Braam Stefan R, Zeinstra Laura, Litjens Sandy, Ward-van Oostwaard Dorien, van den Brink Stieneke, van Laake Linda, Lebrin Franck, Kats Peter, Hochstenbach Ron, Passier Robert, Sonnenberg Arnoud, Mummery Christine L
Leiden University Medical Centre, Postal Zone S-1-P, P.O. Box 9600, 2300 RC Leiden, The Netherlands.
Stem Cells. 2008 Sep;26(9):2257-65. doi: 10.1634/stemcells.2008-0291. Epub 2008 Jul 3.
Defined growth conditions are essential for many applications of human embryonic stem cells (hESC). Most defined media are presently used in combination with Matrigel, a partially defined extracellular matrix (ECM) extract from mouse sarcoma. Here, we defined ECM requirements of hESC by analyzing integrin expression and ECM production and determined integrin function using blocking antibodies. hESC expressed all major ECM proteins and corresponding integrins. We then systematically replaced Matrigel with defined medium supplements and ECM proteins. Cells attached efficiently to natural human vitronectin, fibronectin, and Matrigel but poorly to laminin + entactin and collagen IV. Integrin-blocking antibodies demonstrated that alphaVbeta5 integrins mediated adhesion to vitronectin, alpha5beta1 mediated adhesion to fibronectin, and alpha6beta1 mediated adhesion to laminin + entactin. Fibronectin in feeder cell-conditioned medium partially supported growth on all natural matrices, but in defined, nonconditioned medium only Matrigel or (natural and recombinant) vitronectin was effective. Recombinant vitronectin was the only defined functional alternative to Matrigel, supporting sustained self-renewal and pluripotency in three independent hESC lines.
特定的生长条件对于人类胚胎干细胞（hESC）的许多应用至关重要。目前，大多数特定培养基都与基质胶联合使用，基质胶是一种源自小鼠肉瘤的部分特定的细胞外基质（ECM）提取物。在此，我们通过分析整合素表达和ECM产生来确定hESC对ECM的需求，并使用阻断抗体确定整合素功能。hESC表达所有主要的ECM蛋白和相应的整合素。然后，我们用特定的培养基补充剂和ECM蛋白系统地替代基质胶。细胞能有效地附着于天然人玻连蛋白、纤连蛋白和基质胶，但与层粘连蛋白+巢蛋白和IV型胶原的附着较差。整合素阻断抗体表明，αVβ5整合素介导对玻连蛋白的黏附，α5β1介导对纤连蛋白的黏附，α6β1介导对层粘连蛋白+巢蛋白的黏附。饲养层细胞条件培养基中的纤连蛋白在所有天然基质上部分支持生长，但在特定的非条件培养基中，只有基质胶或（天然和重组）玻连蛋白有效。重组玻连蛋白是基质胶唯一特定的功能替代物，可支持三个独立hESC系的持续自我更新和多能性。
