Establishment and stage characterization of an early differentiation model for human inner ear organoids

TANG Zhengyu; WU Xiaonan; WANG Qiuju

doi:10.3969/j.issn.1672-2922.2026.03.003

Abstract：Objective To validate the feasibility of a stepwise differentiation protocol for generating human inner ear organoids(hIEOs)from human induced pluripotent stem cells(iPSCs).Methods A urine-derived iPSCs line from a healthy male donor was subjected to directed differentiation into hIEOs by recapitulating the sequential activation and inhibition of key signaling pathways involved in human inner ear development,including BMP(Bone Morphogenetic Protein),TGF-β(Transforming Growth Factor-beta),FGF(Fibroblast Growth Factor),WNT(Wingless-related integration site),and SHH(Sonic hedgehog)pathways.During early differentiation,morphological progression was monitored by bright-field microscopy,and stage-specific molecular markers were assessed via immunofluorescence staining.Results(1)Concurrent activation of FGF and BMP signaling coupled with TGF-β inhibition efficiently induced iPSCs toward non-neural ectoderm(NNE)on day 3,confirmed by co-expression of NNE markers EYA1+GATA3+TFAP2A+ECAD+.(2)Subsequent sequential modulation—FGF activation followed by BMP and TGF-β inhibition,and co-activation of WNT and SHH—promoted to the formation of otic placode by day 10 and further progression to otic pit by day 15.(3)Sustained WNT inhibition combined with SHH activation promoted the emergence of three-dimensional,spherical otic vesicles by day 42.Within these vesicles,early hair cell-like cells expressing MYO7A,F-actin,and POU4F3 were observed,potentially early connected by NFL+neuronal-like cells.Conclusions This study demonstrated the reproducibility and efficacy of the protocol for iPSCs-derived early hIEOs,which contained early hair cell-like cells and potential early sensory-neural connectivity.The hIEO is expected to provide a human-derived in vitro model for further studies on otogenesis and pathogenesis.