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Survival of pig cardiac xenografts in a non-human primate (NHP) model has improved significantly over the last 4 years with the introduction of costimulation blockade based immunosuppression (IS) and genetically engineered (GE) pig donors. The longest survival of a cardiac xenograft in the heterotopic (HHTx) position was almost 3 years and only rejected when IS was stopped. Recent reports of cardiac xenograft survival in a life-sustaining orthotopic (OHTx) position for 6 months is a significant step forward. Despite these achievements, there are still several barriers to the clinical success of xenotransplantation (XTx). This includes the possible transmission of porcine pathogens with pig donors and continued xenograft growth after XTx. Both these concerns, and issues with additional incompatibilities, have been addressed recently with the genetic modification of pigs. This review discusses the spectrum of issues related to cardiac xenotransplantation, recent progress in preclinical models, and its feasibility for clinical translation.

Upregulation of tissue factor (TF) expression on activated donor endothelial cells (ECs) triggered by the immune response (IR) has been considered the main initiator of consumptive coagulopathy (CC). In this study, we aimed to identify potential factors in the development of thrombocytopenia and CC after genetically engineered pig liver transplantation in baboons. Baboons received a liver from either an α1,3-galactosyltransferase gene-knockout (GTKO) pig (n = 1) or a GTKO pig transgenic for CD46 (n = 5) with immunosuppressive therapy. TF exposure on recipient platelets and peripheral blood mononuclear cell (PBMCs), activation of donor ECs, platelet and EC microparticles, and the IR were monitored. Profound thrombocytopenia and thrombin formation occurred within minutes of liver reperfusion. Within 2 h, circulating platelets and PBMCs expressed functional TF, with evidence of aggregation in the graft. Porcine ECs were negative for expression of P- and E-selectin, CD106, and TF. The measurable IR was minimal, and the severity and rapidity of thrombocytopenia were not alleviated by prior manipulation of the IR. We suggest that the development of thrombocytopenia/CC may be associated with TF exposure on recipient platelets and PBMCs (but possibly not with activation of donor ECs). Recipient TF appears to initiate thrombocytopenia/CC by a mechanism that may be independent of the IR.

Although human complement-dependent cytotoxicity (CDC) of alpha1,3-galactosyltransferase gene-knockout (GTKO) pig cells is significantly weaker than that of wild-type (WT) cells, successful xenotransplantation will require pigs with multiple genetic modifications. Sera from healthy humans were tested by (i) flow cytometry for binding of IgM/IgG, and (ii) CDC assay against peripheral blood mononuclear cells and porcine aortic endothelial cells from five types of pig - WT, GTKO, GTKO transgenic for H-transferase (GTKO/HT), WT transgenic for human complement regulatory protein CD46 (CD46) and GTKO/CD46. There was significantly higher mean IgM/IgG binding to WT and CD46 cells than to GTKO, GTKO/HT, and GTKO/CD46, but no difference between GTKO, GTKO/HT, and GTKO/CD46 cells. There was significantly higher mean CDC to WT than to GTKO, GTKO/HT, CD46, and GTKO/CD46 cells, but no difference between GTKO and GTKO/HT. Lysis of GTKO/CD46 cells was significantly lower than that of GTKO or CD46 cells. CD46 expression provided partial protection against serum from a baboon sensitized to a GTKO pig heart. GTKO/CD46 cells were significantly resistant to lysis by human serum and sensitized baboon serum. In conclusion, the greatest protection from CDC was obtained by the combination of an absence of Gal expression and the presence of CD46 expression, but the expression of HT appeared to offer no advantage over GTKO. Organs from GTKO/CD46 pigs are likely to be significantly less susceptible to CDC.

The production of alpha1,3-galactosyltransferase gene-knockout (GT-KO) pigs has overcome the barrier of preformed anti-Galalpha1,3Gal (Gal) antibodies that has inhibited progress in pig-to-primate organ xenotransplantation for many years. Survival of GT-KO pig organs in nonhuman primates is currently limited by the development of a thrombotic microangiopathy that results in increasing ischemic injury of the transplanted organ over weeks or months. Potential causative factors include vascular endothelial activation from preformed anti-nonGal antibodies or cells of the innate immune system that recognize nonGal pig antigens directly, and coagulation dysregulation associated with molecular incompatibilities between pig and primate. Carefully isolated pancreatic islets from wild-type (genetically unmodified) adult pigs express minimal Gal epitopes, allowing survival sometimes for weeks or months after transplantation into nonhuman primates receiving immunosuppression directed only at T-cell function. However, there is a considerable immediate loss of islets, probably related to activation of coagulation and complement cascades. Further genetic manipulation of organ-source pigs is therefore required to overcome these problems. GT-KO pigs expressing a human complement-regulatory protein, e.g. decay-accelerating factor, and/or an 'anti-coagulant' gene, e.g. human tissue factor pathway inhibitor, might prevent the change in vascular endothelium from an anti-coagulant to a procoagulant phenotype, and protect the islets from early loss.