig. 1f). Reducing factors downstream of p53 also increased reprogramming efficiency. For example, p21 shRNA increased reprogramming by approximately threefold (Fig. 1h). This probably underestimates the magnitude to which p21 induction suppresses reprogramming as p21-shRNA-expressing cells still responded toNutlin-3a treatment as discussed earlier. We also noted a modest induction of the proapoptotic factor Bax, another p53-inducible gene, in three-factor experiments (data not shown). Consistent with a limiting role of the p53-induced apoptotic response during reprogramming, overexpression of the Bax antagonist Bcl2 suppressed apoptosis in two, three and four factor experiments, and increased the frequency of colonies expressing the pluripotency factor Nanog by fourfold