中国组织工程研究

中国组织工程研究 ›› 2011, Vol. 15 ›› Issue (27): 4969-4974.doi: 10.3969/j.issn.1673-8225.2011.27.007

• 骨髓干细胞 bone marrow stem cells • 上一篇    下一篇

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

蔡  耘1，2，张绪超2，3，陈惠芹1，2，黄绍良2   

  1. 1中山大学附属第三医院儿科，广东省广州市510630
    2中山大学附属第二医院干细胞研究中心，广东省广州市  510120
    3广东省人民医院医学研究中心，广东省广州市  510080
  • 收稿日期:2011-01-23 修回日期:2011-03-09 出版日期:2011-07-02 发布日期:2011-07-02
  • 通讯作者: 张绪超，博士，副研究员，广东省人民医院医学研究中心，广东省广州市510080 zhangxuchao@263.net 并列通讯作者：黄绍良，教授，博士生导师，中山大学附属第二医院干细胞研究中心，广东省广州市510120 hshl@gzsums.edu.cn
  • 作者简介:蔡耘★，女，1974年生，广东省广州市人，汉族，2006年中山大学毕业，博士，主治医师，主要从事儿科血液/肿瘤临床科研工作。 wcyf@163.com
  • 基金资助:

    国家自然科学基金资助项目(30300377)；广东省科技计划项目(2008B030301046) 。

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

Cai Yun1, 2, Zhang Xu-chao2, 3, Chen Hui-qin1, 2, Huang Shao-liang2   

  1. 1Department of Pediatrics, Third Affiliated Hospital, Sun Yat-sen University, Guangzhou  510630, Guangdong Province, China
    2Center for Stem Cell Research, Second Affiliated Hospital, Sun Yat-sen University, Guangzhou  510120, Guangdong Province, China
    3Research Center of Medical Sciences, Guangdong Provincial People’s Hospital, Guangzhou  510080, Guangdong Province, China
  • Received:2011-01-23 Revised:2011-03-09 Online:2011-07-02 Published:2011-07-02
  • Contact: Zhang Xu-chao, Doctor, Associate researcher, Center for Stem Cell Research, Second Affiliated Hospital, Sun Yat-sen University, Guangzhou 510120, Guangdong Province, China; Research Center of Medical Sciences, Guangdong Provincial People’s Hospital, Guangzhou 510080, Guangdong Province, China zhangxuchao@263.net Correspondence to: Huang Shao-liang, Professor, Doctoral supervisor, Center for Stem Cell Research, Second Affiliated Hospital, Sun Yat-sen University, Guangzhou 510120, Guangdong Province, China hshl@gzsums.edu.cn
  • About author:Cai Yun☆, Doctor, Attending physician, Department of Pediatrics, Third Affiliated Hospital, Sun Yat-sen University, Guangzhou 510630, Guangdong Province, China; Center for Stem Cell Research, Second Affiliated Hospital, Sun Yat-sen University, Guangzhou 510120, Guangdong Province, China wcyf@163.com
  • Supported by:

    the National Natural Science Foundation of China, No. 30300377*; the Scientific Research Project of Guangdong Province, No. 2008B030301046*

PDF

379

摘要：

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6 d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。

关键词: 主动脉-性腺-中肾区, 肝脏, 骨髓, 胚胎干细胞, 造血干细胞

Abstract:

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.

中图分类号: