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Different tissue types are characterized by varying stiffness and biochemical ligands. Increasing substrate stiffness has been shown to trigger Yes-associated protein (YAP) translocation from the cytoplasm to the nucleus, yet the role of ligand density in modulating mechanotransduction and stem cell fate remains largely unexplored. Using polyacrylamide hydrogels coated with fibronectin as a model platform, we showed that stiffness-induced YAP translocation occurs only at intermediate ligand densities. At low or high ligand densities, YAP localization is dominated by ligand density independent of substrate stiffness. We further showed that ligand density-induced YAP translocation requires cytoskeleton tension and αVβ3-integrin binding. Finally, we demonstrate that increasing ligand density alone can enhance osteogenic differentiation regardless of matrix stiffness. Together, the findings from the present study establish ligand density as an important parameter for modulating stem cell mechanotransduction and differentiation, which is mediated by integrin clustering, focal adhesion, and cytoskeletal tension.

Biochemical Ligand Density Regulates Yes-Associated Protein Translocation in Stem Cells through Cytoskeletal Tension and Integrins