Reprogramming somatic cells to induced pluripotent stem (iPS) cells has been accomplished by expressing pluripotency factors and oncogenes1–8, 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. We show that reprogramming factors can activate the p53 pathway. Reducing signaling to p53 by expressing a mutated version of one of its negative regulators, by deleting or silencing p53 or its target gene, p21, or by antagonizing apoptosis enhanced three factor (Oct4/Sox2/Klf4)-mediated reprogramming of mouse fibroblasts. Notably, decreasing p53 protein levels enabled fibroblasts to give rise to iPS cells capable of generating germline transmitting chimeric mice using only Oct4 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.

Human pluripotent stem cells, such as embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), have the unique abilities of differentiation into any cell type of the organism (pluripotency) and indefinite selfrenewal. Here, we show that the Rem2 GTPase, a suppressor of the p53 pathway, is up-regulated in hESCs and, by loss-and gain-of-function studies, that it is a major player in the maintenance of hESC self-renewal and pluripotency. We show that Rem2 mediates the fibroblastic growth factor 2 (FGF2) signaling pathway to maintain proliferation of hESCs. We demonstrate that Rem2 effects are mediated by suppressing the transcriptional activity of p53 and cyclin D 1 to maintain survival of hESCs. Importantly, Rem2 does this by preventing protein degradation during DNA damage. Given that Rem2 maintains hESCs, we also show that it is as efficient as c-Myc by enhancing reprogramming of human somatic cells into iPSCs eightfold. Rem2 does this by accelerating the cell cycle and protecting from apoptosis via its effects on cyclin D 1 expression/localization and suppression of p53 transcription. We show that the effects of Rem2 on cyclin D 1 are independent of p53 function. These results define the cell cycle and apoptosis as a rate-limiting step during the reprogramming phenomena. Our studies highlight the possibility of reprogramming somatic cells by imposing hESC-specific cell cycle features for making safer iPSCs for cell therapy use.[Keywords: Rem2; cyclin D1; p53; reprogramming; self-renewal] Supplemental material is available at http://www.genesdev.org. Received October 20, 2009; revised version accepted February 3, 2010. In recent years, the field of pluripotent human embryonic stem cells (hESCs), including the discovery of induced pluripotent stem cells (iPSCs), has moved rapidly in the direction of finding a safe application for clinical use, such as cell replacement therapy and modeling for drug discovery. However, relatively little has been done to advance our mechanistic insights into the properties of self-renewing hESCs, and even less is known about the mechanisms governing iPSC formation. A better understanding of the molecular mechanisms controlling pluripotency and self-renewal would be essential for the clinical translation of hESCs and iPSCs.hESCs were first derived from the pluripotent cells of the blastocyst inner cell mass and can be maintained in vitro indefinitely with the addition of fibroblastic growth factor 2 (FGF2) and other unknown factors secreted from feeder cell layers (Thomson et al. 1998). The pluripotency of hESCs is regulated by a set of unique transcription factors including Oct4, Sox2, and Nanog (Chambers and Smith 2004). It has been shown that a combination of three or four factors of Oct4, Sox2, and KLf4, with or without Myc, can reprogram somatic cells to generate iPSCs (Takahashi and Yamanaka 2006;Takahashi et al. 2007). Analysis of partially reprogrammed iPSCs reveals temporal and separable contributions of the four factors and indicates that ectopic c-Myc ac...

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In vitro, high-risk human papillomavirus E6 proteins have been shown, in conjunction with E6-associated protein (E6AP), to mediate ubiquitination of p53 and its degradation by the 26S proteasome by a pathway that is thought to be analogous to Mdm2-mediated p53 degradation. However, differences in the requirements of E6/E6AP and Mdm2 to promote the degradation of p53, both in vivo and in vitro, suggest that these two E3 ligases may promote p53 degradation by distinct pathways. Using tools that disrupt ubiquitination and degradation, clear differences between E6-and Mdm2-mediated p53 degradation are presented. The consistent failure to fully protect p53 protein from E6-mediated degradation by disrupting the ubiquitin-degradation pathway provides the first evidence of an E6-dependent, ubiquitin-independent, p53 degradation pathway in vivo.

SummarySince the first generation of induced pluripotent stem cells (iPSCs), several reprogramming systems have been used to study its molecular mechanisms. However, the system of choice largely affects the reprogramming efficiency, influencing our view on the mechanisms. Here, we demonstrate that reprogramming triggered by less efficient polycistronic reprogramming cassettes not only highlights mesenchymal-to-epithelial transition (MET) as a roadblock but also faces more severe difficulties to attain a pluripotent state even post-MET. In contrast, more efficient cassettes can reprogram both wild-type and Nanog−/− fibroblasts with comparable efficiencies, routes, and kinetics, unlike the less efficient reprogramming systems. Moreover, we attribute a previously reported variation in the N terminus of KLF4 as a dominant factor underlying these critical differences. Our data establish that some reprogramming roadblocks are system dependent, highlighting the need to pursue mechanistic studies with close attention to the systems to better understand reprogramming.