A Preliminary Study About Immune Regulation Of Placenta-derived Mesenchymal Stem Cells On Allogeneic B Lymphocytes

Background and Objective:Mesenchymal stem cells (MSCs) are important members of stem cell family. Theyhave the ability to self-renew and differentiate into multiple tissues. MSCs in recent yearshave been increasingly concerned because placenta-derived mesenchymal stem cells,(PMSCs) has a rich source, no ethical issues, and is more convenient to access, besides, theexpression of phenotype is similar to the bone marrow source of MSCs (BMSCs). MSCshave low immunogenicity and unique immunomodulatory effects. MSCs can be widelyused for clinical application in autoimmune diseases, tissue repair, organ transplantationand other areas. But their specific immunomodulatory effects and mechanism remains tobe clarified.In recent years, more research about immune regulation of MSCs on T cell could beobserved. Meanwile, the immunomodulatory effects of MSCs on B cells remain unclear.And different researchers have different perspectives. Furthermore, it is little reported inthe research area about immune regulation of PMSCs on B cell. In this study, we firstlyisolated and cultivated mesenchymal stem cells from human placenta tissue in vitro, thenmade a preliminary study about PMSCs on allogeneic B lymphocyte proliferation,activation and related cell surface expression of costimulatory molecules, to explore theregulation of PMSCs on B lymphocyte in order to provide the experimental basis forfurther clinical applications. Methods:Form uniform adherent cells were isolated and cultured from human placenta. Then weidentified the cells by detection of cell surface antigen expression, differentiation potentialand other characteristics to determine that the proceeds of cells of mesenchymal stem cells.CpG2006, the recombinant CD40L, human IL-2, IL-4and IgM+IgG (H+G)(short forCpG), stimulated B cells, and treated PMSCs cultured in vitro with mitomycin. ThenPMSCs were co-cultured with the activated B lymphocytes. The ratio of PMSCs to Blymphocytes is1:5. Culture supernatant groups and no stimulus groups were comparedwith MSCs. Cells specific grouped into unactivated B cells group(B), activated B cellsgroup(B+CpG), activated B cells and PMSCs co-cultured group(B+CpG+PMSCs),activated B cells and the culture supernate of PMSCs co-cultured group[B+CpG+PMSCs(sup)], unactivated B cells and PMSCs co-cultured group(B+PMSCs), unactivatedB cells and the culture supernate of PMSCs co-cultured group[B+PMSCs(sup)],the controlgroup(PMSCs).The proliferation and surface positive and negative costimulatory moleculesof CD80, CD86and PD-L1of B lymphocytes was detected by immunofluorescencelabeling and flow cytometry, after co-cultured for72hours.Results:1. MSCs isolated from placenta tissue had the similar morphology and cell surfacemarkers with bone marrow mesenchymal stem cells.2. PMSCs did not express HLA-DR and positive or negative costimulatory moleculesincludingCD80, CD86, CD483, PD-1and B7H4while highly expressed negativecostimulatory molecule PD-L1.3. In an ex vivo culture system, PMSCs could be induced to develop into adipocytes.In adipogenic induction medium, fat globules could be observed in the cytoplasm under thelight microscope, and Oil Red O staining was positive. PMSCs could also be induced todevelop into osteoblast. Von Kossa-positive staining showed calcium deposition.4. B lymphocyte in the B,+CpG group agglomerated and activated significantly24hours after adding stimulus include CpG2006, the people of the restructuring of CD40L, human IL-2, IL-4and IgM+IgG (H+G). Activated cell mass were lager in the B+CpG+PMSCs group, However, Activated cell mass was significantly smaller in the B+CpG+PMSCs (sup) group than that of the B+CpG+PMSCs group.There were not activatedmass in groups with no stimulus.5. The expression of nuclear proliferation antigen Ki67in group B+CpG+PMSCs(41.60±13.81)%was significantly higher than in group B+CpG (19.23%±2.79)%. Theexpression in group B+CpG+of MSCs (sup)(16.00%±1.57)%was lower comparedwith group B+CpG (19.23%±2.79)%. There were statistically significant differencesamong the three groups.6. The expression of positive co-stimulatory molecule CD86between the B+CpG+PMSCs (sup) group (4.21±1.10)%and the B+CpG group (3.89±0.43)%were notsignificantly different. The expression of CD86in Two sets were both lower than B+CpG+PMSCs group (10.87±1.86)%. There are statistically significant.7. The expression of Positive costimulatory molecule CD80was (17.33±9.60)%, itwas lower. CD80in B+CpG group (51.17±6.53)%, B+CpG+PMSCs group (49.37±19.45)%and group B+CpG+PMSCs (sup)(54.74±13.59)%, higher expressed thanthose not stimulated groups. However, there was no significant statistical difference amongthe three groups.8. The expression of the negative costimulatory molecule PD-L1was lower in the notstimulate group (3.24±0.25)%., There were higher expression of PD-L1in group B+CpG (37.80±4.67)%, group B+CpG+PMSCs (37.85±6.65)%and group B+CpG+PMSCs (sup)(32.65±5.65)%by adding CpG stimulation. However, there was nosignificant statistical difference among the three groups.Conclusions:1. Placental tissue derived mesenchymal stem cells have rich source. PMSCs andBMSCs have similar immune phenotype and multilineage differentiation capacity.2. PMSCs under certain circumstances, can promote the activation and proliferationof B lymphocytes. This facilitation effect might be related to the direct intercellular contact.3. The mechanisms about PMSCs promoting the activation and proliferation on Blymphocyte may be related to the surface co-stimulatory molecules CD86, but probablynot by the costimulatory molecule PD-L1and CD80-mediated pathway.