Bryan R. Kuo, Emily M. Baldwin, Wanda S. Layman, Makoto Mark Taketo, Jian Zuo

The mammalian cochlea exhibit minimal spontaneous regeneration, and loss of sensory hair cells (HCs) results in permanent hearing loss. In nonmammalian vertebrates, spontaneous HC regeneration occurs through both proliferation and differentiation of surrounding supporting cells (SCs). HC regeneration in postnatal mammalian cochleae in vivo remains limited by the small HC number and subsequent death of regenerated HCs. Here, we describe in vivo generation of 10-fold more new HCs in the mouse cochlea than previously reported, most of which survive to adulthood. We achieved this by combining the expression of a constitutively active form of β-catenin (a canonical Wnt activator) with ectopic expression of Atoh1 (a HC fate determination factor) in neonatal Lgr5(+) cells (the presumed SC and HC progenitors of the postnatal mouse cochlea), and discovered synergistic increases in proliferation and differentiation. The new HCs were predominantly located near the endogenous inner HCs, expressed early HC differentiation markers, and were innervated despite incomplete alignment of presynaptic and postsynaptic markers. Surprisingly, genetic tracing revealed that only a subset of Lgr5(+) cells that lie medial to the inner HCs respond to this combination, highlighting a previously unknown heterogeneity that exists among Lgr5(+) cells. Together, our data indicate that β-catenin and Atoh1 mediate synergistic effects on both proliferation and differentiation of a subset of neonatal cochlear Lgr5(+) cells, thus overcoming major limitations of HC regeneration in postnatal mouse cochleae in vivo. These results provide a basis for combinatorial therapeutics for hearing restoration. Hearing loss in humans from aging, noise exposure, or ototoxic drugs (i.e., cisplatin or some antibiotics) is permanent and affects every segments of the population, worldwide. However, birds, frog, and fish have the ability to recover hearing, and recent studies have focused on understanding and applying what we have learned from them for restoring hearing in humans. However, studies have been hampered by low efficiency, limited cell numbers, and subsequent death of these newly generated auditory cells. Here, we describe a combinatorial approach, which results in the generation of auditory cells in greater numbers than previously reported, with most of them surviving to adult ages in vivo. These results provide a basis for combinatorial therapeutics for hearing restoration efforts.