Laboratory for Vascular Biology and Stem Cell, Korea Advanced Institute of Science and Technology, Daejeon, Korea.
Blood. 2012 Sep 27;120(13):2733-44. doi: 10.1182/blood-2012-04-421610. Epub 2012 Aug 14.
Successful differentiation and expansion of endothelial cells (ECs) from embryonic stem cell (ESC)-derived Flk1(+) mesodermal precursor cells (MPCs) requires supplementation of vascular endothelial growth factor-A (VEGF-A). While analyzing VEGF-A/VEGFR2 downstream signaling pathway that underlies the VEGF-A-induced differentiation and expansion of ECs, we fortuitously found that Rho-associated protein kinase (ROCK) inhibitor Y27632 profoundly promoted the differentiation and expansion of ECs from Flk1(+) MPCs while reducing the differentiation and expansion of mural cells. The ROCK suppression-induced expansion of ECs appears to have resulted from promotion of proliferation of ECs via activation of PI3-kinase-Akt signaling. The ECs obtained by the combination of ROCK suppression and VEGF-A supplementation faithfully expressed most pan-EC surface makers, and phenotypic analyses revealed that they were differentiated toward arterial EC. Further incubation of the ICAM2(+) ECs with Y27632 and VEGF-A for 2 days promoted expansion of ECs by 6.5-fold compared with those incubated with only VEGF-A. Importantly, the ROCK suppression-induced ECs displayed neovasculogenic abilities in vitro and in vivo. Thus, supplementation of ROCK inhibitor Y27632 along with VEGF-A in 2D Matrigel culture system provides a simple, efficient, and versatile method for obtaining ample amount of ESC-derived ECs at high purity suitable for use in therapeutic neovascularization.
成功地从胚胎干细胞（ESC）衍生的 Flk1(+)中胚层前体细胞（MPC）中分化和扩增内皮细胞（ECs）需要补充血管内皮生长因子-A（VEGF-A）。在分析 VEGF-A/VEGFR2 下游信号通路的过程中，该信号通路是 VEGF-A 诱导 ECs 分化和扩增的基础，我们偶然发现 ROCK 抑制剂 Y27632 可显著促进 Flk1(+) MPC 向 EC 的分化和扩增，同时减少壁细胞的分化和扩增。ROCK 抑制诱导的 EC 扩增似乎是通过激活 PI3-kinase-Akt 信号通路促进 EC 增殖而实现的。通过 ROCK 抑制和 VEGF-A 联合使用获得的 EC 忠实表达了大多数 pan-EC 表面标志物，表型分析显示它们向动脉 EC 分化。进一步将 ICAM2(+) EC 与 Y27632 和 VEGF-A 孵育 2 天，与仅用 VEGF-A 孵育相比，EC 的扩增增加了 6.5 倍。重要的是，ROCK 抑制诱导的 EC 在体外和体内显示出新生血管生成能力。因此，在 2D Matrigel 培养系统中补充 ROCK 抑制剂 Y27632 和 VEGF-A 为获得大量高纯度适合用于治疗性血管新生的 ESC 衍生 EC 提供了一种简单、高效和通用的方法。
