ABSTRACT
Mammalian auditory hair cells are few in number, experimentally inaccessible, and do not proliferate postnatally or in vitro. Immortal cell lines with the potential to differentiate into auditory hair cells would substantially facilitate auditory research, drug development, and the isolation of critical molecules involved in hair cell biology. We have established two conditionally immortal cell lines that express at least five characteristic hair cell markers. These markers are the transcription factor Brn3.1, the alpha 9 subunit of the acetylcholine receptor, the stereociliary protein fimbrin and the myosins VI and VIIA. These hair cell precursors permit functional studies of cochlear genes and in the longer term they will provide the means to explore therapeutic methods of stimulating auditory hair cell regeneration.
Subject(s)
Cell Line, Transformed , Hair Cells, Auditory/cytology , Microfilament Proteins , Animals , Cell Differentiation , DNA-Binding Proteins/biosynthesis , Dyneins , Epithelial Cells/metabolism , Female , Hair Cells, Auditory/metabolism , Male , Membrane Glycoproteins/biosynthesis , Mice , Mice, Inbred C57BL , Myosin Heavy Chains/biosynthesis , Myosin VIIa , Myosins/biosynthesis , Receptors, Cholinergic/biosynthesis , Stem Cells/cytology , Stem Cells/metabolism , Transcription Factor Brn-3 , Transcription Factor Brn-3A , Transcription Factors/biosynthesisABSTRACT
The aim of this work was to culture conditionally immortalized cells that possess the potential to differentiate into mechanosensory hair cells. Utricular epithelia at embryonic stage E16 were cultured from the vestibular system of the H2kbtsA58 transgenic mouse (Immortomouse) that carries a conditionally expressed immortalizing gene derived from the simian virus 40. Immunolabelling showed that the immortalizing transgene product, the T antigen (Tag), was expressed in utricular cells under permissive conditions and that it was inactivated under non-permissive conditions. Several morphologically distinct cell types proliferated when Tag was expressed, including those that resembled fibroblasts, nerve cells and epithelial cells. Mixed cultures of cells from the utricle, passaged up to 50 times every 3-4 days over a period of 5 months, were subsequently allowed to differentiate for 10 days by transferring them to non-permissive conditions. Monoclonal antibody markers were used to locate expression of hair cell specific antigens. One antibody that normally labels stereociliary bundles from postnatal stage P4-6 labelled cellular projections from a population of spheroid cells that were distributed across the culture surface. A second antibody that normally labels stereociliary bundles did not label the same structures. We conclude that utricular hair cell progenitors can be derived from the H2kbtsA58 transgenic mouse but that under the experimental conditions used they do not follow the normal pattern of differentiation.
