Laminins are major components of basement membranes that sustain a wide variety of stem cells. Among 15 laminin isoforms, laminin-511 and its E8 fragment (LM511E8) have been shown to strongly promote the adhesion and proliferation of human pluripotent stem cells. The aim of this study was to endow the cell-adhesive activity of laminin-511 on collagen matrices, thereby fabricating collagen-based culture scaffolds for stem cells with defined composition. To achieve this goal, we utilized the collagen-binding domain (CBD) of fibronectin to immobilize LM511E8 on collagen matrices. CBD was attached to the N-termini of individual laminin chains (α5E8, β1E8, γ1E8), producing LM511E8s having one, two, or three CBDs. While LM511E8 did not bind to collagen, CBD-attached LM511E8s (CBD-LM511E8s) exhibited significant collagen-binding activity, dependent on the number of attached CBDs. Human iPS cells were cultured on collagen-coated plates preloaded with CBD-LM511E8s. Although iPS cells did not attach or grow on collagen, they robustly proliferated on CBD-LM511E8-loaded collagen matrices, similar to the case with LM511E8-coated plates. Importantly, iPS cells proliferated and yielded round-shaped colonies even on collagen gels preloaded with CBD-LM511E8s. These results demonstrate that CBD-attached laminin E8 fragments are promising tools for fabrication of collagen-based matrices having the cell-adhesive activity of laminins.

中文翻译：

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具有胶原结合活性的重组层粘连蛋白片段：一种将层粘连蛋白样细胞粘附活性赋予胶原蛋白基质的工具。

层粘连蛋白是维持多种干细胞的基底膜的主要成分。在15种层粘连蛋白同工型中，层粘连蛋白511及其E8片段（LM511E8）已显示出可强烈促进人多能干细胞的粘附和增殖。这项研究的目的是赋予层粘连蛋白511在胶原蛋白基质上的细胞粘附活性，从而制造具有确定成分的干细胞胶原蛋白培养支架。为了实现此目标，我们利用纤连蛋白的胶原结合域（CBD）将LM511E8固定在胶原基质上。CBD连接到单个层粘连蛋白链的N末端（α5E8，β1E8，γ1E8），产生具有一个，两个或三个CBD的LM511E8。尽管LM511E8不与胶原蛋白结合，但附有CBD的LM511E8s（CBD-LM511E8s）表现出明显的胶原蛋白结合活性，取决于连接的CBD的数量。将人iPS细胞培养在预装有CBD-LM511E8s的胶原蛋白包被板上。尽管iPS细胞没有附着或生长在胶原蛋白上，但它们在载有CBD-LM511E8的胶原蛋白基质上仍能强劲增殖，这与LM511E8涂覆的板的情况相似。重要的是，即使在预装了CBD-LM511E8s的胶原蛋白凝胶上，iPS细胞也可以增殖并产生圆形菌落。这些结果表明，附有CBD的层粘连蛋白E8片段是用于制造具有层粘连蛋白的细胞粘附活性的基于胶原的基质的有前途的工具。与LM511E8涂层板的情况相似。重要的是，即使在预装了CBD-LM511E8s的胶原蛋白凝胶上，iPS细胞也可以增殖并产生圆形菌落。这些结果表明，附有CBD的层粘连蛋白E8片段是用于制造具有层粘连蛋白的细胞粘附活性的基于胶原的基质的有前途的工具。与LM511E8涂层板的情况相似。重要的是，即使在预装了CBD-LM511E8s的胶原蛋白凝胶上，iPS细胞也可以增殖并产生圆形菌落。这些结果表明，附有CBD的层粘连蛋白E8片段是用于制造具有层粘连蛋白的细胞粘附活性的基于胶原的基质的有前途的工具。