Abstract
Due to the lack of regenerative capacity of the mammalian auditory epithelium, sensory hair cell loss results in permanent hearing deficit. Nevertheless, a population of tissue resident stem/progenitor cells has been recently described. Identification of methods to trigger their activity could lead to exploitation of their potential therapeutically. Here we validate the use of transgenic mice reporting cell cycle progression (FUCCI), and stemness (Lgr5-GFP), as a valuable tool to identify regulators of cell cycle re-entry of supporting cells within the auditory epithelium. The small molecule compound CHIR99021 was used to inhibit GSK3 activity. This led to a significant increase in the fraction of proliferating sphere-forming cells, labeled by the FUCCI markers and in the percentage of Lgr5-GFP + cells, as well as a selective increase in the fraction of S-G2-M cells in the Lgr5 + population. Using whole mount cultures of the organ of Corti we detected a statistically significant increment in the fraction of proliferating Sox2 supporting cells after CHIR99021 treatment, but only rarely appearance of novel MyoVIIa+/Edu + hair cells. In conclusion, these tools provide a robust mean to identify novel regulators of auditory organ regeneration and to clarify the contribution of stem cell activity.
Highlights
Sound perception in mammals relies on the function of specialized mechano-sensitive hair cells located within the organ of Corti (OC)
Our work identifies that proliferation of otic stem/progenitor cells can be triggered by a small molecule inhibitor targeting GSK3: CHIR99021
Using whole organ cultures of FUCCI reporter lines we detected a significant increase in the proliferation of Sox2 + supporting cells
Summary
Sound perception in mammals relies on the function of specialized mechano-sensitive hair cells located within the organ of Corti (OC). Mechano-sensory hair cells are organized in a mosaic structure with non-sensory supporting cells within the epithelium The latter have been recently recognized as dormant stem/progenitor cells of this organ[2,3,4,5,6]. Genetic ablation of hair cells in vivo was shown to drive stem cell activity in the Lgr5 + cell pool, contributing to some extent to spontaneous hair cell regeneration This occurred though, at very low levels and only in early postnatal stages[5]. Translation of these findings towards therapeutic application will require identification of selective small molecule inhibitors able to induce stem cell activity by driving re-expression of positive cell cycle regulators or by triggering developmental genes. In combination with the stem cell reporter Lgr[5], the FUCCI system allows for analysis of cell cycle re-entry and progression of Lgr5 + OC supporting cells
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