Single cell gene expression profile of adult cochlear explants over the course of hair cell regeneration stimulated by chemical reprogramming [CocktailVehicleAtoh1]

Strategies to overcome irreversible cochlear hair cell (HC) damage and loss are of vital importance to develop a treatment for hearing loss. HC regeneration in adult cochlea relies on a two-phase process: 1) Reprogramming mature cochlear (SCs) to regain the properties of their younger selves; 2) Activating Atoh1, a gene responsible for HC fate-determining, in the reprogrammed adult SCs for HC regeneration. We have shown that, by transient co-activation of Myc and NICD (Notch1 intracellular domain), the adult mouse cochlea can be successfully reprogrammed to a relatively younger stage and regain progenitor capacity, with the regeneration of HCs following Atoh1 overexpression in vitro and in vivo. We identified a combination (the cocktail) of drug-like molecules composing of small molecules and siRNAs to activate the pathways of Myc, Notch1, Wnt and cAMP. To identify molecules to reprogram mature cochlear SCs and HC regeneration, we utilized single-cell RNA sequencing and uncovered the pathways and their target genes underlying chemical-mediated reprogramming. We used an in-house adult cochlea explant culture system and carried out single-cell RNA sequencing to examine the gene expression profiles of cochlear explants, in response to chemical-induced reprogramming. We compared gene expression profiles between Vehicle/ad.Atoh1 activation vs. Cocktail (chemical reprogramming)/ad.Atoh1 activation. Overall design: In chemical reprogramming group, we treated whole adult WT C57BL/6J cochlear explants with VPA (1.5 mM), LiCl (8mM), FSK (20µM), siFIR (0.02 µM), and siMxi1 (0.02 µM) for 4 days, followed by 2 weeks ad.Atoh1 overexpression, before dissecting sensory epithelium to create cDNA libraries subjected to 10X Genomics pipeline barcoding, library preparation, and sequencing. In control group, we treated whole adult WT C57bl/6j cochlear explants with sterile water with 0.1%DMSO in vitro for four days, followed by 2 weeks ad.Atoh1 overexpression, before dissecting sensory epithelium to create cDNA libraries. Vehicle (sterile water containing 0.1% DMSO)_ad.Atoh1, n=5; Cocktail (chemical reprogramming)_ad.Atoh1 group, n=5.