Activin/Nodal/TGF-β Pathway Inhibitor Accelerates BMP4-Induced Cochlear Gap Junction Formation During in vitro Differentiation of Embryonic Stem Cells

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.

Modelling inner ear development and disease using pluripotent stem cells – a pathway to new therapeutic strategies

“…In culture, cells expressing supporting cell markers have been generated from stem cell-derived ectodermal cells cultured on mouse-derived trypsin-resistant inner ear feeder cells. Ectodermal cells were initially induced in suspension culture from mouse iPSCs and ESCs by the simultaneous addition of Bmp4 and inhibition of Tgf-β ( Fukunaga et al, 2016 , 2021a ), and from human iPSCs by the addition of insulin ( Fukunaga et al, 2016 , 2021a , b ). In a protocol designed to generate specifically outer sulcus supporting cells, Hosoya et al (2017) induced otic progenitor cells by adding an FGF signalling cocktail (FGF2/FGF3/FGF10/FGF19) and BMP4, alongside weak alkaline conditions.…”

“…4 E) ( Fukunaga et al, 2021b ). Here, the conserved disease phenotypes in cultures produced from a mouse ESC line ( Fukunaga et al, 2021a ) and multiple mouse iPSC ( Fukunaga et al, 2016 ) and patient iPSC ( Fukunaga et al, 2021b ) lines indicate a level of reliability in these PSC-derived disease modelling systems. However, further investigation is required to definitively characterise whether GJB2 -related deafness is caused by impaired intercellular communication, and how affected supporting cells might interact with other cell types of the sensory epithelium.…”