5-Azacytidine induces sensory hair cell marker expression in human inner ear cells in vitro

Adult human inner ear hair cells have extremely limited regenerative ability, and the inaccessibility of human inner ear tissue makes studying regeneration challenging. In this study, we examined whether 5-azacytidine (5-aza) could induce hair cell-like differentiation using our previously established human utricular cell (HUC) line. Our results showed that treatment with 40-80 μM 5-aza was non-toxic, as confirmed by cell counting and calcein/propidium iodide assays. BrdU incorporation analysis showed that fewer treated HUCs entered the S-phase, indicating reduced cell proliferation following 5-aza treatment. Reverse transcription PCR revealed that 5-aza induced the expression of hair cell genes (Myo7a, Pou4f3, Atoh1, and Myo6), which were absent in untreated cells. Immunofluorescence confirmed the expression of hair cell proteins in 5-aza-treated cells, including Myosin VIIa, Pou4f3, Atoh1, Myosin VI, and Calretinin. Additionally, FM1-43 uptake assays indicated the emergence of functional mechanotransduction-like channels in 5-aza-treated HUCs. Together, these results demonstrate that 5-aza reduces proliferation and promotes differentiation of HUCs toward a hair cell-like phenotype. This finding provides new insight into the molecular mechanisms regulating human sensory hair cell differentiation in vitro and highlights the potential of HUCs as a model for studying human inner ear hair cell regeneration.