RNA-sequence facilitates analyze changes of signaling pathways after cell reprogramming

Cochlear hair cells are the sensory cells responsible for transduction of acoustic signals. In mammals, damaged hair cells do not regenerate, resulting in permanent hearing loss. Reprogramming of the surrounding supporting cells to functional hair cells represent a novel strategy to hearing restoration. However, cellular processes governing the efficient and functional hair cell reprogramming are not completely understood. Employing the mouse cochlear organoid system, we performed detailed metabolomic characterizations of the expanding and differentiating organoids. We found that hair cell differentiation is associated with increased mitochondrial electron transport chain (ETC) activity and reactive oxidative species generation. Transcriptome and metabolome analyses indicate reduced expression of oxidoreductases and tricyclic acid (TCA) cycle metabolites. The metabolic decoupling between ETC and TCA cycle limits the availability of the key metabolic cofactors, α-ketoglutarate and NAD+. Reduced expression of NAD+ in cochlear supporting cells by PGC1α deficiency further impairs hair cell reprogramming, while supplementation of α-ketoglutarate and NAD+ promotes hair cell reprogramming both in vitro and in vivo. Our findings reveal metabolic rewiring as a central cellular process during hair cell differentiation, and highlight the insufficiency of key metabolites as a metabolic barrier for efficient hair cell reprogramming.