无血清单层细胞诱导法培养猪诱导多能性干细胞定向分化为血管内皮细胞

A serum-free monolayer method for differentiation of porcine induced pluripotent stem cells into vascular endothelial cells

背景:诱导多能性干细胞是一种新型的干细胞来源,具有多向分化潜能。猪作为人类心血管及代谢性疾病研究的良好模型,对其多能性细胞向血管内皮细胞定向分化体系的建立,将为创建心血管疾病模型提供保障。目的:建立一种无血清单层诱导分化方法,使猪诱导多能性干细胞定向分化为CD31阳性血管内皮细胞,并对获得的内皮细胞进行鉴定。方法:使用CHIR99021、骨形态发生蛋白4、碱性成纤维生长因子、血管内皮生长因子等对猪诱导多能性干细胞和人胚胎干细胞进行单层细胞诱导分化,使干细胞经历3个分化阶段最终成为血管内皮细胞,检测并比较分化过程中2种干细胞的胚层标记基因的表达,对流式分选后获得的血管内皮细胞进行鉴定。结果与结论:①通过无血清单层细胞诱导法从猪诱导多能性干细胞定向诱导分化获得的内皮细胞具有与猪永生化主动脉血管内皮细胞类似的基因表达模式及生物学功能——能够吸收低密度脂蛋白并在Matrigel上形成微血管样结构;②该诱导体系下,猪诱导多能性干细胞的分化效率高于人胚胎干细胞的分化效率,这可能与诱导过程中2种细胞谱系基因表达模式上的差异相关。

BACKGROUND:Induced pluripotent stem cells are a cell source with multiple differentiation potentials.Since pig is a suitable model for human cardiovascular and metabolic diseases,induction of porcine pluripotent stem cells into endothelial cells will provide a support for creating a porcine cardiovascular disease model.OBJECTIVE:To establish a serum-free single layer culture method to induce porcine induced pluripotent stem cells into CD31 positive endothelial cells and to identify the obtained endothelial cells.METHODS:We used the serum-free single layer culture method to induce porcine pluripotent stem cells and human embryonic stem cells into endothelial cells under different combinations of CHIR99021,bone morphogenetic protein 4,basic fibroblast growth factor and vascular endothelial growth factor through three stages.The expressions of germ layer markers of the two kinds of stem cells during their differentiation were detected and compared.The endothelial cells obtained after the flow sorting were identified.RESULTS AND CONCLUSION:The endothelial cells differentiated from porcine induced pluripotent stem cells by a single layer culture method showed comparable morphological and functional properties to the immortalized porcine aortic endothelial cells,capable of taking up low-density lipoprotein and forming microvascular-like structure on matrigel.We found that the differentiation efficiency of porcine pluripotent stem cells was higher than that of human embryonic stem cells using the same method,which may be associated with the different expression patterns of lineage-specific markers in the two kinds of cells during the early differentiation.