Culture and characterization of human embryonic stem cells
Culture and characterization of human embryonic stem cells
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DOI：
10.1089/scd.2004.13.325
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发表时间：
2004-08-01
影响因子：
4
通讯作者：
Andrews, PW
中科院分区：
文献类型：
--
作者：
Draper, JS;Moore, HD;Andrews, PW
人胚胎干细胞（ES细胞）为药物发现、毒理学和再生医学提供了大量的机会，但是在限定的条件下大规模培养它们以及控制和指导它们的分化的有效技术的开发提出了巨大的挑战。基于先前对胚胎癌（EC）细胞（其来自畸胎癌的恶性对应物）的研究，用于定义未分化细胞的标记物已被充分建立。这些为监测人类ES培养物及其分化状态提供了有价值的工具。然而，目前的培养技术是次优的，并且涉及使用定义不明确的培养基和使用饲养细胞。随着时间的推移，细胞也可能获得核型变化，反映遗传选择和适应体外培养条件。不过，正在取得进展。最初，人ES细胞是在含有胎牛血清的培养基中衍生和维持的。它们现在在专有的无血清制剂（来自Invitrogen Corp.，卡尔斯巴德，CA），并且最近我们已经在这种不含胎牛血清的培养基中衍生了新的人ES系。人成纤维细胞也可用于代替小鼠胚胎成纤维细胞作为饲养细胞。我们现在已经发现可以在基质胶或纯化的IV型胶原、层粘连蛋白和纤连蛋白上培养人ES细胞的亚系，而不需要饲养层或饲养层条件培养基。然而，这些细胞保留了未分化的人ES细胞的特征，包括分化能力。虽然这些细胞也进行核型变化，进一步的研究集中在了解控制自我更新，细胞凋亡和分化的承诺的机制，将促进定义的培养条件，最大限度地减少遗传变化和优化未分化干细胞的维护的发展。
Human embryonic stem (ES) cells offer substantial opportunities for providing well-defined differentiated cells for drug discovery, toxicology, and regenerative medicine, but the development of efficient techniques for their large-scale culture under defined conditions, and for controlling and directing their differentiation, presents a substantial challenge. Markers for defining the undifferentiated cells are well established, based upon previous studies of embryonal carcinoma (EC) cells, their malignant counterparts from teratocarcinomas. These provide valuable tools for monitoring human ES cultures and their state of differentiation. However, current culture techniques are suboptimal and involve the use of poorly defined culture media and the use of feeder cells. Over time, the cells may also acquire karyotypic changes, reflecting genetic selection and adaptation to in vitro culture conditions. Nevertheless, progress is being made. Originally, human ES cells were derived and maintained in medium containing fetal calf serum. They are now widely cultured in a proprietary serum-free formulation ( serum replacement from Invitrogen Corp., Carlsbad, CA), and recently we have derived a new human ES line in this medium without fetal calf serum. Human fibroblasts can also be used to replace mouse embryo fibroblasts as feeder cells. We have now found it possible to culture a subline of human ES cells on Matrigel, or purified collagen type IV, laminin, and fibronectin, without feeders or feeder-conditioned medium. These cells nevertheless retain the features of undifferentiated human ES cells, including a capacity for differentiation. Although these cells also carried karyotypic changes, further research focused upon understanding the mechanisms that control self-renewal, apoptosis, and commitment to differentiation will facilitate the development of defined culture conditions that minimize genetic change and optimize the maintenance of the undifferentiated stem cells.