Singh Mark D, Kreiner Michaela, McKimmie Clive S, Holt Stephen, van der Walle Christopher F, Graham Gerard J
Division of Immunology Infection and Inflammation, Glasgow Biomedical Research Centre, University of Glasgow, 120 University Place, Glasgow G12 8TA, UK.
Biochem Biophys Res Commun. 2009 Dec 18;390(3):716-21. doi: 10.1016/j.bbrc.2009.10.035. Epub 2009 Oct 13.
We present the first report utilizing, and showing the functional relevance of, self-assembling polyvalent ligands specific for integrin alpha5beta1 in murine embryonic stem (mES) cell adhesion. Di, tri and tetrameric 9th-10th type III fibronectin domains (FIII9'10) were used to generate clustered integrin alpha5beta1 ligand surfaces for mES cell culture. Compared to gelatin, FIII9'10 (monomer), FIII9'10-trimer and -tetramer, the FIII9'10-dimer supported the highest number of mES cell colonies. No evidence of domain unfolding upon surface adsorption was found. Colonies appeared disperse with a spread cell morphology unless subdued back to a tight morphology with increasing concentrations of leukemia inhibitory factor (LIF). In the presence of LIF, mES cells adherent to the FIII9'10-dimer showed transient upregulation of Oct-4, the mesodermal transcription factor, Brachyury, and the ectodermal marker, Nestin. However, dual upregulation of Nanog maintained the mES cells in a pluripotent state, confirmed by alkaline phosphatase staining. Therefore, the behavior of mES cells adherent to dimeric integrin alpha5beta1 ligands is a largely morphological phenomenon conferring pro-differentiation signals towards mesodermal and ectodermal lineages. This work will be of interest to cell and tissue engineering groups aiming to control ES cell behavior through integrin ligand presentation and synthetic substrates.
我们首次报道了利用对整合素α5β1具有特异性的自组装多价配体，并展示其在小鼠胚胎干细胞（mES）黏附中的功能相关性。使用二聚体、三聚体和四聚体的第9-10型III型纤连蛋白结构域（FIII9'10）来生成用于mES细胞培养的聚集整合素α5β1配体表面。与明胶、FIII9'10（单体）、FIII9'10-三聚体和-四聚体相比，FIII9'10-二聚体支持的mES细胞集落数量最多。未发现表面吸附后结构域展开的证据。集落呈现分散状态，细胞形态呈铺展状，除非随着白血病抑制因子（LIF）浓度增加使其恢复为紧密形态。在LIF存在的情况下，黏附于FIII9'10-二聚体的mES细胞显示中胚层转录因子Oct-4、Brachyury以及外胚层标志物Nestin的短暂上调。然而，Nanog的双重上调使mES细胞维持在多能状态，碱性磷酸酶染色证实了这一点。因此，黏附于二聚体整合素α5β1配体的mES细胞的行为在很大程度上是一种形态学现象，赋予向中胚层和外胚层谱系的促分化信号。这项工作将引起细胞和组织工程团队的兴趣，他们旨在通过整合素配体呈现和合成底物来控制ES细胞行为。
