Comparison Of Multi-source MSCs In Regulating GVHD In Severe Bone Marrow-type Radiation Sickness After Haploid Hematopoietic Stem Cell Transplantation

Acute radiation sickness(ARS)is a systemic disease caused by large dose exposure to radiation in a short time.Bone marrow-type ARS(1 Gy<dose < 10 Gy)is the most common damage and could be treated well.The main treatment of Bone marrow-type ARS in clinical is hematopoietic stem cell transplantation(HSCT).It is still restricted due to the sources and severe GVHD after HSCT.Therefore,it is important to seek more sources of hematopoietic stem cells and steady control GVHD after HSCT in ARS.Haploidentical HSCT(Haplo-HSCT)could be gained in a wide range of sources and transplantation of haplo-HSCT can effectively solve the above problem.However,the strategy is still faced with high failure rate,severe GVHD and severe mortality.Therefore,steady control of GVHD is the most urgent problem.Mesenchymal stem cells(MSCs)have the function of immunosuppression and hemopoiesissupporting,and it is more and more important in HSCT.MSCs have been separated frombone marrow,umbilical cord,dermis,fat and other source recently.As a result,we will treat severe bone marrow-type ARS by transplantation of Haplo-HSCTtohether with different MSCs.From this we are going to compare the hematopoietic reconstruction and GVHD prevention as well as the underlying mechanism.I.Methods1.Construction of severe bone marrow-type ARS.F1(h-2b×d)mice were generated by C57BL/6(h-2b)and BALB/C(h-2d)hybrid as recipient.C57BL/6(h-2b)mice wereused as donor.Different doses of irradiation were set.Hematopoietic reconstruction,survival rate,clinical score and chimerical rate were analyzedafter HSCT.2.MSCs respectingly from bone marrow,umbilical cord,leather and adipose were cultured in vitro.Cell cycle,proliferation and cell subsetsin human peripheral blood T cells were analyzed after co-culturing with MSCs.3.Haplo-HSCT combined with MSCs for severe ARSwith GVHD model was constructed.Different doses of MSCs were set up.CM-DIL was used to mark MSCs in homing experiment.4.Comparitive study of four sources of MSCs in GVHD after Haplo-HSCT.Hematopoietic reconstruction ability,survival rate,clinical score andhistopathology were analyzed after Haplo-HSCT.5.RNA were extracted from four sources MSCs.RNA-seqsequencing was performed to analyze the differences in gene expression in different MSCs.II.Results1.Severe bone marrow-type of ARSwas successfullyconstructed with dose of 800 cGy.2.Four types of MSCs were successfully separated andidentified It is showed that all the MSCs conform to the standard marker(CD14/CD20/CD34/CD45-,CD105+,CD73+,CD90+)and could be induced to differentiate into bone cells and fat cells.It isalso showed that all MSCs could inhibit proliferation of T cell and increase the proportion of G1 cells after co-culture.All MSCs could inhibit proliferation of Th1,promote the proliferation of Th2,and increase the proportion of Treg cells.3.Haplo-HSCT combined with MSCs for severe ARSwith GVHD modelwas successfully constructed.The HUCMSCs were marked with CM-DIL before Haplo-HSCT.The results showed that the HUCMSCshomed to the spleen,liver,lung and bone marrow at the beginning and then most homedto bone marrow.4.With increasedsurival rate and decreased clinical score HUCMSCs was significantly better than the other groups(P<0.05)After the Haplo-HSCT.It is showed that GVHDwas workedly deliciated by MSCs.CD4 T cellswith positive infiltrated into skin and lungs of the model mice,while CD8 T cells expressed in the small intestine with large quantities.T cell subsets test showed that the Th1 cells in HUCMSCs and DMSCs groups were dramaticdly lower than the GVHD group(P<0.05)and Th2 cells were significantly increased(P<0.05).The numbers of Treg cell showed a significant increase in MSCs groups compared to the GVHD group(P<0.05).IFN-γ,IL-2 and TNF-alpha were significantly increased in GVHD group(P<0.01)compare with normal group in peripheral blood.And MSCs groups were significantly lower than the GVHD group(P<0.05).The IL-4 in GVHD group was significantly higher than normal group(P<0.05).And IL-4 in the HUCMSCs group,DMSCs group and BMSCs group were all higher than GVHD group(P<0.01).5.RNA-seq results showed that HUCMSCs were significantly different from BMSCs,DMSCs and Ad-MSCs.And the pathway analysis showed that there were significantly difference in cell adhesion,cytokines,and metabolic pathways.III.Conclusion1.Transplantation of Hematopoietic stem cell can effectively treat severe bone marrow-type ARS in mouse model.2.The combined transplantation of MSCs can effectively reduce GVHD and improve survival rate,among which HUCMSCs is the best candidate cell.3.Different gene expression between HUCMSCs and other MSCsmay be the important reason for more effective regulation of GVHD by HUCMSCs.