Human ATOH1 adenoviral particles

Studies of the adult mammalian auditory system, such as regeneration, have been hampered by the lack of an in vitro system in which hypotheses can be effectively tested. This is primarily because the adult inner ear is encased in the body's strongest bone, and removal of the inner ear results in the death of the sensory epithelium in culture. Here, researchers hypothesized that the intact cochlear structure could be exploited to maintain the entire inner ear architecture and improve sensory epithelial survival in culture. Researchers found that by culturing adult mouse cochleae with the (surrounding) skeleton intact, supporting cells (SCs) survived and nearly all hair cells (HCs) degenerated. To assess the utility of the explant culture system, researchers demonstrated that overexpression of the HC fate determinant Atoh1 was sufficient to induce transdifferentiation of adult SCs into HC-like cells (HCLCs). Transdifferentiation-derived HCLCs resembled developing young HCs and were able to attract adult ganglion neurites. Furthermore, using an injury model, degenerating adult ganglia responded to regenerating HCLCs with directed neurite outgrowth resulting in HCLC-neuron contacts, strongly supporting an intrinsic property of HCLCs in establishing HCLC-neuron connections. Adult whole cochlear explant culture is suitable for various studies of the adult inner ear, including regeneration, HC-neuron pathways, and inner ear drug screening.

To test the utility of the explant culture system for regenerating HCs in vitro, researchers cultured adult cochleae from Atoh1-GFP transgenic mice, in which GFP expression is under the control of the Atoh1 3' enhancer and induced by Atoh1 expression. Researchers infected cultured adult Atoh1-GFP transgenic mouse cochleae with adenovirus carrying human ATOH1 (Ad-Atoh1), while adenovirus carrying the V5 tag (Ad-V5) was used as an infection control. In Ad-V5-infected Atoh1-GFP cochleae that were subsequently cultured for 14 days, a few weak GFP+ cells were detected in both surviving IHCs and SCs near surviving IHCs in the explants (Figure 1a1-a3), reflecting weak endogenous ATOH1 activity. In adult Atoh1-GFP cochleae infected with Ad-Atoh1 and cultured for 14 days, a greater number of GFP+ cells were detected in the sensory epithelium and the spiral limbus region in the explants (Figure 1b, e). These GFP+ cells are cells with Atoh1 transcriptional activity due to Ad-Atoh1 infection.

Figure 1. Transdifferentiation of Ad-Atoh1-infected cells and lineage tracing in cultured adult cochleae. (Li W, et al., 2022)

To further determine the origin of HCLCs, the researchers performed lineage tracing using Sox2-CreER-Tm-red mice. After 3 days of daily tamoxifen injection, adult SCs were permanently labeled with tdTomato (Tm-red+). Cochleae from adult mice injected with tamoxifen were cultured and infected with Ad-Atoh1 or Ad-V5. After 14 days of Ad-V5 infection, no PVALB+ HCLCs were observed in the OHC region, and the rest of the existing IHCs were negative for Tm-red (Figure 1c). In contrast, PVALB+ HCLCs were detected in the OHC region after Ad-Atoh1 infection, and all of them were Tm-red+ (Figure 1d). These studies suggest that Atoh1 overexpression leads to regeneration of HCLCs in the OHC region due to transdifferentiation of SOX2+ SCs, as well as regeneration of HCLCs in the limbal region due to transdifferentiation of SOX2-negative cells.