Experimental Study On Suppression Of Immunogenicity Of Human Embryo-derived Cardiac Progenitor Cell By RNA-interference

Stem cells are the basic elements and prerequisites for clinical application of stem celltransplantation, tissue engineering, and other regenerative medicine. Allogeneic stem cellsutilization is limited by ethics, rejection and safety issues. Self-body-derived stem cells aredifficult for industrial production, constraining the clinical application. Nuclear transfer(cloning) and the induced pluripotent stem (iPS) cells technology are the ideal schemes tosolve the problem. However, the former has not yet succeeded in humans and the later hasmet the difficulty of immune rejection and safety. Based on the fact that immune rejectionis determined by the immunogenic molecules on the cell membrane, this study aimed touse the human embryo-derived cardiac progenitor cells as the embryo-derived stem/progenitor cells. First, we detected histocompatibility antigens (transplantation antigens) ofthe cells under undifferentiated state and with IFN-γ and TNF-α added as analogue of invivo microenvironment to determine whether embryo-derived stem/progenitor cells hadimmunogenicity. Secondly, we used the gene chip technology to screen genes closelyrelated to immunogenicity. Last, cDNA microarray and immunogenicity closely gene.Finally, we used RNA interference technology to interfere in the selected genes, aiming toexplore the possibility of reducing immunogenicity, which might provide new ideas forclinical application of embryo-derived stem/progenitor cells.Part I HECPCs immunogenicity and related gene chip analysisPurpose: To study the expression of immunogenic molecules in human embryo-derivedcardiac progenitor cells (HECPCs) and to screen genes closely related to HECPCstransplant rejection by cDNA microarray.Materials and methods:1. Embryos of abortion with4-6weeks age were used andHECPCs were isolated in vitro and cultivated. The flow cytometry was used to determinethe surface immunogenic molecules expression and to observe the immunogenic moleculeexpression with inflammatory factors added.2. The SBC (H1029) gene chip was used todetect the expression of immune-related genes before and after the stimulation of theinflammatory factors. The important genes which were significantly up-regulated and wereclosely related to immunogenicity were selected initially and verified by RT-PCR. Results:1. The state of HECPCs cultured in vitro was stable, consistent with the in vivostate. The flow cytometry showed that after adding IFN-γ and TNF-α, HLA-I moleculesexpression was significantly increased and reached the peak on the fifth day. HLA-DRmolecules were not expressed at different time points.2Gene chips screening showed thatafter adding IFN-γ and TNF-α,116genes were up-regulated and122genes weredown-regulated. Among the up-regulated genes,57had close relationship with immuneregulation. Four genes with significantly increased expression were selected and verified.ASNS, IRF-1and MCP-3were up-regulated genes.Conclusions:1. As the seeding cell in regenerative medicine, HECPCs still hadimmunogenicity problems.2.Genes as ASNS, IRF-1and MCP-3might be closely relatedto immunogenicity of HECPCs.Part II IRF-1interference with siRNA and the impact on HECPCsimmunogenicityPurpose: To establish RNA interference lentiviral vectors targeting IRF-1, reduce theIRF-1expression in HECPCs and obtain the cell model with low IRF-1expression and todetermine their immunogenicity with the one-way mixed lymphocyte reaction test.Materials and methods:1. The selected interferon regulatory factor (IRF)-1gene, whichwas closely related to immunogenicity, was used as the target to construct the lentiviralvector.2. Realtime-PCR and Western blot were used to screen the lentiviral vectors withthe best interference effect.3. The one way mixed lymphocyte reaction test was used todetermine the effects of RNA interference of IRF-1on the immunogenicity of HECPCs.Results:1. Targeting different IRF-1sequences, Lenti-L1, Lenti-L2and Lenti-L3werebuilt.2. After72h of lentiviral infection, realtime-PCR was used to detect the IRF-1mRNA interference efficiency of Lenti-L1, Lenti-L2and Lenti-L3as74%,86%and71%.The western blot was used to detect the IRF-1protein expression, which was consistentwith Lenti-L1, Lenti-L2and Lenti-L3interference results in realtime-PCR. Lenti-L2interference effect was the most significant. The one way mixed lymphocyte reactionindicated that after processed with RNAi, HECPCs stimulating PBMCs proliferation wassignificantly weakened, suggesting that the immunogenicity was compromised.Conclusions:1. RNA interference with IRF-1expression could reduce the immunogenicity of HECPCs.2. The application of RNA interference to modify theimmunogenicity related genes was a possible way of reducing the immunogenicity of theembryo-derived stem/progenitor cells and alleviating transplantation rejection.