INDUCTION OF CELL-PROLIFERATION BY FIBROBLAST AND INSULIN-LIKE GROWTH-FACTORS IN PURE RAT INNER-EAR EPITHELIAL-CELL CULTURES

Proliferation of supporting cells in the inner ear is the early major event occurring during hair cell regeneration after acoustic trauma or aminoglycoside treatment. In the present study, we examined the possi ble influence of 30 growth factors on the proliferation of pure rat ut ricular epithelial cells in culture. Utricular epithelial sheets were separated and partially dissociated from early postnatal rats via a co mbined enzymatic and mechanical method. The cultured utricular epithel ial cells expressed exclusively epithelial cell antigens, but not fibr oblast, glial, or neuronal antigens. With tritiated thymidine incorpor ation assays, we found that several fibroblast growth factor (FGF) fam ily members, insulin-like growth factor-1 (IGF-1), IGF-2, transforming growth factor-alpha (TGF-alpha), and epidermal growth factor (EGF), s timulated proliferation of the utricular epithelial cells. In contrast , neurotrophins and other growth factors did not elicit any detectable mitogenic effects. Among all of the growth factors examined, FGF-2 wa s the most potent mitogen. When FGF-2 was added in combination with IG F-1 or TGF-alpha to the medium, combined effects were seen. These resu lts were confirmed with BrdU immunocytochemistry, Thus, the present cu lture system provides a rapid and reliable assay system to screen nove l growth factors involved in proliferation of mammalian inner ear supp orting cells. Furthermore, immunostainings revealed that the cultured utricular epithelial cells expressed FGF and IGF-1 receptors, and utri cular hair cells produced FGF-2 in vivo. The addition of neutralizing antibodies against FGF-2 or IGF-1 to the cultures significantly inhibi ted the utricular epithelial cell proliferation. This work suggests th at FGF-2 and IGF-1 may regulate the proliferation step during hair cel l development and regeneration.