| Mesenchymal stem cells(MSCs) originated from the mesoderm maintain great ability of self-renewal and multilineage differentiation potential as a kind of multipotent stem cells.They could be differentiated into multiple tissue types under different induced conditions,such as bone,cartilage,adipocyte,myocardial cells etc.MSCs were initially found in bone marrow,while the content of MSCs here was extremely low which was only about 0.01%-0.001%,and only through invasive procedures could bone marrow-derived MSCs be obtained.Therefore,many new sources of MSCs were searched,for example,the placenta,umbilical cord blood,Micronesia-induced bone,fat, muscle ect.Placental tissue is readily available and easily procured,and its use does not elicit ethical debate.In our research we aimed to isolate and culture the MSCs from placenta(PMSCs) in vitro,and focus on immune biological characteristics between PMSCs and MSCs from bone marrow(BMSCs.)Recent studies have shown that MSCs could support hematopoiesis in vitro, promote hematopoietic reconstitution in vivo,inhibit the allogeneic immune response and reduce graft versus host disease(GVHD) to extend the survival time of graft,so MSCs were described as an ideal seed cells for tissue engineering.But the exact mechanism has not been completely clarified.Some report showed that PMSCs could inhibit the activation,and proliferation of T cells,and there was also some reports indicated that PMSCs did not express HLA-DR,and immunogenicity was low,however the reports related about its Immunoregulatory mechanisms was few.In our resarch we isolated and culutre the PMSCs in vitro,and compared its biological characteristics with BMSCs,afterwhich the regulation of PMSCs with high expression of PD-L1 on the cell cycle,activation,and cytokines secretion of and other aspects of T cell were observed, all these layed a theoretical foundation for clinical and experimental research in future.Partâ… The biological characteristics of placenta-derived mesenchymal stem cells and bone marrow mesenchymal stem cells of humanObjective:To compare the different Isolation and culture method of placenta derived from mesenchymal stem cells(PMSCs) with the bone marrow mesenchymal stem cells (BMSCs),and their biological characteristic.Methods:PMSCs were isolated from human placenta by trypsin-digesting, BMSCs were isolated by gradient centrifugation.After 3 passages,PMCSs and BMSCs were observed under inverted phase contrast microscope,their proliferative ability were compared,the immunoflouresence staining and flowcytometer assay were used to detect the surface markers on PMSCs and BMSCs,thereafter,PMSCs and BMSCs were cultured under different induced condition to show their multiable differentiation potential respectively.T cells were stimulated by PHA and then co-cultured with PMSCs and BMSCs separatly to show the interactions between T cells with PMSCs and BMSCs.Mixed lymphocyte reaction,~3H-TdR insert and PD-L1 monoclone antibody block experiment were carried out to study the immunological effect of PMSC and BMSC.Results:(1)Both PMSCs and BMSCs showed plastic adherence and typical fibroblastic morphology by light microscopy,the same surface marker expressions as CD29,CD44,CD73,CD90,CD105,CD106and CD166 were positive,while CD34,CD45, CD80,CD86,and HLA-DR were negative.(2)The same differentiation potential as both of the two kinds of MSCs could be induced into adipocytes,and osteocyte in vitro, which indicate that PMSCs and BMSCs had the similar biological characteristics with each other.(3)Both of the two kinds of MSCs could obviously inhibited T cell activation and proliferation in vitro.Conclusion:PMSCs and BMSCs had similar biologic characteristic,phenotype and multi-differentiation potential,as well as the negative immune-regulation.Thus,it could be an ideal kind of seed cell for tissue engineering. Partâ…¡The expression of co-stimulatory molecule PD-L1 on placenta-derived mesenchymal stem cells and negative immune-regulation on T cell in vitroObjective:To study expression of negative co-stimulatory molecules on PMSCs and the negative co-stimulation on T-cells in in vitro.Method:FCM and immunofluoresecence staining were used to analysis the expression of co-stimulatory molecules on PMSCs and HBMSCs after 3 passages. ~3H-TDR insert was used to detected the effect of PMSCs on proliferation of PHA-stimulated T cells.PD-L1 mAb block were used to analysis the role of PD-L1 in the immune-regulation of PMSCs on T cell proliferation.The effect of PMSCs on the cell cycle of PHA-stimulated T cell and the expression of CD69,a molecule expressed on the early stage of activated T cells,were observed by FCM.The expression level of cell cytokine with or without PD-L1 mAb blockingwere tested by ELISA.Result:(1)Neither PMSCs nor HBMSCs expressed the positive co-stimulatory molecules as CD80,CD83,CD86,etc.PD-L1 was expressed highly on PMSCs,but lowly expressed on HBMSCs.(2) PMSCs could inhibit T cell proliferation in vitro,and arrested the PHA-stimulated T cells in the G0/G1 phase.Meanwhile,It could down-regulated the expression of CD69,and regulated the secretion of IL-2,IFN-r and IL-10.These results indicate that the negative immune regulation of PMSCs is partly through high expression of PD-L1,and PD-L1 is a main effective molecule on PMSC.Conclusion:PMSCs could inhibit T cell activation and proliferation,and PD-L1 expressed on PMSCs had an important role in the immune-regulation.
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