MSCs Derived from Human Amnion Possess a Higher Proliferation Activity and Clearer Stem Cell Properties Than from Bone Marrows and They Exhibit Similar Immunosuppressive Ability In Vitro

Abstract

Objectives: Human bone marrow mesenchymal stem cells (hBMSCs) have been used for the prevention and treatment of acute graft-versus-host disease (aGVHD) after allogeneic hematopoietic stem cells transplantation (allo-HSCT). In this study, we compared the biological characteristics and immunosuppressive activity of human amniotic mesenchymal stem cells (hAMSCs) and hBMSCs to provide experimental evidence for the potential use of hAMSCs for treating aGVHD, thus solving the problem of insufficient hBMSCs sources. Methods: hAMSCs and hBMSCs were isolated by using enzymatic digestion and Ficoll-Hypaque density gradient centrifugation, respectively, were culture-expanded in vitro to obtain the 4th-generation cells. The biological characteristics of both stem cell types were compared by morphological analysis, analysis of cell growth, cell cycle profiling, immunophenotyping, and immunofluorescence assays. An in vitro co-culture of MSCs and peripheral blood mononuclear cells (PBMCs) stimulated with phytohemagglutinin (PHA-PBMSC) was performed. The proliferation and changes of T lymphocyte subsets were measured using the Cell Counting Kit-8(CCK-8) assay and flow cytomety, respectively. The production of Interferon-γ (IFN-γ), interleukin-2 (IL-2) and IL-10 in the co-culture supernatant was determined using enzyme-linked immunosorbent assay (ELISA). Results: Both hAMSCs and hBMSCs had fibroblast-like morphology. hAMSCs could be maintained for at least 15 culture passages, but hBMSCs started to show signs of aging and remarkably reduced proliferation at 6-7 passages. There was no significant difference in the proportion of cells in G2/M phase between hAMSCs and hBMSCs (P > 0.05). Immunophenotyping revealed positive expression of CD105, CD90, and CD73 and negative expression of CD34, CD45, CD11b, CD19, and HLA-DR on the surface of both hAMSCs and hBMSCs. hAMSCs were positive for Oct-3/4, but hBMSCs were not. Both hAMSCs and hBMSCs expressed vimentin. PHA-stimulated PBMCs proliferation was inhibited by hAMSCs and hBMSCs. There were not significant differences between the inhibitory effects of the two MSCs types on PBMCs proliferation (P > 0.05). Co-culture with either hAMSCs or hBMSCs significantly increased the proportions of Treg, Th2 and Tc2 and decreased Th1 and Tc1 cell subsets in the PBMCs as compared with the PBMCs cultured alone (P < 0.05), and the changes in the PBMCs were similar between the two co-culture systems (P>0.05). In both of the two co-culture systems, IFN-γ, IL-2 production by the lymphocytes was significantly lowered (P < 0.05) and IL-10 production was significantly increased (P < 0.05) as compared with their levels in the PBMCs cultured alone; no significant difference was found in IFN-γ, IL-2 or IL-10 levels between the two co-culture systems (P > 0.05).Conclusion: The results of this study demonstrated that MSCs derived from human amnion and bone marrows have similar immunomodulatory effects on the T lymphocytes, however, hAMSCs have higher proliferation activity and clearer stem cell properties than hBMSCs, suggesting the possibility of using hAMSCs in the treatment of aGVHD after allo-HSCT. DisclosuresNo relevant conflicts of interest to declare.