Isolation and culture of hair cell progenitors from postnatal rat cochleae.

Cochlear hair cells are a terminally differentiated cell population that is crucial for hearing. Although recent work suggests that there are hair cell progenitors in postnatal mammalian cochleae, isolation and culture of pure hair cell progenitors from a well-defined cochlear area have not been reported. Here we present an experimental method that allows isolation and culture of hair cell progenitors from postnatal rat cochleae. These progenitor cells are isolated from the lesser epithelial ridge (LER, or outer spiral sulcus cell) area of pre-plated neonatal rat cochlear segments. They express the same markers as LER cells in vivo, including ZO1, Islet1, Hes1, and Hes5. When these cells are induced to express Hath1, they show the potential to differentiate into hair cell-like cells. Interestingly, these cells can be lifted from monolayer cultures and maintained in aggregate cultures in which spheres can be formed. Hair cell progenitors in the spheres display their proliferating capability and express only epithelial markers. Furthermore, when these spheres are mixed with dissociated mesenchymal cells prepared from postnatal rat utricular whole mounts, and replated onto a collagen substratum, the epithelial progenitor cells are able to differentiate into cells expressing markers of hair cells and supporting cells in epithelial islands, which mirrors the inner ear sensory epithelium in vivo. Successful isolation and culture of hair cell progenitors from the mammalian cochlea will facilitate studies on gene expression profiling and mechanism of differentiation/regeneration of hair cells, which are crucial for repairing hearing loss.