Unraveling the Mechanisms of Vestibular Neuron Formation from Human Induced Pluripotent Stem Cells.

The development of in vitro models that accurately recapitulate the complex cellular and molecular interactions of the inner ear is crucial for understanding inner ear development, function, and disease. In this study, we utilized a customized microfluidic platform to generate human induced pluripotent stem cell (hiPSC)-derived three-dimensional otic sensory neurons (OSNs). hiPSC-derived otic neuronal progenitors (ONPs) were cultured in hydrogel-embedded microfluidic channels over a 40-day period. Careful modulation of Wnt and Shh signaling pathways was used to influence dorsoventral patterning and direct differentiation toward a vestibular neuron lineage. After validating the microfluidic platform, OSN spheroid transcription factor and protein expression were assessed using real-time quantitative polymerase chain reaction (RT-qPCR), immunocytochemistry, and flow cytometry. The results demonstrated the successful differentiation of hiPSCs into ONPs and subsequent divergent differentiation into vestibular neuronal lineages, as evidenced by the expression of characteristic markers. Overall, our microfluidic platform provides a physiologically relevant environment for the culture and differentiation of hiPSCs, offering a valuable tool for studying inner ear development, disease and drug screening, and regenerative medicine applications.