Immunobiological Study On Marrow-derived Mesenchymal Stem/Progenitor Cells Modified With MIL-10 Gene In A Model Of Murine Acute Graft-versus-host Disease After H-2 Haploidentical Bone Marrow Transplantation

As an adult stem cell with the ability of multiple lineage differentiations(including osteoblasts,adipocytes,chondrocytes,and myoblasts),mesenchymal stem cells have received considerable attention and been a perfect "seed cell" in regenerative medicine.Recent research has showed that mesenchymal stem cells have some special efficacy on hematopoietic reconstitution and immunomodulation,which making them favourable perspective in clinical application.The encouraging results have been observed in limited clinical trials,co-transplantation with MSC and HSC enhanced hematopoietic engraftment,attenuated transplant-related complications,especially the incidence and severity of GVHD.However,the mechanism of immunomodulation has not been completely resolved,some latest studies suggested that the efficacy of MSC on prophylaxis and treatment for severe acute GVHD was limited,even not confirmable.Interleukin-10 is an ideal anti-inflammatory cytokine,inhibits the activation of T lymphocytes,monocytes/macrophages,and the synthesis of various inflammatory cytokines.IL-10 represents a substancial suppressor of the cellular immune response of Th1 type,which implys its potential effects on preventing the occurrence and development of GVHD.Because of its anti-inflammatory properties and its association with dendritic cells and regulatory T cells,IL-10 should have substantial benefit on the induction of immune tolerance and graft survival.Holler's research showed that decreased IL-10 production correlated with a subsequent high incidence of GVHD.We assumed that with the combination of cell therapy and gene therapy,the co-transplantation with marrow-derived mesenchymal stem/progenitor cells modified with mIL-10 gene will prevent or attenuate GVHD more effectively in coordination with IL-10,but it hard to carry out in human because of the limitation of ethics and technology.In our research,firstly we purified and cultured murine marrow-derived mesenchymal stem/progenitor cells(MSCs/MPCs) with active proliferation activity and multidifferentiation potential,and studied its biological characteristic and corresponding properties.Then,a mlL-10 recombinant adenoviral vector was constructed Successfully and proved to be high efficient in transfecting mMSC/MPCs.The mMSCs/MPCs modified with mIL-10 gene expressed mIL-10 protein efficiently in vitro.Thirdly,we established an eligible model of murine acute GVHD after H-2 haploidentical bone marrow transplantation.On the basis of the model,MSCs/MPCs and MSCs/MPCs modified with mIL-10 gene were transplanted respectively to observe the immunobiologic effects.Section 1:The optimization of culture system ex vivo of mMSC/MPC,the corresponding characterization of mMSC/MPC.Methods:Bone marrow cells were collected by flushing the femurs and tibias from female C57BL/6 mice with medium repeatedly and thoroughly,while scraping the endosteum Of the bone,then the collection were vibrated with the bone fragments.The mononuclear cells were made and plated in the 25cm² culture flasks,at a concentration of 1×10⁶/cm²,with three different complete medium respectively:DMEM-LG medium supplemented with 15%FBS,DMEM -LG/MCDB201 medium supplemented with 15% FBS,and Mesencult basal medium supplemented with 20%MSC Stimulatory Supplement.Flasks were maintained in incubator with 5%CO2 at 37℃.After 24h,nonadherent cells were eliminated by medium changing,then the medium were changed every 3 days.The cells were trypsinized when tending to 70%-80% confluences,and replated at a concentration of 2.5～5×10³/ cm².The characters of different cell passages,such as morphology,the CFU-F with different medium,cell growth curve,cell cycle,phenotype were demonstrated.The ability of multidifferentiation along adipocytic,osteoblastic pathways were also assayed.The expression of IL-10mRNA in the cells were detected by RT-PCR and IL-10 protein in the culture supernatant by ELISA.The calculations and statistical comparisons were performed using the software SPSS 11.5 and the reported P was 2 sided(a =0.05).Results:The cell population consisted of spindle,shaped cells in confluent cultures after three consecutive passaging,and seemed homogeneous by light microscopy.The Mesencult complete medium was the optimal culture system.The biological stabilities were maintained till 10 passages above,and the P3 cells were choosed to be the suitable object for the experiment.Cell-doubling time was 31.1±0.6 hours in log phase.Cell cycle analyses revealed that 80.3%of the P3 cells at G0/G1 phase,12.4%, 7.3%at G2 and S phase respectively,when they tended to 80%-90% confluences.Flow-cytometric analysis showed that the P3 cells were high positive for CD29,CD44,CD105,Sca-1,moderate positive for CD31,and nearly negative for CD45. The cells can be differentiated into osteoblast,adipocyte in vitro.We failed to identify IL-10 mRNA of the cells by RT-PCR and protein of IL-10 in the culture supernatant by ELISASection 2:The construction of mIL-10 recombinant adenoviral vector,the transfection and mIL-10 expression of mMSC/MPCs ex vivoMethods:The mIL-10 cDNA was removed and amplificated from pORF5-mIL-10 plasmid through PCR,then inserted into adenoviral vector pSGCMV to construct the recombinant plasmid,pSGCMV-mIL10.The recombinant plasmid were mixed with human serum type v adenoviral plasmid(pBGHE3) and co-transfected into HEK 293 cell line through Lipofectamine 2000.The replication-incompetent adenovirus containing mIL-10 was harvested from the supernatant of HEK 293 cell line.The adenovirus was produced sufficiently in HEK 293 cell line and purificated by CsCl gradient centrifugation.The titer of the recombinant adenovirus was assayed through the method of TCID50.The optimal MOI for mMSC/MPC was identified by the adenovirus Ad5-EGFP.The expression of IL-10mRNA in the mMSC/MPC transfected was detected by RT-PCR and IL-10 protein in the culture supernatant by ELISA. Results:The amplification production was verified by restrictive endonucleases analysis,PCR,DNA sequencing.The recombinant adenovirus containing mIL-10 was verified by PCR,and the titer of adenovirus was 2.5×¹⁰pfu/ml.the mMSCs/MPCs transfected with mIL-10 recombinant adenoviral vector expressed mIL-10 protein efficiently ex vivo at the MOI of 200.Section 3:The establishment of a model of murine acute GVHD after H-2 haploidentical bone marrow transplantation,the immunobiologic effects of mMSCs/MPCs modified with mIL-10 gene in the model when co-transplanted.Methods:The female C57BL/6 mice as donor,male CB6F1 mice were used as recipients to establish aGVHD model of murine after H-2 haploidentical BMT.The recipients received 12Gy(100cGy/min) total body irradiation(6mv,X-ray) as the lethal conditioning.Bone marrow MNC(1×10⁷/a recipient mouse) and splenic MNC(0,1×10⁷,2×107,3 10⁷/a recipient mouse,respectively) were transplanted by tail vein infusion into irradiated recipients in 4 hours.Four groups were divided(n=15) according to the different MNC mixture.The general state,survival time,loss of weight,WBC counting in peripheral blood,chimerism,clinical signs were observed after BMT regularly.The severity of aGVHDwas assessed with a clinical GVHD scoring system and a semiquantitative system for histologic examination,and the corresponding parameters were determined for an eligible acute GVHD model.On the basis of the model,MSCs/MPCs and mMSC/MPCs modified with mIL-10 gene were transplanted respectively to observe the immunobiologic effects. Four groups were divided according to the different dose of cells(1×10⁵ and 5×10⁵/a recipient mouse).The general state,survival time,loss of weight,WBC counting in peripheral blood,chimerism,clinical signs,were observed regularly,and the severity of aGVHD was assessed similarly.The serum of recipient mice was collected on the day 14,28,35 after transplantation and in extremis,then be used to detect cytokines of mIL-10, mIL-4,mINF-γand mTNF-a by ELISA.Results:Under the condition of TBI(X-ray,6mv,total dose:12Gy,dose rate:100 cGy/min), typical aGVHD clinical signs were induced stably in limited days(+24d～+28d) when recipient mice were infused with 1×10⁷ bone marrow MNCs plus 3×10⁷ splenic MNCs from the donor mice.The clinical and histologic degree was relatively coincident,and the incidence of aGVHD in 35 days were 100%,all the recipient mice died within +27d～+33d.Accordingly,the C57BL/6→CB6F1 model of munne aGVHD after H-2 haploidentical BMT was set up successfully.The co-transplant of 1×10⁵ MSCs/MPCs seemd to decrease the incidence and severity of aGVHD(P＜0.05),while increased cell dose(5×10⁵) failed to enhance the effect significantly(P＞0.05).The co-transplant of 1×10⁵ MSCs/MPCs modified with mIL-10 gene showed the more significant decreasing incidence and severity of aGVHD than MSCs/MPCs(P＜0.01).While it failed to decrease,even augment the incidence and severity of cGVHD,similarly,increased cell dose(5×10⁵) failed to enhance the effect significantly(P＞0.05),The detection of cytokines showed that the co-transplant of MSCs/MPCs up-regulate the level of IL-4 significantly in 14 days after transplant(P＜0.05),decrease the level of INF-γand TNF-a significantly(P＜0.05),while no significant effect on IL-10(P＞0.05).The co-transplant MSCs/MPCs modified with mIL-10 gene showed the significant increase of IL-10(P＜0.05),the corresponding increase of IL-4(P＜0.05),and more dramatic decrease of INF-γ(P＜0.01).Conclusions:1.mMSCs/MPCs from C57BL/6 mice have been isolated,cultured and purified ex vivo successfully.With the stable biologic characterization,active proliferation and multiple differentiated abilities,the mMSCs/MPCs could be used in gene-transfection and animal model experiment.2,mlL-10 recombinant replication-incompetent adenoviral vector system was constructed successfully,and mMSCs/MPCs could be gene-transfected with high efficiency by the adenoviral vector.The mMSCs/MPCs transfected with mIL-10 recombinant adenoviral vector expressed mIL-10 protein efficiently and stably ex vivo.3.The C57BL/6→CB6F1 model of murine aGVHD after H-2 haploidentical BMT was established successfully.The co-transplant of 1×10⁵ MSCs/MPCs could decrease the incidence and severity of aGVHD,while increased cell dose(5×10⁵) failed to enhance the effects significantly.MSCs/MPCs up-regulated the level of IL-4 significantly in 14 days after transplant,decreased the level of INF-γand TNF-a significantly(no significant effect on IL-10),it maybe the one of the mechanisms of MSCs/MPCs alleviating aGVHD.The co-transplant of 1×10⁵ MSCs/MPCs modified with mIL-10 gene showed the more significant effects on decreasing incidence and severity of aGVHD than MSCs/MPCs,while it failed to decrease,even augment the incidence and severity of cGVHD.Similarly,increased cell dose(5×10⁵) failed to enhance the effects significantly.The significant increase of IL-10,the corresponding increase of IL-4 and more dramatic decrease of INF-γmay accounted for the possible reason,while the upregulation of IL-10 may increase the risk of cGVHD.