Such spontaneous Ca transients have

Such spontaneous Ca2+ transients have been reported in postnatal mouse cochlear supporting cells (Tritsch et al., 2007), and the Ca2+ signals were suggested to be propagated via gap junctions and hemichannels containing CX26 and CX30 (Anselmi et al., 2008; Schutz et al., 2010). It was also suggested that such spontaneous Ca2+ signals in supporting cells may play a crucial role in generating the periodic, high-frequency burst of activity observed in the auditory center of the 740 Y-P (Wang et al., 2015). Therefore, iCx26GJCs in the 2D cultures may differentiate into postnatal cochlear supporting cells before the onset of hearing. In iCx26GJC from a CX26-deficient deafness mouse model (CX26f/f P0-Cre), GJP formations showed visible drastic disruption (Figures 6H, 6J, 6L, and 6N), reported to be a primary pathology of GJB2-related hearing loss (Kamiya et al., 2014). This suggests that the iPSCs derived from CX26f/f P0-Cre mouse have the potential to differentiate into iCx26GJC as an in vitro disease model of GJB2-related hearing loss. By using these cells, it is expected to establish the drug screening and inner-ear cell therapy after in vitro restoration of GJPs by the GJB2 gene transfer (Iizuka et al., 2015) targeting GJB2-related hearing loss. In summary, we hypothesize that iPS-derived cells formed floating aggregate in serum-free medium (SFEBq culture), after which the culture conditions including BMP (BMP4) and TGF-β inhibitor (SB-431542) treatment were selected to generate high-CX26/CX30 aggregates. Unlike hair cell differentiation (Koehler and Hashino, 2014; Koehler et al., 2013), these characteristic iPSC aggregates form distinct epithelia and small vesicles attached to the outer epithelium. In 2D culture, these small vesicles colonized on TRIC feeder cells. The small vesicle-derived colony exhibits proliferation potency and contains iCx26GJCs. The iCx26GJCs form functional CX26-GJPs that exhibit spontaneous ATP- and hemichannel-mediated Ca2+ transients typical of the developing cochlea. By using this method, we generated the in vitro disease model cells with GJP disruption for GJB2-related hearing loss (Figure 7). In the present study, we demonstrated that the aggregate formation of iPSCs under several medium conditions followed by adherent culture with cochlear feeder cells induced: (1) the upregulation of mRNAs encoding CX26/CX30; (2) GJP formation composed of CX26/CX30; (3) the ultrastructure typical of gap junctions; (4) functional GJIC networks; and (5) spontaneous ATP- and hemichannel-mediated Ca2+ transients typical of developing cochlea. These are known to be the biological properties of cochlear supporting cells. Cochlear supporting cells are the most CX26-abundant cells that play crucial roles in maintaining proper endocochlear potential via ion transport. Furthermore, the disease model cells with GJB2 mutation showing drastic GJP disruptions in the present study are thought to be the optimum therapeutic target for the treatment of GJB2-related hearing loss, the most typical type of hereditary deafness worldwide. It is expected, then, that these iPS-derived cells, which can be obtained from patients, will be particularly useful for drug screening and inner-ear cell therapies targeting GJB2-related hearing loss.
Experimental Procedures
Author Contributions
Acknowledgments We thank M. Yoshida for help with transmission electron microscopy, K. Karasawa, K. Kobayashi, and Y. Furuta for experimental assistance, and T. Sakurai for help in the use of the facilities. This work was supported in part by JSPS KAKENHI grant number 25462653 (to K.K.), grant number 25293351 (to K.I.), and grant number 15K20229 (to I.F.), MEXT-support program for the Strategic Research Foundation at Private Universities, 2011–2015 (to K.I.), the Research on Intractable Diseases from Japan Agency for Medical Research and Development, AMED grant number 15ek0109125h0001 (to K.K.), Promotion and mutual aid corporation for Private Schools of Japan (to K.K.), the Terumo Life Science Foundation (to K.K.), and Takeda Science Foundation (to K.K.).