Establish GGTA1 Knockout Pigs And Hypoimmunogenic Human Pluripotent Stem Cells

(1)Generation of GGTA1 knockout pigsXenotransplantation is an alternative strategy for the shortage of human organ donors in clinical transplantation.Numerous studies proved that pigs are appropriate xenograft donors.There are many immunologic obstacles that need to be overcomed in pigs xenograft,including HAR,AHXR,ACR,coagulation dysregulation,CR and inflammatory reactions.With the discovery of their mechanisms,gene-modified pigs are currently the main measures for these problems.Pig pluripotent stem cell lines with high quality are still not been established,and gene modification with homologous recombination in pig somatic cells is inefficient.In recent years,some gene editing tools have been established,including ZFN,TALEN and CRISPR/Cas9.These tools have been successfully applied in a variety of organisms for their high efficiency.We established a pig gene modification system based on ZFN and TALEN.Firstly,we used ZFN to produce GGTA1 KO(GTKO)pigs.GGTA1 is involved in the formation of Gal antigens on the pig cell surfaces,which are the main cause of HAR.During evolution this gene was lost in human and Old World monkeys,so their cell surfaces do not have Gal antigens and their blood have lots of natural antibodies targeting Gal antigens.In order to overcome HAR,researchers produced GTKO pigs with somatic cells homologous recombination and somatic cell nuclear transfer(SCNT).The results of xenotransplantation showed GTKO pigs can inhibit HAR significantly.Somatic cells homologous recombination is inefficient,and the exogenous resistance genes introduced are obstacles to the follow-up study.We transfected GGTA1 targeting ZFN plasmids into pig primary fetal fibroblasts by electroporation.After drug screening,we got 18 clones with one copy knockout and 4 clones with double copies knockout,with the efficiency 23.4%and 5.2%,respectively.Then the clones with double copies knockout were used to produce GTKO pigs via SCNT.At last,a total of three GTKO pigs were got.We also established a GTKO pig fetal fibroblasts cell line.The Gal was completely cleared from GTKO pig cell surfaces,which help GTKO pig cells to escape from the complement-mediated immune attack when they were co-cultured with human serum.Secondly,We used TALEN to modifiy pig SLAs system based on GTKO cells.We planned to replace pig SLAs system with human HLAs system.Efficient TALEN targeting SLA-1 and-2 were designed based on a lot of sequencing.PCR results showed that SLA-1 genes are replaced by HLA-A.The effect of this strategy in xenotransplantation should been tested in pig xenotransplantation model.(2)Generation of hypoimmunogenic human pluripotent stem cellsTheoretically hPSCs have the capacity to self-renew and differentiate into all human cell types.Therefore,the greatest promise of hPSCs-based therapy is to replace the damaged tissues of patients suffering from traumatic or degenerative diseases by the exact same type of cells derived from hPSCs.Allograft immune rejection is one of the obstacles for hPSCs-based clinical applications.HLA ? induces CD8+T cells-mediated allograft rejection.HLA ? induces CD4+T cells-mediated allograft rejection.Hence,we focus on optimizing hPSCs for clinic application through gene modification.We already established B2M-/-hPSCs with low expression of HLA ?,which can decrease the immune rejection mediated by T cells.Here we established CIITA-/-hPSCs,which decrease the expression of HLA ?.TALEN was used to target CIITA in hPSCs efficiently.CIITA-/-hPSCs did not show any difference in the differentiation potential and self-renewal capacity.DCs derived from CIITA-/-hPSCs expressed CD83 and CD86 but without the constitutive HLA ?.Fibroblasts derived from CIITA-/-hPSCs did not have the inducible expression of HLA? after IFN-y treatment.And they induced weaker CD4+T cells reaction,when they were co-cultured with CD4+T cells.We generated HLA ? defected hPSCs via deleting CIITA,a master regulator of constitutive and IFN-y inducible expression of HLA ?genes.CIITA-/-hPSCs can differentiate into tissue cells with non-HLA ? expression.It's promising that CIITA-/-hPSCs-derived cells could be used in cell therapy(e.g.,T cells and DCs)and escape the attack of receptors'CD4+T cells,which are the main effector cells of cellular immunity in allograft.