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

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 was higher in embryoid bodies from urine cells derived iPSCs than that in amniotic fluid cells and embryonic stem cells.(4)Embryoid bodies from different cells derived iPSCs and embryonic stem cells all had pluripotency and the ability of three layer-differentiation,but they were distinctive.Urine cells derived iPSCs may replace embryonic stem cells as hematopoietic cell differentiation model in vitro.