Isolation, growth and differentiation of hair cell progenitors from the newborn rat cochlear greater epithelial ridge

Abstract

Mammalian cochlear hair cell loss is irreversible and leads to permanent hearing loss. To restore hearing physiologically, it is necessary to generate new functional hair cells either from endogenous cells or from exogenously transplanted hair cells/progenitors. Previous studies suggest that cochlear greater epithelial ridge (GER) and lesser epithelial ridge (LER) cells are capable of differentiating into hair cells. While it was recently possible to obtain and culture pure LER progenitors, isolation of pure GER progenitors has not been reported. Here we describe a method that allows isolation of pure GER cells from neonatal rat cochleae. The cochlear epithelial sheet (CES) containing GER progenitor cells was mechanically separated from the underlying mesenchymal tissue after digestion with thermolysin. The GER area could then be dissected following mechanical removal of organ of Corti as well as all the lateral area. The isolated GER cells showed significant proliferation and expressed markers for GER cells but not markers for hair cells or LER. When the GER cells were cultured in serum-free medium containing epidermal growth factor, spheres were formed where they continued to proliferate. Furthermore, when GER cells were induced to express Hath1 or co-cultured with mesenchymal cells prepared from neonate rat cochleae, they showed the potential to differentiate into hair cell-like cells. Successful isolation, culture and differentiation of GER hair cell progenitors will shed additional light on the mechanism of hair cell differentiation and potential hair cell replacement.