Mechanical signalling through collagen I regulates cholangiocyte specification and tubulogenesis during liver development

Cholangiocyte dysfunction accounts for a third of liver transplantations, access to which is limited by a shortage of healthy donor organs. A promising alternative is the therapeutic use of human induced pluripotent stem cell (hiPSC)-derived cholangiocytes. However, the use of hiPSCs is impeded by a lack of knowledge regarding intrahepatic cholangiocyte development, limiting the generation of fully functional cells. In this study, we generate hiPSC-derived tubular cholangiocytes using an approach based in synthetic hydrogels. These hydrogels exert control over stiffness and extracellular matrix (ECM) composition and stability, allowing us to address a critical gap in understanding cholangiocyte development. Our findings reveal that stable collagen I functionalisation, particularly on a soft substrate, enhances cholangiocyte differentiation, largely irrespective of substrate stiffness. Furthermore, high collagen I stability on a soft substrate suppresses hepatic identity whilst promoting biliary identity and duct morphogenesis. Our findings highlight the importance of collagen I mechanical signalling in regulating hepatoblast fate determination. Overall, we propose a mechanism by which the ECM modulates cholangiocyte and bile duct development and present a scalable platform for future clinical applications in the understanding and treatment of cholangiopathies.