Human mesenchymal stem cell exosomes have been shown to promote cutaneous wound healing. Their bioactivity is most often attributed to their protein and nucleic acid components, while the function of exosomal lipids remains comparatively unexplored. This work specifically assesses the involvement of lipids and the transmembrane enzyme CD73 in the exosomes’ biological activity in stimulating the cutaneous wound healing process. Since exosome preparation processes are not harmonized yet, certain production and purification parameters are first systematically investigated, enabling the optimization of a standardized protocol delivering high exosome integrity, yield, and purity. An in situ enzymatic assay is introduced to specifically assess the vesicle functionality, and quantitative proteomics is employed to establish the cell culture conditions yielding a stable exosome protein profile. Using a combination of in vitro approaches, CD73 and constitutional lipids of HPV‐16 E6/E7 transformed human bone marrow stromal cell‐derived exosomes are identified as key bioactive components promoting the exosome‐driven acceleration of processes required for wound repair. A pilot wound healing study in mice indeed suggests a role of lipids in the exosomes’ biological activity. Strikingly, the extent of the bioactivity of these exosomal components is found to be dependent on the target cell type.

中文翻译：

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用于伤口愈合的外泌体：生物活性成分的纯化优化和鉴定

人类间充质干细胞外泌体已被证明可以促进皮肤伤口愈合。它们的生物活性通常归因于它们的蛋白质和核酸成分，而外泌体脂质的功能相对而言仍未被探索。这项工作专门评估了脂质和跨膜酶 CD73 在外泌体刺激皮肤伤口愈合过程的生物活性中的参与。由于外泌体制备工艺尚未统一，因此首先系统地研究某些生产和纯化参数，从而能够优化标准化方案，提供高外泌体完整性、产量和纯度。引入原位酶测定来特异性评估囊泡功能，并采用定量蛋白质组学来建立产生稳定的外泌体蛋白质谱的细胞培养条件。通过结合体外方法，CD73 和 HPV-16 E6/E7 转化的人骨髓基质细胞来源的外泌体的组成脂质被确定为关键的生物活性成分，可促进外泌体驱动的伤口修复所需过程的加速。一项针对小鼠的伤口愈合试点研究确实表明了脂质在外泌体生物活性中的作用。引人注目的是，发现这些外泌体成分的生物活性程度取决于靶细胞类型。