Mechanistic Analysis Of Immunomodulation By Human Induced Pluripotent Stem Cells Derived Mesenchymal Stem Cells In Mice

Background:MSCs are usually defined as a group of progenitor cells that are capable of differentiating into a number of mesenchymal lineages such as osteogenesis,chondrogenesis,myogenesis,adipogenesis and endothelial cell.Cell theraphy is an important branch of regeneration medicine.Increasiong data show that MSCs act therapeutic effect by reparing tissue damage.Stem cells are deemed as the seeds of tissue engeering biomaterials and have a promising source for novel therapies.Besides,MSCs exhibit a negative modulation of immune properties.In coculture system in vitro,MSCs suppress T cells response,inhibit dendritic cell maturation and antigen presentation,impair B cell activation and proliferation,reduce proliferation and cytotoxicity of NK cells,and promote the generation of regulatory T cells.However,the resource of MSC is difficult to meet the need of clinical use.Induced pluripotent stem cell(IPSC),reprogrammed from somatic cell,may breakthrough this limitation.IPS-MSC(induced pluripotent stem cell derived mesenchymal stem cell),can strongly inhibit the cytotoxic functions of natural killer.Moreover,iPS-MSCs and ES-MSCs are more resistant than BM-MSCs(bone marrow mesenchymal stem cells)to pre-activated NK cells.CD4+ T helper cells differentiate into several diverse subsets to provide host protection from a variety of pathogens,including T helper 1(Th1)cells and T helper 2(Th2)cells.MSCs cause Thl cells to decrease IFN-y and cause the Th2 cells to increase secretion of IL-4 in co-culture system.Lacking of animal model indeed restrict the research on immunomodulation of MSC in vivo.The effect and the mechanism of MSC regulating immune system in vivo are still unclear.Caspase activation is required for T cell proliferation and early selction.Therefore,Caspase may act roles in MSC inhibited T cell response.Materials and methods:1)We have utilized the example of islet transplantation,and established an animal model by kidney sub-capsular injection of iPS-MSC.Mice were randomly divided into 4 groups,control,HUVEC,MSC and co-grafts groups:i)control group.Mice were operated with same wound and transplanted with equal PBS in volume.ii)Human umbilical vein endothelial cell(HUVEC)group.HUVEC transplantation was positive control because vascular endothelial cells play an important role in the initial step of eosinophil recruitment and activation in immune and inflammatory responses.;iii)iPS-MSC transplantation;iv)co-transplantated the same number of HUVEC and iPS-MSC mix(1:1)(Co-grafts)as group B and group C.2)Histopathological detection.Examine infiltrating inflammatory cells and survival of transplanted graftes by hematoxylin and eosin(H&E)and laser scanning microscope respectively one week post-transplantation.3)T cells subsets analysis.Analyze the proportion of T cells in spleen,and proportion of Thl,Th2 and Treg subsets in CD4+Tcell by flow cytometry(FCM)Analysis;co-culture the purified T cells and HUVEC or iPS-MSC,detect the Thl and Th2 subsets.4)Detect the expression of inflammatory factor in mRNA level and protein level by real-time PCR and ELISA respectively.5)Examination of proliferation of lymphocyte.Isolate the splenocytes of recipent mice and cultured in vitro.The proliferation of splenocytes was detect by BrdU assay and MTT assay.Co-culture the purified T cells and HUVEC or iPS-MSC and detect the activation of T cells by BrdU assay.Culture the purified T cells in the supernatant of HUVEC or iPS-MSC,and dectect the activation of T cells.6)RayBio(?)Human Label-based Antibody Array.RayBio(?)Human label-based antibody array was performed to examine the metabolite of iPS-MSC and HUVEC,by which 507 factors were detected.The results were collected and analyzed by Go oncology analysis.7)Signal pathway examination.The activity of caspase was detected by western blot.Besides,we validated this results by caspase inhibitation.Results:Compared with the HUVEC transplanted group,iPS-MSC transplantation significantly decreased the infiltrating inflammatory cells and have a better survaval in the sub-capsular of kidney.2)iPS-MSC transplantation significantly declined the proportion of T cells and CD4+Th1 and Th2,increased the number of Tregs.3)In co-culture system,iPS-MSC reduced the CD4+ Thl and TH2 proportion.4)iPS-MSC inhibited the proliferation of splenocytes of recipent mice and the proliferation of purified T cells in vitro.5)IPS-MSC transplantation impaired the expression of pro-inflammatory factors and promoted the the expression of anti-inflammatory factors in mRNA and protein level.6)RayBio(?)Human label-based antibody array reveals that iPS-MSC high expressed and secreted 276 soluble factors than HUVEC,including IL-10,TGF-?,TSG-6,et cl.which have been reported as immunomodulatory factors.Go oncology analysis showed that 137 of the 276 factors enriched in immune system process,51 factors enriched in cytokine production.There are many factor enriched in immunocyte regulation.7)The activition of caspase 3,caspase 8 and PARP in spleen of recipent mice was inhibited by iPS-MSC transplantation.Inhibitation of caspase 3 and totale capapse lead to a reduction of Thland Th2 proportion and T cell respones induced by ConA exposure.Adiministration of TGF-P inhibited the proliferation of both splenocytes and T cells as well as the Thl and Th2 subset.Exposure of splenocyes to TGF-? inactived capase 3,but,in contrast,actived caspase 8 and PARP.Conclusion:1)iPS-MSC transplantation significantly decreased the infiltrating inflammatory cells and have a better survaval in the sub-capsular of kidney;2)iPS-MSC transplantation decreased the expression and secretion of pro-inflammatory factors and increased the expression and secretion of anti-inflammatory factors;3)iPS-MSC transplantation declined the proportions of T helper cells and negetively regulated T cells response in a paracrine way.4)iPS-MSCs expressed and secreted masses of soluble factors which involved in immune response.5)iPS-MSC transplantation inhibited immune resonse in spleen of recipent mice by,at least partly by,inactivating caspase 3,caspase 8 and PARP.6)Adiministration of TGF-? inhibited ConA induced proliferation of both splenocytes and T cells,impaired Th1 and Th2 proportion.Exposure of splenocyes to TGF-? inactived capase 3,but,in contrast,actived caspase 8 and PARP,which suggest that iPS-MSCs inhibited T cell mediated immune response not completely through TGF-? pathway.