Fukunaga Ichiro, Oe Yoko, Chen Cheng, Danzaki Keiko, Ohta Sayaka, Koike Akito, Ikeda Katsuhisa, Kamiya Kazusaku
Department of Otorhinolaryngology, Juntendo University Faculty of Medicine, Tokyo, Japan.
Front Cell Dev Biol. 2021 Apr 21;9:602197. doi: 10.3389/fcell.2021.602197. eCollection 2021.
Mutations in gap junction beta-2 (), the gene that encodes connexin 26 (CX26), are the most frequent cause of hereditary deafness worldwide. We recently developed an model of 2-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 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 -related deafness may be possible.
缝隙连接蛋白β-2（）基因发生突变，该基因编码连接蛋白26（CX26），是全球遗传性耳聋最常见的病因。我们最近利用基于骨形态发生蛋白4（BMP4）信号的悬浮培养（无血清培养类胚体样聚集体并进行快速聚集培养；以下简称SFEBq培养）和贴壁培养，从小鼠诱导多能干细胞（iPSC）中建立了一种与2相关的耳聋模型（诱导形成CX26缝隙连接的细胞；iCX26GJC）。然而，要将这些细胞用作高通量药物筛选或再生治疗的疾病模型平台，必须大幅提高细胞产量。除了BMP4外，其他因素也可能诱导CX26缝隙连接的形成。在SFEBq培养中，BMP4与激活素/节点/TGF-β信号通路抑制剂SB431542（SB）联合使用，比单独使用BMP4处理产生了更多可分离的表达CX26的细胞团（CX26囊泡）和更高的mRNA水平，这表明SB可能以剂量依赖的方式促进BMP4介导的CX26囊泡的产生，从而提高高度纯化的iCX26GJC的产量。这是第一项证明SB在干细胞悬浮培养过程中加速BMP4诱导的iCX26GJC分化的研究。通过控制SB添加浓度并结合CX26囊泡纯化，有可能大规模生产适用于与2相关耳聋的高通量药物筛选或再生治疗的高度纯化的iCX26GJC。
