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:

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by defined factors. However, the low efficiency and slow kinetics of the reprogramming process have hampered progress with this technology. Here we report that a natural compound, vitamin C (Vc), enhances iPSC generation from both mouse and human somatic cells. Vc acts at least in part by alleviating cell senescence, a recently identified roadblock for reprogramming. In addition, Vc accelerates gene expression changes and promotes the transition of pre-iPSC colonies to a fully reprogrammed state. Our results therefore highlight a straightforward method for improving the speed and efficiency of iPSC generation and provide additional insights into the mechanistic basis of the reprogramming process.

Human induced pluripotent stem cells (iPSCs) have been generated with varied efficiencies from multiple tissues. Yet, acquiring donor cells is, in most instances, an invasive procedure that requires laborious isolation. Here we present a detailed protocol for generating human iPSCs from exfoliated renal epithelial cells present in urine. This method is advantageous in many circumstances, as the isolation of urinary cells is simple (30 ml of urine are sufficient), cost-effective and universal (can be applied to any age, gender and race). Moreover, the entire procedure is reasonably quick--around 2 weeks for the urinary cell culture and 3-4 weeks for the reprogramming--and the yield of iPSC colonies is generally high--up to 4% using retroviral delivery of exogenous factors. Urinary iPSCs (UiPSCs) also show excellent differentiation potential, and thus represent a good choice for producing pluripotent cells from normal individuals or patients with genetic diseases, including those affecting the kidney.

Induced pluripotent stem cell (iPS) technology appears to be a general strategy to generate pluripotent stem cells from any given mammalian species. So far, iPS cells have been reported for mouse, human, rat, and monkey. These four species have also established embryonic stem cell (ESC) lines that serve as the gold standard for pluripotency comparisons. Attempts have been made to generate porcine ESC by various means without success. Here we report the successful generation of pluripotent stem cells from fibroblasts isolated from the Tibetan miniature pig using a modified iPS protocol. The resulting iPS cell lines more closely resemble human ESC than cells from other species, have normal karyotype, stain positive for alkaline phosphatase, express high levels of ESC-like markers (Nanog, Rex1, Lin28, and SSEA4), and can differentiate into teratomas composed of the three germ layers. Because porcine physiology closely resembles human, the iPS cells reported here provide an attractive model to study certain human diseases or assess therapeutic applications of iPS in a large animal model. Induced nuclear reprogramming through induced pluripotent stem cell (iPS)2 technology is an amazing achievement full of challenge to the intellect and important practical implications (1, 2). Overexpression of exogenous factors that are highly enriched in embryonic stem cell (ESC) can rearrange the genetic program of different cell types, including somatic and adult stem cells, and induce a long lasting ESC-like pluripotent state (3-7). The repercussions of iPS technology are vast: it provides a way to create patient-specific stem cells that bypasses ethical and technical issues surrounding human ESC derivation and somatic cell nuclear transfer (8, 9), a state of the art model for studying genetic diseases in vitro (10, 11), and an incredible backwards route that can crystallize our current understanding of developmental and stem cell biology. Many questions, especially mechanistic, remain unanswered, but the current rhythm of research may bring iPS to clinical application sooner than expected. However, before jumping onto such extraordinary endeavor, safety must be scrupulously tested in an animal model close enough to humans. Nowadays that iPS technology is expanding, with improved delivery systems, chemical additions, new tissue culture conditions, and multiple cell sources being reported regularly, such animal model is essential to set up quality standards (12-18). Mice, and maybe rats, will possibly continue unrivalled as the easier ways to learn about reprogramming machinery and improve methodology, but their size, physiology, and reduced lifespan are handicaps for making serious assumptions regarding safety in humans. Given philogenetic similarity, monkeys are theoretically an excellent alternative, but in practice ethical concerns remain to at least some extent, and they are neither easy to maintain nor to breed. Swine, a regular source of food whose farming humans have adapted over myriads of years and whose physiology is r...

The umbilical cord and placenta are extra-embryonic tissues of particular interest for regenerative medicine. They share an early developmental origin and are a source of vast amounts of cells with multilineage differentiation potential that are poorly immunogenic and without controversy. Moreover, these cells are likely exempt from incorporated mutations when compared with juvenile or adult donor cells such as skin fibroblasts or keratinocytes. Here we report the efficient generation of induced pluripotent stem cells (iPSCs) from mesenchymal cells of the umbilical cord matrix (up to 0.4% of the cells became reprogrammed) and the placental amniotic membrane (up to 0.1%) using exogenous factors and a chemical mixture. iPSCs from these 2 tissues homogeneously showed human embryonic stem cell (hESC)-like characteristics including morphology, positive staining for alkaline phosphatase, normal karyotype, and expression of hESC-like markers including Nanog, Rex1, Oct4, TRA-1-60, TRA-1-80, SSEA-3, and SSEA-4. Selected clones also formed embryonic bodies and teratomas containing derivatives of the 3 germ layers, and could as well be readily differentiated into functional motor neurons. Among other things, our cell lines may prove useful for comparisons between iPSCs derived from multiple tissues regarding the extent of the epigenetic reprogramming, differentiation ability, stability of the resulting lineages, and the risk of associated abnormalities.

Somatic cell reprogramming by exogenous factors requires cooperation with transcriptional co-activators and co-repressors to effectively remodel the epigenetic environment. How this interplay is regulated remains poorly understood. Here, we demonstrate that NCoR/SMRT co-repressors bind to pluripotency loci to create a barrier to reprogramming with the four Yamanaka factors (OCT4, SOX2, KLF4 and c-MYC), and consequently, suppressing NCoR/SMRT significantly enhances reprogramming efficiency and kinetics. The core epigenetic subunit of the NCoR/SMRT complex, histone deacetylase 3 (HDAC3), contributes to the effects of NCoR/SMRT by inducing histone deacetylation at pluripotency loci. Among the Yamanaka factors, recruitment of NCoR/SMRT-HDAC3 to genomic loci is mostly facilitated by c-MYC. Hence, we describe how c-MYC is beneficial for the early phase of reprogramming but deleterious later. Overall, we uncover a role for NCoR/SMRT co-repressors in reprogramming and propose a dual function for c-MYC in this process.

The deepest rocks known from within Earth are fragments of normal mantle (Ϸ400 km) and metamorphosed sediments (Ϸ350 km), both found exhumed in continental collision terranes. Here, we report fragments of a highly reduced deep mantle environment from at least 300 km, perhaps very much more, extracted from chromite of a Tibetan ophiolite. The sample consists, in part, of diamond, coesite-after-stishovite, the high-pressure form of TiO 2, native iron, high-pressure nitrides with a deep mantle isotopic signature, and associated SiC. This appears to be a natural example of the recently discovered disproportionation of Fe 2؉ at very high pressure and consequent low oxygen fugacity (fO2) in deep Earth. Encapsulation within chromitite enclosed within upwelling solid mantle rock appears to be the only vehicle capable of transporting these phases and preserving their low-fO 2 environment at the very high temperatures of oceanic spreading centers.boron nitride ͉ coesite after stishovite ͉ Luobasa chromitite ͉ TiO2 II ͉ titanium nitride-osbornite U ntil recently, the deepest rock samples recovered from Earth's interior were xenoliths carried to the surface in explosive eruptions of kimberlite and related rocks, with maximum depth of Ϸ300 km (1). However, the advent of microstructural analysis of rocks from continental collision zones has established exhumation from still greater depths [peridotites from Ͼ300-400 km (2-5) and metamorphosed sediments from Ϸ350 km (6)]. More recently, a small fraction of diamonds from kimberlitic rocks has been found to carry inclusions that strongly suggest even greater depths, perhaps 1,700 km (7), but rocks that previously incorporated those diamonds at depth have not been recognized. We report here discovery of unexpected mineral assemblages from an ocean-spreading center environment, representing another window into Earth's deep interior.The discovery consists of very high-pressure and highly reduced phases extracted from chromitite within the mantle portion of an only lightly altered (e.g., small amounts of serpentine) fossil cross-section of oceanic crust and uppermost mantle (an ophiolite) in Tibet. Initial description of this material (8) documented diamonds and coesite-after-stishovite, establishing a minimum depth of origin of Ϸ300 km. This surprising and curious discovery was strengthened by observation of coesite and diopside lamellae within chromite collected from the same outcrop (9) and, very importantly, discovery of diamonds and indicators of very low oxygen fugacity from similar chromitite in a second ophiolite in the Polar Ural Mountains (10), establishing that the Tibetan occurrence is not unique. This discovery strongly enhances the conclusion of ref. 8 that this occurrence of very high-pressure minerals is not the result of meteorite impact. Thus, a subset of ophiolites (currently of unknown abundance) contains high-pressure and reduced phases, despite the overwhelming evidence that the ophiolites themselves formed at oceanic spreading centers under oxidizing conditions...

Recent pre-clinical and clinical studies have suggested that endogenous cardiospheres (eCS) are potentially safe and effective for cardiac regeneration following myocardial infarction (MI). Nevertheless the preparation of autologous eCS requires invasive myocardial biopsy with limited yield. We describe a novel approach to generate induced cardiospheres (iCS) from adult skin fibroblasts via somatic reprogramming. After infection with Sox2, Klf4, and Oct4, iCS were generated from mouse adult skin fibroblasts treated with Gsk3b inhibitor-(2 0 Z,0 -oxime and Oncostatin M. They resembled eCS, but contained a higher percentage of cells expressing Mesp1, Isl1, and Nkx2.5. They were differentiated into functional cardiomyocytes in vitro with similar electrophysiological properties, calcium transient and contractile function to eCS and mouse embryonic stem cell-derived cardiomyocytes. Transplantation of iCS (1 3 10 6 cells) into mouse myocardium following MI had similar effects to transplantation of eCS but significantly better than saline or fibroblast in improving left ventricular ejection fraction, increasing anterior/septal ventricular wall thickness and capillary density in the infarcted region 4 weeks after transplantation. No tumor formation was observed. iCS generated from adult skin fibroblasts by somatic reprogramming and a cocktail of Gsk3b inhibitor-6-Bromoindirubin-3 0 -oxime and Oncostatin M may represent a novel source for cell therapy in MI. STEM CELLS 2016;34:2693-2706 SIGNIFICANCE STATEMENTThis study provides the first proof-of-principle results that demonstrate the feasibility of generating donor-specific induced cardiospheres via a somatic reprogramming process, providing a new source of cell therapy for treatment of myocardial infarction