一种人诱导多能干细胞向内皮细胞定向分化方案的建立及鉴定  

Establishment and identification of an efficient protocol for differentiation of endothelial cells from human induced pluripotent stem cells

作　　者：黄文俊 王洁 周亚飞[1,2,3,4,5] 李环[1,2,3,4,5] 蒋丛姗 张艳敏 周锐[1,2,3,4,5] Huang Wenjun;Wang Jie;Zhou Yafei;Li Huan;Jiang Congshan;Zhang Yanmin;Zhou Rui(Key Laboratory of Precision Medicine to Pediatric Diseases of Shaanxi Province,Xi'an 710002,Shaanxi Province,China;Xi'an Key Laboratory of Children's Health and Diseases,Xi'an 710002,Shaanxi Province,China;Shaanxi Institute for Pediatric Diseases,Xi'an 710002,Shaanxi Province,China;Xi'an Children's Hospital,Xi'an 710002,Shaanxi Province,China;Affiliated Children's Hospital of Xi'an Jiaotong University,Xi'an 710002,Shaanxi Province,China)

机构地区：[1]陕西省儿童疾病精准医学重点实验室,陕西省西安市710002 [2]西安市儿童健康与疾病重点实验室,陕西省西安市710002 [3]陕西省儿科疾病研究所,陕西省西安市710002 [4]西安市儿童医院,陕西省西安市710002 [5]西安交通大学附属儿童医院,陕西省西安市710002

出　　处：《中国组织工程研究》2023年第10期1553-1559,共7页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金面上项目(81974014),项目负责人:张艳敏;西安市卫生健康委员会卫生科研人才项目(2022ms08),项目负责人:周锐;西安市卫生健康委员会卫生科研人才项目(2022ms09),项目负责人:黄文俊;西安市儿童医院院级课题(2021A01),项目负责人:周锐;西安市儿童医院院级课题(2021B02),项目负责人:黄文俊。

摘　　要：背景:人诱导多能干细胞是一种与胚胎干细胞性状高度类似的多能干细胞,其具备三胚层分化潜能和持续自我更新能力,因此能够为临床细胞替代治疗提供足量目的细胞。目的:构建并鉴定一种高效的基于人诱导多能干细胞的内皮细胞定向分化实验方案。方法:采用免疫荧光技术对人诱导多能干细胞干性和增殖活性进行鉴定。采用转录因子重组蛋白和小分子化合物时序性调控系列信号通路,即在第0,1,2,5天顺序加入激活素A、GSK通路抑制剂CHIR99021/骨形态发生蛋白4、碱性成纤维细胞生长因子/血管内皮生长因子/骨形态发生蛋白4、碱性成纤维细胞生长因子/血管内皮生长因子/CHIR99021,人诱导多能干细胞经历心脏中胚层、内皮前体细胞命运转变并最终分化成为有功能的内皮细胞。采用明场显微镜、实时定量PCR、免疫荧光动态观察不同阶段细胞形态特征和分子标记物,采用血管成环实验验证内皮细胞的体外血管形成功能。结果与结论:(1)人诱导多能干细胞内源性高表达诱导多能干细胞干性特异标记物(OCT4、NANOG、SSEA4)和增殖标记物(Ki67);(2)开始分化后的人诱导多能干细胞经历了诱导多能干细胞、心脏中胚层、内皮前体细胞、内皮细胞等阶段显著的形态改变;(3)qPCR结果显示,随着分化进展,内皮前体细胞标记物(KDR、CD34)呈现先升高再下调趋势,而内皮细胞标记物(VE-CADHERIN、ICAM1、PECAM1)则呈现出逐渐递增趋势,免疫荧光在蛋白水平进一步证实了内皮细胞标记物的表达递增趋势;(4)血管成环实验显示,内皮细胞血管形成数量随血管内皮生长因子质量浓度增加而增多;(5)综上提示:成功建立了人诱导多能干细胞定向分化内皮细胞的实验方案,有望为未来血管构建提供细胞基础和实验依据。BACKGROUND:Similar to the human induced embryonic stem cells,human induced pluripotent stem cell is a kind of pluripotent stem cells.By virtue of the differentiation potential of three germ layers and self-renewal ability,it could serve as a means to generate a scalable source of cells for therapeutic applications.OBJECTIVE:To establish a protocol for the directed differentiation of human induced pluripotent stem cells into functional endothelial cells.METHODS:The authenticity of human induced pluripotent stem cells was verified by the immunofluorescence method.Human induced pluripotent stem cells were then differentiated into cardiac mesoderm,endothelial progenitor cells and finally functional endothelial cells through the manipulation of signaling pathways using recombinant transcription factors and small molecule compounds by adding activin A,GSK pathway inhibitor CHIR99021/bone morphogenetic protein 4,fibroblast growth factor/vascular endothelial growth factor/bone morphogenetic protein 4,basic fibroblast growth factor/vascular endothelial growth factor/CHIR99021 on days 0,1,2,and 5 sequentially.The induced endothelial cells were characterized by the morphology and the expression of endothelial cells-specific markers using brightfield microscope,real-time quantitative PCR and immunofluorescence method.Moreover,the tube formation assay was performed to test the angiogenetic ability of induced endothelial cells.RESULTS AND CONCLUSION:(1) The human induced pluripotent stem cells expressed high-levels of the induced pluripotent stem cell-specific markers(OCT4,NANOG,and SSEA4) and self-renewal marker(Ki67) as well.(2) The human induced pluripotent stem cells were successfully induced sequentially into to cardiac mesoderm cells,endothelial progenitor cells and final typical endothelial cells.(3) Real-time quantitative PCR results showed that with the progression of the differentiation,endothelial progenitor cell markers(KDR and CD34) were increased,and then gradually decreased;and endothelial cell markers(VE-CADHER

关 键 词：人诱导多能干细胞 内皮细胞 分化 信号通路 内皮前体细胞 

分 类 号：R459.9[医药卫生—治疗学] R318[医药卫生—临床医学] R-331