Howard Hughes Medical Institute, Ansary Stem Cell Institute, Department of Genetic Medicine, Weill Cornell Medical College, New York, New York, USA.
Nat Biotechnol. 2010 Feb;28(2):161-6. doi: 10.1038/nbt.1605. Epub 2010 Jan 17.
Previous efforts to differentiate human embryonic stem cells (hESCs) into endothelial cells have not achieved sustained expansion and stability of vascular cells. To define vasculogenic developmental pathways and enhance differentiation, we used an endothelial cell-specific VE-cadherin promoter driving green fluorescent protein (GFP) (hVPr-GFP) to screen for factors that promote vascular commitment. In phase 1 of our method, inhibition of transforming growth factor (TGF)beta at day 7 of differentiation increases hVPr-GFP(+) cells by tenfold. In phase 2, TGFbeta inhibition maintains the proliferation and vascular identity of purified endothelial cells, resulting in a net 36-fold expansion of endothelial cells in homogenous monolayers, which exhibited a transcriptional profile of Id1(high)VEGFR2(high)VE-cadherin(+) ephrinB2(+). Using an Id1-YFP hESC reporter line, we showed that TGFbeta inhibition sustains Id1 expression in hESC-derived endothelial cells and that Id1 is required for increased proliferation and preservation of endothelial cell commitment. Our approach provides a serum-free method for differentiation and long-term maintenance of hESC-derived endothelial cells at a scale relevant to clinical application.
先前将人类胚胎干细胞（hESC）分化为内皮细胞的努力并未实现血管细胞的持续扩增和稳定。为了定义血管生成发育途径并增强分化，我们使用内皮细胞特异性 VE-cadherin 启动子驱动绿色荧光蛋白（GFP）（hVPr-GFP）筛选促进血管承诺的因素。在我们方法的第 1 阶段，在分化的第 7 天抑制转化生长因子（TGF）β可使 hVPr-GFP（+）细胞增加 10 倍。在第 2 阶段，TGFβ 抑制维持了纯化的内皮细胞的增殖和血管特性，导致同质单层中内皮细胞的净 36 倍扩增，其表现出 Id1（高）VEGFR2（高）VE-cadherin（+） EphrinB2（+）的转录谱。使用 Id1-YFP hESC 报告基因系，我们表明 TGFβ 抑制维持 hESC 衍生的内皮细胞中的 Id1 表达，并且 Id1 是增加增殖和维持内皮细胞承诺所必需的。我们的方法提供了一种无血清方法，可用于分化和长期维持与临床应用相关规模的 hESC 衍生的内皮细胞。
