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. 2021 Aug;18(4):537-548.
doi: 10.1007/s13770-020-00316-x. Epub 2021 Feb 5.

Human Adipose Mesenchymal Stem Cell-Derived Exosomes: A Key Player in Wound Healing

June Seok Heo  1 , Sinyoung Kim  2 , Chae Eun Yang  3 , Youjeong Choi  1 , Seung Yong Song  4 , Hyun Ok Kim  5   6
Affiliations

Human Adipose Mesenchymal Stem Cell-Derived Exosomes: A Key Player in Wound Healing

June Seok Heo et al. Tissue Eng Regen Med. 2021 Aug.

Abstract

Background: Human adipose-derived mesenchymal stem cells (AMSCs) are an attractive resource for wound healing because their regenerative capacity improves injury repair. Recently, stem cell-derived exosomes have been shown to play a positive role in stem cell-based therapies. However, the effects of exosomes derived from AMSCs (AEXOs) on wound healing are unclear. In this study, we aimed to examine the role of AEXOs in attenuating inflammation and explore their effects in normal wound healing.

Methods: We isolated exosomes from AMSCs and established a cellular model of inflammation by treatment with the inflammatory cytokines, interferon gamma and tumor necrosis factor alpha, to determine whether AEXOs can inhibit inflammation. We examined the wound healing effects of AEXOs in in vitro wound healing models and performed a miRNA array to understand the role of AEXOs in inflammation and wound healing.

Results: A significant difference was observed in wound closure and the expression of anti-inflammatory and wound-healing-related factors between control and AEXO-treated cells.

Conclusion: Our results showed that besides alleviating the inflammation response, AEXOs also promote wound healing. Thus, AEXOs represent a novel, stem-cell-based, therapeutic strategy for wound healing.

Keywords: Adipose-derived stem cells; Exosomes; Inflammation; Wound healing.

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Conflict of interest statement

The authors declare that they no conflict of interest.

Figures

Fig. 1
Fig. 1
Treatment with a mixture of IFNγ and TNFα induced inflammation in fibroblasts. A Representative phase-contrast images of fibroblasts stimulated with 20 ng/mL of IFNγ and TNFα for 48 h (×200 magnification, scale bar = 100 μm). B Cell proliferation was measured by using a EZ-cytox assay kit with WST. C The relative mRNA levels of inflammatory IL-6 and CCL2 were analyzed by real-time PCR. The data are expressed as the mean ± SD of three independent experiments. *Significant difference from untreated control cells, p < 0.05. ##Significant difference from untreated control cells, p < 0.01
Fig. 2
Fig. 2
Characterization of AMSCs and exosomes derived from AMSCs. A The morphology and expression of cell surface markers of AMSCs. Cells cultured from adipose tissue displayed the typical fibroblast-like morphology of MSCs (×100 magnification, scale bar = 200 μm). Flow cytometry analysis revealed that cells were positive for CD73, CD90, and CD105, all MSC markers, but negative for hematopoietic markers, CD34 and CD45. B Transmission electron microscopy image of AEXOs (scale bar = 200 nm). C Exosome particle size was analyzed by using Nanosight; the mean diameter was 76.3 nm. D CD9 and CD63, both exosomal markers, were detected by western blotting. E Fluorescence images were obtained after treating PBMCs and fibroblasts with CFSE-labeled (green) exosomes for 12 h. Unlabeled exosomes were used as negative control (×200 magnification, scale bar = 100 μm)
Fig. 3
Fig. 3
Exosomes derived from AMSCs induced an anti-inflammatory M2 phenotype in PBMCs. A Relative expression levels of TNFα, Arg1, and CD206 were determined by real-time PCR. PBMCs were co-cultured with IFNγ-and-TNFα-treated fibroblasts. Untreated fibroblasts were used as control. B Expression of miR-34a-5p, miR-124-3p, and miR-146a-5p was upregulated in exosomes compared to that in AMSCs. Exosomes derived from AMSCs exerted immunomodulatory effects and suppress inflammation. C The suppression of the proliferation of PHA-stimulated PBMCs was evaluated by using a proliferation assay kit. Unstimulated PBMCs were used as control. D The mRNA levels of IL-6, TSG-6, and TGF-β1 in PBMCs co-cultured with IFNγ-and-TNFα-stimulated fibroblasts were analyzed by real-time PCR. PBMCs co-cultured with unstimulated fibroblasts were used as control. The data are expressed as the mean ± SD of three independent experiments. *Significant difference from untreated control cells, p < 0.05. ##Significant difference from untreated control cells, p < 0.01
Fig. 4
Fig. 4
Effects of exosomes derived from AMSCs on proinflammatory-cytokine-treated fibroblasts. A Changes in gene expression of TNFα, IL-6, and IL-8 were detected by real-time PCR after IFNγ-and-TNFα treatment. Untreated fibroblasts were used as control. B A multiplex assay was performed to determine the changes in inflammation-related factors at protein levels in conditioned media. The conditioned media of unstimulated fibroblasts were used as control. The data are expressed as the mean ± SD of three independent experiments. *Significant difference from untreated control cells, p < 0.05. ##Significant difference from untreated control cells, p < 0.01
Fig. 5
Fig. 5
Exosomes derived from AMSCs promoted proliferation and migration of fibroblasts. A Proliferation rates of fibroblasts treated with exosomes were analyzed by using a proliferation assay kit. B Migration of fibroblasts treated with exosomes was evaluated by an in vitro scratch wound healing assay. A representative of three independent experiments is shown (×100 magnification, scale bar = 200 μm). Fibroblast migration was quantified. C Real-time PCR analysis of the relative mRNA levels of type III collagen, fibronectin, and type I collagen genes was performed in fibroblasts after treatment with exosomes and compared to the respective mRNA levels in untreated cells. D Fibronectin, collagen, and VEGF concentrations were measured in conditioned media by multiplex assay. E The expression of miR-132, miR-21, and miR-29a was analyzed in exosomes produced by AMSCs by real-time PCR. The data are expressed as the mean ± SD of three independent experiments. *Significant difference from untreated control cells, p < 0.05. ##Significant difference from untreated control cells, p < 0.01
Fig. 6
Fig. 6
Differential expression of miRNAs. The distribution of differentially expressed miRNAs in exosomes released by AMSCs compared with control AMSCs. A Scatter plots of miRNA expression in AEXOs versus AMSCs. B Hierarchical clustering analysis of miRNA expression related to wound regeneration in AEXOs and AMSCs. C Gene ontology analysis of top 10 upregulated miRNAs AEXOs versus AMSCs
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