In Vitro Culture And Induced Differentiation Of Greater Epithelial Ridge Cells And Establishment Of Immortalized Greater Epithelial Ridge And Lesser Epithelial Ridge Cell Lines From Rat Cochlea

In the mammalian auditory system, cochlea hair cell loss resulting from aging, ototoxic drugs, infections, overstimulation and other causes is irreversible and leads to permanent sensorineural hearing loss. Sensorineural hearing loss affects millions of people worldwide. Although hair cell replacement occurs spontaneously following injury in birds and lower vertebrates, the regeneration of new hair cells in mature mammalian cochlea is currently unavailable. As a matter of fact, cochlear hair cell loss is one of the major causes of hearing impairment.Recent work suggests that there are hair cell progenitors in postnatal mammalian cochlea and the GER (greater epithelial ridge) and LER (lesser epithelial ridge) cells may serve as hair cell progenitor cells. A very recent study on the LER cells has been performed, whereas the islation and in vitro culture of pure GER cell progenitors from a well-defined cochlear area have not been reported. Here we developed an experimental method that allows isolation and culture of GER cells from postnatal rat cochleae using a combinatorial approach of enzymatic digestion and mechanical separation. The dissociated GER cells express Islet1, Islet2, ZO1, Phalloidin, Hesl and BrdU, but not myosin VIIa and Calretinin which are mature and immature marker of hair cell respectively. They grow in islands-like patches and assume a polygonal morphology in medium containing 10% fetal bovine serum while forming spheres in serum-free medium. The GER cell cultures show significant ability to proliferate in both conditions and tend to form dome which is a kind of specific structure of epithelial cells. Successful isolation and culture of hair cell progenitors from the mammalian cochlea will facilitate studies on gene expression profiling and mechanism ofdifferentiation/ regeneration of hair cells, which are crucial for repairing hearing loss.It is generally belived that hair cell fate determination in the inner ear is the result of both cell-cell interaction and regulation of specific genes. In particular, a bHLH transcription factor, Mathl, is necessary and sufficient for hair cell differentiation. For example, when GER and LER cells that normally differentiate into fluid-forming cells in postnatal rat cochlear explants are induced to express Mathl or its human homolog Hathl, they become hair cell-like cells. In the present experiments, we explored the differentiation in pure GER cell cultures. When these cells are induced to express Hathl, they show the potential to differentiate into hair cell-like cells. Surprisingly, we found that there are some sterocilliary bundle-like structures in the edge of the majority of GER cell islands. Furthermore, when GER cells are mixed with dissociated mesenchymal cells prepared from postnatal rat cochlear whole mounts, the epithelial progenitor cells are able to differentiate into cells expressing markers of mature or immature hair cells and supporting cells in epithelial islands. This study provides direct support for the notion that the GER cells may serve as hair cell progenitors.Identification of the molecular and cellular mechanisms underlying the development and regeneration of hair cells has been hampered by the small tissue size, the complicated in vivo bony structures of the inner ear, and by the lack of culture systems composed primarily of hair cell progenitors. Establishment of immortalized cell lines from hair cell progenitors is ideal for studies of hair cell regeneration because of solving the problem of difficult microdissection and the limited number of cells. Here we present a retrovirus-mediated SV40 large T antigen gene transfer technique which made the immortalization of GER and LER cells from postnatal rat cochlear realized. The rat cochlear GER and LER cell lines (GLERC-6) kept the phenotype characterization of the primary GER andLER cells and stably maintained continuous ability to proliferate. Furthermore, the GLERC-6 cells underwent a differentiation into hair cell phenotype when forced to express Hathl. This study may provide experimental approach for the study on hair cell differentiation and regeneration as well as an alternative way to replace lost hair cells by introducing exogenous progenitor/stem cells into the inner ear.