Density-dependent differentiation in nontransformed human retinal progenitor cells in response to basic fibroblast growth factor- and transforming growth factor-α
Density-dependent differentiation in nontransformed human retinal progenitor cells in response to basic fibroblast growth factor- and transforming growth factor-α
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DOI：
10.1089/104454903770238085
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发表时间：
2003-10-01
影响因子：
3.1
通讯作者：
Dutt, K
中科院分区：
文献类型：
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作者：
Ezeonu, I;Wang, M;Dutt, K
多电位视网膜前体产生多层视网膜中所有类型的细胞。神经细胞类型分化和多样性的产生是由外源性调控信号和内源性遗传程序共同决定的。我们之前报道过，人视网膜前体细胞（SV-40T）的细胞承诺可以在外源性生长因子、碱性成纤维细胞生长因子和转化生长因子α （bFGF和TGFalpha）的作用下发生改变。我们在本研究中报道，未转化的人视网膜前体细胞通过细胞密度依赖机制分化为光感受器，并且bFGF和TGFalpha单独或联合作用可增强其作用。与在无血清条件下以3-5 × 10(4)个细胞/cm(2)和1 × 10(5)个细胞/cm(2)的密度镀的细胞相比，以1 × 10(4)个细胞/cm(2)的密度镀的多电位前体分化为神经元（光感受器）的比例更大，并且根据生长因子的反应，效果被放大了7到8倍。碱性成纤维细胞生长因子（bFGF）和TGFa可以在较低细胞密度下诱导90%的细胞呈现光感受器表型，而在中等和较高细胞密度下，只有30%和25%的细胞获得光感受器表型。此外，在较低的细胞密度下，60 - 70%的细胞含有溴脱氧尿苷（Brdu），这表明细胞周期中的细胞可能对神经营养素作出特定的反应。具有光感受器表型的神经元对三组不同的棒/锥抗体呈阳性。细胞还表现出其他蛋白质的上调，如光感受器中表达的D4受体蛋白，杆状双极体和蓝锥细胞中表达的蛋白激酶Calpha (PKCalpha)，以及其他一些神经元细胞类型。Western blot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细胞死亡前可存活数天。总之，前体的分化依赖于密度，生长因子放大了这种效应。
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 TGFa 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.