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Mesenchymal stem cell (MSC)-derived exosomes have diverse functions in regulating wound healing and inflammation; however, the molecular mechanism of human umbilical cord MSC (hUCMSC)-derived exosomes in regulating burn-induced inflammation is not well understood. We found that burn injury significantly increased the inflammatory reaction of rats or macrophages exposed to lipopolysaccharide (LPS), increased tumor necrosis factor α (TNF-α) and interleukin-1β (IL-1β) levels and decreased IL-10 levels. hUCMSC-exosome administration successfully reversed this reaction. Further studies showed that miR-181c in the exosomes played a pivotal role in regulating inflammation. Compared to control hUCMSC-exosomes, hUCMSC-exosomes overexpressing miR-181c more effectively suppressed the TLR4 signaling pathway and alleviated inflammation in burned rats. Administration of miR-181c-expressing hUCMSC-exosomes or TLR4 knockdown significantly reduced LPS-induced TLR4 expression by macrophages and the inflammatory reaction. In summary, miR-181c expression in hUCMSC-exosomes reduces burn-induced inflammation by downregulating the TLR4 signaling pathway., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

Objective: To investigate the effects of different concentrations of lipopolysaccharide (LPS) on proliferation and apoptosis of human umbilical cord mesenchymal stem cells (hUCMSCs) in vitro, and to explore their possible mechanism., Methods: hUCMSCs from umbilical cord tissue of full-term healthy fetus delivered by caesarean section were isolated and cultured in vitro using tissue attachment method. The 3rd passage hUCMSCs were used in the study. Cells were divided into groups A, B, C, D, and E, which were treated with DMEM/F12 medium containing 0, 0.1, 1.0, 10.0, and 100.0 µg/mL of LPS respectively. In groups B, C, D, and E, methyl-thiazole-tetrazolium assay was used to detect proliferative activity of hUCMSCs at post treatment hour (PTH) 12, 24, and 48 (denoted as absorption value), with 5 samples in each group at each time point; apoptosis of hUCMSCs at PBH 24 was identified with acridine orange-ethidium bromide (AO-EB) staining, with 4 samples in each group; apoptotic rate of hUCMSCs was determined by flow cytometer, with 5 samples in each group. Above-mentioned indexes were determined in group A at the same time points. Data were processed with analysis of variance and LSD- t test., Results: (1) There was no statistically significant difference in proliferative activity of hUCMSCs at PTH 12 among groups A, B, C, D, and E (with t values from -1.67 to 1.33, P values above 0.05). Compared with that of group A, proliferative activity of hUCMSCs was increased in groups B, C, and D at PTH 24 and 48 (with t values from -13.42 to 17.34, P < 0.05 or P < 0.01), especially so in group C. Proliferative activity of hUCMSCs was lower in group E at PTH 24 and 48 than in group A (with t values respectively 8.64 and 17.34, P values below 0.01). (2) Obvious apoptosis of hUCMSCs was observed in group E but not in the other 4 groups with AO-EB staining. (3) Apoptosis rates of hUCMSCs in groups A, B, C, D, and E were respectively (3.1 ± 0.6)%, (2.6 ± 0.7)%, (2.9 ± 0.8)%, (3.1 ± 0.4)%, (25.1 ± 2.7)% (F = 272.19, P < 0.01). Apoptotic rate of hUCMSCs in group B, C, or D was respectively close to that in group A (with t values respectively 1.22, 0.57, -0.14, P values above 0.05), but it was higher in group E than in group A (t = -17.63, P < 0.01)., Conclusions: hUCMSCs proliferation may be promoted by low concentration of LPS. hUCMSCs proliferation is inhibited or induced to apoptosis along with the increase in concentration of LPS, and it may be related to activation of different major molecular signaling pathways by different concentrations of LPS.

Objective: To explore the effects of lipopolysaccharide (LPS) pretreatment on endotoxin tolerance of human umbilical cord mesenchymal stem cells (hUCMSCs) and its possible mechanism., Methods: hUCMSCs (1×10(4) cells/well) were exposed to 0, 0.1, 1.0, 10.0, 20.0, 30.0, 40.0, 50.0 µg/ml LPS for 24 h respectively. And the cell viability of hUCMSCs was detected by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT). 1 µg/ml and 50.0 µg/ml LPS were used as pretreatment and apoptosis induction concentrations respectively. Pyrrolidine dithiocarbamate (PDTC) (20 µmol/L, pretreatment for 20 min) was used as a specific inhibitor of nuclear transcription factor NF-κB. hUCMSCs were randomly divided by Stata software into 7 groups: control (A), LPS induction (B), pretreatment + LPS induction (C), PDTC (D), PDTC+ pretreatment + LPS induction (E), pretreatment (F) and PDTC + pretreatment (G). The apoptosis of hUCMSCs was measured by Hoechst 33258 staining and flow cytometry (FCM). The expressions of NF-κB p65 and cellular FLICE-inhibitory protein (c-FLIP) were measured by Western blot., Results: The cell viability of 0, 0.1, 1.0, 10.0, 20.0, 30.0, 40.0, 50.0 µg/ml LPS groups were 100%, (117.0 ± 8.8)%, (134.7 ± 6.9)%, (105.3 ± 8.3)%, (99.2 ± 8.3)%, (84.2 ± 9.3)%, (66.4 ± 6.6)% and (59.2 ± 8.0)% respectively. In comparison with 0 µg/ml LPS group, the cell viability of 1.0 µg/ml LPS group increased significantly (P = 0.004) while decreased in 40 and 50 µg/ml LPS groups (P = 0.005, 0.002). Hoechst 33258 staining indicated that chromatin of hUCMSCs was distributed evenly in group A; the apoptotic cell in group B dramatically increased; and the apoptotic cell in group C significantly decreased in comparison with that in group B. Apoptotic rates of groups A, B, C, D and E were (2.8 ± 0.8)%, (29.7 ± 3.4)%, (17.8 ± 3.0)%, (2.9 ± 0.4)% and (23.2 ± 2.6)% respectively. Compared with group A, apoptosis rate significantly increased in group B (P < 0.001). The apoptotic rate in group C significantly decreased than that in group B (P < 0.001) while group E was higher than group C (P = 0.015). The levels of NF-κB p65 and c-FLIP in group F (0.851 ± 0.031, 0.534 ± 0.053) was higher than that in group A (0.220 ± 0.021, 0.049 ± 0.009) (both P < 0.001), G (0.418 ± 0.007, 0.299 ± 0.061) (P < 0.001, P = 0.007)., Conclusions: LPS pretreatment can resist LPS-induced hUCMSCs apoptosis and enhance the ability of endotoxin tolerance. And the mechanism may be related with activating the NF-κB signaling pathway and up-regulating the expression of c-FLIP.

Wound healing is a dynamic and complicated process, which generally takes three overlapping phases: inflammation, proliferation, and remodeling. If wounds complicated by severe trauma, diabetes, vascular dysfunction disease, or a massive burn injury failed to pass through the three normal phases of healing, they might end up as chronic and refractory wounds. Mesenchymal stem cells (MSCs) play different important roles in the regulation of all the phases of wound healing. MSCs can be recruited into wound and differentiated into wound repair cells, as well as promote wound healing by exerting functions like anti-inflammation, anti-apoptosis, and neovascularization. This review focuses on the role and mechanism of MSCs in each phase of the wound healing process.

Objective: To investigate the effect of the serum from severe burn patients on the biology characteristics of human umbilical cord mesenchymal stem cells (hUCMSCs) in vitro, so as to explore the feasibility of hUCMSCs transplantation for treating severe burn., Methods: The 3rd passage of hUCMSCs were randomly divided into 3 groups: 10% fetal bovine serum group (group A), 10% normal serum group (group B), and 10% burn serum group (group C). At 24 hours, 72 hours, and 6 days after culture, the cell morphology and density were observed by inverted microscope; the cell proliferation was assessed by MTT; after 6 days of culture, the cell cycle by propidium iodide staining and flow cytometry, the apoptosis by acridine orange/ ethidium bromide staining, and the cell senescence by beta-galactosidase staining; the levels of tumor necrosis factor alpha (TNF-alpha), interleukin 1 (IL-1), platelet-derived growth factor (PDGF), and insulin-like growth factor 1 (IGF-1) in serum were detected by a double-antibody sandwich ELISA kit., Results: hUCMSCs were long spindle/polygon in 3 groups. The cell fusion of group C was obviously faster than that in group A and group B. The cell proliferation curves showed that the velocity and number of cell proliferation in group C were significantly higher than those in group A and group B at 2-6 days after culture (P < 0.05). The rates of proliferation period (S) of hUCMSCs were 9.21% +/- 1.02%, 11.79% +/- 1.87%, and 20.54% +/- 2.03%, respectively in groups A, B, and C at 6 days, and group C was significantly higher than that of group A and group B (P < 0.05). The hUCMSCs showed normal morphology and structure in 3 groups, and no apoptosis cells was observed. The positive cells percentage of group C (2.6% +/- 0.1%) was significantly lower than that of group A (4.8% +/- 0.2%) and group B (3.8% +/- 0.4%) (P < 0.05). The levels of TNF-alpha, IL-1, PDGF, and IGF-1 in group C were significantly higher than those in group B (P < 0.05)., Conclusion: The higher levels of cytokines in serum from the severe burn patients can significantly stimulate hUCMSCs proliferation, prevent cells apoptosis, and reduce cells senescence. Therefore, it is feasible to use hUCMSCs transplantation for treating severe burn patients.

Objectives. To investigate whether hUC-MSCs attenuated severe burn-induced ALI and the effects were based on TSG-6 secreted from hUC-MSCs. Method. A rat model was established and evaluated as follows: cytokine expression was measured by ELISA, and both inflammatory cell infiltration and lung injury were assessed by immunohistochemistry assay. Results. In vitro, TSG-6 levels in serum from the burn group were significantly increased compared with those from the sham group. In vivo, TSG-6 levels of lung tissues and serum in the burn+hUC-MSC group were significantly increased compared with those in the burn group. Both in lung tissues and in serum, increased levels of proinflammatory cytokines (TNF-α, IL-1β, and IL-6) were remarkably decreased, but the anti-inflammatory cytokine IL-10 increased after hUC-MSC administration (p < 0.05). These significant positive effects after hUC-MSC transplantation did not occur in the burn+siTSG-6 group. Conclusion. The intratracheal implantation of hUC-MSCs has been an effective treatment for severe burn-induced ALI via promoting TSG-6 secretion and inhibiting inflammatory reaction in lung tissue. [ABSTRACT FROM AUTHOR]

Background: Severe burns are a common and highly lethal trauma. The key step for severe burn therapy is to promote the wound healing as early as possible, and reports indicate that mesenchymal stem cell (MSC) therapy contributes to facilitate wound healing. In this study, we investigated effect of human umbilical cord MSCs (hUC-MSCs) could on wound healing in a rat model of severe burn and its potential mechanism. Methods: Adult male Wistar rats were randomly divided into sham, burn, and burn transplanted hUC-MSCs. GFP labeled hUC-MSCs or PBS was intravenous injected into respective groups. The rate of wound closure was evaluated by Image Pro Plus. GFP-labeled hUC-MSCs were tracked by in vivo bioluminescence imaging (BLI), and human-specific DNA expression in wounds was detected by PCR. Inflammatory cells, neutrophils, macrophages, capillaries and collagen types I/III in wounds were evaluated by histochemical staining. Wound blood flow was evaluated by laser Doppler blood flow meter. The levels of proinflammatory and anti-inflammatory factors, VEGF, collagen types I/III in wounds were analyzed using an ELISA. Results: We found that wound healing was significantly accelerated in the hUC-MSC therapy group. The hUC-MSCs migrated into wound and remarkably decreased the quantity of infiltrated inflammatory cells and levels of IL-1, IL-6, TNF-α and increased levels of IL-10 and TSG-6 in wounds. Additionally, the neovascularization and levels of VEGF in wounds in the hUC-MSC therapy group were markedly higher than those in other control groups. The ratio of collagen types I and III in the hUC-MSC therapy group were markedly higher than that in the burn group at indicated time after transplantation. Conclusion: The study suggests that hUC-MSCs transplantation can effectively improve wound healing in severe burned rat model. Moreover, these data might provide the theoretical foundation for the further clinical application of hUC-MSC in burn areas. [ABSTRACT FROM AUTHOR]