A Low Dose of Rapamycin Promotes Hair Cell Differentiation by Enriching SOX2+ Progenitors in the Neonatal Mouse Inner Ear Organoids

To investigate the impact of rapamycin on the differentiation of hair cells. Murine cochlear organoids were derived from cochlear progenitor cells. Different concentrations of rapamycin were added into the culture medium at different proliferation and differentiation stages. Rapamycin exhibited a concentration-dependent reduction in the proliferation of these inner ear organoids. Nevertheless, organoids subjected to a 10-nM dose of rapamycin demonstrated a markedly increased proportion of hair cells. Furthermore, rapamycin significantly upregulated the expression of markers associated with both hair cells and supporting cells, including ATOH1, MYO7A, and SOX2. Mechanistic studies revealed that rapamycin preferentially suppressed cells without Sox2 expression during the initial proliferation stage, thereby augmenting and refining the population of SOX2+ progenitors. These enriched progenitors were predisposed to differentiate into hair cells during the later stages of organoid development. Conversely, the use of the mTOR activator MHY 1485 demonstrated opposing effects. Our findings underscore a practical strategy for enhancing the generation of inner ear organoids with a low dose of rapamycin, achieved by enriching SOX2+ progenitors in an in vitro setting.