Ezeonu Ifeoma, Wang Meir, Kumar Ravindra, Dutt Kamla
Department of Pathology, Morehouse School of Medicine, Atlanta, Georgia 30310, USA.
DNA Cell Biol. 2003 Oct;22(10):607-20. doi: 10.1089/104454903770238085.
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.
多能视网膜前体细胞可分化为多层视网膜中所见的所有细胞类型。神经元细胞类型的分化和多样性的产生由外在调节信号和内源性遗传程序共同决定。我们之前报道过，人视网膜前体细胞（SV - 40T）的细胞定向可因外源性生长因子、碱性成纤维细胞生长因子和转化生长因子α（bFGF和TGFα）而发生改变。我们在本研究中报告，未转化的人视网膜前体细胞通过细胞密度依赖性机制分化为光感受器，bFGF和TGFα单独或联合使用可增强这种效应。与在无血清条件下以3 - 5×10⁴/cm²和1×10⁵细胞/cm²接种的细胞相比，以1×10⁴细胞/cm²的密度接种的多能前体细胞中，有更大比例分化为神经元（光感受器），并且生长因子可使这种效应放大7至8倍。与在中等和较高细胞密度下分别只有30%和25%的细胞获得光感受器表型相比，碱性成纤维细胞生长因子（bFGF）和TGFα在较低细胞密度下可诱导90%的细胞呈现光感受器表型。此外，在较低细胞密度下，60 - 70%的细胞掺入溴脱氧尿苷（Brdu），这表明处于细胞周期中的细胞可能会因神经营养因子而定向分化为特定命运的细胞。具有光感受器表型的神经元对用于视杆细胞/视锥细胞的三组不同抗体呈阳性反应。细胞还表现出其他蛋白质的上调，如在光感受器中表达的D4受体蛋白、在视杆双极细胞和蓝色视锥细胞以及其他一些神经元细胞类型中表达的蛋白激酶Cα（PKCα）。蛋白质印迹分析也证实了这一点。在无血清条件下，新产生的光感受器在显著细胞死亡发生前可存活数天。总之，前体细胞的分化依赖于密度，生长因子可增强这种效应。
