Oncogenic transcription factors are known to mediate the conversion of somatic cells to tumour or induced pluripotent stem cells (iPSCs). Here we report c-Jun as a barrier for iPSC formation. c-Jun is expressed by and required for the proliferation of mouse embryonic fibroblasts (MEFs), but not mouse embryonic stem cells (mESCs). Consistently, c-Jun is induced during mESC differentiation, drives mESCs towards the endoderm lineage and completely blocks the generation of iPSCs from MEFs. Mechanistically, c-Jun activates mesenchymal-related genes, broadly suppresses the pluripotent ones, and derails the obligatory mesenchymal to epithelial transition during reprogramming. Furthermore, inhibition of c-Jun by shRNA, dominant-negative c-Jun or Jdp2 enhances reprogramming and replaces Oct4 among the Yamanaka factors. Finally, Jdp2 anchors 5 non-Yamanaka factors (Id1, Jhdm1b, Lrh1, Sall4 and Glis1) to reprogram MEFs into iPSCs. Our studies reveal c-Jun as a guardian of somatic cell fate and its suppression opens the gate to pluripotency.

Generation of induced pluripotent stem cells from somatic cells using defined factors has potential relevant applications in regenerative medicine and biology. However, this promising technology remains inefficient and time consuming. We have devised a serum free culture medium termed iSF1 that facilitates the generation of mouse induced pluripotent stem cells. This optimization of the culture medium is sensitive to the presence of Myc in the reprogramming factors. Moreover, we could reprogram meningeal cells using only two factors Oct4/Klf4. Therefore, iSF1 represents a basal medium that may be used for mechanistic studies and testing new reprogramming approaches.The discovery that four transcription factors Oct4, Sox2, Klf4, and Myc can revert differentiated somatic cells to a pluripotent state resembling embryonic stem cells (ESCs) 3 (1-4) not only demonstrates the remarkable plasticity of the mammalian genome, but also offers a unique opportunity to investigate the mechanisms associated with cell fate determination at the molecular level (5, 6). Besides, human induced pluripotent stem cells (iPSCs) overcome ethical issues associated with human ESCs and the risk of immune rejection (7,8). However, the reprogramming process remains largely an art form, with low efficiency and inconsistency among different experimental settings (1,3,4,6). Nowadays, the mouse system remains the model in which many mechanistic/technical breakthroughs are achieved. Therefore, albeit more robust than the human setting, it is important to improve mouse iPSC generation to achieve a better understanding of nuclear reprogramming. Here, we report an optimized method to generate mouse iPSCs consistently with high efficiency that is potentially useful for multiple applications including screening for small molecules, dissection of molecular mechanisms, and testing of new methods. EXPERIMENTAL PROCEDURESCell Culture-Mouse embryonic fibroblasts (MEFs) were derived from day 13.5 embryos (e13.5) hemizygous for the Oct4-GFP transgenic allele (9, 10) and Rosa26 allele and were maintained in fibroblast medium: DMEM supplemented with 10% fetal bovine serum (FBS), L-glutamine, and non-essential amino acid. iPSCs and ESCs were routinely expanded on MEF feeder layers (MEFs inactivated with mitomycin C) in both FBS-containing medium (mES) or KSR medium. mES medium consisted of DMEM supplemented with 15% FBS, L-glutamine, NEAA, sodium pyruvate, penicillin/streptomycin, ␤-mercaptoethanol, and 1000 units/ml leukemia inhibitory factor (Millipore). KSR medium consisted of knock-out DMEM supplemented with 15% knock-out Serum Replacement (SR), L-glutamine, NEAA, penicillin/streptomycin, ␤-mercaptoethanol, and 1000 units/ml LIF. fSF1 consisted of DMEM/F12 supplemented with 10% SR, 1/200 N2, L-glutamine, NEAA, penicillin/streptomycin, 1000 units/ml LIF, and 5 ng/ml basic FGF. iSF1 was like fSF1 but contained high glucose DMEM. Unless otherwise indicated, all reagents were purchased from Invitrogen. The FBS lot number was 709778, which was used in our previo...

Cell fate determination is a major unsolved problem in cell and developmental biology. The discovery of reprogramming by pluripotent factors offers a rational system to investigate the molecular mechanisms associated with cell fate decisions. The idea that reprogramming of fibroblasts starts with a mesenchymal-epithelial transition (MET) suggests that the process is perhaps a reversal of epithelial to mesenchymal transition (EMT) found frequently during early embryogenesis. As such, we believe that investigations into MET-EMT may yield detailed molecular insights into cell fate decisions, not only for the switching between epithelial and mesenchymal cells, but also other cell types.