In Vivo Generation of Immature Inner Hair Cells in Neonatal Mouse Cochleae by Ectopic Atoh1 Expression

Regeneration of auditory hair cells (HCs) is a promising approach to restore hearing. Recent studies have demonstrated that induced pluripotent stem cells/embryonic stem cells or supporting cells (SCs) adjacent to HCs can be converted to adopt the HC fate. However, little is known about whether new HCs are characteristic of outer or inner HCs. Here, we showed in vivo conversion of 2 subtypes of SCs, inner border cells (IBs) and inner phalangeal cells (IPhs), to the inner HC (IHC) fate. This was achieved by ectopically activating Atoh1, a transcription factor necessary for HC fate, in IBs/IPhs at birth. Atoh1+ IBs/IPhs first turned on Pou4f3, another HC transcription factor, before expressing 8 HC markers. The conversion rate gradually increased from similar to 2.4% at 1 week of age to similar to 17.8% in adult. Interestingly, new HCs exhibited IHC characteristics such as straight line-shaped stereociliary bundles, expression of Fgf8 and otoferlin, and presence of larger outward currents than those of outer HCs. However, new HCs lacked the terminal differentiation IHC marker vGlut3, exhibited reduced density of presynaptic Cbtp2 puncta that had little postsynaptic GluR2 specialization, and displayed immature IHC outward currents. Our results demonstrate that the conversion rate of IBs/IPhs in vivo by Atoh1 ectopic expression into the IHC fate was higher and faster and the conversion was more complete than that of the 2 other SC subtypes underneath the outer HCs; however, these new IHCs are arrested before terminal differentiation. Thus, IBs/IPhs are good candidates to regenerate IHCs in vivo.