Investigation and validation of stereocilia development in homogenic cochlea progenitor cell organoid using bulk and single-cell RNA sequencing analysis

The hair cell (HC)s exhibit mechanoelectrical transduction that is enabled by stereocilia. Leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5) is known to be involved in the generation of HCs. The stripping method is widely used to differentiate LGR5 progenitor cells (LPC) into inner-ear organoids. However, cells separated via stripping are heterotypic, making it impossible to identify specific cell–cell interactions that affect differentiation. Therefore, methods are needed to differentiate homotypic LPCs. We performed homotypic differentiation of LPCs, and eventually observed HC like cells. However, in further analysis of cellular morphology, immature stereocilia was identified from organoids grown from homotypic LPCs. Using bulk RNA-seq, downregulation of morphogenesis-related genes was identified in organoid by homotypic LPCs. We assessed the effects of an SHH agonist and found that it induced the generation of further differentiated stereocilia. This was confirmed by electron microscopy and significantly heightened expression of stereocilia-related genes ( Pls , LMO7 , LRBA ). Using scRNA-seq, we concentrated on various cochlear markers, including stereocilia formation, to identify cell types that shared a similar developmental trajectory with HCs. Among them, cluster 11 showed robust expression of stereocilia-related genes including Espn , Lhfpl5 , Loxhd1 , and Tmc1 . Further functionality of the cells with this mature stereocilia was confirmed by electrophysiology using multielectrode array.