Associate Professor Osaka University Suita, Osaka, Japan
Abstract: Three-dimensional (3D) gel culture systems bridge the gap between in vivo microenvironments and conventional two-dimensional in vitro culture, enabling more physiologically relevant cell behaviors. Fibrin, a hydrogel widely used in surgical applications, is also used in regenerative medicine as a scaffold for 3D culture of stem cells because of its biocompatibility and biodegradability. However, fibrin gels exhibit limited cell adhesion, except for platelets, restricting their utility in cell-based therapies. To overcome this limitation, we engineered a bi-functional fusion protein, Chimera-511, by linking the N-terminal self-polymerization domain of fibrinogen to the C-terminal integrin-binding domain of laminin-511 via their heterotrimeric coiled-coil domains. Chimera-511 binds fibrinogen in a thrombin-dependent manner and retains its integrin-binding activity in a fibrinogen/fibrin-bound form. Fluorescence imaging showed that GFP-labeled Chimera-511 colocalized with fibrin, confirming its integration into the gel. When cultured in the Chimera-511-functionalized fibrin gel, human iPS cells stably proliferated into a sphere-like structure and maintained their undifferentiated state (Oct3/4, Nanog, SSEA-4 expression) and pluripotency (trilineage differentiation potential) for at least five passages. These findings demonstrate that Chimera-511-functionalized fibrin gel provides a 3D microenvironment suitable for stem cell cultivation, offering a versatile platform for both basic research and clinical applications involving stem cell manipulation.
