Development of a Pericyte-Based Cell-Free Strategy to Recover Aged Skeletal Muscle After Disuse

Our laboratory previously demonstrated that perivascular stem/stromal cells (CD146+ pericytes) can effectively recover muscle mass after a period of immobilization in young adult mice. However, cell-based therapies are problematic in aged mouse models due to lack of viability upon transplantation. Therefore, the purpose of this study was to develop a pericyte-based, cell-free strategy to recover muscle mass after disuse in aged mice. Single-cell RNA sequencing (scRNA-Seq) was performed on adult mouse skeletal muscle after two weeks of unilateral hindlimb immobilization, which revealed that muscle-resident pericytes uniquely upregulate the long noncoding RNA Malat1, a negative regulator of Nrf2, and fail to induce antioxidant gene expression in response to reactive oxygen species (ROS; H2O2). This information was used to guide the design of a strategy in which healthy donor pericytes were stimulated with ROS to produce small extracellular vesicles (EVs) that were subsequently transplanted into 4- and 24-26-month-old C57BL/6 mice after two weeks of unilateral hindlimb immobilization. H2O2-primed healthy muscle-derived pericytes produced EVs in culture that effectively reduced restored myofiber CSA in both adult (p=0.009) and aged (p=0.006) muscle after disuse. In contrast, unprimed pericyte-derived EVs did not influence myofiber size. Neither primed, nor unprimed EVs recovered capillary density, yet both stimulated collagen turnover. Healthy ROS-primed pericyte-derived small EVs effectively improve skeletal muscle recovery after immobilization, representing a novel cell-free approach to rebuild muscle mass in older adults after a period of disuse. Six mice were subjected to unilateral hindlimb immobilization for 2 weeks. The tibialis anterior (TA) muscles from mobile (“Mobile”; control) and previously immobilized (“Immobile”) limbs were excised and pooled according to treatment.