The Role Of Stromal Cell Derived Factor-1 In The Induction Of Transplant Immune Tolerance Induced By Endometrial Regenerative Cells

Background : Organ transplantation is an effective treatment for end-stage organ failure and congenital organ dysfunction,such as heart,liver,kidney and so on.With the continuous improvement of preoperative preparation,surgical techniques,and postoperative immunosuppressive treatment,the survival time of transplanted organs prolongs gradually.However,with the use of immunosuppressive drugs,the corresponding side effects also emerge.The main manifestations are the infections and malignancies caused by over-immunosuppression,and organ rejections caused by under-immunosuppression.Therefore,an antigen-specific transplant tolerance therapy is urgently needed.The combination therapies based on the mesenchymal stromal cells(MSCs)have shown great potential on the induction of immune tolerance.However,the large-scale use of MSCs for the clinical has been limited by many factors,such as invasive procedures,limited proliferation capacity,as well as potential tumorigenicity and the promotion of tumor development and metastasis.Thus,there is a need to find another type of stromal cell with a better risk-to-benefit profile.Endometrial regenerative cells(ERCs)are mesenchymal-like stromal cell obtained from menstrual blood,with the characteristics of the phenotype of adult stem cells and the ability of various lineages differentiation.They also have the advantages of non-invasively obtained method,abundant resources,highly proliferative rate and lack of immunogenicity.ERCs play critical roles of immunosuppression and organ protection in the inflammation and organ transplantation models.However,their roles and mechanisms in the inhibition of transplant rejection and induction of allograft tolerance have not been evaluated.Stromal cell derived factor-1(SDF-1)is a chemotactic factor expressed in multiple tissues,and play important roles in various physiological and pathological processes,including embryonic development,inflammatory response,immune surveillance,maintaining homeostasis,angiogenesis,tumor growth and metastasis.Moreover,ERCs can secrete SDF-1 in a substantial amount.Further,SDF-1 keeps highly conservative in evolution,the putative mature amino acid sequences and coding region nucleotide sequences of human and mouse SDF-1 are more than 90% identical.Therefore,it is necessary to study the role of SDF-1 secreted by ERCs.Objective: The objective of this study was to conform that ERCs could secret SDF-1,as well as to investigate the effect of human ERCs and the SDF-1 secreted by ERCs on the differentiation of mouse lymphocytes,and to investigate whether human ERC-based therapy with rapamycin could induce cardiac allograft immune tolerance,and the contribution of SDF-1 secreted by ERCs to their immunoregulation.Methods:This study was divided into three parts.Part one,in vitro isolation and culture of ERCs,then the ERCs' growth morphology,phenotype identification and the concentration of SDF-1 secreted in the culture medium were detected.Part two,ERCs were co-cultured with mouse splenocytes,and the effects of ERCs and SDF-1 secreted by ERCs on the generation of tolerogenic dendritic cells(Tol-DCs),macrophage type 2 cells(M2),regulatory T cells(Tregs),regulatory B cells(Bregs)were analyzed by fluorescence-activated cell sorting analysis.Part three was an in vivo study in a mouse allogeneic cardiac transplantation model,to investigate whether ERCs in combination with rapamycin could induce antigen-specific immune tolerance and the influence on the generation of immune cells in splenocytes,as well as to explore the effect of SDF-1 secreted by ERCs played in it by using AMD3100,which is the SDF-1 receptor's inhibitor.Results: Part one,ERCs were successfully isolated and cultured,and the quantity of SDF-1 secreted by the third and fourth generations of ERCs was larger,with 11831.827±341.042pg/ml and 10820.150±530.024pg/ml,respectively,and which were used for the follow-up experiments.Part two,ERCs could induce the mouse splenocytes differentiation to Tol-DCs,M2,Tregs and Bregs,and SDF-1 secreted by ERCs played a critical role in it.Part three,ERCs could reduce the severity of rejection and prolong the survival time of the allografts in mice allogenic cardiac transplantation.And the combination therapy of ERCs and rapamycin could induce donor-specific immune tolerance.The positive effects were correlated with decreasing the CD4+T and CD8+T cells infiltration and donor-reactive Ig M and Ig G antibodies deposition in the grafts,increasing the percentages of tolerogenic immune cells including CD11c+MHC class IIlowCD86lowCD40lowDC(Tol-DC),CD68+CD206+macrophage(M2),CD4+CD25+Foxp3+T cell(Treg),CD19+CD1dhighCD5highCD83lowIL-10 highB cell(Breg)in the splenocytes,and decreasing the percentages of total macrophages,CD3+CD4+T cell,CD3+CD8+T cell in the splenocytes,and SDF-1 secreted by ERCs played a critical role in it.Conclusion: Human ERC-based therapy could induce allogenic cardiac allograft tolerance through affecting the proportion of immune cells in mice,and SDF-1 play critical role in it.