scRNA-seq of P2 mouse cochlea

Purpose and Methods: Cochlear sensory hair cells (HCs) are essential for our sense of hearing. They are embedded in the organ of Corti that lacks regenerative capacity, which is a major cause of hearing loss. However, some neonatal non-sensory cells in the organ of Corti display a limited regenerative ability. We used fluorescence-activated cell sorting (FACS) to isolate different cochlear cell types from postnatal day 2 (P2) mice to assess the individual cell groups’ potential to grow organoids and to generate new HCs. Single-cell RNAseq validated the composition of the cell types in the sorted cell groups. Results and Conclusions: The greater epithelial ridge (GER), a transient tissue that only exists in the neonatal inner ear, harbored the most potent organoid-forming cells. GER-derived organoids expanded into large colonies when cultured adherently and gave rise to new hair cell marker-expressing cells in a sensory epithelia-resembling organization. The organoid forming ability of GER cells was synergistically enhanced when they were cultured at increasing density. The synergistic effect relied on direct cell-to-cell contact rather than released soluble factors. We used Sox2-GFP, Lfng-GFP, Fgfr3-CreERT2, and Ai14-tdTomato two-day-old transgenic mice to isolate five distinctive groups of cochlear cells. Single-cell RNAseq was performed on these cells using Illumina NextSeq 500 high output flow cell configuration with a 150 bp paired-end sequencing.