Identification of microRNA-Related Target Genes for the Development of Otic Organoids

Mammalian hearing loss is typically permanent due to the inability to replace damaged cochlear hair cells. However, the neonatal mice inner ear demonstrates regenerative capacity, with cochlear floor cells proliferating and differentiating into organoids containing new hair cells and supporting cells, yet the governing molecular mechanisms remain poorly understood. Here, we isolated extracellular vesicles (EVs) from inner ear organoids at proliferation and differentiation stages, characterized their EV miRNA profiles through sequencing, and validated findings using public transcriptomic datasets to elucidate miRNA-mediated regulatory mechanisms during inner ear development. Inner ear organoids were successfully developed from cochlear duct cells, expressing otic progenitor marker SOX2 and hair cell marker Myo7A and demonstrating functional mechano-transduction activity through FM1-43 uptake. Small RNA sequencing identified 35 differentially expressed EV miRNAs between developmental stages. Integrated analysis with public transcriptome datasets revealed 18 genes with significant differential expression, leading to identification of three key regulatory genes-Trp53, Ezh2, and Zbtb4-that exhibited dynamic spatiotemporal expression during inner ear maturation. Pathway analysis demonstrated that these genes are associated with DNA Repair, P53, and Wnt/β-Catenin signaling with remarkable cell-type specificity. Our results demonstrate that EV miRNAs are temporally regulated during organoid development, with predominant downregulation during differentiation. These findings provide crucial insights into developmental mechanisms that could optimize organoid-based models and guide EV miRNA-based therapeutic strategies for hearing restoration.