As with reprogramming factors Oct Klf

As with reprogramming factors (Oct4, Klf2, Sox2, and c-Myc), overexpression of E-cadherin enhances the efficiency of iPSC generation from mouse embryonic fibroblasts (Chen et al., 2010). We demonstrated that E-cadherin overexpression affects both attenuation of β-catenin signaling and enhancement of LIF-Stat3 signaling. These changes might underlie efficient reprogramming. We consider blocking nuclear localization of β-CATENIN to be a major factor because Wnt inhibitors also promoted reprogramming. We also demonstrated that although innate E-cadherin acetylcholine chloride levels vary among EpiSC lines, E-cadherin overexpression supported reprogramming independent of innate expression levels. This may suggest that, rather than absolute β-catenin signal intensity, the relative change in β-catenin signaling is important. Consistent with reports indicating such a role for E-CADHERIN (Conacci-Sorrell et al., 2003; Sasaki et al., 2000; Stockinger et al., 2001), our data confirmed negative regulation of nuclear translocation of β-CATENIN through E-cadherin overexpression in mouse EpiSCs. As expected, we could also demonstrate that blocking nuclear localization of β-CATENIN by the small-molecule inhibitors IWP-2 or XAV939 confers high efficiency in conversion of mouse EpiSCs to naive-like PSCs that can contribute to chimeras. This suggests that, as was the case with upregulation of E-cadherin expression, the combination of blocking nuclear localization of β-CATENIN and LIF signaling activation primes mouse EpiSCs for reprogramming. Absent upregulation of E-cadherin, small-molecule inhibitors of Wnt signaling can significantly amplify reprogramming frequency. One can infer that upregulation of E-cadherin leads efficient reprogramming by blocking nuclear localization of β-CATENIN. In this way, we succeeded in establishing culture conditions for efficient conversion of primed PSCs to naive-like PSCs. To our surprise, we demonstrated that E-cadherin overexpression and Wnt inhibitor treatments did not affect to TCF/LEF-mediated transcriptional activity though nuclear localization of β-CATENIN was significantly blocked. Kim et al. (2013) previously reported that subcellular localization of β-CATENIN does not implicate Wnt-downstream transcriptional activity but has some role in maintenance of self-renewal. Our results suggested that TCF/LEF-independent β-catenin signaling is stimulated by blocking nuclear localization or subcellular localization of β-CATENIN, thereby also promoting conversion from primed state to naive state pluripotency. Pluripotentiality in primate PSCs resembles pluripotentiality in mouse EpiSCs; primate PSCs are not thought to contribute to chimeras (Nichols and Smith, 2009). For Gene dosage reason, the use of primate PSCs for knockout and transgenic studies is rare. Although our work was done only in mice, our findings, with efficient conversion of primed to naive PSCs, may provide the key to establishing naive PSCs derived from other animals. We consider that requirements for β-catenin signaling must be reassessed, because the culture conditions employed in reports of establishment of human naive PSCs all were set to promote β-catenin signaling (Gafni et al., 2013; Hanna et al., 2010; Li et al., 2009; Ware et al., 2014). Further study would be required to clarify the effect of β-catenin signaling for the conversion to naive-like state in other animals and human PSCs.
Experimental Procedures
Author Contributions
Acknowledgments We thank H. Niwa for providing the EB3DR ESC line; A. Smith for providing the Rex1-GFP ESC line; Randall Moon for providing the Super 8x TOPFlash plasmid; and M. Kasai, A. Knisely, Y. Yamazaki, Y. Ishii, and M. Watanabe for supporting this work. This work was supported by grants from Japan Science and Technology Agency (JST), Exploratory Research for Advanced Technology (ERATO), Nakauchi Stem Cell and Organ Regeneration Project, Tokyo, Japan. H.N. is a founder and shareholder of iCELL and a founder, shareholder, and a scientific advisor for Megakaryon Corporation and ReproCELL.