Tissue-like Viscoelasticity Drives Muscle Stem Cell Expansion Through Mechanical Memory Formation

Mechanical cues are potent regulators of stem cell fate, with even transient exposure to substrates with varied elastic stiffness driving persistent changes in cell fate. Such elastic mechanical memory contributes to the loss of regenerative potential in skeletal muscle stem cells (MuSCs) when cultured on rigid substrates, hampering the translation of myogenic cell replacement therapies. Beyond elastic mechanical properties, cells also respond to time-dependent, viscoelastic mechanical properties, such as stress relaxation. However, whether tissue-like viscoelasticity can impart a mechanical memory that directs stem cell fate remains an open question. Accordingly, dual-network hydrogel biomaterials with decoupled control of stiffness, stress relaxation, and adhesive ligand presentation were engineered to recapitulate the viscoelastic mechanical properties of native muscle. Muscle-like viscoelastic substrates promote in vitro MuSC activation and accelerate stem cell expansion, matching in vivo activation timescales. Mechanistically, physiological viscoelasticity promotes focal adhesion stabilization and enhances Rac-Akt signaling, establishing a viscoelastic mechanical memory that supports progenitor expansion and persistent maintenance of a stem-like phenotype. These findings identify matrix viscoelasticity as a potent and persistent regulator of MuSC fate and provide a framework for engineering biomaterials that preserve MuSC stemness and expansion ex vivo.