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

Abstract

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.

Impact statement

This study introduces a customized microfluidic platform capable of accommodating three-dimensional human vestibular neuronal spheroids differentiated from human induced pluripotent stem cells (hiPSCs). By precisely controlling the culture conditions and signaling molecule gradients, this platform enables the faithful differentiation of hiPSCs into vestibular neuronal progenitors and subsequent maturation into functional vestibular neurons. The successful generation of human vestibular neurons provides a valuable model system to investigate the development and function of the vestibular system, as well as to study vestibular disorders and develop potential therapeutic interventions. This breakthrough technology opens new avenues for understanding the intricate mechanisms underlying vestibular biology and offers promising opportunities for the development of personalized medicine approaches in the field of vestibular research and treatment.