Epithelial-to-mesenchymal transition (EMT) is a developmental process important for cell fate determination. Fibroblasts, a product of EMT, can be reset into induced pluripotent stem cells (iPSCs) via exogenous transcription factors but the underlying mechanism is unclear. Here we show that the generation of iPSCs from mouse fibroblasts requires a mesenchymal-to-epithelial transition (MET) orchestrated by suppressing pro-EMT signals from the culture medium and activating an epithelial program inside the cells. At the transcriptional level, Sox2/Oct4 suppress the EMT mediator Snail, c-Myc downregulates TGF-beta1 and TGF-beta receptor 2, and Klf4 induces epithelial genes including E-cadherin. Blocking MET impairs the reprogramming of fibroblasts whereas preventing EMT in epithelial cells cultured with serum can produce iPSCs without Klf4 and c-Myc. Our work not only establishes MET as a key cellular mechanism toward induced pluripotency, but also demonstrates iPSC generation as a cooperative process between the defined factors and the extracellular milieu. PAPERCLIP:
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...
A new method for the synthesis of δ- and α-carbolines through Ni-catalyzed [2 + 2 + 2] cycloaddition of ynamide-nitriles or alkyne-cyanamides with alkynes has been developed. The catalytic system of NiCl(DME)/dppp/Zn with a low-cost Ni(II)-precursor was first utilized in Ni-catalyzed [2 + 2 + 2] cycloaddition reactions, and the in situ generated Lewis acid may play an important role for the successful transformation. Not only internal alkynes but also terminal alkynes undergo the desired cycloaddition reactions efficiently to furnish the carboline derivatives with wide diversity and functional group tolerance.
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