体外诱导小鼠胚胎干细胞血管形成

In Vitro Vasculogenesis and Angiogenesis of Mouse Embryonic Stem Cells

目的探讨体外定向诱导胚胎干细胞向内皮细胞分化的条件,模拟体内血管生成和血管新生过程,为研究新血管的形成提供一种新思路。方法体外培养小鼠胚胎干细胞诱导形成胚胎小体,将11d的胚胎小体种植到胶原中诱发出芽性血管新生;通过免疫组织化学染色方法检测胚胎小体及其出芽向内皮细胞和功能血管的分化。结果胚胎干细胞在体外能自发形成胚胎小体,其内部含有血管样结构,并伴有平滑肌的分化;种植到胶原中的胚胎小体能再现出芽性血管新生。结论胚胎干细胞在体外不但能定向分化成内皮细胞,还能再现体内血管生成、血管新生及动脉生成过程。

Objective To explore an optional condition to induce mouse embryonic stem(ES)cells to differentiate into endothelial cells and to establish in vitro models of vasculogenesis and angiogenesis.Methods Mouse ES cells were cultured in differentiation medium containing a cocktail of vascular endo-thelial growth factor(VEGF), basic fibroblast growth factor(bFGF), interleukin-6(IL-6)and erythropoietin (EPO)in 1% methylcellulose to induce formation of embryoid bodies(EBs). At day 11, EBs were harvested and suspended in rat-tail collagen type I with the same cocktail of cytokines cultured for three additional days. The differentiation of ES cells into endothelial cells, processes of vasculogenesis and angiogenesis were examined using immunostaining of EBs slices and whole-mount immunocytochemistry of EBs with monoclonal antibodies(mAbs)against platelet endothelial cell adhesion molecule-1(PECAM-1)and α-smooth muscle actin(SMA). Results Under appropriate culture conditions, ES cells spontaneously differentiated and formed EBs containing vascular structures and tubular channels, which were positive for PECAM-1 co-differentiated with smooth muscle. When not treated with angiogenic growth factors, PECAM-1-positive cells could not organize into vascular structures of 11-day-old EBs. In the presence of angiogenic factors 11-day old EBs embedded into type I collagen, and rapidly developed an endothelial networks. Whole-mount immunocytochemistry of collagen gel with anti-PECAM-1 antibody showed the for-mation of primary vascular structures sprouting from EBs. Quantitative analysis revealed that 100 μg/ml thalidomide significantly reduced the number and length of EBs endothelial sprouting. ConclusionsMouse ES cells can differentiate into endothelial cells combined with smooth muscle differentiation during EBs formation and further develop endothelial outgrowths after EBs embedded into collagen, which respec-tively recapitulate vasculogenesis, angiogenesis, and arteriogenesis processes in vivo. The models provide a useful tool to investigate vasculogenesis, angiogenesis, and arteriogenesis mechanisms and evaluate the effects of angiogenic and angiostatic agents.