Hydrogel biomaterials that stiffen and soften on demand reveal that skeletal muscle stem cells harbor a mechanical memory

Muscle stem cells (MuSCs) are specialized cells that reside in adult skeletal muscle poised to repair muscle tissue. The ability of MuSCs to regenerate damaged tissues declines markedly with aging and in diseases such as Duchenne muscular dystrophy, but the underlying causes of MuSC dysfunction remain poorly understood. Both aging and disease result in dramatic increases in the stiffness of the muscle tissue microenvironment from fibrosis. MuSCs are known to lose their regenerative potential if cultured on stiff plastic substrates. We sought to determine if muscle stem cells harbor a memory of their past microenvironment and if it can be overcome. We tested MuSCs in situ using dynamic hydrogel biomaterials that soften or stiffen on demand in response to light and found that freshly isolated MuSCs develop a persistent memory of substrate stiffness characterized by loss of proliferative progenitors within the first three days of culture on stiff substrates. MuSCs cultured on soft hydrogels had altered cytoskeletal organization and activity of Rho and Rac GTPase and YAP mechanotransduction pathways compared to those on stiff hydrogels. Pharmacologic inhibition identified RhoA activation as responsible for the mechanical memory phenotype, and single cell RNA sequencing revealed a molecular signature of the mechanical memory. These studies highlight that microenvironmental stiffness regulates MuSC fate and leads to MuSC dysfunction that is not readily reversed by changing stiffness. Our results suggest that stiffness can be circumvented by targeting downstream signaling pathways to overcome stem cell dysfunction in aged and disease states with aberrant fibrotic tissue mechanics. Overall design: Primary skeletal muscle stem cells were isolated from the hindlimbs of young (2 mo) or aged (>24 mo) C57BL/6 mice and were subsequently cultured on poly(ethylene glycol) hydrogels with either 12 kPa or 40 kPa stiffness for 1, 3, or 7 days. A subset of cells were treated with rhosin, a RhoA inhibitor, for three days prior to collection. Cells were enzymatically detached from the hydrogels and processed for single cell sequencing according to the Parse Biosciences Evercode WT version 1 protocol.