The Distribution Of Bone Marrow-derived Mesenchymal Stem Cells And The Impact On GVHD And GVT Effect After Hematopoietic Stem Cells Transplantation

Background and ObjectivesAllogenic hematopoietic stem cells transplantation(allo-HSCT) is the principle and effective protocol for hematopoietic malignancy,non-hematopoietic malignancy, aplastic anemia,autoimmune diseases and some genetic diseases. Graft-versus-host-disease(GVHD),infection induced by delayed immunological reconstitution,relapse of malignancy and the second malignancy are major complications and death causes after HSCT.Therefore,to reduce the severity and incidence of GVHD and to improve Graft-versus-tumor(GVT) effect and to accelerate immunological reconstitution are important approaches to inerease the long time survival rate and improve living quality for recipients after HSCT.Mesenchymal stem cells(MSCs) are multipotential nonhematopoietic progenitor cells capable of differentiating into multiple lineages of the mesenchyme.Although MSCs originally were isolated from BM,similar populations have been isolated from other tissues,including adipose tissue,placenta,amniotic fluid,and fetal tissues such as fetal lung and blood.The capacity to differentiate into multiple mesenchymal lineages,including bone,fat,and cartilage,is being used as a functional criterion to define MSCs.At present no specific marker or combination of markers has been identified that specifically defines MSCs.Phenotypically,ex vivo expanded MSCs express a number of nonspecific markers,including CD29,CD73(SH3 or SH4), CD90(Thy-1),CD105(SH2 or endoglin),CD166(VCAM-1) and CD44.Meanwhile, MSCs are considered to be hypoimmunogenic,displaying low expression levels of human leukocyte antigen(HLA) major histocompatibility complex(MHC) classⅠand,importantly,no expression of costimulatory molecules such as CD40,CD40L, B7-1,B7-2.Therefore,MSCs are not able to trigger T-cell activation.Moreover, MSCs are capable,as third-party cells,of inhibiting allostimulated T-cell proliferation and expanding CD4~+CD25~+ T cells.These properties have been reported to be mediated by secretion of soluble mediators,such as prostaglandin E2(PGE2), transforming growth factor-β1(TGF-β1),interleukin-10(IL-10),and hepatocyte growth factor(HGF),as well as through indoleamine 2,3-dioxygenase(IDO).MSCs also inhibit the differentiation of monocytes to dendritic cells(DC) and modulate B-cell functions.Finally,MSCs have been shown to suppress natural killer(NK) cytotoxicity.In this respect,MSCs offer new perspectives to prevent rejection in human transplantation settings.To date immunosuppressive treatments using MSCs infusions to prevent or treat GVHD after HSCT is under investigation.Also,MSCs may be used in both HSCT and solid transplantation as a novel therapeutic approach to modulate immune rejection and improve GVT effect.Also,the ability of MSCs to migrate to areas of injury and to tumors has encouraged investigation of MSCs as therapeutic tools.For example,systemically administered MSCs have been shown to improve recovery in animal models of stroke and myocardial infarction.MSCs have also been used for targeted delivery of therapeutic gene products to the tumor microenvironment in animal models,and the therapeutic use of MSCs is being explored for various disease conditions.Although the migratory behavior of MSCs has now been extensively documented,distinct signals that guide migration of MSCs to specific in vivo targets are unknown.In this study,we have established models of syngenic BALB/c mice and nude mice received MSCs intravenously,quantification of SRY(sex-determining region of Y-chromosome) gene expression in different organs was performed to assess whether the mechanisms of homing and distribution of MSCs are different in recipients normal and with severe immunodeficiency or irradiation.Meanwhile,we have established another models of allogenic/third party MSCs castrated-B16 bearing murine HSCT. Assays of ratio of CD4~+/CD8~+ and proportions of CD4~+CD25~+ regulatory T cells and levels of IFN-γ,IL-2,IL-4 and TGF-βin peripheral blood were also detected to investigate the immunemodulatory effects of MSCs.Materials and Methods1.To establish model 1 of syngenic BALB/c mice and nude mice received MSCs intravenously.2.Experiment groups:The female recipients were killed at 24h,72h,1w,2w and 1w,2w,4w,6w respectively after MSCs infusion,then spleen,lung,intestine,liver, thymic,blood and BM were collected.3.Real-time PCR with SYBR GreenⅠtechnique was used for detecting SRY gene expression levels in different organs.4.To establish model 2 of allogenic/the third party MSCs castrated-B16 bearing murine HSCT.5.Experiment groups:Mice were divided into 7 groups:Group A of mice treated with low dose of third party MSCs;Group B of mice treated with median dose of third party MSCs;Croup C of mice treated with high dose of third party MSCs; Group D of mice treated with high dose of allogenic MSCs;Group E of mice treated with allogenic HSCT;Group F of B16-bearing mice treated irradiation and Group G of B16-bearing mice were control groups.6.Criteria of hematopoiesis reconstitution:Peripheral white blood cell count is over 1.0×10~9/L,and the detection of donor-host chimera reveals that the transplantation is successful.7.The detection of donor-host chimera:Chromogene Y was detected in peripheral blood by PCR.8.Manifestation and scores of aGVHD:To investigate the weight,appetite, consciousness,locomotor activity and the condition of skin and mucosa,and diarrhea.At the same time,aGVHD scores based on the situations of body weight loss, stance,locomotor activity,hairs and skin were evaluated.The severity of aGVHD was also evaluated by the manifestations and aGVHD scores combining with the characteristics of pathology of skin,liver and intestine.9.Assays of ratio of CD4~+/CD8~+ and proportions of CD4~+CD25~+ regulatory T cells were determined with double fluorescent-labeled antibodies and flow cytometry (FACS) each week.10.The examinations of levels of IFN-γ,IL-2,IL-4 and TGF-βin peripheral blood:On +14d the average levels of different cytokines were evaluated by enzyme linked immunosorbant assay(ELISA).11.Effects of MSCs on GVT:Criteria of death caused by tumor:peripheral white blood cell count was over 1.0×10~9/L while the recipients were dead,the size of tumor significantly increased even ulcer or necrosis without manifestation of aGVHD. Marked the size and appear time of tumor 2 times a week until the mice were dead.12.Statistical analysis:Values are mean±SD.Univariate analysis was used to value the expression of SRY gene in different organs,whereas for multiple group comparisons,one way ANOVA(LSD/Dunnett's T3) was used.For ratio of CD4~+/CD8~+ and proportions of CD4~+CD25~+ regulatory T cells analysis,repeated measures was used.The linear regression test was used for regression analysis.A P value of＜0.05 was considerd statistically significant.Moreover,Kaplan-Meier curve was used to express the survival of different groups of mice after HSCT.Results1.Recipients were all alive after MSCs infusion,there was no acute toxicity and there were no signs of ectopic tissue after transplant.2.There was the diversity of SRY mRNA expression levels in different groups and times.In the syngenic BALB/c mice group,the expression of SRY mRNA was significantly different after 24h MSCs infusion.Specific localization accounts in peripheral blood and BM were the highest at 24h,then decreased in peripheral blood until it could not be detected after 1w.Lung,intestine,liver and BM significantly increased as time going,except for the decreased expression of lung after 1w,another organs decreased slowly until 2w.Meanwhile,the levels of SRY mRNA could not be detected in spleen during the experimental periods.In the nude mice group,all organs could be detected the expression of SRY gene except for peripheral blood,and the levels were significantly higher than that in the syngenic BALB/c mice group.The decrease in expression of lung and intestine was delayed until 4w but was still able to be detected after 6w.The account of spleen,liver,thymic and BM kept increasing during the experimental periods.3.The establishment of models of allogenic/the third party MSCs castrated-B16 bearing murine HSCT:Firstly,all mice as recipients received B16 cells(1.0×10~6), then were lethally irradiated as condition regimen(8.0Gy,0.SGy/min) and subsequently transplanted with bone marrow cells(5.0×10~6) and splenic cells (1.0×10~7) from donors.All groups attained hematopoiesis reconstitution and GVHD in each group was induced with the incidence of 100%.4.Hematopoiesis reconstitution:On +3d,the WBC counts from group A～E were in the lowest levels.The day on which the WBC counts were over 1.0×10~9/L were (9.50±0.58)d,(8.25±0.50)d,(7.05±0.50)d,(7.00±0.82)d,(10.75±0.96)d,respectively. The days of hematopoiesis reconstitution which seemed to be dose-dependent in groups A～D were much earlier than that in group E,furthermore,there was no significant difference between group C and group D(P＞0.05).5.The detection of donor-host chimera:Chromogene Y was detected in peripheral blood of recipients by PCR on +14d,which revealed that the transplantation was successful.6.Manifestations of aGVHD:Mice in group E presented with somnolence, diarrhea,body weight loss,cicinnus and depilation from +6d～+8d while it was significantly postponed in group C and group D.The manifestations of aGVHD in group A appeared from +9d～+10d which hardly differed from group E.Group B had aGVHD from +9d～+11d but the manifestations were gradually improved.It indicated that MSCs may be effectively used to modulate immune rejection and that function is dose-dependent.All mice in group A～E had aGVHD with the incidence of 100%. aGVHD scores were:1.50±0.55,1.33±0.52,0.67±0.52,0.50±0.55 and 1.83±0.41.The scores in Group E was significantly higher than that in group A～D. 7.Pathology for aGVHD:In group A and group E,the characteristics of pathology showed dermic edema with macrophage and neutrophil infiltrate in skin, and hepatocyte edema with destructure and necrosis in liver,and the epithelium necrosis with schistic exuviations were seen in intestine.The severity of pathology for aGVHD was lower in group B～D.8.The survival of mice:The groups A～G of mice were:(21.88±6.88)d, (24.38±5.83)d,(31.63±9.35)d,(32.25±8.62)d,(14.50±1.78)d,(15.13±1.81)d and (17.50±1.85)d,respectively.The survival days among group A～D were significantly longer than group E(P＜0.01) which seemed to be dose-dependent.All recipients mice died of aGVHD at final.9.The analysis of ratio of CD4~+/CD8~+ and proportions of CD4~+CD25~+ regulatory T cells:The ratios of CD4~+/CD8~+ in groups A～D all increased and at +21d reached the highest,the average levels were:1.25±0.18,2.16±0.19,3.23±0.53 and 3.52±1.29, then decreased as time going but the ratios in group A～D were still higher than that in group E all the time.The proportions of CD4~+CD25~+ regulatory T cells also reached the highest at +21d,the average levels were:1.62±0.33,3.18±0.69,3.72±0.57 and 3.67±1.11,respectively.Meanwhile,the proportions of CD4~+CD25~+ regulatory T cells among group A～D were significantly higher than group E.10.The examinations of levels of IFN-γ,IL-2,IL-4 and TGF-βin peripheral blood:On +14d,the average levels of IFN-γin 5 groups were(13.62±0.59)ng/ml, (10.91±0.55)ng/ml,(6.91±0.28)ng/ml,(6.78±0.44)ng/ml and(19.85±1.05)ng/ml, respectively.The average levels of IL-2 were(14.52±1.18)pg/ml,(11.09±0.83)pg/ml, (8.35±0.61)pg/ml,(8.46±0.83)pg/ml and(20.40±1.92)pg/ml,respectively.The average levels of IL-4 were(7.41±0.54)pg/ml,(7.41±0.58)pg/ml,(7.32±0.43)pg/ml, (7.47±0.56)pg/ml and(4.83±0.62)pg/ml,respectively.The average levels of TGF-βwere(8.36±0.68)pg/ml,(10.90±1.42)pg/ml,(16.69±0.93)pg/ml,(16.46±0.82)pg/ml and(5.69±0.71)pg/ml,respectively.There were significant differences in the multiple comparisons of mean levels of cytokines among group A～E except for IL-4.The analysis of linear regression showed the average levels of IL-2 significantly associated with aGVHD scores:the average levels of IL-2 paralled with aGVHD scores(P=0.000).11.Effects of MSCs on GVT:we successfully established the B16-bearing mice models.The appear time of tumor in groups A～G were(8.25±0.50)d,(8.75±0.50)d, (8.75±0.96)d,(8.75±0.50)d,(6.75±0.50)d,(5.75±0.50)d and(5.25±0.96)d, respectively.The sizes of tumor were(2.187±0.196)cm~3,(2.193±0.148)cm~3, (2.142±0.180)cm~3,(2.178±0.169)cm~3,(2.153±0.077)cm~3,(10.947±0.328)cm~3 and (8.434±0.631)cm~3,respectively.The appear time of tumor in group A～D were significantly put off compared with control group F and group G.Meanwhile,the sizes of tumor were also much more smaller than that in control groups but there was no significant difference in the multiple comparisons of mean among group A～E. These results suggested effects of MSCs on GVT which did not relate to the dose of MSCs.DiscussionAllogenic hematopoietic stem cells transplantation(allo-HSCT) is the principle and effective protocol for hematopoietic malignancy,non-hematopoietic malignancy, aplastic anemia,autoimmune diseases and some genetic diseases. Graft-versus-host-disease(GVHD),infection induced by delayed immunological reconstitution,relapse of malignancy and the second malignancy are major complications and death causes after HSCT.Therefore,to reduce the severity and incidence of GVHD and to improve Graft-versus-tumor(GVT) effort and to accelerate immunological reconstitution are important approaches to increase the long time survival rate and improve living quality for recipients after HSCT.MSCs are multipotential nonhematopoietic progenitor cells capable of differentiating into multiple lineages of the mesenchyme.MSCs are considered to be hypoimmunogenic,displaying low expression levels of human leukocyte antigen (HLA) major histocompatibility complex(MHC) classⅠand,importantly,no expression of costimulatory molecules such as CD40,CD40L,B7-1,B7-2.Therefore, MSCs are not able to trigger T-cell activation.Moreover,MSCs are capable,as third-party cells,of inhibiting allostimulated T-cell proliferation and expanding CD4~+CD25~+ T cells.The ability of MSCs to prevent or reverse GVHD may be via secretion of soluble factors or direct cell-cell contact on alloreactive T cells or by suppressing DC or NK function.To date immunosuppressive treatments using MSCs infusions to prevent or treat GVHD after HSCT is under investigation.Also,MSCs may be used in both HSCT and solid transplantation as a novel therapeutic approach to modulate immune rejection and improve GVT effort.Also,the ability of MSCs to migrate to areas of injury and to tumors has encouraged investigation of MSCs as therapeutic tools.For these purposes concerned,MSCs have also been used for targeted delivery of therapeutic gene products to the tumor microenvironment in animal models,and the therapeutic use of MSCs is being explored for various disease conditions.In this study,we have established models of syngenic BALB/c mice and nude mice received MSCs intravenously.Comparison of SRY gene expression levels in different organs showed that the mechanisms of homing and distribution of MSCs were different in recipients normal and with severe immunodeficiency.Moreover,we have established another models of allogenic/the third party MSCs castrated-B16 bearing murine HSCT.Comparison of ratios of CD4~+/CD8~+ and proportions of CD4~+CD25~+ regulatory T cells and levels of IFN-γ,IL-2,IL-4 and TGF-βin peripheral blood among experiment groups indicated that third-party MSCs were as effective as allogenic MSCs.This finding had practical implications and suggests that third-party cells can be prepared and stored frozen to be used for GVHD therapy.It was concluded that MSCs may prevent the lethal aGVHD after allo-HSCT and raise the survival rate by acting on the ratios of CD4~+/CD8~+ and Th1/Th2 and proportions of CD4~+CD25~+ regulatory T cells in vivo which seemed to be dose-dependent. Furthermore,we drew a conclusion by comparison of appear time and sizes of tumors among experiment groups that MSCs had significant effects on GVT which seemed to be nothing to do with the dose of MSCs.However,the mechanisms underlying the immunosuppressive effects of MSCs need further investigation.Conclusion 1.No side-effects or acute toxicities were seen after MSCs infusions and there were no signs of ectopic tissue after transplant.2.The mechanisms of homing and distribution of MSCs were different in recipients normal and with severe immunodeficiency.3.The third party MSCs could as effectively as allogenic MSCs enhance hematopoietic recovery and T cell reconstitution after allo-HSCT,which seemed to be dose-dependent.4.The third party MSCs could as significantly as allogenic MSCs improve the survival rate and decrease in the incidence or severity of aGVHD which also seemed to relate to the dose of MSCs.5.MSCs had significant effects on GVT which seemed to be nothing to do with the dose of MSCs.