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[在源自自身的饲养层上培养人类胚胎干细胞]

[Growing of human embryonic stem cells on feeders derived from themselves].

作者信息

An Shi-Min, Zeng Qiao, Teng Xiang-Yun, Long Zhi-Gao, Li Juan, Pan Qian, Wu Ling-Qian, Liang De-Sheng, Xia Kun, Xia Jia-Hui, Zhang Zhuo-Hua

机构信息

National Lab of Medical Genetics, Central South University, Changsha 410078, China.

出版信息

Yi Chuan. 2008 Dec;30(12):1567-73. doi: 10.3724/sp.j.1005.2008.01567.

DOI:10.3724/sp.j.1005.2008.01567
PMID:19073571
Abstract

This study was carried out to determine whether mesenchymal stem cells (MSCs) derived from teratoma of human embryonic stem cells (hESCs) function as feeder cells to support hESCs growth. Approximately 5x10(6) hESCs were injected into the hind limb muscle of each SCID-beige mouse to form teratoma. After 8 weeks, the MSCs were isolated from the teratoma and cultured in Mesencult medium. Purified MSCs were then used as the feeder cells for hESCs culture. High purity MSCs derived from teratoma were isolated. The cells were morphologically similar to bone marrow MSCs (bMSCs). The teratoma-derived MSCs were negative for CD34 and CD45 but positive for CD29, CD49b, CD105, CD73, and CD90, which resembled those expressed by bMSCs. After passaged on MSCs feeder cells more than 10 passages, hESCs maintained hESC characteristics in morphology. Reverse PCR showed the expression of Oct4 and Nanog. SSEA-1 was negative and SSEA-4, TRA-1-60, and TRA-1-81 were positive. Alkaline phosphatase staining showed positive results.The karyotype remained normal. Moreover, the hECSs cultured on teratoma-derived MSCs formed teratoma in vivo and embryoid body in vitro confirmed their pluripotency. Accordingly, MSCs derived from hESCs by in vivo differentiation can be used as the feeder cells for hESCs culture.

摘要

本研究旨在确定源自人胚胎干细胞(hESCs)畸胎瘤的间充质干细胞(MSCs)是否可作为饲养层细胞来支持hESCs的生长。将约5×10⁶个hESCs注射到每只SCID-米色小鼠的后肢肌肉中以形成畸胎瘤。8周后,从畸胎瘤中分离出MSCs并在Mesencult培养基中培养。然后将纯化的MSCs用作hESCs培养的饲养层细胞。分离出了源自畸胎瘤的高纯度MSCs。这些细胞在形态上与骨髓MSCs(bMSCs)相似。畸胎瘤来源的MSCs CD34和CD45呈阴性,但CD29、CD49b、CD105、CD73和CD90呈阳性,这与bMSCs表达的相似。在MSCs饲养层细胞上传代超过10代后,hESCs在形态上保持hESCs特征。逆转录PCR显示Oct4和Nanog的表达。SSEA-1呈阴性,SSEA-4、TRA-1-60和TRA-1-81呈阳性。碱性磷酸酶染色呈阳性结果。核型保持正常。此外,在畸胎瘤来源的MSCs上培养的hESCs在体内形成畸胎瘤,在体外形成胚状体,证实了它们的多能性。因此,通过体内分化从hESCs获得的MSCs可作为hESCs培养的饲养层细胞。

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