Mesenchymal Stem Cell Aggregation-Released Extracellular Vesicles Induce CD31+ EMCN+ Vessels in Skin Regeneration and Improve Diabetic Wound Healing.

The blood vessel system is essential for skin homeostasis and regeneration. While the heterogeneity of vascular endothelial cells has been emergingly revealed, whether a regeneration-relevant vessel subtype exists in skin remains unknown. Herein, a specialized vasculature in skin featured by simultaneous CD31 and EMCN expression contributing to the regeneration process is identified, the decline of which functionally underlies the impaired angiogenesis of diabetic nonhealing wounds. Moreover, enlightened by the developmental process that mesenchymal condensation induces angiogenesis, it is demonstrated that mesenchymal stem/stromal cell aggregates (CAs) provide an efficacious therapy to enhance regrowth of CD31+ EMCN+ vessels in diabetic wounds, which is surprisingly suppressed by pharmacological inhibition of extracellular vesicle (EV) release. It is further shown that CAs promote secretion of angiogenic protein-enriched EVs by proteomic analysis, which directly exert high efficacy in boosting CD31+ EMCN+ vessels and treating nonhealing diabetic wounds. These results add to the current knowledge on skin vasculature and help establish feasible strategies to benefit wound healing under diabetic condition.

Isolation, Characterization, and Therapeutic Application of Extracellular Vesicles from Cultured Human Mesenchymal Stem Cells.

Extracellular vesicles (EVs) are heterogeneous membrane nanoparticles released by most cell types, and they are increasingly recognized as physiological regulators of organismal homeostasis and important indicators of pathologies; in the meantime, their immense potential to establish accessible and controllable disease therapeutics is emerging. Mesenchymal stem cells (MSCs) can release large amounts of EVs in culture, which have shown promise to jumpstart effective tissue regeneration and facilitate extensive therapeutic applications with good scalability and reproducibility. There is a growing demand for simple and effective protocols for collecting and applying MSC-EVs. Here, a detailed protocol is provided based on differential centrifugation to isolate and characterize representative EVs from cultured human MSCs, exosomes, and microvesicles for further applications. The adaptability of this method is shown for a series of downstream approaches, such as labeling, local transplantation, and systemic injection. The implementation of this procedure will address the need for simple and reliable MSC-EVs collection and application in translational research.