Defining developmental trajectories of prosensory cells in human inner ear organoids at single-cell resolution

The inner ear sensory epithelia contain mechanosensitive hair cells and supporting cells. Both cell types arise from SOX2-expressing prosensory cells, but the mechanisms underlying the diversification of these cell lineages remain unclear. To determine the transcriptional trajectory of prosensory cells, we established a SOX2-2A-ntdTomato human ES cell line using CRISPR/Cas9, and performed single-cell RNA-sequencing analysis with SOX2-positive cells isolated from inner ear organoids at various time points between differentiation days 20 and 60. Our pseudotime analysis suggests that hair cells arise primarily from supporting cells, rather than bi-fated transitional cells in organoids. Moreover, ion channel- and ion transporter-related gene sets were enriched in supporting cells vs. prosensory cells, whereas Wnt signaling-related gene sets were enriched in hair cells vs. supporting cells. These findings provide valuable insights into how prosensory cells give rise to hair cells and supporting cells during human inner ear development, and may provide a clue to promote hair cell regeneration from resident supporting cells in individuals with hearing loss and balance disorders. Yoshitomo, Ueda