Abstract
PURPOSE: Permanent hearing loss primarily results from the inability of the mammalian cochlea to replace lost inner ear hair cells. However, neonatal mice exhibit a unique capacity: isolated cochlear floor cells can efficiently proliferate in vitro and form organoids that harbor new hair cells and supporting cell populations. In this study, we isolated extracellular vesicles (EVs) from organoids and analyzed the miRNAs derived from them to identify gene regulatory elements that coordinate proliferation and regeneration. METHOD: We utilized cochlear floor cells from postnatal day two mice and optimized the culture conditions to efficiently grow organoids that exhibit progenitor properties. Next, we isolated EVs from the culture media of organoids in their proliferative state. We analyzed miRNAs contained in these EVs to identify potential regulators that drive or modulate organoid cell proliferation. The miRNA sequencing data from organoid EVs were compared with miRNAs identified in EVs obtained from the culture supernatant of P2 mouse cochlear ducts. RESULTS: We identified 184 miRNAs in organoid EVs and 176 miRNAs in cochlear duct EVs. A total of 122 miRNAs differed more than twofold between these groups, with 12 miRNAs (10 upregulated and 2 downregulated in organoid EVs) exhibiting statistically significant differences. The target genes of these twelve differentially expressed miRNAs are associated with pathways related to pluripotent stem cell regulation, cell proliferation, ear development, and cell fate modulation. This indicates that the miRNAs in organoid-derived EVs may impact processes associated with cell proliferation and the generation of inner ear cell types. CONCLUSION: Our study comprehensively inventoried miRNAs contained in EVs released by growing inner ear organoids. Our differential miRNA expression analysis provides insight into regulatory mechanisms that promote cochlear floor cell proliferation and organoid formation, which could be leveraged in miRNA-based therapeutic approaches.