Immunomodulatory Activity of Human Bone Marrow and Adipose-Derived Mesenchymal Stem Cells Prolongs Allogenic Skin Graft Survival in Nonhuman Primates

Abstract

Objective: In the present study, we examined the tolerance-inducing effects of human adipose-derived mesenchymal stem cells (hAD-MSCs) and bone marrow-derived MSCs (hBM-MSCs) on a nonhuman primate model of skin transplantation.

Materials and methods: In this experimental study, allogenic and xenogeneic of immunomodulatory properties of human AD-MSCs and BM-MSCs were evaluated by mixed lymphocyte reaction (MLR) assays. Human MSCs were obtained from BM or AD tissues (from individuals of either sex with an age range of 35 to 65 years) and intravenously injected (2×106 MSCs/kg) after allogeneic skin grafting in a nonhuman primate model. The skin sections were evaluated by H and E staining for histopathological evaluations, particularly inflammation and rejection reaction of grafts after 96 hours of cell injection. At the mRNA and protein levels, cellular mediators of inflammation, such as CD4+IL-17+ (T helper 17; Th17) and CD4+INF-γ+ (T helper 1, Th1) cells, along with CD4+FoxP3+ cells (Treg), as the mediators of immunomodulation, were measured by RT-PCR and flow cytometry analyses.

Results: A significant Treg cells expansion was observed in MSCs-treated animals which reached the zenith at 24 hours and remained at a high concentration for 96 hours; however, Th1 and Th17 cells were significantly decreased. Our results showed that human MSCs significantly decrease Th1 and Th17 cell proliferation by decreasing interleukin-17 (IL-17) and interferon-γ (INF-γ) production and significantly increase Treg cell proliferation by increasing FoxP3 production. They also extend the allogenic skin graft survival in nonhuman primates. Histological evaluations showed no obvious presence of inflammatory cells or skin redness or even bulging after MSCs injection up to 96 hours, compared to the group without MSCs. There were no significant differences between hBM-MSCs and hAD-MSCs in terms of histopathological scores and inflammatory responses (P<0.05).

Conclusion: It seems that MSCs could be regarded as a valuable immunomodulatory tool to reduce the use of immunosuppressive agents.

Keywords: Adipose; Allogenic; Bone Marrow; Immunomodulation; Mesenchymal Stem Cells.

Fig.1

Allogenic immunomodulatory properties of in vitro expanded hAD-MSCs obtained from the aspirate of adipose tissue. A-C. Immunomodulatory properties of hAD-MSCs were assessed in the MLR medium following co-culture of hAD-MSCs and responder (R) and stimulator (S) human T cells (R+S+AD-MSCs) at different ratios (1:10, 1:5, 1:1, and 2:1) and different time periods (24, 48, 72, and 96 hours). The cell ratio of 1:1 and culture period of 72 hours were selected as the optimal conditions. The comparison of the immunomodulatory properties of D. hAD-MSCs (n=5 patients) and E. hBM-MSCs (n=8 patients), was made under the optimal conditions in the MLR medium. No significant difference was observed among R+S and R+S+BM-MSCs (patients 1, , and 5), and R+S+AD-MSCs (patient 1). F. Also, no significant difference was found between hAD-MSCs and hBM-MSCs. Data are presented as the mean ± standard deviation. *; P<0.05, **; P<0.01, ***; P<0.001, hAD-MSCs; Human adipose-derived mesenchymal stem cells, hBM-MSCs; Human bone marrow-derived mesenchymal stem cells. MLR; Mixed lymphocyte reaction, BM-MSCs; Bone marrow-derived mesenchymal stem cells, and AD-MSCs; Adipose-derived mesenchymal stem cells.

Fig.2

Xenogeneic immunomodulatory properties of hAD-MSCs and hBM-MSCs. A. Xenogeneic immunomodulatory properties of human AD-MSCs and B. BMMSCs were evaluated in the MLR medium following co-culture of hAD-MSCs and responder (R) and stimulator (S) monkey T cell (R+S+AD-MSCs) under the optimal conditions (i.e., at the ratio of 1:1 for 72 hours). The comparison of the immunomodulatory properties of hAD-MSCs (patients 2, , , and 5) and hBM-MSCs (patients 2, , , 7 and 8) was made under the optimal conditions in the MLR medium. C. Significant differences were found among R+S and R+S+BM-MSCs, and R+S+AD-MSCs in all groups. Also, no significant difference was observed in xenogeneic immunomodulatory properties between hBM-MSCs and hAD-MSCs. D. In vitro immunomodulatory effects of hAD-MSCs and hBM-MSCs on rhesus T cells subset were assessed. There were significant differences in the mRNA level of IL-17 (Th17), IFN-γ (Th1) and Treg (FoxP3) among hBM-MSCs and hAD-MSCs, and R+S. Data are presented as the mean ± standard deviation. *; P<0.05, **; P<0.01, ***; P<0.001, hAD-MSCs; Human adipose-derived mesenchymal stem cells, and hBM-MSCs; Human bone marrow-derived mesenchymal stem cells.

Fig.3

Immunomodulatory effects of hAD-MSCs and hBM-MSCs transplantation on T cell subsets in a healthy monkey recipient and immunomodulatory effects of hAD-MSCs transplantation on rhesus T cell subsets after skin grafting. A. A schematic overview of in vivo cell transplantation in a healthy monkey recipient. B, C. No significant differences in xenogeneic immunomodulatory properties were found between hBM-MSCs and hAD-MSCs in a healthy monkey recipient, 6-96 hours after cell transplantation. However, a significant difference was found in xenogeneic immunomodulatory properties when comparing hBM-MSCs and hAD-MSCs in terms of Foxp3+ T cells and CD4+CD25+ T cells (*P<0.05), 24-96 hours after cell transplantation. Also, a significant difference was observed in xenogeneic immunomodulatory properties between hBM-MSCs and hAD-MSCs in terms of Foxp3+ T cells (*P<0.05) and CD4+CD25+ T cells (***P<0.001), before 24 hours of cell transplantation. D. Schematic overview of in vivo hAD-MSCs transplantation with skin graft. E. Immunomodulatory effect of hAD-MSCs transplantation on monkey T cells subsets 48 hours after skin grafting in a monkey model. F. As a control, at the protein level, T cells of rhesus after skin graft and before hAD-MSCs transplantation were exposed to TGF-β (10 and 20 ng/ml) as an immunosuppressive factor, and the percentage of IL17 and IFN-γ were decreased. G-I. At the protein level, after hAD-MSCs IV transplantation, 6-48 hours after cell transplantation, the percentage of CD4+IL-17+ and CD4+INF-γ+ cells, as the cellular mediators of inflammation, was decreased, while the number of CD4+FoxP3+ cells, as the mediators of immunomodulation, was increased. Data are presented as the mean ± standard deviation. *; P<0.05, ***; P<0.001, hAD-MSCs; Human adipose-derived mesenchymal stem cells, and hBMMSCs; Human bone marrow-derived mesenchymal stem cells. h; Hour; IL; Interleukin ,and IFN; Interferon.

Fig.4

Immunomodulatory effect of human adipose-derived mesenchymal stem cells (hAD-MSCs) and human bone marrow-derived mesenchymal stem cells (hBM-MSCs) transplantation on rhesus T cell subsets after allogenic skin grafting. A. A schematic overview of in vivo cell transplantation with skin grafting. Immunomodulatory effects of hAD-MSCs and hBM-MSCs transplantation on monkey T cells subsets 24, 48, 72, and 96 hours after skin grafting in a monkey model. B. Inflammatory features, such as redness and bulging can be detected around skin graft area without cell transplantation. Visual inspection and histopathological analysis of transplanted tissues showed mild inflammation around allogenic skin graft after cell transplantation compared with monkeys receiving no cell transplantation and showed severe inflammation. C. At the mRNA level, after IV transplantation of hBM-MSCs and hADMSCs, the percentages of Th1 and Th17, as the cellular mediators of inflammation, were decreased, while the percentage of Treg, as mediators of immunomodulation, was increased. D. Also, at the protein level, after IV transplantation of hBM-MSCs and hAD-MSCs, the percentages of Th1 and Th17 were decreased, while the number of Tregs was increased.