Objective To investigate the isolation and culture of porcine bone marrow mesenchymal stem cell (BMSC) with α-1, 3-galactosyltransferase (GGTA1) gene knockout (GTKO), GTKO/ human CD46 (hCD46) insertion and cytidine monopho-N-acetylneuraminic acid hydroxylase (CMAH)/GGTA1 gene knockout (Neu5GC/Gal), and the protective effect of co-culture with porcine islets on islet cells. Methods Bone marrow was extracted from different transgenic pigs modified with GTKO, GTKO/hCD46 and Neu5GC/Gal. Porcine BMSC were isolated by the whole bone marrow adherent method and then cultured. The morphology of BMSC was observed and the surface markers of BMSC were identified by flow cytometry. Meantime, the multi-directional differentiation induced by BMSC was observed, and the labeling and tracing of BMSC were realized by green fluorescent protein (GFP) transfection. The porcine BMSC transfected with GFP were co-cultured with porcine islet cells. Morphological changes of porcine islet cells were observed, and compared with those in the porcine islet cell alone culture group. Results BMSC derived from pigs were spindle-shaped in vitro, expressing biomarkers of CD29, CD44, CD73, CD90, CD105 and CD166 rather than CD34 and CD45. These cells were able to differentiate into adipocytes, osteoblasts and chondrocytes. Porcine BMSC with GFP transfection could be labeled and traced, which could be stably expressed in the daughter cells after cell division. Porcine BMSC exerted certain protective effect on islet cells. Conclusions GFP-labeled porcine BMSC modified with GTKO, GTKO/hCD46 and Neu5GC/Gal are successfully established, which exert certain protective effect upon islet cells.

Objective To investigate the differences and the immunocompatibility of wild-type (WT), four-gene modified (TKO/hCD55) and six-gene modified (TKO/hCD55/hCD46/hTBM) pig erythrocytes with human serum. Methods The blood samples were collected from 20 volunteers with different blood groups. WT, TKO/hCD55, TKO/hCD55/hCD46/hTBM pig erythrocytes, ABO-compatible (ABO-C) and ABO-incompatible (ABO-I) human erythrocytes were exposed to human serum of different blood groups, respectively. The blood agglutination and antigen-antibody binding levels (IgG, IgM) and complement-dependent cytotoxicity were detected. The immunocompatibility of two types of genetically modified pig erythrocytes with human serum was evaluated. Results No significant blood agglutination was observed in the ABO-C group. The blood agglutination levels in the WT and ABO-I groups were higher than those in the TKO/hCD55 and TKO/hCD55/hCD46/hTBM groups (all P<0.001). The level of erythrocyte lysis in the WT group was higher than those in the ABO-C, TKO/hCD55 and TKO/hCD55/hCD46/hTBM groups. The level of erythrocyte lysis in the ABO-I group was higher than those in the TKO/hCD55 and TKO/hCD55/hCD46/hTBM groups (both P<0.01). The pig erythrocyte binding level with IgM and IgG in the TKO/hCD55 group was lower than those in the WT and ABO-I groups. The pig erythrocyte binding level with IgG and IgM in the TKO/hCD55/hCD46/hTBM group was lower than that in the WT group and pig erythrocyte binding level with IgG was lower than that in the ABO-I group (all P<0.05). Conclusions The immunocompatibility of genetically modified pig erythrocytes is better than that of wild-type pigs and close to that of ABO-C pigs. Humanized pig erythrocytes may be considered as a blood source when blood sources are extremely scarce.

Effective preoperative matching of organ transplantation is essential for the success of organ transplantation.The current methods of preoperative matching for xenotransplantation are derived from human allogeneic organ transplantation.However,these methods are simple and cannot accurately predict whether rejection will occur after transplantation.A noteworthy feature of xenotransplantation lies in the stability of the donor source and the clarity of its genotype,which facilitate convenient sampling and ensure robust repeatability.The multi-link and multi-dimensional preoperative matching is conducive to accurately screening suitable donors and reducing the occurrence of postoperative rejection after xenotransplantation,including making full use of the advantages of donor control in xenotransplantation,expanding tissue level matching methods on the basis of improving traditional cytological matching,paying attention to the role of endothelial cells in matching,and developing organic-level matching methods.Therefore,this article reviews the methods of allogeneic organ transplantation matching,current methods,existing problems and possible breakthroughs of xenotransplantation matching,in order to provide reference for further research on xenotransplantation matching.

Objective To discuss the perioperative care and wound protection of xenotransplantation from genetically edited pigs to monkeys, with the goal of improving the success rate of such experimental procedures. Methods From October 2022 to October 2023, perioperative care and wound protection were performed on 7 recipient rhesus monkeys undergoing xenotransplantation of genetically edited pig tissues and organs. Customized wound protective garments were designed based on monkeys' size and surgical area to protect the wounds, alongside meticulous perioperative care. This included preoperative preparation and medication, intraoperative monitoring of physiological indicators and anesthesia management, and postoperative care comprising wound protection, observation and monitoring, and nutritional support. Results All seven monkeys successfully underwent xenotransplantation. With the aid of protective garments and detailed care, all surgical wounds healed by first intention, and postoperative recovery was satisfactory. Conclusion Proper care and wound protection during xenotransplantation from genetically edited pigs to monkeys not only promote wound healing, but also alleviate pain and harm to animals. This has significant implications for advancing experimental research in pig-monkey xenotransplantation and enhancing animal welfare.

Objective:To investigate the changing trends in cardiac function following xenogeneic heterotopic heart transplantation of multi-gene edited pig hearts and assess the impact of recipient immune responses on donor heart, laying experimental groundwork for the clinical application of gene editing technology.Methods:On December 16, 2023, xenogeneic heterotopic heart transplantation was performed between pigs and rhesus monkeys. Functional status of the graft under post-transplantation load conditions and recipient immune indicators were observed.Results:The recipient monkeys survived for 40 days with satisfactory functionality of both donor and recipient hearts, and no hyperacute or acute immune rejection reactions were observed.Conclusion:Multi-gene editing technology provides potential for xenotransplantation, yet further exploration is needed for its clinical application.

Objective To investigate the establishment of a six-gene-edited pig-to-non-human primate kidney xenotransplantation model. Methods The kidney of humanized genetically-edited pig (GTKO/β4GalNT2KO/CMAHKO/hCD55/hCD46/hTBM) was transplanted into a cynomolgus monkey. The survival of the recipient and kidney condition after blood perfusion were observed. The parenchymal echo, blood flow changes, and size of the kidney were monitored on a regular basis. Routine blood test, kidney function test and electrolyte assessment were carried out. Dynamic changes of urine, feces and body mass were monitored. At the end of life, the transplant kidney, heart, liver, spleen, lung, and cecum were collected for pathological examination. Results The recipient died at postoperative 7 d. After blood flow was restored, the kidney was properly perfused, the organ was soft and the color was normal. At the end of the recipient's life, a slight amount of purulent secretion was attached to the ventral side of the kidney, with evident congestion and swelling, showing the appearance of "red kidney". Postoperatively, the echo of renal parenchyma was increased, blood flow was decreased, the cortex was gradually thickened, and a slight amount of effusion surrounded the kidney and abdominal cavity over time. In the recipient, the amount of peripheral red blood cells, hemoglobin, albumin, and platelets was progressively decreased, and serum creatinine level was increased to 308 μmol/L at postoperative 7 d, whereas the K⁺ concentration did not significantly change. Light yellow urine was discharged immediately after surgery, diet and drinking water were resumed within postoperative 3 h, and light yellow and normal-shape stool was discharged. The reddish urine was gradually restored to normal color within postoperative 1 d, which were consistent with the results of the routine urine test. A large amount of brown bloody stool was discharged twice in the morning of 2 d after surgery. Omeprazole was given for acid suppression, and the stool returned to normal at postoperative 4 d. The β₂-microglobulin level was increased to 0.75 mg/L at postoperative 7 d. The body mass was increased by 1.7 kg. Autopsy pathological examination showed interstitial edema and bleeding of the transplant kidney, a large amount of infiltration of lymphocytes and macrophages, infiltration of lymphocytes in the arteriole wall and arterial cavity, accompanied by arteritis changes, lymphocyte infiltration in the cecal stroma and congestion in the spleen tissues. No significant abnormal changes were observed in other organs. Conclusions The humanized genetically-edited pig-to-non-human primate kidney xenotransplantation model is successfully established, and postoperative survival of the recipient is 1 week.

Liver transplants are in huge demand in China, but facing the problem of extreme shortage of donor organs. Xenogeneic (pig) liver transplantation may be a potential way to alleviate the shortage of donors. The liver is a detoxifying organ with synthetic functions, and when genetically modified pigs are used as donors, it faces the dual problem of overcoming immune rejection and resolving physiological function mismatches. Therefore, suppressing the innate immune response can better alleviate the immune rejection of xenotransplantation. In addition, the use of chimeras of humanized cell livers may resolve the problem of physiological function mismatch. Moreover, with the development of gene editing technology, it has become possible to obtain multiple gene edited pigs and chimeras. Therefore, exploring and researching from these two aspects will hopefully solve the current problems of liver xenotransplantation and promote the further development of the field of liver xenotransplantation.

Objective To validate whether the expression of human cluster of differentiation 55 (hCD55) protein in porcine islet cells could inhibit the activation of complement components in human serum. Methods Four adult pigs with WT (wild type), GTKO [α-1, 3-galactosyltransferase (GGTA1) knockout], GTKO/hCD55 and hCD55 genotypes were selected. Islet cells were isolated from WT, GTKO and GTKO/hCD55 pigs, and the purity and insulin secretion function were detected. The expression of hCD55 at the DNA, RNA and protein levels was analyzed by agarose gel electrophoresis, reverse transcription polymerase chain reaction (RT-PCR) and flow cytometry, respectively. Complement-dependent cytotoxicity assay and complement deposition assay were performed under the incubation conditions with fresh human serum. Results The purity of isolated porcine islet cells from three genotype pigs was > 75%, and the glycemic index was > 1. The expression of hCD55 messenger RNA(mRNA) and protein in GTKO/hCD55 porcine islet cells decreased the deposition of human complement component C3c and membrane-attacking complex C5b-9, and reduced the cytotoxicity. Conclusions The expression of hCD55 protein in porcine islet cells could inhibit the activation of human complement and reduce complement-mediated killing effect, indicating that hCD55 protein could exert complement protection effect on porcine islet cells. These findings provide theoretical basis for the application of hCD55 in islet xenotransplantation.

Objective To investigate the effect of human CD47 (hCD47) in inducing the immune tolerance of human macrophages to porcine endothelial cells. Methods The porcine iliac endothelial cell (PIEC) transfected with pCDH-hCD47-FLAG plasmid was assigned into the pCDH-hCD47 group, PIEC transfected with pCDH-FLAG empty vector plasmid was assigned into the pCDH group, PIEC transfected with hCD47-dN was assigned into the pCDH-hCD47-dN group and human umbilical vein endothelial cell (HUVEC) was assigned into the positive control group. The cells were co-cultured with human macrophages to detect and analyze the phosphorylation of signal regulatory protein α (SIRPα) and the killing effect of human macrophages on PIEC. Furthermore, porcine arteriae endothelial cell (PAEC) was isolated from GT^－/－ and GT^－/－/ hCD 47 gene editing pigs to analyze the phosphorylation of SIRPα and the killing effect of human macrophages on PAEC. Results The pCDH group cells could not induce the phosphorylation of SIRPα, whereas the pCDH-hCD47 group cells could activate the phosphorylation of SIRPα after 10 min co-culture with human macrophages, and the degree of phosphorylation of SIRPα was increased with the prolongation of the co-culture time. The pCDH-hCD47-dN group cells failed to activate the phosphorylation of SIRPα. Human macrophages exerted significant effect on killing the pCDH group cells. The pCDH-hCD47 group cells could evidently inhibit the killing effect of human macrophages (P < 0.05), whereas the pCDH-hCD47-dN cells failed to suppress the killing effect of human macrophages. GT^－/－-PAEC could not activate the phosphorylation of SIRPα after co-culture with human macrophages. However, GT^－/－/hCD47-PAEC significantly activated the phosphorylation of SIRPα after co-culture with human macrophages. Human macrophages exerted significant killing effect on GT^－/－-PAEC, and GT^－/－/hCD47-PAEC could obviously inhibit the killing effect of human macrophages (P < 0.05). Conclusions The expression of hCD47 in the porcine endothelial cells can inhibit the killing effect of human macrophages on endothelial cells by activating the phosphorylation of SIRPα.

To investigate the changes in the expression levels of scavenger receptor class B member 1 (SCARB 1) in the liver tissues before and after liver xenotransplantation and analyze the relationship between the variations in the SCARB1 expression and coagulation regulating dysfunction in the recipients.Methods The Wuzhishan miniature pig with α-1,3-galactosyltransferase gene-knockout(GTKO) was utilized as the donor and Macaca thibetana was chosen as the recipient.Heterotopic auxiliary liver xenotransplantation models were established.The liver tissue specimen was collected before and after liver xenotransplantation.Primary hepatocytes were extracted from the pig using collagenase digestion method.Human peripheral blood mononuclear cells were obtained by immunomagnetic bead sorting.These two types of cells were co-cultured and supplemented with human plasma to establish cell models with coagulation regulating dysfunction following liver xenotransplantation.Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) and Western blot were performed to quantitatively measure and statistically compared the expression levels of messenger ribonucleic acid (mRNA) and protein of SCARB1 in the tissue and cell samples.At the cellular level,the expression of SCARB 1 was interfered by lentiviral vector.The coagulation time was detected to validate the effect upon coagulation function.Results The expression levels of SCARB1 mRNA and protein were significantly down-regulated after liver xenotransplantation (both P＜0.05).In the cell models,the expression levels of SCARB1 mRNA and protein in the porcine hepatocytes co-cultured with human monocytes were significantly down-regulated compared with those in porcine hepatocytes without intervention (both P＜0.05).Compared with the non-intervention group,the coagulation time was significantly prolonged after the expression of SCARB1 was interfered by lentiviral vector (P＜0.05).Conclusions The down-regulated expression of SCARB1 in the liver graft is one of the main causes of mediating coagulation regulating dysfunction.Intervention of SCARB1 expression contributes to resolve the coagulation regulating dysfunction in the recipients after liver xenotransplantation.