诱导小鼠胚胎干细胞分化获取血管平滑肌和内皮细胞

Derivation of vascular smooth muscle and endothelial cells from mouse embryonic stem cells

目的探讨从诱导分化的小鼠胚胎干细胞中获取体外构建组织工程化血管种子细胞的可行性。方法利用Flk-1作为小鼠胚胎干细胞分化来源的血管前体细胞标志,流式细胞仪分选分化的类胚体中Flk-1阳性细胞,分别添加血小板源生长因子(PDGF)、血管内皮生长因子(VEGF),诱导血管前体细胞向血管平滑肌细胞和血管内皮细胞分化。免疫荧光染色鉴定诱导分化后细胞的相关标志表达;流式细胞仪分析诱导分化后细胞的分化效率和纯度。结果流式细胞仪分析显示,在分化第4天类胚体中有50%左右的细胞表达Flk-1,经分选的Flk-1阳性细胞通过进一步诱导,可以得到(95.9±3.5)%α-SMA阳性的血管平滑肌细胞和(59.1±4.8)%CD31阳性的血管内皮细胞。结论成功应用流式细胞分选法从诱导分化的小鼠胚胎干细胞中获取血管平滑肌细胞和血管内皮细胞,实验为组织工程化血管构建中种子细胞来源提供了新途径。

Objective To investigate the feasibility of obtaining smooth muscle and endothelial cells from differentiated mouse embryonic stem cells （ESCs）. Methods Flk-1 was used as a marker for vascular precursors. Flk-1 positive cells were then sorted from embryoid bodies by flow cytometery, and induced to vascular smooth muscle cells and endothelial cells with platelet derived growth factor and vascular endothelial growth factor, respectively. After induction, the markers for smooth muscle and endothelial cells were analyzed by immunofluorescent staining, and the differentiation efficiency and purity were analyzed by flow eytometery. Results It was revealed by flow cytometry that about 50% of cells in day 4 embryoid bodies expressed Flk-1 ,and （95.9 ±3.5） % of α-SMA positive smooth muscle cells and （59.1 ±4.8） % of CD31 positive endothelial cells were obtained from differentiation of sorted Flk-1 positive cells. Conclusion Vascular smooth muscle and endothelial cells can be obtained from differentiated mouse ESCs, providing a new cell source for tissue engineered blood vessel construction.