Cochlear Organoids for the Study of Hair Cell Development and Regeneration

Cochlear hair cells (HC) are susceptible to noise exposure, ototoxic treatments, and age. Currently, no treatment exists to promote HC regeneration in the cochlea and deafness is therefore irreversible once HCs are lost. Many efforts are put on identifying genes, pathways and molecules that would make cochlear HC regeneration possible. However, the low number of cochlear HC as well as the size of the cochlea and its inaccessibility limit in vivo studies. Thus, there is a critical need for a reliable model to examine HC development and to screen for treatments that could stimulate hair cell regeneration.
To develop such a model, we generated a mouse strain expressing mCherry under the Atoh1 promoter and combined it with a mouse strain expressing GFP under the Lgr5 promoter. We obtained mice in which a subset of supporting cells and all HCs were labeled in different colors.
Application in this new mouse strain of a previously established protocol that allows for clonal expansion of HC progenitors and subsequent HC differentiation, results in labeled cochlear cells in organoids: Lgr5-expressing supporting cells which include hair cell progenitors are labeled in green and HCs in red. Using flow cytometry from dissociated organoids, we can track HC differentiation from Lgr5+ cells to Atoh1+ cells and assess both the number of HC progenitors and the number of HCs at progressive differentiation times.
Finally, our model helps to track HC differentiation and to easily assess the number of HC progenitors and HC in the organoids. Additionally, we can sort these populations and assess gene expression (RNA-sequencing), protein expression (immunostaining), and protein-chromatin interactions (CUT&RUN). As the effect of treatments during proliferation or differentiation can be easily determined, the two-color model provides an easy-to-use and reliable tool for assessing the effect of treatments on HC generation, and we are currently using the model to screen small molecules and genes critical for HC development and regeneration.