Interplay of matrix sti ness and protein tethering in stem cell di erentiation

Stem cells regulate their fate by binding to, and contracting against, the extracellular matrix. Recently, it has been proposed that in addition to matrix sti ness and ligand type, the degree of coupling of fibrous protein to the surface of the underlying substrate, that is, tethering and matrix porosity, also regulates stem cell di erentiation. By modulating substrate porosity without altering sti ness in polyacrylamide gels, we show that varying substrate porosity did not significantly change protein tethering, substrate deformations, or the osteogenic and adipogenic di erentiation of human adipose-derived stromal cells andmarrow-derivedmesenchymal stromal cells. Varying protein–substrate linker density up to 50-fold changed tethering, but did not a ect osteogenesis, adipogenesis, surface–protein unfolding or underlying substrate deformations. Di erentiationwas also una ected by the absence of protein tethering. Our findings imply that the sti ness of planar matrices regulates stem cell di erentiation independently of protein tethering and porosity.