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 3D otic sensory neurons (OSNs). Careful modulation of Wnt and Shh signaling pathways was used to direct differentiation toward a vestibular neuron lineage. hiPSC-derived otic neuronal progenitors were cultured in hydrogel-embedded microfluidic channels over a 40-day period. Variable concentrations of Wnt3a and Shh were used to influence dorsoventral patterning prior to neuronal differentiation. Spheroid viability and differentiation were assessed using viability/cytotoxicity assays, immunocytochemistry, reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). Additionally, intracellular flow cytometry was employed to analyze specific intracellular markers of interest. The results demonstrated the successful differentiation of hiPSCs into ONPs and subsequent divergent differentiation into vestibular neuronal lineages, as evidenced by the expression of specific 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.