Generation of two iPSC lines from siblings of a homozygous patient with hearing loss and a heterozygous carrier with normal hearing carrying p.G45E/Y136X mutation in GJB2

GJB2 c.35del variant up-regulates GJA1 gene expression and affects differentiation of human stem cells

“…The GJB2 gene is expressed in human embryonic stem cells (ESCs), induced pluripotent stem cells (IPSCs), and derived embryonic bodies and neural cells ( Jin et al , 2015 ). IPSCs obtained from human individuals with GJB2 c.109G>A/(p.Val37Ile) differentiated into the major cells of the three germ layers ( Lu et al , 2020 ; Fukunaga et al , 2021 ; Colbert et al , 2022 ). IPSCs harboring a frameshifting pathogenic variant 200 nucleotides downstream of c.35del ( GJB2 , c.235del, p.Leu79CysfsTer3) were able to differentiate into neural progenitor cells and neurons in vitro and overexpressed the GJB1 mRNA ( Degen et al , 2011 ; Jin et al , 2015 ).…”

Induced Pluripotent Stem Cells, a Stepping Stone to In Vitro Human Models of Hearing Loss

“…Mutations in the gap junction beta 2 ( GJB 2) gene, which codes for the connexin 26 (CX26) protein, are responsible for a very large proportion of the cases of non-syndromic HL in the world; this protein is found in gap junction channels in various non-sensory cell types in the cochlea, and it plays a role in K + recycling and cochlear homeostasis. Fukunaga and colleagues (2021) differentiated hiPSCs to CX26 gap junction-forming cells that exhibited similar characteristics to SCs [ 68 ]. To do this, they prepared SFEBq (serum-free floating culture of EB-like aggregates with quick reaggregation) cultures to initiate differentiation.…”

“…To do this, they prepared SFEBq (serum-free floating culture of EB-like aggregates with quick reaggregation) cultures to initiate differentiation. Differently from the method established by Koehler and co-workers (2017) [ 31 ] to generate HC-like cells within human inner ear organoids, Fukunaga et al (2021) observed that TGFβ inhibition during the induction process led to reduced appearance of CX26-positive cells within day 7 aggregates [ 68 ]. Therefore, they only applied BMP and saw an increase in the expression of the otic progenitor markers PAX2, PAX8, GATA3, GJB2 and GJB6.…”

“…Moreover, this group observed that addition of insulin to the cultures in the presence or absence of BMP was sufficient to promote their growth and survival and the induction of CX26-expressing vesicles within the aggregates; in those cells, CX26 protein was localized to gap junctions at the cell–cell borders. Subsequent culture of CX26-expressing vesicles on a layer of murine cochlear feeder cells yielded proliferative human gap junction-forming CX26-positive cells that resembled cochlear SCs, according to their mRNA and protein expression profiles [ 68 ]. Also important is the hiPSC-based model developed by Hosoya et al (2017) that gives rise to outer sulcus cell (OSC)-like cells [ 36 ].…”

“…Several hiPSC lines are also available to study the detrimental effects of certain mutations on the physiology of otic SC types. A number of these carry mutations in the Gap Junction Beta 2 (GJB 2) gene that codes for the CX26 protein and is associated with over half of the cases of hereditary HL in the world with more than 340 pathogenic variants [ 68 , 89 , 91 , 108 , 109 ]. Fukunaga and colleagues (2021) have recently reported that absence of the CX26 protein in gap junction-forming inner ear SC-like cells generated from patients’ hiPSCs results in deficient gap junction-mediated intercellular communication, associated with shortened gap junction plaques in those cells [ 37 ]; in this case, the authors have reported similar findings to those obtained in GJB 2 mutant mice [ 110 ].…”