Generation of Functional CX26-Gap-Junction-Plaque-Forming Cells with Spontaneous Ca Transients via a Gap Junction Characteristic of Developing Cochlea.

Mutation of the gene GJB2, encoding connexin 26 (CX26; also known as gap junction beta 2), is the most frequent cause of hereditary deafness worldwide. CX26 is expressed in cochlear nonsensory cells, such as cochlear supporting cells, and forms gap junction plaques (GJPs) at cell-cell borders. Cochlear CX26-GJP-forming cells (Cx26GJCs) are thought to be an important therapeutic target for treatment of hereditary deafness. Nevertheless, the generation of Cx26GJCs-such as cochlear supporting cells-from embryonic stem/induced pluripotent stem (ES/iPS) cells has not been reported to date. Here, we detail a novel strategy for differentiating iPS cells into functional Cx26GJCs such as are found in cochlea. Several assays to characterize the phenotype of iPS-derived Cx26GJCs are described, including qRT-PCR, immunohistological analysis, morphological analysis, a scrape-loading and dye transfer assay, and calcium imaging. This in vitro model has applications in the establishment of inner-ear cell therapies and in drug screening to target GJB2-related hearing loss. © 2019 by John Wiley & Sons, Inc. Basic Protocol: Induction of mouse stem cells to create CX26-GJP-forming cells Support Protocol 1: Maintenance and passage of mouse induced pluripotent stem cells Support Protocol 2: Screening for high GJB2 and GJB6 expression in SFEBq culture using quantitative real-time PCR Support Protocol 3: Characterization of cells at different stages of differentiation by immunostaining Support Protocol 4: Ultrastructural analyses of cells at different stages of CX26-GJP-forming cell induction Support Protocol 5: Functional analyses of stem cell-derived CX26-GJP-forming cells.