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. Overall design: 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.

本实验室既往研究证实，血管周干细胞/基质细胞（CD146阳性周细胞，CD146+ pericytes）可在青年成年小鼠肢体制动后有效恢复肌肉质量。然而，基于细胞的治疗方案在老年小鼠模型中存在缺陷，因其移植后存活率不足。因此，本研究旨在开发一种基于周细胞的无细胞策略，以实现老年小鼠废用后肌肉质量的恢复。本研究对单侧后肢制动2周后的成年小鼠骨骼肌进行了单细胞RNA测序（single-cell RNA sequencing, scRNA-Seq），结果显示，肌肉驻留周细胞特异性上调长链非编码RNA Malat1——该分子是核因子红细胞2相关因子2（nuclear factor erythroid 2-related factor 2, Nrf2）的负调控因子——且无法响应活性氧（reactive oxygen species, ROS；过氧化氢H₂O₂）诱导抗氧化基因表达。基于上述发现，我们设计了如下策略：用ROS刺激健康供体周细胞，以制备小型细胞外囊泡（extracellular vesicles, EVs），随后将其移植至单侧后肢制动2周的4月龄及24~26月龄C57BL/6小鼠体内。经H₂O₂预处理的健康肌肉来源周细胞在培养中分泌的细胞外囊泡，可在废用后有效增加成年（p=0.009）及老年（p=0.006）小鼠的肌纤维横截面积（cross-sectional area, CSA）。与之相反，未预处理的周细胞来源细胞外囊泡对肌纤维大小无显著影响。无论是预处理还是未预处理的细胞外囊泡，均未恢复毛细血管密度，但均可促进胶原周转。经ROS预处理的健康周细胞来源小型细胞外囊泡可有效改善制动后骨骼肌的恢复过程，为老年人群废用后肌肉量重建提供了一种全新的无细胞治疗方案。整体实验设计：6只小鼠接受单侧后肢制动2周。采集活动侧（“活动组”；对照）与既往制动侧（“制动组”）的胫骨前肌（tibialis anterior, TA），按处理组分别混合后进行后续实验。