Hagemann Kyle N, McColl Rhys S, Lovett Jason A C, Snyman Celia, Myburgh Kathryn H
Department of Physiological Sciences, Stellenbosch University, Stellenbosch, 7600, South Africa.
J Muscle Res Cell Motil. 2025 Aug 18. doi: 10.1007/s10974-025-09705-y.
Muscle injury activates satellite cells and fibroblasts, with extracellular vesicles (EVs) mediating the related intercellular communication. The influence of EVs released by either cell type on recipient cell behaviour is still unclear. This study investigated the uptake and effects of EVs derived from C2C12 myoblasts (myo-EVs) and L929 fibroblasts (fibro-EVs) on proliferating myoblasts. Both cell lines were cultured in media largely depleted of FBS-derived EVs. Myo-EVs and fibro-EVs isolated from conditioned media were characterised using conventional methods. Effects of these EVs on myoblast function were assessed via PKH67-labelled EV uptake, proliferation, scratch closure, leading front migration rate and individual cell trajectories and western blot analysis for MyoD and Myogenin. Myoblasts preferentially internalised myo-EVs at 5 h (myo-EVs: 3.41 ± 1.34 vs fibro-EVs: 1.25 ± 1.13 puncta per cell) and 48 h (myo-EVs 16.55 ± 12.60 vs fibro-EVs 9.67 ± 4.88) (both p < 0.05). Under proliferative EV-depleted conditions, added EVs did not change myoblast proliferation. However, the elevated expression of Myogenin indicating a subtle shift toward differentiation. Myo-EVs increased myoblast migration rate into a scratch, compared to controls (13.77 ± 1.7 vs 11.08 ± 2.23 µm/h, p < 0.01), but had no effect under conditions of FBS EV depletion. On the other hand, fibro-EVs increased the speed of individual cells, but negatively affected leading front migration due to impaired myoblast persistence. These findings highlight the importance of cell-specific EV-mediated communication in muscle regeneration. Further, tissue explants used to generate cell-specific EVs for treatment should be free of contaminating cell types.
肌肉损伤会激活卫星细胞和成纤维细胞，细胞外囊泡（EVs）介导相关的细胞间通讯。两种细胞类型释放的细胞外囊泡对受体细胞行为的影响仍不清楚。本研究调查了源自C2C12成肌细胞的细胞外囊泡（肌源性细胞外囊泡，myo-EVs）和L929成纤维细胞的细胞外囊泡（成纤维细胞源性细胞外囊泡，fibro-EVs）对增殖中的成肌细胞的摄取及影响。两种细胞系均在基本不含胎牛血清衍生细胞外囊泡的培养基中培养。使用传统方法对从条件培养基中分离出的肌源性细胞外囊泡和成纤维细胞源性细胞外囊泡进行表征。通过PKH67标记的细胞外囊泡摄取、增殖、划痕闭合、前沿迁移率和单个细胞轨迹以及对MyoD和肌细胞生成素进行蛋白质印迹分析，评估这些细胞外囊泡对成肌细胞功能的影响。成肌细胞在5小时（肌源性细胞外囊泡：每个细胞3.41±1.34个斑点，而成纤维细胞源性细胞外囊泡：每个细胞1.25±1.13个斑点）和48小时（肌源性细胞外囊泡16.55±12.60，而成纤维细胞源性细胞外囊泡9.67±4.88）时优先内化肌源性细胞外囊泡（两者p<0.05）。在增殖性无细胞外囊泡条件下，添加的细胞外囊泡不会改变成肌细胞的增殖。然而，肌细胞生成素表达升高表明向分化有细微转变。与对照组相比，肌源性细胞外囊泡增加了成肌细胞向划痕内的迁移率（13.77±1.7对11.08±2.23μm/h，p<0.01），但在无胎牛血清细胞外囊泡的条件下没有影响。另一方面，成纤维细胞源性细胞外囊泡提高了单个细胞的速度，但由于成肌细胞持久性受损而对前沿迁移产生负面影响。这些发现突出了细胞特异性细胞外囊泡介导的通讯在肌肉再生中的重要性。此外，用于生成细胞特异性细胞外囊泡进行治疗的组织外植体应不含污染的细胞类型。
