Greater Epithelial Ridge Cells are the Principal Organoid-Forming Progenitors of the Mouse Cochlea

Abstract

In adult mammals, sensorineural hearing loss is irreversible due to the lack of regenerative potential of non-sensory cochlear cells. Some neonatal cochlear cells, however, display a limited regenerative ability that is activated by cochlear cell loss or by culture in non-adherent conditions. These cultured non-sensory cells can grow into inner ear organoids that harbor sensory epithelial cells, including hair cells and supporting cells. Here, we purified different cochlear cell types from postnatal day 2 (P2) mice, validated the composition of the different groups with single-cell RNA-Seq, and assessed the various groups’ potential to grow into inner ear organoids. We found that the greater epithelial ridge (GER), a transient cell population that disappears during postnatal cochlear maturation, harbors the most potent organoid-forming cells. GER cells grew into large organoids that, when attached to an adherent substrate, continued to expand into large colonies with many cells organized in sensory epithelia-resembling structures containing hair cells interspersed with supporting cells. We computationally identified three distinct GER cell groups at P2 that correlate with a specific spatial distribution of marker genes. Interestingly, organoid formation was synergistically enhanced when the GER cells were cultured at increasing density. Our experiments suggest that this synergistic effect is not due to a diffusible signal but instead requires direct cell-to-cell contact. Our findings provide a new basis for the development of cell-based assays to study culture-generated inner ear cell types.