Introthymic Or Intravenous Administration Of Rat MSCs Induce The Immunomodulation In Vivo

Objection:As an adherent,fibroblast-like,pluripotent adult stem cells,bone marrow mesenchymal stem cells(MSCs) display immunosuppressive properties in vitro.It was showed in baboon model that infusion of ex-vivo expanded allergenic MSCs can prolonged the rejection of MHC mismatched skin grafts,and the earlier clinical study also suggested that MSCs can be used to treat refractory acute GVHD.However,the exact mechanism underlying these effects is not completely understood,especially in vivo.Our study designed to explore the in vivo mechanism by detecting phenotype of various T ceils subsets from thymus and spleen after introthymic or intravenous administration of the well-characterized rat MSCs.We also performed allogeneic skin grafting to test the immunomodulation effect.This study may be useful for the future applications for allogeneic MSCs-based therapeutic strategies.Method:Bone marrow was flushed from the tibia and femur of adult Wistar rats into serum-containing L-DMEM.MSCs were isolated by density gradient centrifugation using percoll,and purified by multiple passages.Cultured mesenchymal stem cells were identified by phenotypic analysis,adipocyte and osteogenic differentiation.SD rats were were randomly divided into 3 groups: MSC introthymus inoculation,MSCs intravenous injection and N.S 0.1 ml introthymus inoculation as control.After 3 passages,the trypsinized 2×10⁶ MSCs cells suspended in 0.1ml N.S were injected by introthymic or intravenous.The thymuses and spleens were isolated from rats of different groups,and grinded into mononuclear cells on stainless steel mesh to analysis the phenotypes by flow cytometry at different time point.Meanwhile,to assess tolerance induction, transplanted animals also received allogeneic skin grafts from Wistar rats 7 days after MSCs transplantation. Result:Rat bone marrow-derived MSCs were culture-expanded successfully,and these cells display the following immune phenotype after the third passages: CD106⁺,CD90⁺,CD11b^- and CD34^-,CD45^-.In addition,osteogenic and adipogenic differentiation can be induced under defined culture conditions. Phenotypes analysis of the T cells from spleens and thymuses by FCM after transplantation showed:1,the percentage of CD3⁺CD4⁺cells in thymus and spleens after inoculation of MSCs in vivo have no proliferation with time going on(P＞0.01),while CD3⁺CD8⁺ cells in spleens may reach 62±5%transiently the third day after MSCs introthymic injection.2,Administration of MSCs by introthymic or intravenous can induce the increasing of CD4⁺CD25⁺, CD4⁺CD25^hi in vivo and CD8⁺CD28^- in thymus comparing to control(P＜0.01). 3,We found that CD4⁺CD8⁺ cells may increase with time after introthymic injection of MSCs(P＜0.01),while decrease after intravenous injection(P＜0.01). 4,Animals primed with host MSCs alone failed to induce long term tolerance,and allogeneic skin grafts from animals injected MSCs by thymus survived for 5.5±0.8 days with CD8⁺cell infiltration in the site of rejection,however,animal that received intravenous injection of MSCs can prolong allogeneic skin graft with reduction in CD3⁺ cell infiltration in contranst with control(10±0.7vs8.3±0.9, P＜.0001).Conclusions:Allogenic rat MSCs administrated in vivo may not induce CD3⁺CD4⁺ cells proliferation,while introthymic injection may elicit CD3⁺CD8⁺cells amplification and infiltraion in the site of rejection,elicit rejection of allogeneic skin grafts more quickly although we show here that administration of MSCs in vivo can also increasing of CD4⁺CD25⁺ and CD8⁺CD28^- Treg cell.Moreover,introthymic injection of MSCs also promote the production of CD4⁺CD8⁺,which prove MSCs may play a role in T lymphopoeisis of thymus.The result may provide new clues to explain immunomodulation mechanism of MSCs in vivo.