br Experimental Procedures br Author Contributions M M
Author Contributions M.M. and M.T. designed and supervised the study. S.I. conducted the analysis on NOG-rd mice. H.-Y.T., M.F., and T.M. conducted and analyzed MEA. S.I., H.-Y.T., and M.M. performed 3D image analysis. N.K., T.K., M.G., and R.T. planned and established NOG-rd mice. S.S. performed immunological analysis of NOG-rd mice. G.A.S. supervised modeling and analysis of retinal degeneration. S. Yamasaki, A. Kuwahara, and A. Kishino prepared and characterized hESC retinas. T.W. performed in vivo experiments/analysis. S. Yonemura provided data on electron microscopy. S.I., H.-Y.T., M.M., M.E., and H.T. analyzed and interpreted the data. S.I., H.-Y.T., G.A.S., and M.M. wrote the manuscript. All authors reviewed and approved the final manuscript.
Acknowledgments We thank Kenji Sakamoto at Kitasato University for supplying rd1-2J mice and Hanako Ikeda at Kyoto University for supplying rd10 mice for relocating procedure under Jackson’s approval. We thank Tomoyo Hashiguchi, Yoshiko Takahashi, Junki Sho, Shoko Fujino, and Naoko Hayashi for technical support and members of the M.T. laboratory for discussions. S.I. was supported by the fellowship of Junior Research Associate (JRA) from RIKEN. This study was supported by AMED under grant number 17bm0204002h0005. S. Yamasaki, A. Kuwahara, and A. Kishino are employees of Sumitomo Dainippon Pharma; this work was supported in part by a grant from Sumitomo Dainippon Pharma.
Introduction Embryonic stem vx 765 (ESCs) are derived from the inner cell mass of the blastocyst, which comprises pluripotent cells with the potential to differentiate into all cell types of the body (Williams et al., 1988b). This capacity enables ESCs a wide utilization in regenerative medicine and cell-based therapies. Therefore, a better understanding of the underlying mechanisms that modulate the differentiation of ESCs is essential for their clinical application in the future (Chen et al., 2008). Leukemia inhibitory factor (LIF) was found to be the key growth factor for the culture and maintenance of mouse ESCs (mESCs) in vitro (Smith et al., 1988, Williams et al., 1988a). LIF, a member of the interleukin-6 (IL-6) family of cytokines, binds to gp130/LIFR and results in the phosphorylation on tyrosine 705 residues of STAT3, a member of the STAT gene family identified in the interferon-induced regulatory pathways (Darnell et al., 1994, Fu et al., 1990, Fu et al., 1992, Schindler et al., 1992). STAT3, first identified as a transcription factor (TF) for the IL-6 family of cytokines (Akira et al., 1994, Zhong et al., 1994), was subsequently found to be crucial for ESC pluripotency (Boeuf et al., 1997, Boyer et al., 2005, Niwa et al., 1998, Raz et al., 1999, Ying et al., 2003). Conventional knockout of Stat3 in mice results in embryonic lethality at embryonic day 6.5 (E6.5) (Takeda et al., 1997). By eliminating Stat3 in the mouse oocytes and embryos we found that STAT3 has an essential role in inner cell mass lineage specification and maintenance, and in pluripotent stem cell identity through the OCT4-NANOG circuit (Do et al., 2013). The c-Jun NH2-teminal kinase (JNK) belongs to the mitogen-activated protein (MAP) kinase family, which were initially identified as ultraviolet-responsive protein kinases that activated c-Jun by phosphorylating its NH2-terminal serine/threonine residues (Dérijard et al., 1994, Hibi et al., 1993). In response to growth factors, cytokines, and a number of environmental stresses, JNK is activated through a well-orchestrated cascade of MAP kinase activation (Jaeschke et al., 2006, Sabapathy et al., 2004). In particular, mitogen-activated kinase kinase 4 and 7, isoforms of MAP2K, directly phosphorylate and activate JNK, which in turn leads to the phosphorylation of (TF) c-Jun and switching on of transcriptional regulation exclusively through formation of complex with other TFs, such as c-fos, in the activator protein-1 complex (Davis, 2000, Weston and Davis, 2007). JNK is encoded by two ubiquitously expressed genes (JNK1 and JNK2) and by a third gene (JNK3) that is selectively expressed in neurons. MAPKBP1 is a JNK binding protein and enhances the activation of JNK (Koyano et al., 1999, Lecat et al., 2012). Recent studies indicated that JNK signaling is required for lineage-specific differentiation but not stem cell self-renewal. ESCs lacking JNK1 show transcriptional deregulation of several lineage-commitment genes and fail to undergo neuronal differentiation, as do ESCs lacking JNK pathway scaffold proteins (Xu and Davis, 2010). Studies also found that JNK binds to a large set of active promoters during the differentiation of stem cells and results in histone 3 phosphorylation on chromatin (Tiwari et al., 2011). It is also reported that JNK regulates STAT3 activity via its Ser-727 phosphorylation, showing the crosstalk between STAT3 and JNK pathways (Lim and Cao, 1999).