Division of Matrixome Research and Application, Institute for Protein Research, Osaka University, Suita, Osaka, Japan.
Division of Matrixome Research and Application, Institute for Protein Research, Osaka University, Suita, Osaka, Japan; Division of Research and Development, Matrixome Inc., Suita, Osaka, Japan.
Matrix Biol. 2018 Jan;65:75-90. doi: 10.1016/j.matbio.2017.08.001. Epub 2017 Aug 8.
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.
层粘连蛋白是基底膜的主要成分，维持着各种干细胞的生长。在 15 种层粘连蛋白亚型中，层粘连蛋白-511 及其 E8 片段（LM511E8）已被证明能强烈促进人类多能干细胞的黏附和增殖。本研究旨在赋予层粘连蛋白-511 在胶原蛋白基质上的黏附活性，从而构建具有特定成分的用于干细胞的胶原蛋白基培养支架。为了实现这一目标，我们利用纤连蛋白的胶原结合域（CBD）将 LM511E8 固定在胶原蛋白基质上。CBD 附着在单个层粘连蛋白链的 N 端（α5E8、β1E8、γ1E8）上，产生具有一个、两个或三个 CBD 的 LM511E8。虽然 LM511E8 不与胶原蛋白结合，但附着 CBD 的 LM511E8（CBD-LM511E8）表现出显著的胶原蛋白结合活性，这取决于附着的 CBD 数量。人诱导多能干细胞（iPS 细胞）在预先加载 CBD-LM511E8 的胶原蛋白涂层板上进行培养。尽管 iPS 细胞不附着或在胶原蛋白上生长，但它们在 CBD-LM511E8 负载的胶原蛋白基质上大量增殖，与 LM511E8 涂层板的情况相似。重要的是，即使在预先加载 CBD-LM511E8 的胶原蛋白凝胶上，iPS 细胞也能增殖并形成圆形集落。这些结果表明，附着 CBD 的层粘连蛋白 E8 片段是制备具有层粘连蛋白黏附活性的胶原蛋白基质的有前途的工具。
