中国组织工程研究

中国组织工程研究 ›› 2011, Vol. 15 ›› Issue (10): 1827-1831.doi: 10.3969/j.issn.1673-8225.2011.10.025

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

人胚胎干细胞在无血清mTeSR?1培养基中维持培养和向内皮细胞的诱导分化

李向东，王纪文，魏国峰   

  1. 大连医科大学附属第二医院心血管内科，辽宁省大连市  116023
  • 收稿日期:2010-10-23 修回日期:2011-02-14 出版日期:2011-03-05 发布日期:2011-03-05
  • 通讯作者: 魏国峰，硕士，主任医师，大连医科大学附属第二医院心血管内科，辽宁省大连市 116023 guofeng1204@yahoo.com.cn
  • 作者简介:李向东★，男，1969年生，辽宁省宽甸县人，汉族，1993年大连医科大学毕业，硕士，副主任医师，主要从事心血管方面的研究。 dodo58782002@yahoo.com.cn

Human embryonic stem cells maintained and cultured in serum-free medium mTeSR?1 and induced into endothelial cells differentiation

Li Xiang-dong, Wang Ji-wen, Wei Guo-feng   

  1. Department of Cardiovascular Medicine, the Second Affiliated Hospital of Dalian Medical University, Dalian  116023, Liaoning Province, China
  • Received:2010-10-23 Revised:2011-02-14 Online:2011-03-05 Published:2011-03-05
  • Contact: Wei Guo-feng, Master, Chief physician, Department of Cardiovascular Medicine, the Second Affiliated Hospital of Dalian Medical University, Dalian 116023, Liaoning Province, China Guofeng1204@yahoo.com.cn
  • About author:Li Xiang-dong★ Master, Associate chief physician，Department of Cardiovascular Medicine, the Second Affiliated Hospital of Dalian Medical University, Dalian 116023, Liaoning Province, China dodo58782002@yahoo.com.cn

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摘要：

背景：传统的人胚胎干细胞培养扩增方法中应用含动物血清培养基，并依赖饲养层细胞培养，这种培养方法显著制约了干细胞的体外培养规模；另外异源动物血清成分介入，使病原污染及免疫排斥的概率显著增加。
目的：明确应用无血清培养基mTeSR?1对人胚胎干细胞进行长期体外培养的可行性，并建立诱导人胚胎干细胞分化为血管内皮细胞的相关技术平台。
方法：采用无血清培养基mTeSR?1以非饲养层细胞依赖的方式体外培养、扩增人胚胎干细胞株H9。经过40余次体外传代后，于倒置显微镜下观察其生长形态，并利用免疫荧光染色方法评估其细胞表型。此外，应用条件培养基诱导H9细胞株向内皮细胞方向分化。利用免疫荧光染色技术，定量RT-PCR以及低密度脂蛋白摄取实验对该胚胎干细胞源内皮细胞的表型及功能进行评价、分析。
结果与结论：mTeSR?1培养基能够支持H9细胞株在体外以非饲养层依赖的方式进行长期扩增，同时维持其未分化的干细胞潜能。添加血管内皮细胞的条件培养基能够定向诱导H9细胞向内皮细胞方向分化。该胚胎干细胞源内皮细胞不但表达内皮细胞的标志基因(kdr，pecam)和标记蛋白CD31，而且还能够摄取低密度脂蛋白，形成类似微血管结构。提示实验中所提供的培养及诱导分化体系能够支持胚胎干细胞的增殖与分化行为。

关键词: 无血清培养基, 胚胎干细胞, 内皮细胞, 分化, 血管内皮生长因子

Abstract:

BACKGROUND: Containing fetal bovine serum medium is used in the traditional culture and amplification method of human embryonic stem cells (hESCs), and this method relys on feeder layer cell culture and significantly limits culture formula of stem cells in vitro. In addition, the intervention of heterologous serum components significantly increases pathogen contamination and the probability of immune rejection.
OBJECTIVE: To elucidate the feasibility of serum-free medium mTeSR?1 on the hESCs long term cultured in vitro and to establish a platform of induced hESC differentiate into endothelial cells.
METHODS: Serum-free medium mTeSR?1 was applied to culture in vitro and amplificate hESCs line H9 by non- feeder layer cell dependent. After more than 40 times passage in vitro, the growth morphology of hESCs was observed by inverted microscope, and their phenotype was evaluated by immunofluorescence staining method. Moreover, a conditioned medium was utilized to induce hESCs line H9 to differentiate into endothelial cells. The phenotype and function of ESC-derived endothelial cells were assayed by immunofluorescence staining, quantitative RT-PCR, and low-density lipoprotein (LDL) uptaking experiment.
RESULTS AND CONCLUSION: mTeSR?1 medium can support hESCs line H9 long term amplification in vitro by non- feeder layer cell dependent and maintain its potential of undifferentiated stem cells. When the hESCs were cultivated under a conditioned medium with vascular endothelial cell supplementation, the cells were induced differentiation into H9 endothelial-like cells. These cells not only one of important surface markers of endothelia cells (kdr, pecam) and expressed CD31, but also uptake LDL, formed vascular-like structure during the differentiation. The system of culture and induced differentiation experiment provided can support the proliferation and differentiation behavior of ESCs.

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