Sung Dong Kyung, Chang Yun Sil, Sung Se In, Ahn So Yoon, Park Won Soon
Department of Pediatrics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul 06351, Korea.
Stem Cell and Regenerative Medicine Institute, Samsung Medical Center, Samsung Biomedical Research Institute, Seoul 06351, Korea.
J Clin Med. 2019 Apr 18;8(4):533. doi: 10.3390/jcm8040533.
The aim of this study was to determine the optimal preconditioning regimen for the wound healing therapeutic efficacy of mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs). To this end, we compared various preconditioning regimens for both the quantitative and qualitative production of MSC-derived EVs, and their therapeutic efficacy for proangiogenic activity in vitro and cutaneous wound healing in vivo. After preconditioning with thrombin (40 U), HO (50 μM), lipopolysaccharide (1 μg/mL), or hypoxia (10% O), EV secretion was assessed quantitatively by measuring production per cell and protein quantification, and qualitatively by measuring a proteome profiler and an enzyme-linked immunosorbent assay (ELISA) contained within EVs. The therapeutic efficacy of EVs was assessed in vitro by proliferation, migration and tube formation assays of human umbilical cord blood endothelial cells (HUVECs), and in vivo by quantification of cutaneous wound healing. Thrombin preconditioning optimally boosted EV production and enriched various growth factors including vascular endothelial growth factor and angiogenin contained within EVs compared to other preconditioning regimens. Thrombin preconditioning optimally enhanced proliferation, the migration and tube formation of HUVECs in vitro via ERK1/2 and AKT signaling pathways, and cutaneous wound healing in vivo compared to other preconditioning regimens. Thrombin preconditioning exhibited optimal therapeutic efficacy compared with other preconditioning regimens in promoting proangiogenic activity in vitro and in enhancing cutaneous wound healing in vivo. These preconditioning regimen-dependent variations in therapeutic efficacy might be mediated by boosting EV production and enriching their cargo content.
本研究的目的是确定间充质干细胞（MSC）衍生的细胞外囊泡（EVs）促进伤口愈合治疗效果的最佳预处理方案。为此，我们比较了多种预处理方案对MSC衍生EVs的定量和定性产生的影响，以及它们在体外促血管生成活性和体内皮肤伤口愈合方面的治疗效果。在用凝血酶（40 U）、血红素加氧酶（HO，50 μM）、脂多糖（1 μg/mL）或缺氧（10% O₂）进行预处理后，通过测量每个细胞的产量和蛋白质定量来定量评估EV分泌，并通过测量EVs中包含的蛋白质组分析和酶联免疫吸附测定（ELISA）来定性评估。通过人脐血内皮细胞（HUVECs）的增殖、迁移和管形成试验在体外评估EVs的治疗效果，并通过定量皮肤伤口愈合在体内评估。与其他预处理方案相比，凝血酶预处理能最佳地促进EV产生，并富集EVs中包含的各种生长因子，包括血管内皮生长因子和血管生成素。与其他预处理方案相比，凝血酶预处理通过ERK1/2和AKT信号通路在体外最佳地增强了HUVECs的增殖、迁移和管形成，并在体内促进了皮肤伤口愈合。与其他预处理方案相比，凝血酶预处理在促进体外促血管生成活性和增强体内皮肤伤口愈合方面表现出最佳的治疗效果。这些治疗效果中依赖于预处理方案的差异可能是通过促进EV产生和富集其货物含量来介导的。
