Abstract
The study of an adult mammalian auditory system, such as regeneration, has been hampered by the lack of an in vitro system in which hypotheses can be tested efficiently. This is primarily due to the fact that the adult inner ear is encased in the toughest bone of the body, whereas its removal leads to the death of the sensory epithelium in culture. We hypothesized that we could take advantage of the integral cochlear structure to maintain the overall inner ear architecture and improve sensory epithelium survival in culture. We showed that by culturing adult mouse cochlea with the (surrounding) bone intact, the supporting cells (SCs) survived and almost all hair cells (HCs) degenerated. To evaluate the utility of the explant culture system, we demonstrated that the overexpression of Atoh1, an HC fate-determining factor, is sufficient to induce transdifferentiation of adult SCs to HC-like cells (HCLCs). Transdifferentiation-derived HCLCs resemble developmentally young HCs and are able to attract adult ganglion neurites. Furthermore, using a damage model, we showed that degenerated adult ganglions respond to regenerated HCLCs by directional neurite outgrowth that leads to HCLC-neuron contacts, strongly supporting the intrinsic properties of the HCLCs in establishing HCLC-neuron connections. The adult whole cochlear explant culture is suitable for diverse studies of the adult inner ear including regeneration, HC-neuron pathways, and inner ear drug screening.
Highlights
Hearing deficits affect one in 500 newborn babies and half of the senior population over 70 years of age in the world (Morton and Nance, 2006; Muller and Barr-Gillespie, 2015)
We first tested the dissection of the adult cochlear sensory epithelium according to the protocol established for culturing neonatal mouse cochleae (Parker et al, 2010; Landegger et al, 2017)
What are the characteristics of new HC-like cells (HCLCs)? Are they relatively mature hair cells (HCs) resulting from formation in the adult cochlea, or are they developmentally young HCs due to recent transdifferentiation from supporting cells (SCs)? Using immunostaining with markers including PVALB, PTPRQ, espin (ESPN), and acetylated tubulin, we studied the presence of HC stereocilia in the regenerated HCLCs
Summary
Hearing deficits affect one in 500 newborn babies and half of the senior population over 70 years of age in the world (Morton and Nance, 2006; Muller and Barr-Gillespie, 2015). To study regeneration in mammals, the neonatal mouse cochlear culture system has been developed and used extensively (Zheng and Gao, 1999, 2000; Zheng et al, 2000). A general approach to culture neonatal mouse cochleae involves the isolation of the sensory epithelium, which is cultured by adhering to the surface of a culture dish. A similar approach for adult cochleae requires the disruption of the ossified cochlear bone followed by removing the sensory epithelium for culture. Under this condition, the sensory epithelium inevitably dies shortly later. By culturing adult mouse cochlea with the intact bone, some sensory epithelial cells may survive long-term within well-preserved cochlear architecture suitable for the study of adult mammalian cochleae in culture
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