| Abstract |
We previously have established several inner ear cell lines from a two-
week old transgenic mouse (Immortomouse) harboring the thermolabile
immortalizing gene tsA58. These cell lines are capable of rapid
proliferation when cultured at 33 C in the presence of interferon-gamma
(IFN) and have the potential to differentiate when cultured at 39 C in the
absence of IFN (non-permissive condition). When cultured in the DMEM
medium supplemented with 10% fetal bovine serum (FBS), these cell lines
are able to express the inner ear specific protein OCP-2 but they neither
fully differentiate nor exhibit the structural characteristics of the
cells from which they are derived. For the present study, we selected two
of these cell lines, SVK-1 and SVK-2, which were derived from the stria
vascularis. We cultured them in the presence of the growth factors TGF-
alpha, IGF1, EGF, FGF2, BDNF, NT3, which were applied individually or in
combination in order to determine whether they could induce the
differentiation process. Cultures were monitored by phase contrast light
microscopy and by immunofluorescence using antibodies against Nestin, a
marker for neuroepithelial stem-like cells and against occludin, a tight
junction protein used as a marker for differentiated epithelial cells. The
cells initially were allowed to divide at the permissive condition (33 C +
IFN) and then were maintained in a medium with 5% FBS without IFN for one
week prior to culturing at 39 C. At the permissive condition the cells
were pleomorphic and expressed nestin, but during the first week at the
non-permissive condition they began to show contact inhibition, great
reduction in nestin expression, and exhibited an epithelial-like shape.
Addition of growth factors stimulated the cells to further differentiate.
EGF (100 ng/ml) was the most effective factor for the SVK-1, while the
combination of TGF-alpha (100 ng/ml) and IGF1 (100 ng/ml) was the most
effective for the SVK-2. Under these conditions both cell lines formed
tight junctions and organized into a regular polygonal epithelial
monolayer that resembled the marginal or basal cells of the stria
vascularis. Thus, these new cell lines probably represent true stria
vascularis epithelial cells, and maybe useful for future studies of stria
vascularis function.
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