比较人不同细胞来源诱导多能干细胞与胚胎干细胞拟胚体的分化培养过程  

Comparison of different human cell derived induced pluripotent stem cells and embryonic stem cells in tuning embryoid body differentiation

作　　者：韦云剑 张风波[1,2,3,4] 龙平 江欣星[1,2,3] 马燕琳 孙菲[1,2,3,5] 李崎 Wei Yunjian;Zhang Fengbo;Long Ping;Jiang Xinxing;Ma Yanlin;Sun Fei;Li Qi(Hainan Provincial Key Laboratory for Human Reproductive Medicine and Genetic Research,Department of Reproductive Medicine,Hainan Provincial Clinical Research Center for Thalassemia,the First Affiliated Hospital of Hainan Medical University,Haikou 570102,Hainan Province,China;Key Laboratory of Tropical Translational Medicine of Ministry of Education,Hainan Medical University,Haikou 570102,Hainan Province,China;Haikou Key Laboratory for Preservation of Human Genetic Resource,the First Affiliated Hospital of Hainan Medical University,Haikou 570102,Hainan Province,China;Department of Obstetrics,Anhui Province Maternity&Child Health Hospital,Hefei 230000,Anhui Province,China;Department of Obstetrics and Gynecology,NanFang Hospital,Southern Medical University,Guangzhou 510515,Guangdong Province,China)

机构地区：[1]海南医学院第一附属医院,海南省人类生殖与遗传重点实验室,生殖医学科,海南省地方病(地中海贫血)临床医学研究中心,海南省海口市570102 [2]海南医学院热带转化医学教育部重点实验室,海南省海口市570102 [3]海南医学院第一附属医院,海口市人类遗传资源保藏重点实验室,海南省海口市570102 [4]安徽省妇幼保健院产科,安徽省合肥市230000 [5]南方医科大学南方医院妇产科,广东省广州市510515

出　　处：《中国组织工程研究》2021年第25期4019-4024,共6页Chinese Journal of Tissue Engineering Research

基　　金：海南省重大科技计划(ZDKJ2017007),项目负责人:马燕琳;海南省自然科学基金项目(创新研究团队项目,2019CXTD408),项目负责人:马燕琳;国家自然科学基金项目(81960283,81660433),项目负责人:马燕琳;国家自然科学基金项目(81460034),项目负责人:李崎;国家自然科学基金项目(81460236),项目参与人:江欣星。

摘　　要：背景:通过小分子化合物、质粒转染等方法可成功诱导出无病毒载体整合的诱导多能干细胞。与胚胎干细胞相似,诱导多能干细胞可向造血祖细胞进行自由分化,但外源性因子的转入及不同来源人体细胞是否对诱导多能干细胞的分化及诱导造血祖细胞产生影响,目前尚未清楚。目的:比较人不同细胞来源诱导多能干细胞和胚胎干细胞拟胚体的基因表达差异,为诱导多能干细胞的临床转化进行探索。方法:分别选用羊水细胞及尿细胞来源诱导多能干细胞和人胚胎干细胞进行诱导分化。收集分化不同时间点的拟胚体,检测多能性基因Oct4、Sox2、Nanog以及内胚层标记基因GATA4、中胚层标记基因MSX1、外胚层标记基因PAX6、造血干细胞标记基因CD34和CD43的表达。结果与结论:①羊水细胞来源诱导多能干细胞拟胚体在培养第8天检测不到Oct4、Sox2、Nanog表达,尿细胞来源诱导多能干细胞和胚胎干细胞拟胚体可维持表达至第16天;②羊水细胞来源诱导多能干细胞拟胚体中GATA4、MSX1、PAX6表达量峰值出现在第4天,持续表达到第8-12天,而其他2种细胞拟胚体中GATA4、MSX1、PAX6表达量峰值出现在第4-8天,并且可持续表达至第12-16天;三胚层标记基因在尿细胞来源诱导多能干细胞拟胚体中的表达量明显高于其他2种细胞拟胚体;③在羊水细胞、尿细胞来源诱导多能干细胞拟胚体中造血干细胞比率在第12天达峰值,而胚胎干细胞拟胚体中造血干细胞比率在第4天达峰值,尿细胞来源诱导多能干细胞拟胚体中造血干细胞比率高于其他2种细胞拟胚体;④人不同细胞来源诱导多能干细胞及胚胎干细胞拟胚体分化培养过程中均具有全能性及三胚层分化能力但各具特点。尿细胞来源诱导多能干细胞或许更适合替代胚胎干细胞成为体外研究造血细胞分化的模型。BACKGROUND:Induced pluripotent stem cells(iPSCs)without virus vector integration can be successfully induced by small molecule compounds and plasmid transfection.Similar to embryonic stem cells,iPSCs can differentiate freely into hematopoietic progenitor cells.However,it is not clear whether exogenous factors and human cells from different sources affect the differentiation of iPSCs and hematopoietic progenitor cells.OBJECTIVE:To compare the gene expression differences between iPSCs of different cell sources and embryoid bodies of embryonic stem cells,so as to explore the clinical transformation of iPSCs.METHODS:The iPSCs and human embryonic stem cells were constructed from amniotic cells-derived cells and urine cells-derived cells.The different days of embryoid bodies were collected to test the expression of pluripotent genes Oct4,Sox2 and Nanog,endoderm marker gene GATA4,mesoderm marker gene MSX1,ectoderm marker gene PAX6,hematopoietic stem cell marker genes CD34/CD43.RESULTS AND CONCLUSION:(1)The expression of Oct4/Sox2/Nanog in embryoid bodies from amniotic fluid cells derived iPSCs was almost undetectable at 8 days,which was different from urine cells derived iPSCs and embryoid bodies embryonic stem cells.They could sustain expression until 16 days.(2)The expression peak of GATA4/MSX1/PAX6 in embryoid bodies from amniotic fluid cells derived iPSCs was at 4 days,and sustained expression at 8-12 days.The expression peak of GATA4/MSX1/PAX6 in embryoid bodies from urine cells derived iPSCs and embryonic stem cells was at 4-8 days,and sustained expression at 12-16 days.The expression level of three layer-differentiation markers for embryoid bodies from urine cells derived iPSCs were significantly higher than iPSCs from amniotic fluid cells and embryonic stem cells.(3)At 12 days,the ratio of hematopoietic stem cell was the most in embryoid bodies from amniotic fluid cells and urine cells derived iPSCs.However,the ratio in the embryoid bodies from embryonic stem cells was at 4 days.The hematopoietic stem cell ratio

关 键 词：干细胞 诱导多能干细胞 胚胎干细胞 拟胚体 多能性 造血分化 造血干细胞 羊水细胞 尿细胞 

分 类 号：R459.9[医药卫生—治疗学] R394.2[医药卫生—临床医学]