Retrovirus–Mediated Gene Transfer in Immortalization of Progenitor Hair Cell Lines in Newborn Rat

Abstract

ObjectiveTo present an experimental method that allows isolation of greater epithelial ridge (GER) and lesser epithelial ridge (LER) cells from postnatal rat cochleae using a combinatorial approach of enzymatic digestion and mechanical separation and to investigate a retrovirus–mediated gene transfer technique for its possible utility in immortalization of the GER and LER cell lines, in an effort to establish an in vitro model system of hair cell differentiation. MethodsGER and LER cells were dissected from postnatal rat cochleae and immortalized by transferring the SV40 large T antigen using a retrovirus. The established cell lines were confirmed through morphology observation, immunnocytochemical staining and RT–PCR analysis. The Hath1 gene was transferred into the cell lines using adenovirus–mediated techniques to explore their potential to differentiate into hair cells. ResultsThe established cell lines were stably maintained for more than 20 passages and displayed many features similar to primary GER and LER cells. They grew in patches and assumed a polygonal morphology. Immunostaining showed labeling by SV40 large T antigen and Islet1 (a specific marker for GER and LER). All passages of the cell lines expressed SV40 large T antigen on RT–PCR analysis. The cells also showed the capability to differentiate into hair cell–like cells when forced to express Hath1. ConclusionRetrovirus–mediated gene transfer can be used in establishing immortalized progenitor hair cell lines in newborn rat, which may provide an invaluable system for studying hair cell differentiation and regeneration for new treatment of sensory hearing loss caused by hair cell loss.