TGF-β inhibitor accelerates BMP4-induced cochlear gap junction formation during in vitro differentiation of embryonic stem cells

Mutations in the connexin 26 (CX26)/gap junction beta-2 (GJB2) gene are the most frequent cause of hereditary deafness worldwide. Using mouse induced pluripotent stem cells (iPSCs) and a BMP4 signal-based floating and adherent culture system, we recently produced in vitro responsible for GJB2-related deafness (CX26-gap junction plaque-forming cells, CX26GJCs). However, to use these cells as a disease model platform for high-throughput drug screening or regenerative therapy, cell yields must be substantially increased. In addition to BMP4, presently uncharacterized factors may also induce CX26 gap junction (GJ) formation. A floating culture with embryonic stem cell (ESC) treatment and BMP4/TGF-{beta} inhibitor (SB431542:SB) has been shown to result in greater production of isolatable CX26-positive small vesicles (CX26+ vesicles) and higher Gjb2 mRNA levels than BMP4 treatment alone, suggesting that SB may promote BMP4-mediated production of CX26+ vesicles in a dose-dependent manner, thereby increasing the yield of highly purified CX26GJCs. In the present study, we first demonstrated that SB accelerates BMP4 induced GJ formation during stem cell differentiation. By controlling the concentration and timing of SB supplementation with CX26+ vesicle purification, large-scale production of highly purified CX26GJCs suitable for high-throughput drug screening or regenerative therapy for GJB2-related deafness may be possible.