The Effect Of Rat MSC On GVHD After Allo-gene Bone Marrow Transplantation

Objective: Allogeneic bone marrow transplantation (Allo-BMT) has revolutionized the treatment of hematopoietic malignancies, inherited hematopoietic disorders, and aplastic anemia. Unfortunately, Graft-versus-host disease (GVHD) is still a major complication of Allo-BMT and results in significant morbidity and mortality. The immunosuppressive drugs is effective at controlling GVHD, but these regiments can lead to serious complications from infection and an increased risk of leukemia relapse after Allo-BMT. Many researchers have therefore sought to develop techniques to prevent GVHD that avoid the complications incurred by immunosuppression.Bone marrow mesenchymal stem cells (MSC), as a kind of non-hematopoietic tissue stem cells, are contained in bone marrow and have the capacity of self-renewal, high proliferation and multilineage differentiation, capable of differentiating into osteoblasts, adipocytes and myoblasts, supporting the survivor of hematopoietic stem cells in vitro and promoting reconstitution of hematopoietic function in vivo. Recent researches have shown that MSC are poor antigen-presenting and do not express MHC classⅡand CD80, CD86 co-stimulatory molecules. These cells also exhibit immunoregulatory properties in vitro as demonstrated by their ability to suppress the mixed lymphocyte reaction (MLR). So, theoretically speaking, Transplanting of MSC can prevent acute immune rejection and reduce the incidence and severity of GVHD. This study aims to investigate the effect of rat MSC on GVHD after Allo-BMT by coinfusion of bone marrow cells and culture-expanded MSC .Methods: 1 First, rat bone marrow MSC were isolated and expanded in vitro by the method of density gradient centrifugation in Percoll with adherence and biological characterization of their morphology, cell cycle, proliferation, phenotype (the expression of specific marks-CD34, CD45, CD44, CD105 and molecules that were associated with immunogenicity-MHCⅠ, MHCⅡ, CD80, CD86 ) were analyzed and multiple differentiation potentials were confirmed by lineage-specific induced differentiation to osteoblasts and adipocytes.2 To test the suppressive effect of MSC on T cell proliferation in vitro, MSC were added to the mixed lymphocyte reaction cultures (MLR) at ratios MSC/responder lymphocytes of 2/5, 1/5, 1/10. MSC were autologous to responder lymphocytes.3 The effect of MSC from donor rats on acute GVHD after allo-BMT was investigated in vivo. After Wistar recipient rats were pretreated with lethal irradiation, they were transplanted with bone marrow and T cells from SD donor rats to induce GVHD. Treated rats received either MSC from donor rats. The ratios of MSC/T cells were 1/5, 2/5, 1/1. Clinical GVHD scores, life-span, Pathological change of liver and small intestine were monitored.Results: 1 The progeneration MSC isolated by the method of density gradient centrifugation in Percoll with adherence proliferated in visible symmetric colonies and long-spindle shape. Their generation time was about 2 weeks.After passaging the cells grew faster. About 2 days later, MSC experienced the log phase growth for 3~4 days and then entered the platform time and passaged in 6 days. Homogeneities of cells rose with passage. MSC of passage 15 became ageing.After passage 5, cells cycle status analysis by measuring DNA content revealed that most of MSC were in silence stage (S+G2+M).Phenotype analysis of MSC showed that after passage 5, the MSC specific markers-CD29, CD44 and CD105 were highly expressed, the HSC specific marks-CD34, CD45 were not expressed; the molecules on MSC that were associated with their immunogenicity-MHC classⅠantigen was low expressed, While MHC classⅡand CD80, CD86 co-stimulatory molecules could hardly be identified.The isolated MSC were able to differentiate into adipocytes and osteoblasts in specific induction culture system.2 In vitro, when MSC were added to the mixed lymphocyte cultures, the proliferation of T cells were inhibited markedly in a dose-dependent manner (r=0.942, P<0.01). At ratio MSC/responder lymphocytes of 2/5, the stronger inhibitory effect was seen and inhibitory rate was (83.6±2.3) %. At 1/5 and 1/10 ratios, the inhibitory rate were (60.6±3.3) % and (23.2±2.2) % respectively.3 When MSC and T cells from donor rats were translpanted into recipient rats at 1/5 ratio with bone marrow cells, clinical GVHD scores of recipient rats were about 10 with severe pathologic lesions of liver and small intestine and statistically nonsignificant (P>0.05) compareing with those of control group (GVHD scores were about 11), but life-span of recipient rats were prolonged markedly (P<0.05); when the ratio of MSC/T was 2/5, clinical GVHD scores of recipient rats were about 8 with mild pathologic lesions of liver and small intestine (P<0.05), life-span of recipient rats were prolonged too (P<0.01). At 1/1 ratio, only 20% of recipient rats developed clinical signs of mile GVHD, and after 1~2 weeks the signs were disappeared gradually, the rests survived in the absence of any sign of GVHD. Clinical GVHD scores of recipient rats in this group were only 1.4 without pathologic lesions of liver and small intestine (P<0.01), and all of them survived for a long-time (P<0.01).Conclusion: 1 It is a simple, effective and practical method to separate and obtain higher purity and activity of MSC from rat bone marrow by gradient centrifugation in Percoll with adherence. Their basic biological characteristics were identified with stem cells. Both absence of MHCⅡ, CD80, CD86 co-stimulatory molecules and low-expression of MHCⅠhave shown that MSC are poor antigen-presenting. 2 In vitro, MSC suppressed the T cell proliferation induced by allgeneic mixed lymphocyte reaction in a dose-dependent manner.3 Transplanting donor derived high-dosed MSC with bone marrow could reduce the incidence and severity of GVHD and prolong the survival time of the receptor.