中国组织工程研究

中国组织工程研究 ›› 2023, Vol. 27 ›› Issue (10): 1553-1559.doi: 10.12307/2023.340

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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

黄文俊1，2，3，4，5，王 洁1，2，3，4，5，周亚飞1，2，3，4，5，李 环1，2，3，4，5，蒋丛姗1，2，3，4，5，张艳敏1，2，3，4，5，周 锐1，2，3，4，5   

  1. 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
  • 收稿日期:2022-03-24 接受日期:2022-06-13 出版日期:2023-04-08 发布日期:2022-09-08
  • 通讯作者: 周锐，生物学博士，副研究员，陕西省儿童疾病精准医学重点实验室，陕西省西安市 710002；西安市儿童健康与疾病重点实验室，陕西省西安市 710002；陕西省儿科疾病研究所，陕西省西安市 710002；西安市儿童医院，陕西省西安市 710002；西安交通大学附属儿童医院，陕西省西安市 710002
  • 作者简介:黄文俊，女，1983年生，四川省德阳市人，汉族，2011年四川大学毕业，药理学硕士，助理研究员，主要从事基于诱导多能干细胞的遗传罕见病机制及治疗研究。
  • 基金资助:
    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

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

Huang Wenjun1, 2, 3, 4, 5, Wang Jie1, 2, 3, 4, 5, Zhou Yafei1, 2, 3, 4, 5, Li Huan1, 2, 3, 4, 5, Jiang Congshan1, 2, 3, 4, 5, Zhang Yanmin1, 2, 3, 4, 5, Zhou Rui1, 2, 3, 4, 5   

  1. 1Key Laboratory of Precision Medicine to Pediatric Diseases of Shaanxi Province, Xi’an 710002, Shaanxi Province, China; 2Xi’an Key Laboratory of Children’s Health and Diseases, Xi’an 710002, Shaanxi Province, China; 3Shaanxi Institute for Pediatric Diseases, Xi’an 710002, Shaanxi Province, China; 4Xi’an Children’s Hospital, Xi’an 710002, Shaanxi Province, China; 5Affiliated Children’s Hospital of Xi’an Jiaotong University, Xi’an 710002, Shaanxi Province, China
  • Received:2022-03-24 Accepted:2022-06-13 Online:2023-04-08 Published:2022-09-08
  • Contact: Zhou Rui, PhD, Assistant researcher, 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
  • About author:Huang Wenjun, Master, Assistant researcher, 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
  • Supported by:
    The National Natural Science Foundation of China, No. 81974014 (to ZYM); The Health and Scientific Research Personnel Foundation of Xi’an Health Committee, No. 2022ms08 (to ZR); The Health and Scientific Research Personnel Foundation of Xi’an Health Committee, No. 2022ms09 (to HWJ); The Natural Science Foundation of Xi’an Children’s Hospital, No. 2021A01 (to ZR); The Natural Science Foundation of Xi’an Children’s Hospital, No. 2021B02 (to HWJ)

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摘要：

文题释义：
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，具备三胚层分化潜能和自我更新能力。人诱导多能干细胞制备是通过将多能干细胞转录因子导入终末分化体细胞重编程获得，从而绕开胚胎干细胞制备需使用人类胚胎引发的伦理问题，因此具备更大的临床应用前景。

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；③qPCR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。

https://orcid.org/0000-0001-7638-1442(周锐) 

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

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

Abstract: 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-CADHERIN, ICAM1 and PECAM1) were gradually increased. Immunofluorescence at the protein level further confirmed the increasing trend of endothelial cell marker expression. (4) Tube formation assay showed that the number of endothelial cells-generated blood vessels increased with the increase of the concentration of vascular endothelial growth factor. (5) It is concluded that an experimental protocol for the directed differentiation of human induced pluripotent stem cells into endothelial cells has been successfully established, which is expected to provide a cellular basis and experimental basis for future blood vessel construction.

Key words: human induced pluripotent stem cell, endothelial cell, differentiation, signaling pathway, endothelial progenitor cell

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