Differentiation of mammalian vestibular hair cells from conditionally immortal, postnatal supporting cells

We provide evidence from a newly established, conditionally immortal cell l ine (UB/UE-1) that vestibular supporting cells from the mammalian inner ear can differentiate postnatally into more than one variant of hair cell. A c lonal supporting cell line was established from pure utricular sensory epit helia of H2k (b)tsA58 transgenic mice 2 d after birth. Cell proliferation w as dependent on conditional expression of the immortalizing gene, the "T" a ntigen from the SV40 virus. Proliferating cells expressed cytokeratins, and patch-clamp recordings revealed that they all expressed small membrane cur rents with little time-dependence. They stopped dividing within 2 d of bein g transferred to differentiating conditions, and within a week they formed three defined populations expressing membrane currents characteristic of su pporting cells and two kinds of neonatal hair cell. The cells expressed sev eral characteristic features of normal hair cells, including the transcript ion factor Brn3.1, a functional acetylcholine receptor composed of alpha 9 subunits, and the cytoskeletal proteins myosin VI, myosin VIIa, and fimbrin . Immunofluorescence labeling and electron microscopy showed that the cells formed complex cytoskeletal arrays on their upper surfaces with structural features resembling those at the apices of normal hair cells. The cell lin e UB/UE-1 provides a valuable in vitro preparation in which the expression of numerous structural and physiological components can be initiated or upr egulated during early stages of mammalian hair cell commitment and differen tiation.