Effect Of Induced Pluripotent Stem Cells-derived Chimeric Thymus Transplantation On T Cell Reconstitution And GVHD After Allogeneic Bone Marrow Transplantation In Murine

BackgroundAccelerating T cell reconstruction after allogeneic bone marrow transplantation can reduce the risks of infection and relapse.Thymus transplantation is a method that had been used in the treatment of DiGeorge syndrome,severe combined immunodeficiency disease,AIDS and other diseases in the clinical,to improve the function of thymus and accelerate T cell reconstitution.Previous researches had shown that TT in conjunction with allo-BMT was effective to accelerate T cell reconstruction and promote graft versus tumor effects without increasing the risk of GVHD.However,the lack of thymus source had restricted the application of TT in allo-BMT.Induced pluripotent stem cells have a similar function as embryonic stem cells,and researchers had successfully applied iPS cells to many cell types,such as hematopoietic progenitor cells,neurons,islet cells,liver cells,endothelial cells,etc.iPS cell technology provides a potential way for developing artificial thymus.Nonetheless,then application of of iPS cells into thymus for allogeneic bone marrow transplantation,had not been reported.ObjectiveOur previous researches had established a green fluorescent protein-expressing C57BL/6 murine iPS cell line and produced chimeras by injecting iPS cells into ICR murine blastocysts.The chimeras could produced thymus that derived from murine iPS cells in bodies.This study used the chimera thymus in allo-BMT for the first time,explored its influence on the T cell reconstitution and GVHD.Chimeric thymus transplantation may provide a viable strategy for the source of TT and accelerating T cell reconstitution.Methods1.IBM-BMT,TT and DLIIBM-BMT:Recipient BALB/c mice were irradiated 24 hours before BMT.Then these mice were injected with 1×107 C57BL/6 BMCs by IBM-BMT.BMCs were collected from the femurs and tibias of C57BL/6 mice.Briefly,a micro-syringe was inserted into the joint surface of the tibia through the patellar tendon and then inserted into the bone cavity.TT:One piece of the chimera thymus(2×2×1mm)was transplanted under the renal capsule of the left kidney in some mice.DLI:Spleens were collected from donor C57BL/6 mice.Single cells were prepared by milling and filtering through a mesh and injected intravenously into the tail vein of recipient mice.2.Observation of the structure of chimera thymus and GVHD after transplantationThe recipient mice were divided into three groups:IBM-BMT group,IBM-BMT+TT group and IBM-BMT+DLI group.Four weeks after BMT,recipients were killed by cervical dislocation and the thymic structure was observed histopathologically to detect the morphology characteristics.The sections were stained with hematoxylin and eosin photographed.Those mice were weighted 2-3 times per week and observed for the clinical features of GVHD.Histological studies were performed in the livers,intestines and skins for the evaluation of GVHD.The tissues were fixed in 10%formaldehyde and embedded in paraffin.Serial tissue sections were prepared and stained using hematoxylin and eosin.3.Analysis of the T cells subsets by flow cytometrySurface markers on lymphocytes from the blood and spleens were detected by 3-color fluorescence staining using a FACScan system.Percentages of CD4,CD8 and intracytoplasmic FoxP3 was analyzed.Results1.The percentage of CD8+ T cells of the peripheral blood in IBM-BMT+TT group,IBM-BMT+DLI group and IBM-BMT group respectively was(11.10±1.49)%,(8.49±0.82)%and(5.52±0.83)%,there were significant differences between each group(P<0.05).The percentage of CD4+ T cells in IBM-BMT+TT group(9.60±0.69)%significantly higher than those in IBM-BMT+DLI group(8.07±0.65)%and IBM-BMT group(6.42±1.40)%(P<0.05).2.Percentages of Foxp3+ in CD4+ T cells in IBM-BMT+TT group(1.86±0.36)%and IBM-BMT group(2.29 ±0.23)%were higher than IBM-BMT+DLI group(0.07±0.05)%(P<0.05).3.IBM-BMT group and IBM-BMT+TT group showed less clinical and histopathological scoring of GVHD than IBM-BMT+DLI group.Conclusion1.iPS cells-derived chimeric thymus transplantation could effectively accelerate the CD4 + and CD8 + T cell subsets to recover and support the development of Foxp3+ CD4 + T cells(Treg cells).2.The mice of IBM-BMT + TT group only had mild GVHD.Probably because chimeric thymus could support the development of Foxp3+CD4+ T cells which reflect the immunosuppressive activity.3.iPS cells-derived chimeric thymus transplantation could accelerate T cell reconstitution and not induce severe GVHD.It could become a viable method for solving the issues of the limited source of thymus,delayed immune reconstitution and immunological rejection after allo-BMT.