Density-dependent differentiation in nontransformed human retinal progenitor cells in response to basic fibroblast growth factor- and transforming growth factor-alpha.

Multipotential retinal precursors give rise to all cell types seen in multilayered retina. The generation of differentiation and diversity of neuronal cell types is determined by both extrinsic regulatory signals and endogenous genetic programs. We have previously reported that cell commitment in human retinal precursor cells (SV-40T) can be modified in response to exogenous growth factors, basic fibroblast growth factor, and transforming growth factor alpha (bFGF and TGFalpha). We report in this study that nontransformed human retinal precursors differentiate into photoreceptors by a cell density-dependent mechanism, and the effects were potentiated by bFGF and TGFalpha alone or in combination. A larger proportion of multipotential precursors plated at a density of 1 x 10(4) cells/cm(2) differentiated into neurons (photoreceptors) compared to cells plated at 3-5 x 10(4)/cm(2) and 1 x 10(5) cells/cm(2) under serum-free conditions and the effects were amplified seven- to eightfold in response to growth factors. Basic fibroblast growth factor (bFGF) and TGFalpha can induce 90% of the cells to assume a photoreceptor phenotype at a lower cell density, compared to only 30 and 25% of the cells acquiring a photoreceptor phenotype at intermediate and higher cell densities. Furthermore, at a lower cell density, 60-70% of the cells incorporate Bromodeoxyuridine (Brdu), suggesting that cells in a cell cycle may make a commitment to a specific fate in response to neurotrophins. Neurons with a photoreceptor phenotype were positive for three different sets of antibodies for rods/cones. Cells also exhibited upregulation of other proteins such as a D4 receptor protein expressed in photoreceptors, protein kinase Calpha (PKCalpha) expressed in rod bipolars and blue cones, and some other neuronal cell types. This was also confirmed by Western blot analysis. Newly derived photoreceptors survive for a few days before significant cell death ensues under serum-free conditions. To summarize, differentiation in precursors is density dependent, and growth factors amplify the effects.