含人AGM区、胎肝及骨髓基质细胞培养体系程序化诱导小鼠胚胎干细胞向造血干细胞的分化

背景：前期已分别制备人主动脉-性腺-中肾区基质细胞系及胎肝基质细胞系,发现前者可促进小鼠胚胎干细胞定向分化为造血干细胞。目的：模拟胚胎发育过程中永久造血发育的时空顺序,探讨人主动脉-性腺-中肾（AGM）区、胎肝（FL）及骨髓（BM）基质细胞对小鼠胚胎干细胞体外诱导分化为造血干细胞的支持作用,以寻求更佳的诱导条件。方法：将小鼠E14胚胎干细胞诱导为拟胚体（EB）,并利用Transwell非接触共培养体系依次在人主动脉-性腺-中肾区、胎肝及骨髓基质细胞饲养层上进一步诱导分化,按不同诱导阶段分为拟胚体对照、EB/AGM、EB/AGM＋FL和EB/AGM＋FL＋BM共4组。共培养6d后分别收获各组拟胚体来源细胞,以流式细胞仪检测Sca-1＋c-Kit＋细胞含量,进行各系造血细胞集落形成单位分析并观察细胞形态。结果与结论：①EB/AGM＋FL组和EB/AGM＋FL＋BM组收获细胞涂片均发现原始造血细胞。②拟胚体来源细胞经AGM区基质细胞诱导后Sca-1＋c-Kit＋细胞明显升高（P〈0.05）。③拟胚体对照组造血细胞集落形成单位低于其他各组（P〈0.05）,而EB/AGM＋FL、EB/AGM＋FL＋BM组造血细胞集落形成单位计数亦较EB/AGM组明显增高。提示AGM＋FL和AGM＋FL＋骨髓基质细胞微环境对原始造血干细胞的扩增效应均明显高于单纯主动脉-性腺-中肾饲养层。

Effects of sequential inductive systems with feeder cells from human aorta-gonad-mesonephros region,fetal liver and bone marrow on the differentiation of mouse embryonic stem cells into hematopoietic stem cells

BACKGROUND：Previous studies have prepared human aorta-gonad-mesonephros（AGM） region stromal cell line and fetal liver stromal cell line,and found that AGM can promote directional differentiation of mouse embryonic stem cells（ESCs） into hemopoietic stem cells（HSCs）.OBJECTIVE：To simulate the spatial and temporal hematopoietic microenvironment changes in embryonic development,investigate the supportive effects of sequential inductive systems with feeder cells from human AGM region and fetal liver and bone marrow on the differentiation of mouse ESCs into HSCs,and design more effective conditions for HSCs output.METHODS：E14 mouse ESCs were induced into embryoid body firstly.Then the cells from embryoid body were further co-cultured with human AGM region,fetal liver and bone marrow stromal cells in Transwell non-contact system in sequential orders.The induced embryoid body cells were divided into four groups according to different culture stages：embryoid body control group;embryoid body/AGM group;embryoid body/AGM＋ fetal liver group;embryoid body /AGM＋ fetal liver ＋ bone marrow group.On day 6 of co-cultured,cells derived from embryoid body in all groups were collected for Sca-1＋c-Kit＋ cells analysis by flow cytometry,colony forming unit assay and observed.RESULTS AND CONCLUSION：①Primitive hematopoietic cells were detected in embryoid body/AGM＋fetal liver group and embryoid body/AGM＋fetal liver＋bone marrow group.②Sca-1＋c-Kit＋ cell proportion of embryoid body/AGM group was much higher than pre-treated embryoid body cells（P 0.05）.③Embryoid body/AGM group,embryoid body/AGM＋fetal liver group and embryoid body/AGM＋fetal liver＋bone marrow group had more colony forming unit amount than embryoid body control group（P 0.05）.And the cells collected from embryoid body /AGM＋ fetal liver group and embryoid body/AGM＋ fetal liver ＋ bone marrow group had better ability of forming multiple lineage hematopoietic colonies than that from embryoid body/AGM group.It suggested that microenvironment of AGM＋ fetal liver or AGM＋ fetal liver ＋ bone marrow stromal cells can significantly expand much more primitive hematopoietic stem cells than AGM stromal cells only.