Institute of Health and Biomedical Innovation, Queensland University of Technology, Kelvin Grove, Australia.
Stem Cells Dev. 2010 Sep;19(9):1297-305. doi: 10.1089/scd.2009.0504.
The therapeutic use of human embryonic stem (hES) cells is severely limited by safety concerns regarding their culture in media containing animal-derived or nondefined factors and on animal-derived feeder cells. Thus, there is a pressing need to develop culture techniques that are xeno-free, fully defined, and synthetic. Our laboratory has discovered that insulin-like growth factor (IGF) and vitronectin (VN) bind to each other resulting in synergistic short-term functional effects in several cell types, including keratinocytes and breast epithelial cells. We have further refined this complex into a single chimeric VN:IGF-I protein that functionally mimics the effects obtained upon binding of IGF-I to VN. The aim of the current study was to determine whether hES cells can be serially propagated in feeder-cell-free and serum-free conditions using medium containing our novel chimeric VN:IGF-I protein. Here we demonstrate that hES cells can be serially propagated and retain their undifferentiated state in vitro for up to 35 passages in our feeder-cell-free, serum-free, chemically defined media. We have utilized real-time polymerase chain reaction (PCR), immunofluorescence, and fluorescence-activated cell sorter (FACS) analysis to show that the hES cells have maintained an undifferentiated phenotype. In vitro differentiation assays demonstrated that the hES cells retain their pluripotent potential and the karyotype of the hES cells remains unchanged. This study demonstrates that the novel, fully defined, synthetic VN:IGF-I chimera-containing medium described herein is a viable alternative to media containing serum, and that in conjunction with laminin-coated plates facilitates feeder-cell-free and serum-free growth of hES.
人胚胎干细胞（hES）的治疗用途受到其在含有动物源性或非定义因子的培养基中和在动物源性饲养细胞上培养的安全性问题的严重限制。因此，迫切需要开发无动物源、完全定义和合成的培养技术。我们实验室发现胰岛素样生长因子（IGF）和玻连蛋白（VN）相互结合，导致几种细胞类型（包括角质形成细胞和乳腺上皮细胞）产生协同的短期功能效应。我们进一步将这种复合物精制为一种单一的嵌合 VN:IGF-I 蛋白，该蛋白在功能上模拟 IGF-I 与 VN 结合所获得的效应。本研究的目的是确定 hES 细胞是否可以在无饲养细胞和无血清条件下，使用含有我们新型嵌合 VN:IGF-I 蛋白的培养基进行连续传代。在这里，我们证明 hES 细胞可以在无饲养细胞、无血清、化学定义的培养基中连续传代，在我们的无饲养细胞、无血清、化学定义的培养基中，最多可传代 35 代，并且保持未分化状态。我们利用实时聚合酶链反应（PCR）、免疫荧光和荧光激活细胞分选（FACS）分析表明，hES 细胞保持未分化表型。体外分化实验表明，hES 细胞保留其多能性潜能，并且 hES 细胞的核型保持不变。这项研究表明，本文所述的新型、完全定义的合成 VN:IGF-I 嵌合蛋白培养基是一种替代含有血清的培养基的可行选择，并且与层粘连蛋白包被的平板结合使用，有利于 hES 的无饲养细胞和无血清生长。
