Introduction: Efficient induction of the otic placode, the developmental origin of the inner ear from human pluripotent stem cells (hPSCs), provides a robust platform for otic development and sensorineural hearing loss modelling. Nevertheless, there remains a limited capacity of otic lineage specification from hPSCs by stepwise differentiation methods, since the critical factors for successful otic cell differentiation have not been thoroughly investigated. In this study, we developed a novel differentiation system involving the use of a three-dimensional (3D) floating culture with signalling factors for generating otic cell lineages via stepwise differentiation of hPSCs. Methods: We differentiated hPSCs into preplacodal cells under a two-dimensional (2D) monolayer culture. Then, we transferred the induced preplacodal cells into a 3D floating culture under the control of the fibroblast growth factor (FGF), bone morphogenetic protein (BMP), retinoic acid (RA) and WNT signalling pathways. We evaluated the characteristics of the induced cells using immunocytochemistry, quantitative PCR (qPCR), population averaging, and single-cell RNA-seq (RNA-seq) analysis. We further investigated the methods for differentiating otic progenitors towards hair cells by overexpression of defined transcription factors. Results: We demonstrated that hPSC-derived preplacodal cells acquired the potential to differentiate into posterior placodal cells in 3D floating culture with FGF2 and RA. Subsequent activation of WNT signalling induced otic placodal cell formation. By single-cell RNA-seq (scRNA-seq) analysis, we identified multiple clusters of otic placode- and otocyst marker-positive cells in the induced spheres. Moreover, the induced otic cells showed the potential to generate hair cell-like cells by overexpression of the transcription factors ATOH1, POU4F3 and GFI1. Conclusions: We demonstrated the critical role of FGF2, RA and WNT signalling in a 3D environment for the in vitro differentiation of otic lineage cells from hPSCs. The induced otic cells had the capacity to differentiate into inner ear hair cells with stereociliary bundles and tip link-like structures. The protocol will be useful for in vitro disease modelling of sensorineural hearing loss and human inner ear development and thus contribute to drug screening and stem cell-based regenerative medicine.
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