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INTRODUCTION
Organ transplantation is the only alternative treatment for end-stage organ failure. However, the number of available organ donors is significantly lower than the demand for transplants. To address this disparity, research into xenotransplantation, the process of transplanting organs between different species, has been conducted for several decades. Nonhuman primates (NHPs) are particularly valuable in this research due to their close immunological and physiological similarities to humans, which are crucial for predicting human immune responses to pig organ transplants. The pig-primate model, enhanced by the pig's attributes such as ease of breeding, short gestation periods, large litter sizes, and organ size and physiological similarities to humans, has become an essential tool in xenotransplantation studies [1]. However, initial studies encountered significant challenges, notably hyperacute rejection (HAR), which seemed to underscore the limitations of xenotransplantation. HAR occurs within minutes to hours posttransplantation when pre-existing antibodies in the recipient recognize and bind to xenogeneic antigens on the pig's vascular endothelium. This binding triggers a series of reactions including complement activation, endothelial cell lysis, thrombosis, hemorrhagic necrosis, and ultimately, graft failure [2]. Since the development of galactose 1,3-galactose (-Gal) knockout (GTKO) pigs in 2003 [3], there have been significant strides in genetic modifications. These modifications either remove genes responsible for producing pig glycans that trigger HAR or introduce human genes that help regulate complement activation and blood coagulation. Coupled with various tested immunosuppressive protocols, these advancements have significantly prolonged the survival of xenografts in NHP models. Remarkably, the longest recorded survival times for kidney and heart xenografts from gene-edited pigs now stand at 499 days [4] and 265 days [5], respectively.
Significant progress has been made in genetic editing technologies, particularly with CRISPR-Cas9, enabling the use of pig organs for human transplantation. These advancements involve removing pig antigens that trigger human immune rejection and incorporating human genes to enhance compatibility [6]. Gene-edited pigs now represent a promising source of transplantable organs, addressing the critical shortage of human donors. In 2022, the first-ever transplant of a gene-edited porcine heart into a human was performed in the United States. The recipient, a 57-year-old man with terminal heart disease, survived for 2 months following the surgery [5]. In 2024, a gene-edited porcine kidney was transplanted into a 62-year-old human patient with end-stage renal disease in the United States. The organ began functioning immediately, producing urine and effectively clearing creatinine, which are primary indicators of kidney function. However, the patient passed away nearly 2 months after the transplant [7]. These trials have demonstrated the potential to save lives. In 2024, a genetically edited pig liver was transplanted into a 50-year-old brain-dead individual in China. The transplanted liver functioned normally and produced bile for up to 10 days without signs of rejection [8]. This marks the first reported case of a pig liver being transplanted into a human. Although specific details regarding the survival period beyond this point have not yet been disclosed, liver xenotransplantation holds immense potential, especially for patients with acute liver failure, where timely transplantation is often the only viable treatment. Despite the remarkable advancements, the success rate of liver xenotransplantation remains relatively poor. To date, the longest survival time for liver xenografts from genetically modified pigs is 29 days [9]. In this study, we aim to examine the reasons and limitations for the significantly lower survival rates in liver xenotransplantation compared to heart and kidney transplants, focusing primarily on hematological aspects, and discuss future prospects.
CURRENT STATUS OF LIVER XENOTRANSPLANTATION RESEARCH
Research on pig liver transplantation into NHP has lagged behind that of other organs, primarily due to the notably poor survival rates of the grafts.
Studies Using Wild-Type Pigs and Gene-Edited Pigs With Human Genes
Since the first pig-to-NHP liver transplant in 1968 [10], most recipients have succumbed within a day of the procedure due to HAR, which is characterized by widespread intravascular coagulation in the liver and other organs, severe thrombocytopenia, and uncontrollable bleeding [11]. Before 2000, only two studies reported survival times exceeding 3 days in pig-to-NHP liver xenotransplantation. In one study, pig-to-baboon orthotopic liver xenotransplantation resulted in one out of three baboons surviving for 3 days after receiving human fibrinogen [10]. The other study involved pig-to-NHP orthotopic liver xenotransplantation, where a baboon and a cynomolgus monkey each survived for 3 days following the removal of xenoreactive antibodies through ex vivo perfusion using a pig liver from a donor different from the transplant donor [12]. In 2000, Ramirez et al. [13] achieved survival beyond 3 days using gene-edited pigs that expressed human complement regulatory genes. In their experiments, three wild-type (WT) pig livers transplanted into baboons resulted in death within 12 hours posttransplant due to HAR. However, two hCD55 transgenic pig livers managed to extend survival to 4 and 8 days, respectively [13]. In this study, concentrated red blood cells (RBCs) were administered, although it remains unclear if this intervention contributed to the extended survival.
Studies Using GTKO Pigs
The first orthotopic liver xenotransplantation using GTKO pigs was performed in 2010. This procedure involved transplanting eight GTKO/hCD46 (membrane cofactor protein) pig livers into baboons, with survival times ranging from 1 to 7 days. Prior to transplantation, two of the pigs were treated with clodronate liposomes to eliminate macrophages, including Kupffer cells (KCs); however, toxicity issues prevented a favorable outcome [14]. In 2012, Kim et al. [15] conducted pig-to-baboon orthotopic liver xenotransplantations using three GTKO pigs. The baboons survived between 6 and 9 days. During these procedures, the plasmin inhibitor aminocaproic acid was administered to suppress fibrinolysis, concentrated RBCs were transfused, and anti-CD154 monoclonal antibody (mAb) was utilized for the first time in this context. In 2016, Shah et al. [16] managed to extend survival to 25 days in a pig-to-baboon orthotopic liver xenotransplantation. This was achieved through continuous infusion of human prothrombin complex concentrate to address coagulopathy and the administration of cytotoxic T-lymphocyte-associated antigen 4 immunoglobulin (CTLA-4 Ig) belatacept [16,17]. In 2017, the same team replaced belatacept with anti-CD40 mAb, setting a new record with the longest survival time of 29 days [9].
Studies Using Multi-Gene-Edited Pigs
There are currently no published papers on liver xenotransplantation using multi-gene edited pigs. However, a personal communication from the Dou team, as reported by Cross-Najafi et al. [11] and later cited by Huai et al. [18], described a liver from a pig with 13 genetic modifications (four knockout genes: PERV/GalT/4GaNT2/CMAH, and nine transgenes: hCD46/hCD55/hCD59/hβ2M/hHLA-E/hCD47/hTHBD/hTFPI/hCD39) being transplanted into a rhesus monkey, which survived for 26 days. Although this was not a case of whole liver xenotransplantation, Lee et al. [19] recently conducted five instances of left auxiliary liver xenotransplantation in a pig-to-cynomolgus model. Two of these instances resulted in survival of more than 3 weeks without uncontrolled thrombocytopenia or anemia. Notably, one case achieved a graft survival of 34 days, the longest reported to date. The left auxiliary approach used in these cases offers distinct advantages, such as supporting liver function while reducing the physiological demands on the xenograft. This technique could potentially act as a bridge therapy for acute liver failure or augment native liver function in cases of partial failure [19].
The findings discussed are summarized in Table 1 [9,13–17,19–23]. From these results, we can draw some preliminary conclusions. Survival beyond 1 day following WT pig liver xenotransplantation is challenging, primarily due to HAR. This condition is marked by congestion and hemorrhage after reperfusion, which are caused by the activation of endothelial cells and the complement system [13]. Therefore, to ensure survival beyond 1 day, it is essential to: (1) use GTKO pigs or gene-edited pigs with human genes to control complement activation, avoiding endothelial cell damage caused by natural antibody binding; (2) administer antithymocyte globulin to reduce ischemia-reperfusion injury and suppress T cell immunity; (3) use cobra venom factor to suppress complement activation; (4) perform splenectomy to maximize the immunosuppressive effect, as the spleen stores T cells, B cells, and macrophages, all critical for complement activation; (5) administer concentrated RBCs or blood transfusions at appropriate times to avoid ischemia; (6) administer human prothrombin complex to stabilize platelet levels; and (7) block CD40-CD40L costimulation in immunosuppressive treatments, as this seems to have an additive effect; however, it remains unclear whether this is due to the classical T cell-antigen presenting cell costimulation blockade, the CD40-CD40L interaction between macrophages and platelets, or both.
HEMATOLOGICAL ISSUES SPECIFIC TO LIVER XENOTRANSPLANTATION
Coagulation Dysregulation: Molecular Incompatibility Between Species
One of the primary reasons for the short survival times observed in liver xenotransplantation is severe thrombocytopenia [14,24] and coagulation dysfunction, which can lead to fatal bleeding [24–26]. Unlike other organs such as the heart and kidneys, the liver is responsible for producing many proteins that are crucial for blood coagulation. Consequently, differences in the amino acid sequences of these proteins between pigs and NHPs can lead to complications in coagulation and its regulation. In the analysis of coagulation factors, fibrinogen (Factor I) is essential for the formation of fibrin, a critical component of blood clot formation. Prothrombin (Factor II) is converted into thrombin, which in turn converts fibrinogen into fibrin. Factors V, VII, IX, X, XI, XII, and XIII play roles in the coagulation cascade to enhance clotting [27]. The molecular incompatibilities between the coagulation factors of pigs and humans in the intrinsic pathway of the coagulation cascade have not yet been explored. However, it is theorized that interspecies molecular incompatibilities involving fibrinogen and prothrombin, which are crucial to the intrinsic pathway, could lead to either incomplete thrombus formation or excessive clot formation. Specifically, if the fibrinogen produced by pigs is not effectively cleaved by thrombin from NHPs, thrombus formation may be incomplete. On the other hand, excessive activation of prothrombin could result in excessive clot formation. Regarding anticoagulant factors, antithrombin serves to inhibit thrombin and other activated coagulation factors (e.g., Factors IXa, Xa) to prevent excessive clotting. Protein C and protein S become activated when thrombin binds to thrombomodulin. Activated protein C, in conjunction with protein S, helps to degrade Factors Va and VIIIa, thereby regulating coagulation [27]. Additionally, factors related to fibrinolysis include plasminogen, which is activated into plasmin to break down formed fibrin clots, and α2-antiplasmin, which inhibits plasmin to prevent excessive fibrinolysis [28].
Many coagulation and anticoagulation factors are synthesized in the liver and are essential for maintaining blood balance, preventing both excessive bleeding and thrombus formation. The molecular incompatibility due to differences in protein sequences between species can lead to severe bleeding or thrombus formation. This makes liver transplantation more challenging compared to other organs like the heart and kidneys, where the primary concerns are immunological (Fig. 1).
Sequestration and Phagocytosis of Blood Cells by Kupffer Cells and Liver Sinusoidal Endothelial Cells
Thrombocytopenia observed in liver xenotransplantation arises when pig liver cells identify primate platelets as abnormal, leading to their sequestration or phagocytosis. Additionally, excessive platelet activation and depletion due to coagulation dysregulation may also contribute to the loss of platelets [29–32]. It has been suggested that pig liver cells can also recognize and phagocytose primate RBCs, resulting in anemia.
Platelet sequestration/phagocytosis
In an ex vivo perfusion model where human blood was circulated through WT pig liver, 93% of human platelets were removed within 15 minutes, with no evidence of activation in endothelial cells or platelets [33,34]. Liver biopsy showed extensive phagocytosis of platelets by pig KCs and the breakdown of human platelets within hepatocytes [33]. The suppression of β2-integrin (CD18) expression in pig cells, achieved through the use of antibodies or siRNA, significantly reduced the binding and phagocytosis of human platelets. This suggests a central role for β2-integrin in the recognition of human platelets by pig KCs [35]. It has been proposed that KC β2-integrin recognizes β-N-acetyl D-glucosamine on human platelets [35,36]. Additionally, pig KCs express CD40, which enhances the recognition and phagocytosis of activated human platelets via CD40L. Monoclonal antibodies targeting the CD40/CD40L complex have been shown to extend graft survival in pig-to-NHP liver transplantation when used as part of a costimulation blockade therapy [9,37]. Beyond KCs, the involvement of liver sinusoidal endothelial cells (LSECs) in the sequestration of human platelets has also been demonstrated [33]. Pig LSECs recognize and bind the Galβ1-4N-acetylglucosamine (Galβ1,4-NacGlc) glycoprotein on human platelets via the asialoglycoprotein receptor-1 (ASGR1) [38]. When this glycoprotein is removed with asialofetuin or when ASGR1 expression is reduced via siRNA, platelet phagocytosis by LSECs is significantly decreased [38]. Moreover, ex vivo perfusion of ASGR1-deficient pig livers has shown a reduction in human platelet phagocytosis [39]. ASGR1-mediated platelet phagocytosis also occurs in the vascular endothelium of pig aortas and femoral arteries [24,40], suggesting that ASGR1-mediated platelet consumption may be a general mechanism in species-discordant platelet consumption [11]. These findings strongly suggest that the sequestration of recipient platelets by pig LSECs, KCs, and hepatocytes is a major mechanism of thrombocytopenia in liver xenotransplantation (Fig. 1) [11].
Consumptive loss due to excessive platelet activation and coagulation
Molecular incompatibility between species regarding coagulation factors and regulators, coupled with the continuous activation of the coagulation pathway through the extrinsic pathway, has been identified as a major cause of consumptive coagulopathy in liver xenotransplantation [36,41]. Tissue factor (TF) activity is regulated by TF pathway inhibitor (TFPI), which inhibits the TF/VIIa complex by binding to it. However, pig TFPI does not inhibit human TF as effectively as human TFPI does. It has been observed that during heterotopic pig-to-baboon liver xenotransplantation, the increased TF activity in the recipient was not adequately inhibited by the donor's TFPI [20]. Additionally, species incompatibility in the thrombin-thrombomodulin complex can lead to disorders in coagulation regulation during liver xenotransplantation. In vitro studies have demonstrated that although pig thrombomodulin can bind to human thrombin, the resulting thrombin-thrombomodulin complex is ineffective at activating human protein C [42]. To address this issue, gene-edited pig aortic endothelial cells that express human thrombomodulin have been shown to significantly enhance the activity of human-activated protein C in vitro [43]. Furthermore, pig von Willebrand factor (vWF) exhibits stronger binding to human glycoprotein Ib (GpIb), resulting in increased platelet activation in vitro. This enhanced binding is attributed to a higher number of O-linked glycans in pig vWF compared to human vWF [44]. Ex vivo perfusion studies of pig livers with primate blood have shown significantly higher platelet activation, measured by thrombomodulin levels, in the xenoperfusion group compared to the alloperfusion control group. This was accompanied by a significant decrease in platelet count in the xenoperfusion group relative to the control group. These findings suggest that the species incompatibility between pig vWF and primate GpIb, particularly the increased O-linked glycosylation, contributes to heightened platelet activation and consumption. Recent studies have further supported this concept by demonstrating a significant reduction in platelet consumption when human vWF is expressed in pig livers in an ex vivo xenoperfusion model (Fig.1) [45].
Sequestration/destruction of red blood cells
In addition to the sequestration and destruction of platelets by pig liver cells, similar processes involving RBCs are also possible. Studies on pig liver xenoperfusion have shown that over 85% of human RBCs perfused were bound to activated pig KCs and destroyed within 72 hours [46]. Interestingly, pig KCs can bind human RBCs without the need for prior opsonization [46,47]. Following xenotransplantation of liver and lungs, and also in cases involving heart and kidney xenotransplantation, anemia has been observed that is disproportionate to the surgical blood loss [9,36,48,49]. This interaction between pig liver cells and human RBCs is believed to be mediated by Siglec-1, a type of sialoadhesin. When mutant pig cell lines lacking Siglec-1 were tested, they lost the ability to bind human RBCs. Furthermore, treatment with a mAb targeting pig Siglec-1 reduced the binding of human RBCs by nearly 90% [50]. In earlier studies of liver xenotransplantation, a reduction in hematocrit was noted immediately after the transplant, necessitating continuous administration of concentrated RBCs or blood (Table 1). The issues of RBC sequestration, destruction, and the resulting anemia continue to pose significant challenges in liver xenotransplantation.
NECESSITY OF GLYCAN MATCHING AND THE USE OF LIVER-SPECIFIC GENE-EDITED PIGS
Glycan compatibility is a critical factor in the success of xenotransplantation due to the immune system's recognition of nonhuman glycan structures as foreign. In the context of liver transplantation, where the organ performs complex metabolic functions, glycan compatibility becomes even more critical. Incompatible glycans can lead to thrombocytopenia or clotting disorders through interactions between porcine liver glycans and human platelets and coagulation factors. Additionally, porcine glycans can trigger human innate immune responses and inflammation, further exacerbating organ rejection. Therefore, gene editing for glycan synthesis in pigs must be considered to match glycans and glycan receptors across species and improve xenotransplant outcomes. The importance of molecular glycan compatibility in xenotransplantation has been well established through the experience of HAR, which occurs due to the presence of glycans such as alpha-Gal on pig cells, which differ from those in humans. Studies on heart [5,51,52] and kidney xenotransplantation [53–55] using multi-gene-edited pigs that remove two or three types of glycans (DKO [double knockout], TKO [triple knockout]) have been reported. However, to date, there is only one study involving DKO, TKO, and quadruple knockout [19], and its survival rates are not better than those reported in previous studies with GTKO (Table 1). Moreover, as seen in research using hCD55, CD59, H-transferase-introduced pigs, GTKO/hCD46 pigs, GTKO/hCD47, and TKO/hCD46/TBM pigs for liver xenotransplantation, the additive effect of additional introduced genes remains uncertain (Table 1).
Unlike proteins, glycans are not encoded by genetic information but are instead attached to proteins or lipids through a highly complex and less understood process regulated by the activity of sugar-transferring enzymes. Consequently, predicting the outcomes of modifying specific genes related to glycans is challenging. The expression of glycans is influenced by factors such as the activity of each glycosyltransferase or glycosidase at any given time and the concentration of each sugar. This makes it difficult to control glycan expression as straightforwardly as protein expression, which can be regulated through gene knockin or knockout techniques. The glycan profile, including structure and expression levels, of pig cells can change when genes are deleted or human genes are introduced. Additionally, there is a possibility that new glycans may emerge through unknown mechanisms to compensate for the absence of the deleted glycans [56–59]. Except for β2-microglobulin, most of the human genes introduced into gene-edited pigs for xenotransplantation are glycoproteins (Table 2) [60–71]. This could result in a different glycan profile compared to that of pig cells. It remains unclear whether these newly emerged glycans will interact negatively or positively with glycan receptors on recipient macrophages or endothelial cells until the glycan profile of the gene-edited pig cells is confirmed. Nevertheless, targeting liver-specific genes to enhance molecular compatibility between pigs and humans could improve liver-specific coagulation regulatory disorders, as the liver produces many regulatory molecules related to blood coagulation. Key targets may include human TFPI, vWF, protein C, and protein S.
In pig-to-NHP liver transplantation or ex vivo perfusion studies, the sequestration and phagocytosis of platelets and RBCs are driven by interactions between glycans on the surface of pig cells and glycan receptors on NHP cells. Glycan receptors are crucial for distinguishing self from nonself. Therefore, variations in the types or expression levels of glycans on the cells of the donor organ compared to those of the recipient can lead to complications. For instance, LSECs express the glycan receptor ASGR1, which binds to Galβ1,4-NacGlc on human platelets, facilitating their removal [38,39]. Notably, human platelets express this ligand at levels approximately four times higher than those found on pig platelets [72]. This indicates that a certain degree of glycan compatibility between donor and recipient cells is essential for the success of the transplantation.
There is ongoing debate about the use of cytidine monophosphate-N-acetylneuraminic hydroxylase gene knockout (CMAH KO) pigs in pig-old world monkey xenotransplantation research. These pigs lack the enzyme responsible for producing N-glycolylneuraminic acid (Neu5Gc). Old world monkeys, such as baboons, rhesus, and cynomolgus monkeys, express Neu5Gc similarly to pigs, whereas humans do not. This similarity creates a "glycan matching" situation between pigs and old world monkeys, making them suitable preclinical models. However, the use of CMAH KO pigs, which do not produce Neu5Gc, results in "glycan mismatching," raising concerns about their suitability for human xenotransplantation [73–75]. Cui et al. [73] investigated antipig IgM/IgG binding and complement-dependent cytotoxicity using peripheral blood mononuclear cells from GTKO, GTKO/β4GalNT2KO (TKO with the CMAH gene), and TKO (without the CMAH gene) pigs. Their findings indicated that GTKO/β4GalNT2KO pigs exhibited lower antibody binding and cytotoxicity compared to TKO pigs. This led to the suggestion that GTKO/β4GalNT2KO pigs might be more suitable for pig-to-baboon xenotransplantation [73]. Therefore, a dual-track strategy may be necessary: preserving the CMAH gene for preclinical studies and removing it for clinical applications.
CONCLUSION
This study highlights the hematological challenges that impede long-term survival in pig-to-NHP liver xenotransplantation. Key obstacles include coagulation dysregulation due to molecular incompatibilities between species in blood coagulation proteins, and the sequestration or phagocytosis of recipient cells by donor KCs and LSECs, which is triggered by mismatches in glycan structures on the surfaces of platelets and RBCs. Additionally, the anatomical and physiological differences between pig and NHP livers—particularly the incompatibility of numerous liver-derived proteins, whether related to coagulation or not—present even greater challenges compared to the xenotransplantation of other organs such as the heart or kidneys.
To advance this field, future efforts should focus on targeted gene edition, such as introducing human-compatible coagulation regulators (e.g., vWF) or deleting/modulating glycan receptors (e.g., ASGR1) to address coagulation imbalances or platelet sequestration. Further research should also explore strategies for optimizing liver-specific functions, such as modifying noncoagulation-related proteins critical for recipient survival and developing more comprehensive models to evaluate and mitigate these mismatches. By addressing these hematological and molecular challenges, significant progress can be made toward achieving successful and sustainable outcomes in liver xenotransplantation.
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