Ullmann U, In't Veld P, Gilles C, Sermon K, De Rycke M, Van de Velde H, Van Steirteghem A, Liebaers I
Research Centre Reproduction and Genetics, University Hospital and Medical School of the Vrije Universiteit Brussel, VUB, Free University of Brussels, Brussels, Belgium.
Mol Hum Reprod. 2007 Jan;13(1):21-32. doi: 10.1093/molehr/gal091. Epub 2006 Nov 7.
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.
无饲养层的人胚胎干细胞（hESC）培养与集落周边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。使用基质胶以及来自小鼠和人类来源的条件培养基进行长期无饲养层hESC培养，结果显示，无论使用哪种条件培养基，间充质样细胞的出现情况相似。标准表征证实了hESC特性得以保留，但无饲养层培养无法维持超过37代。在将饲养层上培养的hESC转换为无饲养层条件后的短期内，对早期分化过程进行了表征。透射电子显微镜显示hESC集落内部存在上皮样结构，而周边细胞呈现出相当程度的间充质样表型。免疫化学分析表明，集落周边的细胞E-钙黏蛋白表达呈阴性，波形蛋白表达呈阳性，提示上皮-间质转化（EMT）。在间充质样细胞群体中还发现β-连环蛋白的核染色呈阳性、N-钙黏蛋白呈阳性以及连接蛋白43呈阴性。经过逆转录聚合酶链反应（RT-PCR）分析，发现间充质样细胞群体中与EMT相关的转录因子Slug和Snail均上调。综上所述，我们的数据表明，在无饲养层条件下培养hESC会增强一种被确定为EMT的早期分化过程。
