Assistant Professor University of Pennsylvania Philadelphia, Pennsylvania, United States
Introduction:: 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 and during diseases 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.
Materials and Methods:: To independently control the mechanical properties and biochemical composition of cell culture substrates, hydrogel precursors were prepared by separately functionalizing multi-arm poly(ethylene glycol) (PEG) macromers with azides and bicyclononynes (BCN) to enable bioorthogonal crosslinking via strain-promoted azide-alkyne cycloaddition (SPAAC). Purified laminin, fibronectin, and gelatin were separately functionalized with PEG-azides to enable selective incorporation into the hydrogel networks. To determine the temporal effects of matrix stiffness on MuSC fate, two additional PEG-based hydrogel networks were prepared: (1) gels that soften upon exposure to 365 nm light due to incorporation of o-nitrobenzyl (oNB) esters and (2) gels that stiffen upon exposure to 365 nm light due to secondary crosslinking by photo-triggered SPAAC. MuSCs were isolated from the hindlimb muscles of either young (2 mo) or aged ( >24 mo) mice by digesting the tissues with collagenase and dispase and enriching for cells expressing α7-integrin and CD34 by FACS. MuSCs were cultured on hydrogels for up to 7 days post-isolation, and cell fate and mechanotransduction pathways were characterized by immunofluorescence. Identified mechanosensitive pathways were interrogated by small molecule inhibitors. To characterize persistent mechanosensitive changes in MuSC transcriptional state, a SPLiT-seq single cell RNA sequencing approach was employed.
Results, Conclusions, and Discussions:: 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 employed hydrogels that could be softened or stiffened on demand by light exposure, using either oNB esters or photoactivated 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 revealed that MuSCs cultured on soft vs. stiff gels exhibited altered cytoskeletal architecture and activity of mechanosensitive Rho/Rac GTPases and YAP/TAZ signaling. A small molecule inhibitor screen identified 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. Inhibition of the dominant mechanotransduction pathway resulted in a transcriptional state resembling early stages of MuSC activation, thereby blocking formation of the mechanical memory. These studies reveal that microenvironmental mechanics regulate the process of MuSC activation, and that activation in an excessively stiff environment leads to MuSC dysfunction and acquisition of a mechanical memory. These results additionally point to potential therapeutic strategies to improve MuSC function in aged and disease states with aberrant tissue mechanics.