(568f) Elucidating Mechanisms of Muscle Stem Cell Mechanical Memory Using Hydrogel Biomaterials with on-Demand Tuning of Mechanical Properties

Muscle stem cells (MuSCs), also known as satellite cells, are specialized cells in adult skeletal muscle that are responsible for the maintenance and repair of muscle tissue throughout life. The ability of MuSCs to regenerate damaged tissues declines markedly with aging, as well as during progressive muscle wasting disorders such as Duchenne muscular dystrophy. This diminished stem cell function leads to increased morbidity and mortality and decreased quality of life, imposing significant burdens on individual patients and the healthcare system as a whole. Despite the central role of MuSCs in maintaining muscle function, the underlying causes of MuSC dysfunction in aging and disease remain poorly understood. A major limitation is the heterogeneous nature of aging in model organisms, which makes assigning causal relationships between changes in the tissue microenvironment and stem cell behavior challenging. Biomaterial cell culture platforms with tunable biophysical and biochemical cues may overcome these challenges by recapitulating key changes in the tissue microenvironment that regulate cell fate without the presence of confounding systemic variables present in living organisms. MuSCs are known to be exquisitely mechanosensitive, rapidly losing the ability to engraft in and repair damaged muscle tissue after culture on hydrogels with elastic moduli greater than healthy muscle tissue (~12 kPa). As fibrosis, or excess extracellular matrix deposition, is a common pathology in muscle wasting from aging and disease, we hypothesized that increased stiffness in fibrotic tissues will impair the ability of MuSCs to properly activate and undergo myogenesis. Indeed, MuSCs cultured on hydrogels with stiffness resembling fibrotic muscle (~35-40 kPa) exhibited impaired expansion and premature differentiation and return to quiescence, whereas MuSCs cultured on healthy-like (12 kPa) hydrogels expanded extensively as myogenic progenitors. These results contrast with previous studies that reported increased proliferation in myoblasts (MuSCs previously expanded on rigid tissue culture plastic) as stiffness is increased. This contradiction suggests that MuSCs exhibit a “mechanical memory” of the microenvironmental stiffness they experience during the process of activation after being isolated from muscle tissues as quiescent cells. To determine the temporal window during which this mechanical memory is acquired, we developed synthetic poly(ethylene glycol) (PEG) hydrogels that could be softened or stiffened on demand by exposure to 365 nm light, using either orthonitrobenzyl esters or photoactivated strain-promoted azide-alkyne cycloaddition (SPAAC) to decrease or increase crosslink density, and therefore stiffness, respectively. By three days of culture on stiff substrates, MuSCs commit to a “stiff” phenotype, even if the substrates are subsequently softened. This “stiff” phenotype consists of a diminished fraction of cells undergoing proliferation, increased fractions of quiescent-like (Pax7+) and committed (myogenin+) cells, and a decreased fraction of MyoD+ progenitors. By functionalizing the hydrogels with different extracellular matrix proteins, this mechanosensitive response was shown to be laminin-dependent, with “stiff” phenotypes occurring on gels that presented fibronectin or gelatin adhesive cues regardless of stiffness. Fluorescence microscopy and biochemical approaches were employed to identify the molecular nature of this mechanical memory. MuSCs cultured on soft vs. stiff gels exhibited altered cytoskeletal architecture and activity of mechanosensitive Rho/Rac GTPases and YAP/TAZ signaling. Small molecule inhibitors were employed to identify the mechanosensitive pathway responsible for the “stiff” phenotype, and single cell RNA sequencing was used to track changes in the transcriptional profile of MuSCs during acquisition of the mechanical memory. These studies highlight the crucial role of microenvironmental mechanics on the proper activation of MuSCs and point to potential therapeutic strategies to improve MuSC function in aged and disease states with aberrant tissue mechanics.