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. 2020 Jun 24:7:119.
doi: 10.3389/fmolb.2020.00119. eCollection 2020.

Differential Wound Healing Capacity of Mesenchymal Stem Cell-Derived Exosomes Originated From Bone Marrow, Adipose Tissue and Umbilical Cord Under Serum- and Xeno-Free Condition

Diem Huong Hoang  1   2 , Tu Dac Nguyen  1   3 , Hoang-Phuong Nguyen  1   2 , Xuan-Hung Nguyen  1   2 , Phuong Thi Xuan Do  1   4 , Van Duc Dang  1   4 , Phuong Thi Minh Dam  1   2 , Hue Thi Hong Bui  1   2 , Mai Quynh Trinh  1   2 , Duc Minh Vu  1   2 , Nhung Thi My Hoang  1   4 , Liem Nguyen Thanh  1   2 , Uyen Thi Trang Than  1   2
Affiliations

Differential Wound Healing Capacity of Mesenchymal Stem Cell-Derived Exosomes Originated From Bone Marrow, Adipose Tissue and Umbilical Cord Under Serum- and Xeno-Free Condition

Diem Huong Hoang et al. Front Mol Biosci. .

Abstract

Exosomes are nano-scale and closed membrane vesicles which are promising for therapeutic applications due to exosome-enclosed therapeutic molecules such as DNA, small RNAs, proteins and lipids. Recently, it has been demonstrated that mesenchymal stem cell (MSC)-derived exosomes have capacity to regulate many biological events associated with wound healing process, such as cell proliferation, cell migration and blood vessel formation. This study investigated the regenerative potentials for cutaneous tissue, in regard to growth factors associated with wound healing and skin cell proliferation and migration, by exosomes released from primary MSCs originated from bone marrow (BM), adipose tissue (AD), and umbilical cord (UC) under serum- and xeno-free condition. We found crucial wound healing-mediated growth factors, such as vascular endothelial growth factor A (VEGF-A), fibroblast growth factor 2 (FGF-2), hepatocyte growth factor (HGF), and platelet-derived growth factor BB (PDGF-BB) in exosomes derived from all three MSC sources. However, expression levels of these growth factors in exosomes were influenced by MSC origins, especially transforming growth factor beta (TGF-β) was only detected in UCMSC-derived exosomes. All exosomes released by three MSCs sources induced keratinocyte and fibroblast proliferation and migration; and, the induction of cell migration is a dependent manner with the higher dose of exosomes was used (20 μg), the faster migration rate was observed. Additionally, the influences of exosomes on cell proliferation and migration was associated with exosome origins and also target cells of exosomes that the greatest induction of primary dermal fibroblasts belongs to BMMSC-derived exosomes and keratinocytes belongs to UCMSC-derived exosomes. Data from this study indicated that BMMSCs and UCMSCs under clinical condition secreted exosomes are promising to develop into therapeutic products for wound healing treatment.

Keywords: ADMSC-derived exosomes; BMMSC-derived exosomes; UCMSC-derived exosomes; exosomes; growth factors; mesenchymal stem cells; wound healing.

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Figures

Figure 1
Figure 1
Human primary mesenchymal stem cells isolated from AD, BM, and UC at passage 3 of cell culture. (A) Typical morphology of MSCs was captured under Nikon Inverted Microscope Eclipse Ti-S. (B) Expression of MSC markers (n = 5) were analyzed using flow cytometry approach and Human MSC Analysis Kit (BD Biosciences). Positive markers include CD90, CD105, and CD73, and negative markers include CD45, CD34, CD11b, CD19, and HLA-DR. ADMSC, Adipose tissue-derived MSCs; BMMSC, Bone marrow-derived MSCs; UCMSC, Umbilical cord-derived MSCs. Error bars indicate ± SD.
Figure 2
Figure 2
Cellular senescence characteristics of human primary ADMSCs, BMMSCs, and UCMSCs at the passage three under serum- and xeno-free culture condition. Three cell types were analyzed for the cellular senescence using Senescence Cells Histochemical Staining Kit (Sigma-Aldrich). Results indicated that only UCMSCs expressed 0.18% cells with senescence signals and BMMSCs and UCMSCs did not expressed any cellular senescence signal.
Figure 3
Figure 3
Morphology and marker analysis of exosomes derived from human primary ADMSCs, BMMSCs, and UCMSCs. (A) Morphology analysis of exosomes observed under TEM showed all exosomes have a cup-shape morphology (Scale bar: 100 nm): (A1) A representative of ADMSC-derived exosomes, (A2) A representative of BMMSC-derived exosomes, (A3) A representative of UCMSC-derived exosomes. (B) Exosomal proteins, 10 μg total protein were loaded each lane, including CD9, CD63, AGO2 and Tubulin, enriched in exosomes released from ADMSCs, BMMSCs, and UCMSCs. ADMSC-EX, ADMSC-derived exosomes; UCMSC-EX, UCMSC-derived exosomes; BMMSC-EX, BMMSC-derived exosomes. Data are representative of at least three independent experiments. Scale bars indicate 100 nm.
Figure 4
Figure 4
Capacity of primary dermal fibroblast and keratinocyte proliferation under stimulation of exosomes released by BMMSCs, ADMSCs, and UCMSCs cultured under serum- and xeno-free condition. Percentage of cell proliferation was normalized to the control (100%). (A) Fibroblast proliferation under exosome stimulation: There were differences between exosomes released from three sources of BMMSCs, ADMSCs, and UCMSCs within the same dose, in which BMMSC-derived exosomes have the greatest enhancement in the primary dermal fibroblasts. There was no difference in cell proliferation stimulation associated with doses, especially different doses of BMMSC-derived exosomes and ADMSC-derived exosomes, excepted for UCMSC-derived exosomes that dose of 20 μg expressed lower stimulation on cell proliferation compared to dose of 1 μg. (B) Keratinocyte proliferation under exosome stimulation: A stronger induction of keratinocyte proliferation was observed in the cell treated with the higher dose of exosomes, especially BMMSC-derived exosomes. With the dose of 1 μg exosomal protein, ADMSC- and UCMSC-derived exosomes exhibited a stronger capacity to enhance keratinocyte proliferation in comparison with UCMSC-derived exosomes which exhibited the lowest capacity to stimulate the proliferation. BMMSC-EX, BMMSC-derived exosomes; ADMSC-EX, ADMSC-derived exosomes; UCMSC-EX, UCMSC-derived exosomes. Statistical significance was determined by ANOVA and post-hoc Tukey HSD tests, and is indicated by: * where p < 0.05; ** where p < 0.01; *** where p < 0.001; **** where p < 0.0001.
Figure 5
Figure 5
Primary dermal fibroblast and keratinocyte migration under stimulation of exosomes released from BMMSCs, ADMSCs, and UCMSCs. Exosomes released by three cell sources were added to scratched primary dermal fibroblast and keratinocyte cultures to final total exosomal protein concentration of 1 μg (A,D), 10 μg (B,E) or 20 μg (C,F) / 0.1 mL depleted culture media. The primary fibroblasts and keratinocytes were incubated at 5% CO2 and 37°C and allowed to migrate with images captured at different time points. Image analysis was performed using ImageJ and data are presented as mean percent area of wound coverage in μm2 ± SD, from at least 3 independent biological replicates. Data showed that BMMSC-derived exosomes exhibited a greater induction on fibroblast migration within the doses of 10 and 20 μg (B,C), whereas UCMSC-derived exosomes exhibited the greater induction on fibroblast migration within the dose of 20 μg exosomal protein (A). Regarding to keratinocyte migration, UCMSC-derived exosomes showed a strongest stimulation on cell migration (D,F), in addition to the higher doses of exosomal protein derived from all three MCSs exhibited the higher stimulation on keratinocyte migration. Statistical significance was determined by ANOVA and post-hoc Tukey HSD tests, and is indicated by: * where p < 0.05; ** where p < 0.01; *** where p < 0.001; **** where p < 0.0001. BMMSC-EX, BMMSC-derived exosomes; ADMSC-EX, ADMSC-derived exosomes; UCMSC-EX, UCMSC-derived exosomes.
Figure 6
Figure 6
Representative images of fibroblast and keratinocyte migration under stimulation of BMMSC-derived exosomes within the dose of 20 μg exosomal protein. The primary fibroblasts and keratinocytes were seeded with 2 × 105 and 3 × 105 cells/well, respectively, and inhibited cell proliferation using mitomycin C. A wound scratch was created for each well and cells were imaged at different time points. In comparison to keratinocytes, fibroblasts migrated faster to close the wound. Scale bars indicate 100 μm.
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