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One of the prerequisites for successful organ xenotransplantation is a reasonable size match between the porcine organ and the recipient's organ to be replaced. Therefore, the selection of a suitable genetic background of source pigs is important. In this study, we investigated body and organ growth, cardiac function, and genetic diversity of a colony of Auckland Island pigs established at the Center for Innovative Medical Models (CiMM), LMU Munich. Male and female Auckland Island pig kidney cells (selected to be free of porcine endogenous retrovirus C) were imported from New Zealand, and founder animals were established by somatic cell nuclear transfer (SCNT). Morphologically, Auckland Island pigs have smaller body stature compared to many domestic pig breeds, rendering their organ dimensions well-suited for human transplantation. Furthermore, echocardiography assessments of Auckland Island pig hearts indicated normal structure and functioning across various age groups throughout the study. Single nucleotide polymorphism (SNP) analysis revealed higher runs of homozygosity (ROH) in Auckland Island pigs compared to other domestic pig breeds and demonstrated that the entire locus coding the swine leukocyte antigens (SLAs) was homozygous. Based on these findings, Auckland Island pigs represent a promising genetic background for organ xenotransplantation., (© 2024 The Authors. Xenotransplantation published by John Wiley & Sons Ltd.)

Heart transplantation is the only cure for patients with terminal cardiac failure, but the supply of allogeneic donor organs falls far short of the clinical need 1-3 . Xenotransplantation of genetically modified pig hearts has been discussed as a potential alternative 4 . Genetically multi-modified pig hearts that lack galactose-α1,3-galactose epitopes (α1,3-galactosyltransferase knockout) and express a human membrane cofactor protein (CD46) and human thrombomodulin have survived for up to 945 days after heterotopic abdominal transplantation in baboons 5 . This model demonstrated long-term acceptance of discordant xenografts with safe immunosuppression but did not predict their life-supporting function. Despite 25 years of extensive research, the maximum survival of a baboon after heart replacement with a porcine xenograft was only 57 days and this was achieved, to our knowledge, only once 6 . Here we show that α1,3-galactosyltransferase-knockout pig hearts that express human CD46 and thrombomodulin require non-ischaemic preservation with continuous perfusion and control of post-transplantation growth to ensure long-term orthotopic function of the xenograft in baboons, the most stringent preclinical xenotransplantation model. Consistent life-supporting function of xenografted hearts for up to 195 days is a milestone on the way to clinical cardiac xenotransplantation 7 .

Significant progress in understanding and overcoming cardiac xenograft rejection using a clinically relevant large animal pig-to-baboon model has accelerated in recent years. This advancement is based on improved immune suppression, which attained more effective regulation of B lymphocytes and possibly newer donor genetics. These improvements have enhanced heterotopic cardiac xenograft survival from a few weeks to over 2 years, achieved intrathoracic heterotopic cardiac xenograft survival of 50 days and orthotopic survival of 57 days. This encouraging progress has rekindled interest in xenotransplantation research and refocused efforts on preclinical orthotopic cardiac xenotransplantation., (Copyright © 2015 IJS Publishing Group Limited. All rights reserved.)

Background: Among other mismatches between human and pig, incompatibilities in the blood coagulation systems hamper the xenotransplantation of vascularized organs. The provision of the porcine endothelium with human thrombomodulin (hTM) is hypothesized to overcome the impaired activation of protein C by a heterodimer consisting of human thrombin and porcine TM., Methods: We evaluated regulatory regions of the THBD gene, optimized vectors for transgene expression, and generated hTM expressing pigs by somatic cell nuclear transfer. Genetically modified pigs were characterized at the molecular, cellular, histological, and physiological levels., Results: A 7.6-kb fragment containing the entire upstream region of the porcine THBD gene was found to drive a high expression in a porcine endothelial cell line and was therefore used to control hTM expression in transgenic pigs. The abundance of hTM was restricted to the endothelium, according to the predicted pattern, and the transgene expression of hTM was stably inherited to the offspring. When endothelial cells from pigs carrying the hTM transgene--either alone or in combination with an aGalTKO and a transgene encoding the human CD46-were tested in a coagulation assay with human whole blood, the clotting time was increased three- to four-fold (P<0.001) compared to wild-type and aGalTKO/CD46 transgenic endothelial cells. This, for the first time, demonstrated the anticoagulant properties of hTM on porcine endothelial cells in a human whole blood assay., Conclusions: The biological efficacy of hTM suggests that the (multi-)transgenic donor pigs described here have the potential to overcome coagulation incompatibilities in pig-to-primate xenotransplantation.

Background: Heterotopic thoracic heart transplantation may be an alternative to the established heterotopic abdominal or orthotopic cardiac xenotransplantation model as it combines the safety of heterotopic transplantation with the benefit of a working heart model., Methods: In a first series of two animals, we tested the surgical feasibility of this procedure with non-transgenic pig hearts transplanted into pre-sensitized baboons (killed after weaning from cardiopulmonary bypass). In a second group (n = 2), double-transgenic alpha(1,3)galactosyl-transferase knock out/hCD46 pig hearts were transplanted into naïve baboons after immunoadsorption. The immunosuppressive regimen consisted of anti-CD20-mAb, tacrolimus, sirolimus, MMF and steroids., Results: The first baboon was successfully transplanted, but died of an air embolism, while in the second animal graft survival was 50 days with the recipient in good physical condition. One rejection reaction was successfully treated by immunoadsorption, ATG and the proteasome inhibitor bortezomib. Post-mortem histopathology showed no evidence for humoral or cellular rejection of the cardiac xenograft., Conclusions: This is the first description of a heterotopic thoracic pig-to-baboon heart transplantation. The procedure combines the advantages of a working heart model with the safety of heterotopic transplantation. In contrast to orthotopic transplantation, the recipient heart can assist the donor heart during episodes of rejection. We believe that the heterotopic thoracic model may accelerate the progress into the clinical application of cardiac xenotransplantation. However, successful combination of this heterotopic transplantation with an experimental model of cardiac failure may be needed before this technique can be promoted to clinical trials.

Background: Only limited data are available on the physiological functional compatibility of cardiac xenografts after orthotopic pig to baboon transplantation (oXHTx). Thus we investigated hemodynamic parameters including cardiac output (CO) before and after oXHTx., Methods: Orthotopic xenogeneic heart transplantation from nine hDAF transgeneic piglets to baboons was performed. We used femoral arterial thermodilution for the invasive assessment of CO and stroke volume., Results: Baseline CO of the baboons after induction of anesthesia was 1.36 (1.0-1.9) l/min. 30 to 60 min after termination of the cardiopulmonary bypass, CO of the cardiac xenograft was significantly increased to 1.72 (1.3-2.1) l/min (P < 0.01). The stroke volumes of the baboon heart before transplantation and the cardiac xenograft was comparable [14.9 (11-26) vs. 11.8 (10-23) ml]. Thus the higher CO was achieved by an increase in heart rate after oXHTx [75.0 (69-110) vs. 140.0 (77-180)/min; P < 0.01]. Despite the increased CO, oxygen delivery was reduced [256 (251-354) vs. 227 (172-477) ml/min; P < 0.01] due to the inevitable hemodilution during the cardiopulmonary bypass and the blood loss caused by the surgical procedures., Conclusion: Our results demonstrate that in the early phase after orthotopic transplantation of hDAF pig hearts to baboons, cardiac function of the donor heart is maintained and exceeds baseline CO. However, in the early intraoperative phase this was only possible by using inotropic substances and vasopressors due to the inevitable blood loss and dilution by the priming of the bypass circuit.

Background: Long-term survival of transgenic cardiac xenografts is currently limited by a form of humoral rejection named acute vascular rejection. Preformed and elicited cytotoxic antibodies against Galalpha(1,3)Gal terminating carbohydrate chains, known as the primary cause of hyperacute rejection, are crucial for this process. We investigated whether GAS914, a soluble, polymeric form of a Galalpha(1,3)Gal trisaccharide would sufficiently minimize xenograft rejection of hDAF-transgenic pig hearts orthotopically transplanted into baboons., Methods: Orthotopic heart transplantations were performed using hDAF transgenic piglets as donors and four non-splenectomized baboons as recipients. Baseline immunosuppression consisted of tacrolimus, sirolimus, ATG, steroids. In addition two animals received low-dose GAS914, and two animals high-dose GAS914. One of these baboons received high dose GAS914 and cyclophosphamide induction therapy. Serum levels of anti-Galalpha(1,3)Gal IgM and IgG antibodies, and anti-pig antibodies were controlled daily by anti-Galalpha(1,3)Gal enzyme-linked immunosorbant assay and anti-pig hemolytic assays. Histomorphological (hematoxylin and eosin, elastic van Gieson) and immunohistochemical (IgM, IgG) evaluations were performed on tissue specimens., Results: Following low-dose GAS914 therapy survival time was 1 and 9 days, respectively. In baboons treated with high dosages of GAS914 a survival of 30 h and 25 days could be obtained. GAS914 caused an immediate and significant reduction of both anti-Galalpha(1,3)Gal IgM and IgG antibodies. However, sufficient antibody reduction was independent of dosage and form of application of GAS914. A pre-transplant GAS914 treatment was not necessary to effectively reduce antibody levels and prevent hyperacute rejection. In the early postoperative period preformed anti-pig antibodies corresponded predominantly to anti-Galalpha(1,3)Gal antibodies making them susceptible to GAS914. Subsequently, while anti-Galalpha(1,3)Gal antibodies remained low, anti-pig antibodies increased despite of GAS914 application. Corresponding to increased anti-pig antibody titers depositions of IgM and IgG immunoglobulins were detected, which were possibly non-Galalpha(1,3)Gal-specific., Conclusions: Following orthotopic transplantation of hDAF-transgenic pig hearts into baboons, GAS914 is able to maintain a sufficient reduction of Galalpha(1,3)Gal-specific cytotoxicity to the graft. GAS914 therefore is able to prevent not only hyperacute rejection, but also acute vascular rejection at its beginning, when serum cytotoxicity to the pig heart appears to be predominantly Galalpha(1,3)Gal-specific. A sustained prevention of acute vascular rejection, however, still requires the identification of antibody specificities other than to Galalpha(1,3)Gal.

Xenotransplantation, like allotransplantation, is usually associated with microchimerism, i.e., the presence of cells from the donor in the recipient. Microchimerism was reported in first xenotransplantation trials in humans, as well as in most preclinical trials in nonhuman primates (for review, see Denner, Viruses 2023, 15, 190). When using pigs as xenotransplantation donors, their cells contain porcine endogenous retroviruses (PERVs) in their genome. This makes it difficult to discriminate between microchimerism and PERV infection of the recipient. Here, we demonstrate the appropriate virological methods to be used for the identification of microchimerism, first by screening for porcine cellular genes, and then how to detect infection of the host. Using porcine short interspersed nuclear sequences (SINEs), which have hundreds of thousands of copies in the pig genome, significantly increased the sensitivity of the screening for pig cells. Second, absence of PERV RNA demonstrated an absence of viral genomic RNA or expression as mRNA. Lastly, absence of antibodies against PERV proteins conclusively demonstrated an absence of a PERV infection. When applying these methods for analyzing baboons after pig heart transplantation, microchimerism could be demonstrated and infection excluded in all animals. These methods can be used in future clinical trials. [ABSTRACT FROM AUTHOR]

Background: Many genetically multi‐modified donor lines for xenotransplantation have a background of domestic pigs with rapid body and organ growth. The intrinsic growth potential of porcine xeno‐organs may impair their long‐term function after orthotopic transplantation in non‐human primate models. Since growth hormone is a major stimulator of postnatal growth, we deleted its receptor (GHR‐KO) to reduce the size of donor pigs in one step. Methods: Heart weight and proteome profile of myocardium were investigated in GHR‐KO and control pigs. GHR‐KO mutations were introduced using CRISPR/Cas9 in an α1,3‐galactosyltransferase (GGTA1)‐deficient background expressing the human cluster of differentiation (hCD46) and human thrombomodulin (hTHBD) to generate quadruple‐modified (4GM) pigs. Results: At age 6 months, GHR‐KO pigs had a 61% reduced body weight and a 63% reduced heart weight compared with controls. The mean minimal diameter of cardiomyocytes was 28% reduced. A holistic proteome study of myocardium samples from the two groups did not reveal prominent differences. Two 4GM founder sows had low serum insulin‐like growth factor 1 (IGF1) levels (24 ± 1 ng/mL) and reached body weights of 70.3 and 73.4 kg at 9 months. Control pigs with IGF1 levels of 228 ± 24 ng/mL reached this weight range three months earlier. The 4GM sows showed normal sexual development and were mated with genetically multi‐modified boars. Offspring revealed the expected Mendelian transmission of the genetic modifications and consistent expression of the transgenes. Conclusion: GHR‐KO donor pigs can be used at an age beyond the steepest phase of their growth curve, potentially reducing the problem of xeno‐organ overgrowth in preclinical studies. [ABSTRACT FROM AUTHOR]

Abstract: Background: In pig‐to‐human xenotransplantation, early cellular rejection reactions are mediated by natural killer cells (NK cells). Human NK cells are inhibited by HLA‐E via CD94/NKG2A receptors. To protect porcine grafts against human NK cell responses, transgenic GTKO pigs expressing hCD46 and HLA‐E have been generated. The aim of this study was to test the effect of this genetic modification on xenogeneic, and in particular human NK cell response, using an ex vivo perfusion model of pig hearts with human blood. Methods: Cardiopleged and explanted genetically modified (gm) pig hearts (GTKO/hCD46/HLA‐E/hβ2‐microglobulin) and wild‐type (wt) controls (n = 6 each) were reperfused and tested in an 8 hours ex vivo perfusion system using freshly drawn human blood. Cardiac function was evaluated during a 165‐minute period in working heart mode. Myocardial damage, antibody deposition, complement activation, and coagulation parameters were evaluated histologically at the end of perfusion. The number of NK cells in the perfusate was determined by flow cytometry at baseline and at 8 hours; tissue infiltration by NK cells was quantified by immunofluorescence microscopy using NKp46 staining of frozen sections. Results: Deposition of IgG (1.2 ± 1 × 107 vs 8.8 ± 2.9 × 106; P < .01), IgM (4.4 ± 3.7 × 106 vs 1.7 ± 1.2 × 106; P < .01), and the complement activation product C4b/c (3.5 ± 1.3 × 106 vs 2.3 × 106 ± 9.4 × 105; P > .01) was lower in gm than wt hearts. NK cell percentages of leukocytes in the perfusate decreased from 0.94 ± 0.77% to 0.21 ± 0.25% (P = .04) during xenoperfusion of wt hearts. In contrast, the ratio of NK cells did not decrease significantly in the gm hearts. In this group, NK cell myocardial infiltration after 480 minutes of perfusion was lower than in wt organs (2.5 ± 3.7 × 104/mm3 vs 1.3 ± 1.4 × 105/mm3; P = .0001). The function of gm hearts was better preserved compared to wt organs, as demonstrated by higher cardiac index during the first 2 hours of ex vivo perfusion. Conclusion: GTKO, hCD46, and HLA‐E expression in porcine hearts reduced complement deposition, complement dependent injury, and myocardial NK cell infiltration during perfusion with human blood. This tested combination of genetic modifications may minimize damage from acute human‐anti‐pig rejection reactions and improve myocardial function after xenotransplantation. [ABSTRACT FROM AUTHOR]

Abstract: For clinical xenotransplantation, transplants must be free of porcine cytomegalovirus (PCMV). Piglets become infected primarily in the perinatal period by the mother sow. While individual donor animals can be protected from infection by isolation husbandry, success is not guaranteed and this strategy poses the risk of undetected infections and raises animal welfare questions. Here, we present the establishment of a completely PCMV‐negative pig herd for breeding donor animals for xenotransplantation. Eleven pregnant DanAvl Basic hybrid sows were purchased from a designated pathogen‐free (DPF), PCMV‐positive colony and transferred to a new pig facility at the Centre for Innovative Medical Models (CiMM) 4 weeks prior to farrowing. At the age of 24 hours, piglets were early‐weaned and transferred to a commercially available Rescue Deck system dedicated to motherless rearing of piglets. Sows were removed from the facility. The PCMV status of F1‐generation animals was determined at regular intervals over a period of 14 months by a sensitive real‐time PCR‐based detection method testing blood, nasal swabs and cultured peripheral blood mononuclear cells (PBMCs). F1 sows were used as recipients of genetically modified embryos to generate a xenotransplant donor herd. Offspring were tested for PCMV accordingly. All offspring have remained PCMV negative over the whole observation period of 14 months. A completely PCMV‐negative pig herd for xenotransplantation has thus been successfully established. [ABSTRACT FROM AUTHOR]

Background The complement system plays a crucial role in acute xenogeneic reactions after cardiac transplantation. We used an ex vivo perfusion model to investigate the effect of Cp40, a compstatin analog and potent inhibitor of complement at the level of C3. Methods Fifteen wild-type pig hearts were explanted, cardiopleged, and reperfused ex vivo after 150 minutes of cold ischemia. Hearts were challenged in a biventricular working heart mode to evaluate cardiac perfusion and function. In the treatment group (n=5), the complement cascade was blocked at the level of C3 using Cp40, using diluted human blood. Untreated human and porcine blood was used for controls. Results Throughout the perfusion, C3 activation was inhibited when Cp40 was used (mean of all time points: 1.11 ± 0.34% vs 3.12 ± 0.48% control activation; P<.01). Compared to xenoperfused controls, the cardiac index improved significantly in the treated group (6.5 ± 4.2 vs 3.5 ± 4.8 mL/min/g; P=.03, 180 minutes perfusion), while the concentration of lactate dehydrogenase as a maker for cell degradation was reduced in the perfusate (583 ± 187 U/mL vs 2108 ± 1145 U/mL, P=.02). Histological examination revealed less hemorrhage and edema, and immunohistochemistry confirmed less complement fragment deposition than in untreated xenoperfused controls. Conclusions Cp40 efficiently prevents C3 activation of the complement system, resulting in reduced cell damage and preserved function in wild-type porcine hearts xenoperfused ex vivo. We suggest that this compstatin analog, which blocks all main pathways of complement activation, could be a beneficial perioperative treatment in preclinical and in future clinical xenotransplantation. [ABSTRACT FROM AUTHOR]

Bauer A, Renz V, Baschnegger H, Abicht J-M, Beiras-Fernandez A, Brenner P, Thein E, Schmoeckel M, Reichart B, Christ F. Microcirculatory alterations after orthotopic pig-to-baboon heart transplantation. Xenotransplantation 2011; 18: 232-238. © 2011 John Wiley & Sons A/S. Abstract: Background: Whilst macrohemodynamic function of porcine xenografts transplanted into baboons has been assessed perioperatively, the ability of the xenograft to maintain systemic microcirculatory perfusion has not been investigated after pig-to-baboon xenotransplantation so far. Methods: We investigated the sublingual microcirculation of six baboons undergoing orthotopic transplantation of hCD46-transgenic pig hearts using orthogonal polarization spectral imaging. Microvascular measurements were performed after induction of anesthesia, in the early phase of cardiopulmonary bypass (CPB), during reperfusion of the porcine heart and 1 h after the xenograft had resumed its life-supporting function. Microvascular blood flow was analyzed semiquantitatively and the number of visualized cell-to-cell interactions was counted. Results: The proportion of continuously perfused microvessels was 97 (96 to 97) % at baseline and 95 (94 to 97) % in the early phase of CPB. It decreased significantly (P < 0.05) during CPB to 89 (84 to 91), and alterations were still present (P < 0.05) when CPB was terminated and the xenograft had taken over systemic perfusion 83 (81 to 85) %. The microcirculatory changes correlated with the lactate levels ( y = 18.1-0.18 x; r2 = 0.55; P < 0.001), but no correlation with macrohemodynamic parameters was found. Conclusion: Microvascular blood flow is altered after orthotopic pig-to-baboon heart transplantation, despite systemic hemodynamic parameters being well maintained by the porcine xenograft. These changes are moderate but persist after termination of CPB. Further studies need to elucidate whether these changes are transient or add to the mortality associated with cardiac xenotransplantation. [ABSTRACT FROM AUTHOR]

Porcine circovirus 3 (PCV3) is a newly described member of the virus family Circoviridae. PCV3 is highly distributed among pigs and wild boars worldwide. A sudden introduction of PCV3 was recently observed in a herd of triple genetically modified pigs generated for xenotransplantation. These animals were used as donor pigs for orthotopic heart transplantation into baboons. In four cases, PCV3-positive hearts were transplanted, and transmission of PCV3 to the recipient was observed. PCV3 was found in all organs of the recipient baboons and a higher virus load was found in animals with a longer survival time of the transplant, indicating replication of the virus. This is the first report showing trans-species transmission of PCV3 to baboons by transplantation of a heart from a PCV3-positive donor pig. Sequence analysis showed that PCV3a and PCV3b were present in the infected pigs and were transmitted. Experiments to infect human 293 cells with PCV3 failed. [ABSTRACT FROM AUTHOR]

An abstract of the study "Heterotopic thoracic cardiac pig-to-baboon xenotransplantation," by Stefan Buchholz and colleagues is presented.

Background: Diabetes mellitus causes microcirculatory rarefaction and may impair the responsiveness of ischemic myocardium to proangiogenic factors.Objectives: This study sought to determine whether microvascular destabilization affects organ function and therapeutic neovascularization in diabetes mellitus.Methods: The authors obtained myocardial samples from patients with end-stage heart failure at time of transplant, with or without diabetes mellitus. Diabetic (db) and wild-type (wt) pigs were used to analyze myocardial vascularization and function. Chronic ischemia was induced percutaneously (day 0) in the circumflex artery. At day 28, recombinant adeno-associated virus (rAAV) (5 × 1012 viral particles encoding vascular endothelial growth factor-A [VEGF-A] or thymosin beta 4 [Tβ4]) was applied regionally. CD31+ capillaries per high power field (c/hpf) and NG2+ pericyte coverage were analyzed. Global myocardial function (ejection fraction [EF] and left ventricular end-diastolic pressure) was assessed at days 28 and 56.Results: Diabetic human myocardial explants revealed capillary rarefaction and pericyte loss compared to nondiabetic explants. Hyperglycemia in db pigs, even without ischemia, induced capillary rarefaction in the myocardium (163 ± 14 c/hpf in db vs. 234 ± 8 c/hpf in wt hearts; p < 0.005), concomitant with a distinct loss of EF (44.9% vs. 53.4% in nondiabetic controls; p < 0.05). Capillary density further decreased in chronic ischemic hearts, as did EF (both p < 0.05). Treatment with rAAV.Tβ4 enhanced capillary density and maturation in db hearts less efficiently than in wt hearts, similar to collateral growth. rAAV.VEGF-A, though stimulating angiogenesis, induced neither pericyte recruitment nor collateral growth. As a result, rAAV.Tβ4 but not rAAV.VEGF-A improved EF in db hearts (34.5 ± 1.4%), but less so than in wt hearts (44.8 ± 1.5%).Conclusions: Diabetes mellitus destabilized microvascular vessels of the heart, affecting the amplitude of therapeutic neovascularization via rAAV.Tβ4 in a translational large animal model of hibernating myocardium. [ABSTRACT FROM AUTHOR]