We recently found that E cad function cannot

We recently found that E-cad function cannot be replaced by N-cad during the formation of the trophectoderm, the first polarized epithelium formed during mammalian development. In the blastocyst, the E-cad extracellular domain provides a unique and indispensable survival signal by facilitating Igf1r signaling, in addition to its adhesive function (Bedzhov et al., 2012; Kan et al., 2007). Here, we investigated whether E-cad has a similarly unique function and governs a related process to promote ES cell identity and induced pluripotency. Our data confirmed and extended previous observations regarding the crucial function of E-cad during iPS cell deprivation and ES cell ground state maintenance (Chen et al., 2010; Redmer et al., 2011). However, although indispensable for these processes, our results suggest that E-cad function can be replaced by N-cad to rescue an undifferentiated pluripotent ES cell state. In addition, N-cad supports iPS cell generation, indicating that the establishment of cell contact formation and adhesion, rather than an E-cad specific induction of reprogramming, is crucial for this process.
Materials and methods
Results
Discussion Unraveling the ground state of ES cell pluripotency and the ability to reprogram differentiated endothelin receptor paved the way for regenerative medicine with the potential to use patient-derived cells with ES cell properties. The pathways and regulators that maintain ES cell pluripotency are still ill defined, and a better understanding of the mechanisms underlying pluripotency and differentiation is necessary (Ying et al., 2008). E-cad is important for the maintenance of proper ES cell pluripotency and is able to enhance the generation of iPS cells, in which it can replace Oct4 in the Yamanaka cocktail of pluripotency factors (Chen et al., 2010; Redmer et al., 2011; Soncin et al., 2009). Reprogramming requires an initial MET. This essential step seems to be highly dynamic with phases of both EMT and MET and cadherins play a very important role during this process (Liu et al., 2013). In fibroblast, directly after viral transduction of the pluripotency genes, temporal activation of Snai2 expression was observed. Consequently, further activation of N-cad and repression of E-cad in an EMT-like process was detectable, important for efficient reprogramming, before MET is stabilized during days 3–5. Here, we investigated the specific requirements of E-cad in ES cell pluripotency and during reprogramming. Our results show that pluripotency and differentiation capacity are maintained in ES cells that exclusively express N-cad or chimeric E-cad/N-cad proteins with swapped extracellular domains. The proper induction of pluripotency, which requires the exogenous expression of Oct4, Sox2 and Nanog, was detected in genetically modified fibroblasts that switch from endogenous N-cad to N-cad expression from a ki allele during initial MET. In contrast, E-cad ko ES cells lost key features of pluripotency and differentiation capacity. Consistent with these findings, no iPS cells were obtained from E-cad ko/∆ fibroblasts. Thus, pluripotency is ultimately connected to cell–cell adhesion for ES cell maintenance and induced pluripotency during the initiation of MET. Moreover, our results show that N-cad-mediated adhesion can replace the function of E-cad in ES and iPS cells, suggesting that the maintenance or establishment of cell adhesion and a specific cytoskeletal architecture that permits cells to cluster and form compact colonies is a fundamental process. This phenomenon blocks differentiation in ES cells and allows efficient reprogramming. The presence of E-cad stabilizes the expression and controls the localization of ZO-1, Eph receptors and the 5T4 oncofetal antigen, supporting the role of E-cad in guiding and coordinating protein localization to remodel the cellular architecture (Orsulic and Kemler, 2000; Spencer et al., 2007). Klf4 was shown to bind to the E-cad promoter and directly activate E-cad transcription during the initial events of fibroblast reprogramming (Chen et al., 2010; Li et al., 2010). In line with this observation, epithelial cells that already express E-cad and Klf4 endogenously, such as keratinocytes, show increased reprogramming capabilities, with greater than 100-fold higher efficiency than mesenchymal cell-originated fibroblasts (Aasen et al., 2008). This indicates that, in fibroblasts, the initial expression of endogenous N-cad is not sufficient to support reprogramming, whereas the N-cad expressed from our ki allele is capable of efficiently promoting MET during this process.