小鼠胚胎干细胞定向血管内皮和造血细胞分化体系的建立

Establishment of the System for Inducing Embryonic Stem Cells to Differentiate into Vascular Endothelial and Hematopoietic Cells in Vitro

目的:建立小鼠胚胎干细胞体外定向分化为血管内皮细胞和造血细胞的体系,并验证诱导后2种细胞的表面分子特征。方法:以小鼠胚胎成纤维细胞为饲养层,首先在无血清培养基StemPro中加入骨形态发生蛋白4(BMP4)、激活素A、碱性成纤维细胞生长因子(FGF-Basic)和血管内皮细胞生长因子(VEGF),诱导小鼠胚胎干细胞系R1/E 4 d后形成拟胚体;再将拟胚体消化后与OP9-DL1基质细胞共孵育,分别用干细胞因子(SCF)、VEGF和SCF、FLt3、白细胞介素3(IL-3)诱导向内皮和造血2个方向分化,并以CD31、CD45、CD144、Kit、CD201作为表面标志,流式检测诱导后细胞的表面分子特征和诱导效率;诱导10 d后免疫组化染色,进行内皮细胞的形态学鉴定。结果:诱导分化10 d后,免疫组化染色观察到多个内皮管状结构,流式检测CD31^+的内皮细胞比例为1.35%±0.05%,进一步分析CD31^+CD144^+CD45^-群体,有3.0%±0.2%的细胞表型为Kit^+CD201^+,提示该部分细胞可能是处于分化上游的内皮干祖细胞;CD45^+的造血细胞比例为35.0%±0.5%,其中0.35%±0.05%的细胞表达Kit和CD201,提示该部分细胞可能是处于分化上游的造血干祖细胞。结论:本研究将胚胎干细胞诱导为内皮细胞和造血细胞,并且能诱导出具有内皮、造血干祖细胞分子特征的细胞,可作为理想的体外诱导分化体系。

Objective:To establish the system for directional differentiation of mouse embryonic stem cells into vascular endothelial cells and hematopoietic cells in vitro,and verify the surface molecular characteristics of the two cells after induction.Methods:Mouse embryonic fibroblasts were used as feeder layer,and four factors including bone morphogenetic protein 4(BMP4),activin A,fibroblast growth factor basic(FGF-Basic)and vascular endothelial growth factor(VEGF)were added into serum-free medium StemPro to induce mouse embryonic stem cell line R1/E 4 d to form embryoid body.After digestion,the embryoid was co-cultured with OP9-DL1 stromal cells,and induced to differentiate into endothelial and hematopoietic directions by stem cell factor(SCF),VEGF and SCF,FLt3 and interleukin-3(IL-3),respectively.CD31,CD45,CD144,Kit and CD201 were used as surface markers,and the surface molecular characteristics and induction efficiency of the induced cells were detected by flow cytometry.Immunohistochemical staining was performed after 10 days induction to identify the morphology of endothelial cells.Results:After 10 days of induced differentiation,a number of endothelial tubular structures were observed by immunohistochemical staining.The ratio of CD31+endothelial cells detected by flow cytometry was 1.35%±0.05%.Further analysis of CD31^+CD144^+CD45^-population showed that 3.0%±0.2%of the cell phenotypes were Kit+CD201^+,suggesting that these cells may be endothelial stem and progenitor cells upstream of differentiation.The proportion of hematopoietic cells with CD45^+is 35.0%±0.5%,of which 0.35%±0.05%express Kit and CD201,suggesting that this part of cells may be hematopoietic stem and progenitor cells upstream of differentiation.Conclusion:The embryonic stem cells have been successfully induced into endothelial cells and hematopoietic cells,and cells with molecular characteristics of endothelial and hematopoietic stem and progenitor cells can be induced,which can be used as an ideal in vitro induced differentiation system.