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. 2021 Mar 12;22(6):2889.
doi: 10.3390/ijms22062889.

Comparisons of Extracellular Vesicles from Human Epidural Fat-Derived Mesenchymal Stem Cells and Fibroblast Cells

Soo-Eun Sung  1   2 , Kyung-Ku Kang  1 , Joo-Hee Choi  1 , Si-Joon Lee  1 , KilSoo Kim  1   3 , Ju-Hyeon Lim  4   5 , Seung Yun Yang  2 , Seul-Ki Kim  6 , Min-Soo Seo  1 , Gun Woo Lee  5
Affiliations

Comparisons of Extracellular Vesicles from Human Epidural Fat-Derived Mesenchymal Stem Cells and Fibroblast Cells

Soo-Eun Sung et al. Int J Mol Sci. .

Abstract

Extracellular vesicles (EVs) are generated and secreted by cells into the circulatory system. Stem cell-derived EVs have a therapeutic effect similar to that of stem cells and are considered an alternative method for cell therapy. Accordingly, research on the characteristics of EVs is emerging. EVs were isolated from human epidural fat-derived mesenchymal stem cells (MSCs) and human fibroblast culture media by ultracentrifugation. The characterization of EVs involved the typical evaluation of cluster of differentiation (CD antigens) marker expression by fluorescence-activated cell sorting, size analysis with dynamic laser scattering, and morphology analysis with transmission electron microscopy. Lastly, the secreted levels of cytokines and chemokines in EVs were determined by a cytokine assay. The isolated EVs had a typical size of approximately 30-200 nm, and the surface proteins CD9 and CD81 were expressed on human epidural fat MSCs and human fibroblast cells. The secreted levels of cytokines and chemokines were compared between human epidural fat MSC-derived EVs and human fibroblast-derived EVs. Human epidural fat MSC-derived EVs showed anti-inflammatory effects and promoted macrophage polarization. In this study, we demonstrated for the first time that human epidural fat MSC-derived EVs exhibit inflammatory suppressive potency relative to human fibroblast-derived EVs, which may be useful for the treatment of inflammation-related diseases.

Keywords: chemokine; cytokine; epidural fat; extracellular vesicle; fibroblast; inflammation; mesenchymal stem cell.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Characteristics of human epidural fat mesenchymal stem cells (MSCs). (A) Representative bright field microscopy images of human dermal fibroblasts and human epidural fat MSCs (original magnification 100x, scale bar = 25 µm). (B) Flow cytometric analysis showing expressed (CD73, CD90, and CD105) and non-expressed (CD14, CD34, and CD45) human epidural fat MSC. The horizontal axis is the fluorescence intensity and the vertical axis is the cell count.
Figure 2
Figure 2
Identification of isolated human dermal fibroblast (DF)- and epidural fat mesenchymal stem cell (MSC)-derived extracellular vesicles. (A) Morphology of human dermal fibroblasts and human epidural fat MSCs shown by transmission electron microscopy (TEM). (B) Flow cytometric analysis showing the tetraspanin markers displayed on extracellular vesicles, such as CD63 and CD81. The horizontal axis is the fluorescence intensity, and the vertical axis is the cell count. (C) Size distribution and concentration of isolated extracellular vesicles by nanoparticle tracking analysis (NTA).
Figure 3
Figure 3
Cytokine and chemokine levels from human dermal fibroblasts and human epidural fat mesenchymal stem cell (MSC)-derived extracellular vesicles. GM-CSF, MIP-1 alpha, MMP-9, IL-5, GRO, and VEGF decreased in human epidural fat MSC-derived extracellular vesicles. IL-4, IL-10, and IL-13 increased in human epidural fat MSC-derived extracellular vesicles. (* p-value < 0.05, ** ≤ 0.001).
Figure 4
Figure 4
Anti-inflammatory effects of human dermal fibroblasts and human epidural fat MSC-derived extracellular vesicles on THP-1 macrophage cells. (A) Human monocyte THP-1 cells were treated with phorbol 12-myristate 13-acetate (PMA, 100 nM) and differentiated into macrophages. PMA-differentiated THP-1 cells were treated with lipopolysaccharides (LPS) and extracellular vesicles. (B) Quantification of TNF-α, IL-6, and IL-10 secretion levels by PMA-differentiated THP-1 cells either stimulated or untreated with LPS and/or extracellular vesicles (EVs) for 24 h. (* p-value < 0.05, *** < 0.0001, n.s. non-significant).
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