Mechanism Of Murine Mesenchymal Stem Cells To Inhibit Acute Graft-versus-Host Disease

Mesenchymal stem cells(MSCs)are firstly isolated from bone marrow and characterized by intrinsic self-renewal capacity and multilineage differentiation potentials.Recent investigations reported that MSCs can be successfully isolated from almost all the tissue and organs of body,such as muscal,fat,compact bone,pacrease, lung,umbilical cord,placeata.In vitro and in vivo experiments demonstrate that MSCs and their differentiated cells have hematopoitiC supporting capacity through providing a system of structural and functional supports for hematopoitic cells.These functionalities of MSCs make them a hotspot in organ engineering and bone marrow transplantation for cell therapy.Hematopoietic stem cell transplantation is the effective but only approach to treat some refractory hematopoietic diseases,such as leukeamia,aplastic anemia,immune impfecta,most severe radiation desease.Acute graft-versus-host disease(aGvHD),which is still a familiar syndrome and major cause of mortality post allogeneic hematopoietic stem cell transplantation(allo-HSCT).The potent immune regulation capacities of MSCs enhance their therapeutic appeal of moving from bench to clinic in the management of aGvHD which has got curative effect and gone into phaseâ…¢clinical trails in America.The bottleneck of the difficulty in the culture of murine MSCs confined the utilization of MSCs in murine model to investigate the immune mechanism of MSCs exerting in vivo.The main object of this study here is to investigate the biological character of MSCs derived from murine compact bone and the mechanism through which MSCs inhibite aGvHD.In this study,a new method was developed to culture murine MSCs from collagenase-digested compact bone fragments.Cells were authenticated in common morphology,immune phenotype and capabilities of differentiation, hemotopoitic supporting and immunoregulation.Murine MSCs were intravenously co-infused into mice of aGvHD model.Mean survival time and pathologic changes were observed to assure the effect of MSCs and futher experiments were performed to investigate the mechanism through which MSCs inhibit aGvHD.In the present study,murine MSCs from compact bone fragments were cultured and expanded.The cells population exhibited homogenous surface antigen profile, which are non-hemotopoitic(CD45~-),non-macrophagic(CD11b~-),non-endothelial (CD31~-),only express some adhesive molecules(CD29~+,CD44~+,CD105~+)and stem cell antigen(Sca-1~+),the profile maintained comparatively stable during passaging. These cells had comparatively stable clone forming activity.The cells presented in vitro multipotential differentiation capacities along osteocyte,chondrocyte and adipocyte lineages.Futher functional activity detecting testified that these cells had similar in vitro hematopoiesis supporting ability as bone marrow stormal cells.They could inhibit T lymphocyte proliferation elicited by ConA or allogeneic cells. Prolonged mean survival time of transplanted allogeneic skin demonstrated that MSCs have immunomodulatory function in vivo.It is by now generally accepted that aGvHD can be summarized in a three-step process.Phaseâ… is defined as effect of conditioning,of which irradiation and chemotherapy injury to host epithelium and endothelial generates APC maturation; During phaseâ…¡,activated APC and inflammatory cytokines render donor T cell activation and expansion;Finally,during phaseâ…¢of aGvHD,activated cells migrate to the target organ and cause further injury.Injuried body tissue can further activate APC,which make the disease go into a malignant circal and very difficult to be controlled.Using a murine splenocyte transfusion model across the major histocompatibility barrier,we have found that MSCs prolonged the mean survival time in a dose dependent way and ameliorated the pathologic damages of the aGvHD mice.Further phenotypic and functional detection on splenocytes illustrated that MSCs decreased the MHCâ…¡molecule and the early activation marker CD69 expression on CD11b~+ cells,lowered CD69 expression on CD3~| T lymphocytes and splenocyte proliferation,restrained splenocyte cytotoxic activity.Further investigations showed that MSCs infusion increased the proportion of intrasplenic CD4~+ subset,giving rise to the remarkable elevation of the ratio of CD4~+ to CD8~+ cells,although obious decrease of CD8~+ subset propotion was only evident at the belated observation time poits.Tregs express FOXP3 when activation.A recent clinical investigation confirms that the number of Tregs is not correlated to the clinical aGvHD grade due to the numerical deficiency of Tregs population and massice donor lymphocyte proliferation.Because of the overall effect of Tregs depends on their number in relation to CD8~+ effector T cells,the ratio can serve as a good parameter for the sevetity of aGvHD.Our result testify it each other.MSCs only slightly increased the proportion of CD4~+CD25~+ regulatory T cells(Tregs),but there were significant difference in the ratio of Tregs to CD8~+ cells between the two groups of mice.It was generally accepted that the development of aGvHD needs three requirements called Billingham' tenets including:The host must be incapable of rejecting the graft,the graft must contain immunocompetent cells,antigen incompatibilities exist between host and donor.The revised tenet emphasized the requirement of the effector cells migrating into the target tissues,which was proposed at 1989 and enlisted to the Billingham's classical criteria at 1993.Interestingly,in murine aGvHD model,we found that MSCs infusion increased the number of T lymphocytes in the secondary lymphoid organs(SLOs).Since the expression of CD62L and CCR7 is prerequisite for lymphocyte migration into SLOs,the in vitro experiments revealed that in the presence of MSCs,T lymphocytes prefered to take the naive-like phenotype(CD62L~+/CCR7~+)in mixed lymphocyte reaction and maintained migratory activity elicited by secondary lymphoid tissue chemokine(SLC). Dendritic cells(DCs)are the initiator of immune response.CCR7 expression is pivotal for DCs' maturation and migration into SLOs.However,CCR7 expression and SLC-driven migratory activity of DCs were remarkably suppressed by MSCs'co-culture.The processes above were realized mainly by the soluble factor creted by MSCs.T cell activation and DCs migration is always accompanied by cytoskeleton reorgnazation.Our results showed MSCs co-culture suppress the polarization and pseudopodium formation on T lymphocytes and the polarization and dendriform prominence formation on DCs.Suppressed activation of relative signal pathway proteins Racl and Cdc42 by MSCs might explain that.Consistently,MSCs infusion maintained T lymphocytes to take(naive)CD62L~+/CCR7~+ phenotype and decreased the CCR7 expression and proportion of DCs in SLOs of aGvHD mice. When eGFP-transferred MSCs were traced after intravenously infused,we found that transplanted MSCs could be readily observed in peripherial organs and scarcely detected in SLOs in situ by fluorescent microscope.The rarity of CD62L and CCR7 expression on MSCs might be the causation of the observed distribution pattern of MSCs after infusion.In conclusion,MSCs obtained by this simple protocol meet the standard definition and have the functionalities of MSCs;MSCs can restrain the development of aGvHD at different stages,proportional alteration of different T lymphocyte subsets may be one of the mechanisms by which grafted MSCs suppress the ongoing immune responses in vivo;the altered migratory properties of T and DCs might contribute to the immunosuppressive activity of transplanted MSCs in the setting of aGvHD.The innovative points of the study:1.breaking through the choke point of culture difficulty in murine MSCs with the new method;2.transfusion of MSCs into murine aGvHD to investigate the in vivo immumoregulatory effect of MSCs;3. discovering that migratory capability alteration of T lymphocytes and DCs by MSCs is one of mechanism through which MSCs inhibit aGvHD in vivo.The study here provides novel cues on the mechanisms by which transplanted MSCs ameliorate aGvHD which can contribute to the therapeutic utilization of MSCs clinically.