Defined populations of inner ear progenitor cells show limited and distinct capacities for differentiation into hair cells , neurons , and glia ARCHIES

Despite the fact that mammalian hair cells and neurons do not naturally regenerate in vivo, progenitor cells exist within the postnatal inner ear that can be manipulated to generate hair cells and neurons. This work reveals the differentiation capabilities of distinct inner ear progenitor populations and pinpoints cell types that can become cochlear hair cells, vestibular hair cells, neurons, and CNS glia. We expanded and differentiated cochlear and vestibular progenitors from mice (postnatal days 1-3) and analyzed the cells for expression of mature properties by RT-PCR, immunostaining, and patch clamping. Whereas previous reports suggested that inner ear stem cells may be pluripotent and/or revert to a more neural stem cell fate, we find that cells from each organ type differentiated into cells with characteristics of the respective organ. Only cochlear-derived cells expressed the outer-hair-cell protein, prestin, while only vestibularderived cells expressed the vestibular extracellular matrix marker, otopetrin. Since Atohi expression is consistently found in new hair cells, we used an Atohl-nGFP mouse line to identify hair cell candidates. We find that cells expressing Atohl also expressed key transduction, hair bundle, and synaptic genes needed for proper function. Whole-cell patch clamp recordings showed that Atohl-nGFP+ cells derived from both cochlear and vestibular tissue had voltagegated ion channels that were typical of postnatal hair cells. Only vestibular-derived AtohinGFP+ cells, however, had Ih, a hyperpolarization-activated current typical of native vestibular hair cells but not native cochlear hair cells. Lineage tracing studies with known supporting cell and glial cell markers showed that progenitor capacity of cochlear supporting cells positive for Lgr5 (Lgr5+ cells) was limited to differentiation into hair cell-like cells but not neuron-like cells. In contrast, glial cells positive for PLP (PLP1+ cells) from the auditory nerve differentiated into multiple cell types, with properties of neurons, astrocytes, or mature oligodendrocytes but not hair cells. Thus, PLP+ progenitor cells within the auditory nerve are limited to neuronal or glial fates but have greater potency than Lgr5+ progenitors, which only formed hair cell-like cells. In summary, this work identifies distinct populations of post-natal inner ear progenitors and delineates their capacity for differentiation and maturation. Thesis Supervisors: Ruth Anne Eatock, Ph.D. & Albert Edge, Ph.D. Title: Defined populations of progenitor cells show limited and distinct capacity for differentiation into hair cells, neurons, and glia 3