br Results br Discussion We report the development of a

Results
Discussion We report the development of a method to generate and proliferate iCx26GJCs from mouse iPSCs for use as a disease model and in inner-ear cell therapies targeting GJB2-related hearing loss, the most frequent type of hereditary deafness worldwide. Our induction method until day 7 was based on previous studies (Koehler and Hashino, 2014; Koehler et al., 2013). However, after the day-7 procedures, which screen for a CX26/CX30 highly expressing condition, isolation of CX26-positive small vesicles, and transferring CX26-positive small vesicles onto TRIC feeder cells, differ from previous studies (Koehler and Hashino, 2014; Koehler et al., 2013). Previous studies have targeted the generation of inner-ear hair cell-like cells from ESCs/iPSCs (Chen et al., 2012; Koehler and Hashino, 2014; Koehler et al., 2013; Oshima et al., 2010). In contrast, we focused on CX26-GJP-forming cells derived from iPSCs, as the cochlear supporting cell-like cells. It has not been reported that ESCs/iPSCs differentiated into the cells with clear CX26-GJPs characterized by immunolabeling and gene expression for CX26/30, dye transfer assay, and Ca2+ imaging. In the modified SFEBq method, we confirmed that the morphology and outer epithelium thickness of day 7–11 wee1 inhibitor (Figure S2) were similar to those previously reported (Koehler et al., 2013). As these thin outer epithelia did not include the NANOG-GFP cells and were clearly demarcated, they could be separated by dissection for further adherent culture on cochlear feeder cells. In this process (Figure 1B), the medium conditions and the timing for optimal treatments were selected according to mRNA expression and gap junction formation as determined in subsequent experiments. In the CX26 and CX30 mRNA expression analysis it has been suggested that the BMP- or the B/S-treatment condition is more suitable for differentiation into CX26- and CX30-expressing cells compared with the B/S + F/L treatment condition, which has been reported to differentiate into sensory epithelium (Koehler and Hashino, 2014; Koehler et al., 2013). It has also been suggested that the differentiation into neural ectoderm by SFEBq culture might be partially inhibited with respect to differentiation into non-neural ectoderm (Koehler and Hashino, 2014; Koehler et al., 2013), and the CX26/CX30 expression levels were increased by BMP supplementation with some regulation by additional supplements such as the transforming growth factor β (TGF-β) inhibitor, SB-431542 (Figure 1A). BMP signaling has been reported to be critical for induction of the non-neural ectoderm from the definitive ectoderm epithelium induced by SFEBq method (Eiraku and Sasai, 2012). TGF-β inhibitor (SB-431542) suppressed the undesirable induction of mesoderm induced by BMP signaling (Koehler and Hashino, 2014; Koehler et al., 2013). The use of cochlear feeder cells was critical to the proliferation of the iCx26GJCs (Figure 2I) and the increase in CX26 plaque length (Figure 2J) in the dissected small vesicle of the aggregates. In contrast to the cochlear (TRIC) feeder system, a feeder system with chicken embryonic inner ear used for hair cell differentiation (Oshima et al., 2010) and feeder-free culture did not promote the cell proliferation or GJP formation of iCx26GJCs. Therefore, it is suggested that TRIC feeder cells may express some molecules to promote the proliferation and GJP formation of iCx26GJCs. Our working hypothesis is that a small number of cells that differentiated from iPSCs into CX26-expressing cells in modified SFEBq culture were induced to proliferate by the growth factors that were secreted from the cochlear feeder cells. Our observation that iCx26GJCs co-expressed CX26 and CX30 (Figures 2K, 2L, and S4E–S4H) suggested that a number of cells in the aggregates had differentiated into cochlear non-sensory cells (Figures S4A–S4D) and generated CX26/CX30 GJPs; moreover, the data suggested that these cells had proliferated on the cochlear feeder cells after dissection of the small vesicles that were attached to the outer epithelium (Movie S2).