Department of Biomedical Engineering, University of Texas at Austin, Austin, Texas, USA; Department of Kinesiology and Health Education, University of Texas at Austin, Austin, Texas, USA.
Department of Biomedical Engineering, University of Texas at Austin, Austin, Texas, USA.
Cytotherapy. 2023 Apr;25(4):375-386. doi: 10.1016/j.jcyt.2022.11.010. Epub 2022 Dec 20.
Skeletal muscle regeneration after severe damage is reliant on local stem cell proliferation and differentiation, processes that are tightly regulated by macrophages. Peripheral artery disease is a globally prevalent cardiovascular disease affecting millions of people. Progression of the disease leads to intermittent claudication, subsequent critical limb ischemia and muscle injury. Tissue-derived and ex vivo-expanded mesenchymal stromal cells (MSCs) for skeletal muscle regeneration have been studied, but pre-clinical and clinical results have not been consistent. As a result, the potential therapeutic efficacy and associated repair mechanisms of MSCs remain unclear. Numerous studies have demonstrated the vulnerability of delivered MSCs, with a precipitous drop in cell viability upon transplantation. This has prompted investigation into the therapeutic benefit of apoptotic cells, microvesicles, exosomes and soluble signals that are released upon cell death.
In this study, we characterized various components produced by MSCs after cell death induction under different conditions. We discovered anti-inflammatory and pro-regenerative effects produced by cell components following a freeze and thaw (F&T) process on macrophage polarization in vitro. We further investigated the underlying mechanisms of macrophage polarization by those components resulting from severe cell death induction.
We found potent therapeutic effects from F&T-induced cell debris are dependent on the externalization of phosphatidylserine on the plasma membrane. In contrast, effects from the supernatant of F&T-induced cell death primarily depends on the released protein content. We then applied the F&T-induced cell supernatant to an animal model of peripheral artery disease to treat muscle injury caused by severe ischemia. Treatment with the F&T supernatant but not the vulnerable MSCs resulted in significantly improved recovery of muscle function, blood flow and morphology and inflammation resolution in the affected muscles 2 weeks after injury.
This study validates the therapeutic potential of F&T-induced supernatant obviating the need for a viable population from vulnerable MSCs to treat injury, thus providing a roadmap for cell-free therapeutic approaches for tissue regeneration.
严重损伤后骨骼肌的再生依赖于局部干细胞的增殖和分化，这些过程受巨噬细胞的严格调控。外周动脉疾病是一种全球流行的心血管疾病，影响着数百万人。疾病的进展导致间歇性跛行，随后出现严重的肢体缺血和肌肉损伤。已经研究了用于骨骼肌再生的组织来源和体外扩增的间充质基质细胞（MSCs），但临床前和临床结果并不一致。因此，MSCs 的潜在治疗效果和相关修复机制仍不清楚。许多研究表明，递送的 MSCs 很脆弱，移植后细胞活力急剧下降。这促使人们研究细胞死亡时释放的凋亡细胞、微泡、外泌体和可溶性信号的治疗益处。
在这项研究中，我们在不同条件下对细胞死亡诱导后 MSC 产生的各种成分进行了特征描述。我们发现，在体外，经过冻融（F&T）处理后，MSC 死亡产生的细胞成分会影响巨噬细胞的极化，并具有抗炎和促进再生的作用。我们进一步研究了这些成分诱导严重细胞死亡后对巨噬细胞极化的潜在机制。
我们发现，F&T 诱导的细胞碎片的强大治疗效果依赖于质膜上磷脂酰丝氨酸的外化。相比之下，F&T 诱导的细胞死亡上清液的作用主要取决于释放的蛋白质含量。然后，我们将 F&T 诱导的细胞上清液应用于外周动脉疾病的动物模型中，以治疗严重缺血引起的肌肉损伤。与脆弱的 MSCs 相比，用 F&T 上清液处理可显著改善损伤后 2 周时肌肉功能、血流和形态以及受影响肌肉的炎症消退。
这项研究验证了 F&T 诱导的上清液的治疗潜力，避免了脆弱 MSCs 中存活细胞群体治疗损伤的需要，从而为组织再生的无细胞治疗方法提供了路线图。
