中国组织工程研究

中国组织工程研究 ›› 2011, Vol. 15 ›› Issue (36): 6776-6780.doi: 10.3969/j.issn.1673-8225.2011.36.029

• 干细胞基础实验 basic experiments of stem cells • 上一篇    下一篇

人主动脉-性腺-中肾区基质细胞诱导胚胎干细胞分化为造血干细胞及体内造血重建

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:2011-02-19 修回日期:2011-04-12 出版日期:2011-09-03 发布日期:2011-09-03
  • 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 zhxuchao3000@yahoo.com.cn 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)。

Differentiation of murine embryonic stem cells into hematopoietic stem cells induced by stromal cells in human aorta-gonad-mesonephros region and hematopoietic reconstitution in vivo

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

  1. 1Department of Pediatrics, the Third Affiliated Hospital, Sun Yat-sen University, Guangzhou  510630, Guangdong Province, China
    2Center for Stem Cell Research, the 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:2011-02-19 Revised:2011-04-12 Online:2011-09-03 Published:2011-09-03
  • Contact: Zhang Xu-chao, Doctor, Associate investigator, Center for Stem Cell Research, the 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 zhxuchao3000@yahoo.com.cn Correspondence to: Huang Shao-liang, Professor, Doctoral supervisor, Center for Stem Cell Research, the 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, the Third Affiliated Hospital, Sun Yat-sen University, Guangzhou 510630, Guangdong Province, China; Center for Stem Cell Research, the 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*

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摘要：

背景：研究证实多种造血生长因子、基质细胞饲养层及其条件培养液可促进胚胎干细胞向造血干细胞分化。
目的：以人主动脉-性腺-中肾(aorta-gonad-mesonephros,AGM)区基质细胞为饲养层体外诱导小鼠胚胎干细胞分化为造血干细胞，并比较不同移植途径对造血干细胞体内造血重建能力的影响。
方法：将小鼠E14 胚胎干细胞诱导为拟胚体，采用Transwell非接触共培养体系在人AGM区基质细胞饲养层上诱导6 d，接种NOD-SCID小鼠检测体内致瘤性。再将诱导后的拟胚体细胞移植经致死量60Co γ射线辐照的BALB/C雌鼠，受鼠随机分为静脉移植组、骨髓腔移植组、照射对照组及正常对照组。
结果与结论：拟胚体细胞经人AGM区基质细胞诱导后Sca-1+c-Kit+细胞占(13.12±1.30)%。NOD-SCID小鼠皮下接种经人AGM区基质细胞诱导的拟胚体细胞可出现畸胎瘤，经骨髓腔接种未见肿瘤形成。静脉移植组动物全部死亡，骨髓腔移植组生存率为55.6%，移植后21 d外周血象基本恢复，存活受鼠检测到供体来源Sry基因。提示小鼠胚胎干细胞经人AGM区基质细胞诱导分化的造血干细胞通过骨髓腔移植安全并具有一定的造血重建能力。

关键词: 主动脉-性腺-中肾区, 胚胎干细胞, 造血干细胞, 拟胚体, 造血重建

Abstract:

BACKGROUND: Up to now it has been confirmed that under proper conditions of hematopoietic growth factors, feeder layers of stromal cells from hematopoietic niches and their conditioned media, embryonic stem cells (ESCs) can be induced into hematopoietic stem cells (HSCs).
OBJECTIVE: To direct murine ESCs into HSCs in vitro by the support of human aorta-gonad-mesonephros (AGM) region stromal cells, and to compare the function of hematopoietic reconstitution in vivo by different routes of HSCs transplantation in mice.
METHODS: Firstly, E14 murine ESCs were induced into embryoid body (EB). Then the cells from EB were further co-cultured with human AGM region stromal cells in Transwell non-contact system for 6 days. The induced EB cells were collected for Sca-1+c-Kit+ cells analysis by flow cytometry, checked for teratoma formation and transplanted to BALB/C female mice conditioned with lethal dose 60Co γ-ray irradiation. The recipient mice were divided into four groups at random, including intravenous injection group, bilateral tibia intra-bone marrow (IBM) injection group, irradiation control group and normal control group. The survival rates, reconstitution of hematopoietic and engraftment of donor cells of different groups were monitored.
RESULTS AND CONCLUSION: Sca-1+c-Kit+ cells in EB cells after co-cultured with human AGM region stromal cells accounted for (13.12±1.30)%. Teratoma could be detected in NOD-SCID mice after subcutaneous injection of EB cells co-cultured with human AGM region stromal cells, while there was no teratoma in the mice after IBM injection of that cells. The recipients in intravenous injection group all died. The survival rate was 55.6% in IBM group, in which the peripheral blood cell count was near to the normal at day 21 after transplantation and Sry gene copies from donor could be detected. It suggested that feeder cells from human AGM region can induce directed differentiation of murine ESCs into HSCs which can reconstruct hematopoiesis in vivo without teratoma by IBM administration.

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