Abstract
Mutations in gap junction beta-2 (GJB2), the gene that encodes connexin 26 (CX26), are the most frequent cause of hereditary deafness worldwide. We recently developed an in vitro model of GJB2-related deafness (induced CX26 gap junction-forming cells; iCX26GJCs) from mouse induced pluripotent stem cells (iPSCs) by using Bone morphogenetic protein 4 (BMP4) signaling-based floating cultures (serum-free culture of embryoid body-like aggregates with quick aggregation cultures; hereafter, SFEBq cultures) and adherent cultures. 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, other factors may also induce CX26 gap junction formation. In the SFEBq cultures, the combination of BMP4 and the Activin/Nodal/TGF-β pathway inhibitor SB431542 (SB) resulted in greater production of isolatable CX26-expressing cell mass (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 iCX26GJCs. This is the first study to demonstrate that SB accelerates BMP4-induced iCX26GJC differentiation during stem cell floating culture. By controlling the concentration of SB supplementation in combination with CX26+ vesicle purification, large-scale production of highly purified iCX26GJCs suitable for high-throughput drug screening or regenerative therapy for GJB2-related deafness may be possible.
Highlights
Hearing loss is the most common congenital sensory impairment worldwide (Chan et al, 2010)
If connexin 26 (CX26)+ vesicles in SFEBq cultures from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) could be obtained in a substantial quantity, we may have an adequate number of iCX26GJCs in adherent cultures
SB Promoted Bone morphogenetic protein 4 (BMP4)-Induced Gjb2/Gjb6 mRNA Expression in SFEBq Cultures iCX26GJCs were induced from mouse ESCs as described (Fukunaga et al, 2016), and the conditions required for differentiation were assessed
Summary
Hearing loss is the most common congenital sensory impairment worldwide (Chan et al, 2010). The inner ear is much more difficult to treat using human cells and tissues than in other sensory organs (eye, nose, tongue), and research on the pathophysiology and the development of treatment methods has been delayed. For this reason, rodents (mainly mouse) are a powerful tool for researching hearing loss. Using mouse iCX26GJC for drug screening and conducting mouse experiments based on the results will be an important discovery opportunity for future applications to human deafness. The inner ear, which is our target, is derived from the otic placode, which is part of
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