建立并鉴定一种基于人诱导多能干细胞的肝脏细胞定向分化实验方案  

Establishment and identification of a protocol for highly efficient differentiation of hepatocytes from human pluripotent stem cells

作　　者：黄文俊 周亚飞 王洁 李环[1,2,3,4,5] 张艳敏[1,2,3,4,5] 周锐 Huang Wenjun;Zhou Yafei;Wang Jie;Li Huan;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1期28-33,共6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--面上培育项目(2022ms08),项目负责人:周锐。

摘　　要：背景:人诱导多能干细胞是一种与胚胎干细胞特性高度类似的多能干细胞类型,具备三胚层分化潜能和持续自我更新能力。将人诱导多能干细胞定向分化成具备功能的肝脏细胞对临床肝脏替代治疗意义重大。目的:建立一种高效的基于人诱导多能干细胞的肝脏细胞定向分化实验方案。方法:利用明场显微镜、免疫荧光对人诱导多能干细胞特性进行鉴定。通过转录因子重组蛋白手段时序性调控不同信号通路,即在第0,5,10,15天顺序加入激活素A、成纤维细胞生长因子4/骨形态发生蛋白4、肝细胞生长因子、肿瘤抑制因子M,使多能干细胞经历终末内皮层、肝内胚层、肝母细胞(肝脏前体细胞)最终分化成为有功能的肝脏细胞。利用明场显微镜、实时定量PCR(qPCR)、ELISA动态观察不同阶段形态特征和分子标记物。结果与结论:①所使用的人诱导多能干细胞具备经典的诱导多能干细胞形态特征并且高表达诱导多能干细胞特异标记蛋白(SSEA4、SOX2、OCT4);②人诱导多能干细胞发生了终末内胚层、幼稚肝细胞、肝脏细胞不同阶段形态转变;③qPCR结果显示,随着肝细胞分化进展,诱导多能干细胞特异标记物(OCT4)显著下调,终末内胚层(GCS、CXCR4)和肝脏内胚层标记物(HNF4α、HNF1β)则呈现先升高再下调趋势,而肝脏细胞标记物(CYP34A、ALB)则呈现出逐渐递增趋势;④ELISA结果进一步显示,诱导15 d以后的肝脏细胞开始具备肝特异蛋白(白蛋白、尿素)分泌功能,并且随着时间延长其分泌功能进一步增加;⑤上述结果提示,成功建立人诱导多能干细胞向肝脏细胞定向分化的实验方案,为未来临床肝脏细胞替代治疗提供实验基础。BACKGROUND:Similar to the human embryonic stem cells,human induced pluripotent stem cells possess the ability to differentiate into three germ layers and self-renewal capacity.It is of great clinical significance to generate functional hepatocytes from human induced pluripotent stem cells.OBJECTIVE:To establish a protocol for the directed differentiation into hepatocytes from human induced pluripotent stem cells.METHODS:Human induced pluripotent stem cells were characterized by a microscope and immunofluorescence.Pluripotent stem cells were differentiated into definitive endoderm,hepatic endoderm,hepatoblasts,and finally functional hepatocytes via sequential modulation of the signaling pathways using recombinant transcription factors.That was,activin A,fibroblast growth factor 4/bone morphogenetic protein 4,hepatocyte growth factor,and tumor suppressor M were added sequentially on days 0,5,10,and 15.The morphological features and molecular markers at different stages were dynamically observed by microscopy,real-time quantitative PCR,and ELISA.RESULTS AND CONCLUSION:(1)The human induced pluripotent stem cells exhibited the typical morphology of pluripotent stem cells and expressed highlevels of the induced pluripotent stem cells-specific markers,SSEA4,SOX2,and OCT4.(2)The human induced pluripotent stem cells were successfully induced sequentially into definitive endoderm cells,hepatoblasts,and hepatocytes.(3)Real-time quantitative PCR showed that with the progression of hepatocyte differentiation,the expression of induced pluripotent stem cells marker(OCT4)was decreased.The expression levels of definitive endoderm(GCS and CXCR4)and hepatic endoderm(HNF4αand HNF1β)markers were increased and then decreased.The expression of hepatocyte markers(CYP34A and ALB)was gradually increased.(4)ELISA results further showed that the hepatocytes after 15 days of induction began to secrete liver-specific proteins(albumin and urea),and their secretory function further increased with the prolongation of time.(5)We conclude that a

关 键 词：人诱导多能干细胞 肝脏细胞 分化 终末内胚层 肝脏内胚层 肝母细胞 

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