Learning Objectives:

The activation of cochlea progenitor cells is a promising approach for hair cell (HC) regeneration and hearing recovery. The mechanisms underlying the initiation of proliferation of postnatal cochlear progenitor cells and their transdifferentiation to HCs remain to be determined. We show that Notch inhibition initiates proliferation of Wnt-responsive Lgr5 + progenitor cells and mitotic regeneration of HCs in neonatal mouse cochlea in vivo and in vitro. We demonstrate that Notch inhibition removes the brakes on the canonical Wnt signaling and promotes Lgr5 + progenitor cells to mitotically generate new HCs. While, by down-regulating Notch signaling, the proliferated supporting cells (SCs) and mitotic generated HCs mainly located at the apex region of cochlea, which usually lose less hair cells compared to the base region of cochlea. For pursuing the extensive proliferation and hair cell generation needed for hearing recovery, we genetically reprogramed the SCs by activating the β-catenin to up-regulate Wnt signaling, deleting the Notch1 to down-regulate Notch signaling and overexpressing the Atoh1 in Sox2 ⁺ SCs in neonatal mouse cochleae, as we show here that the extensive proliferation of SCs followed by mitotic HC generation is achieved. Our study reveals a new function of Notch signaling in limiting proliferation and regeneration potential of postnatal cochlear progenitor cells, and provides a new strategy to regenerate HCs from progenitor cells by genetically reprograming SCs with defined genes involved in HCs formation.