Epithelial-mesenchymal transition process in human embryonic stem cells cultured in feeder-free conditions
Epithelial-mesenchymal transition process in human embryonic stem cells cultured in feeder-free conditions
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DOI：
10.1093/molehr/gal091
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发表时间：
2007-01-01
影响因子：
4
通讯作者：
Liebaers, I.
中科院分区：
文献类型：
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作者：
Ullmann, U.;In't Veld, P.;Liebaers, I.
无饲养层人胚胎干细胞 (hESC) 培养与集落外围出现的间充质样细胞有关。本研究的目的是确定这种早期分化过程。使用来自小鼠和人类的基质胶和条件培养基进行的长期无饲养层 hESC 培养表明，无论使用何种条件培养基，间充质样细胞的外观都是相似的。标准表征证实了 hESC 特性的保存，但无饲养层培养物的维持时间不能超过 37 代。将饲养层培养的 hESC 转为无饲养层培养条件后，早期分化过程在短期内得到表征。透射电子显微镜显示 hESC 集落内有类似上皮的结构，而外周细胞则显示出获得了类似间充质的表型。免疫化学分析显示集落外围的细胞E-钙粘蛋白表达阴性，波形蛋白表达阳性，提示上皮间质转化（EMT）。在间充质样细胞群中还发现了 β-连环蛋白的核染色、N-钙粘蛋白阳性和连接蛋白 43 阴性表达。经过 RT-PCR 分析，Slug 和 Snail 这两种 EMT 相关转录因子在间充质样细胞群中被发现上调。综上所述，我们的数据表明，在无饲养层条件下培养 hESC 可增强被称为 EMT 的早期分化过程。
Feeder-free human embryonic stem cell (hESC) culture is associated with the presence of mesenchymal-like cells appearing at the periphery of the colonies. The aim of this study was to identify this early differentiation process. Long-term feeder-free hESC cultures using matrigel and conditioned medium from mouse and from human origin revealed that the appearance of mesenchymal-like cells was similar regardless of the conditioned medium used. Standard characterization confirmed the preservation of hESC properties, but the feeder-free cultures could not be maintained longer than 37 passages. The early differentiation process was characterized in the short term after switching hESCs cultured on feeders to feeder-free conditions. Transmission electron microscopy showed an epithelium-like structure inside the hESC colonies, whereas the peripheral cells revealed the acquisition of a rather mesenchymal-like phenotype. Immunochemistry analysis showed that cells at the periphery of the colonies had a negative E-cadherin expression and a positive Vimentin expression, suggesting an epithelial-mesenchymal transition (EMT). Nuclear staining of beta-catenin, positive N-cadherin and negative Connexin 43 expression were also found in the mesenchymal-like cell population. After RT-PCR analysis, Slug and Snail, both EMT-related transcription factors, were detected as up-regulated in the mesenchymal-like cell population. Taken together, our data suggest that culturing hESCs in feeder-free conditions enhances an early differentiation process identified as an EMT.