SummaryMutation of the Gap Junction Beta 2 gene (GJB2) encoding connexin 26 (CX26) is the most frequent cause of hereditary deafness worldwide and accounts for up to 50% of non-syndromic sensorineural hearing loss cases in some populations. Therefore, cochlear CX26-gap junction plaque (GJP)-forming cells such as cochlear supporting cells are thought to be the most important therapeutic target for the treatment of hereditary deafness. The differentiation of pluripotent stem cells into cochlear CX26-GJP-forming cells has not been reported. Here, we detail the development of a novel strategy to differentiate induced pluripotent stem cells into functional CX26-GJP-forming cells that exhibit spontaneous ATP- and hemichannel-mediated Ca2+ transients typical of the developing cochlea. Furthermore, these cells from CX26-deficient mice recapitulated the drastic disruption of GJPs, the primary pathology of GJB2-related hearing loss. These in vitro models should be useful for establishing inner-ear cell therapies and drug screening that target GJB2-related hearing loss.

BackgroundMutations in GJB2, which encodes connexin 26 (Cx26), a cochlear gap junction protein, represent a major cause of pre-lingual, non-syndromic deafness. The degeneration of the organ of Corti observed in Cx26 mutant—associated deafness is thought to be a secondary pathology of hearing loss. Here we focused on abnormal development of the organ of Corti followed by degeneration including outer hair cell (OHC) loss.MethodsWe investigated the crucial factors involved in late-onset degeneration and loss of OHC by ultrastructural observation, immunohistochemistry and protein analysis in our Cx26-deficient mice (Cx26f/fP0Cre).ResultsIn ultrastructural observations of Cx26f/fP0Cre mice, OHCs changed shape irregularly, and several folds or notches were observed in the plasma membrane. Furthermore, the mutant OHCs had a flat surface compared with the characteristic wavy surface structure of OHCs of normal mice. Protein analysis revealed an increased protein level of caveolin-2 (CAV2) in Cx26f/fP0Cre mouse cochlea. In immunohistochemistry, a remarkable accumulation of CAV2 was observed in Cx26f/fP0Cre mice. In particular, this accumulation of CAV2 was mainly observed around OHCs, and furthermore this accumulation was observed around the shrunken site of OHCs with an abnormal hourglass-like shape.ConclusionsThe deformation of OHCs and the accumulation of CAV2 in the organ of Corti may play a crucial role in the progression of, or secondary OHC loss in, GJB2-associated deafness. Investigation of these molecular pathways, including those involving CAV2, may contribute to the elucidation of a new pathogenic mechanism of GJB2-associated deafness and identify effective targets for new therapies.

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

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