Genetically engineered porcine liver: a new era in transplantation

Abstract

The persistent shortage of allogenic donor organs has driven the exploration of xenotransplantation as a viable alternative for patients with end-stage liver disease. This review investigates the progress and challenges in liver xenotransplantation, focusing on the use of genetically engineered animals, particularly pigs, as potential organ donors. This narrative review discusses various sources of xenografts, highlighting their advantages and limitations, and provides a historical overview of liver xenotransplantation, encompassing both clinical and experimental experiences. Key obstacles, such as immunological rejection, coagulation dysfunction, and the risk of zoonotic infections, are analyzed, with emphasis on how these challenges have been addressed through genetic modifications and adjunctive therapies. Recent clinical trials are also reviewed, offering insights into the outcomes and lessons learned from these pioneering efforts. We also explored the regulatory, legal, and ethical considerations critical to advancing liver xenotransplantation into clinical practice, along with future perspectives on the field’s evolution. This comprehensive overview aims to provide a clear understanding of the current state of liver xenotransplantation and the strategies being developed to overcome existing barriers, paving the way for future clinical applications.

Introduction

Liver transplantation is considered the only life-saving procedure for patients suffering from a range of liver conditions, including cirrhosis, decompensated disease, acute liver failure, and hepatocellular carcinoma^[1]. Unfortunately, the shortage of donor organs continues to severely limit this life-saving option. Globally, 34 694 liver transplants were performed in 2021, marking an increase of 6.5% from 2020 and a 20% rise since 2015, yet this remains far below the actual need^[2]. Although more than 100 countries performed at least one liver transplant in 2020–2021, most patients worldwide with life-threatening liver disease still lack realistic access to this treatment^[3]. Moreover, the rising population of patients suffering from end-stage liver diseases continues to exacerbate the gap between available organ donors and those in need, resulting in a substantial waiting list. For example, as of 2024, there were 9986 patients on the waiting list in the United States according to the Organ Procurement and Transplantation Network^[4]. Each year, hundreds of patients on the waiting list are removed due to deteriorating health conditions that render them too sick for the surgical procedure or result in death prior to receiving a life-saving liver transplant^[5]. Vulnerable populations, such as Black patients, those living in poverty or rural areas, and children under 1 year of age, face an even greater risk, with 5% of pediatric candidates dying or becoming too ill to undergo transplantation^[3,6]. Addressing the growing demand for transplantation represents a significant challenge to healthcare systems worldwide.

Xenotransplantation, the practice of transplanting organs, tissues, or cells from one species to another, has emerged as a promising solution to overcome the current deficit of donor organs for patients with end-stage organ failure^[7]. In the last few years, significant progress has been achieved in the field of xenotransplantation due to advancements in immunology, biomedicine, and genetic engineering approaches^[8,9].

This review provides an overview of recent advancements in liver xenotransplantation, with a specific focus on the obstacles contributing to graft failure and recipient mortality. Genetic modifications targeted at overcoming the obstacles will also be discussed. Furthermore, we will discuss the recent in vivo clinical findings, shedding light on the progress and challenges in this evolving field of transplantation research. We will also briefly discuss the regulatory, legal, and ethical issues associated with the proposed application of liver xenotransplantation in the clinical setting. Relevant literature of this narrative review was primarily identified through non-systematic searches of PubMed, Scopus, and Google Scholar. We used keyword combinations including “genetically engineered pig liver,” “liver xenotransplantation,” “porcine xenograft,” “xenozoonosis,” “immunological rejection,” and “coagulation dysfunction.” Priority was given to publications from the last 15–20 years to highlight the latest advances, though older foundational studies were also included to provide historical context. Additional sources were identified by screening the reference lists of key articles. Our most recent search was performed prior to resubmission on 10 January 2026. This work has been reported in line with the TITAN (Transparency In The reporting of Artificial iNTelligence) Guideline Checklist 2025 for the transparent reporting of artificial intelligence use in scholarly manuscripts^[10].

Sources of livers for xenotransplantation

Non-human primates (NHPs) and pigs are the primary candidates for organs used in xenotransplantation. Each of these animal species offers unique advantages and challenges that must be carefully considered to achieve successful xenotransplantation.

Non-human primates

Chimpanzees and monkeys exhibit anatomical and physiological similarities to humans that make them attractive candidates for xenotransplantation. Their genetic profile closely resembles that of humans, reducing the risk of immune rejection and improving graft survival^[11]. However, ethical concerns surrounding the use of NHPs in research, coupled with limited availability and high breeding costs, are considered significant prohibitive challenges to their widespread use as organ donors^[12,13]. Baboons, another species of NHPs, offer similar advantages to chimpanzees and cynomolgus monkeys in terms of genetic compatibility and physiological resemblance to humans^[14]. Additionally, baboons have a relatively short gestation period and rapid reproductive rates, potentially increasing the availability of donor organs.

Despite these potential advantages, the use of NHPs in xenotransplantation has been largely abandoned due to the high risk of viral transmission to humans. For instance, a study by Michaels et al reported the transmission of baboon cytomegalovirus (BCMV) to a human recipient of a baboon liver transplant^[15]. Replication-competent BCMV was detected in blood samples collected on days 29, 36, and 42 post-transplantation, alongside human cytomegalovirus DNA, highlighting the zoonotic transmission risk posed by such procedures. Similarly, Allan et al documented the presence of simian retroviruses, including simian foamy virus and baboon endogenous virus, in multiple tissues of two human recipients of baboon liver transplants^[16]. The persistence of these viruses, along with the detection of baboon mitochondrial DNA, indicated that “passenger leukocytes” from the xenografts had migrated to human tissues, carrying latent or active viral infections. These findings highlight the significant infectious risks associated with xenotransplantation using baboon donors, raising concerns about the potential emergence of new zoonotic diseases. Ethical considerations and documented risks of viral transmission emphasize the limitations of using NHPs as organ donors, further motivating the exploration of alternative xenotransplantation approaches^[17].

Pigs

Pigs are the most promising source of organs for xenotransplantation due to their physiological compatibility with humans, large litter sizes, rapid growth, and relatively short generation times^[18]. Pigs also encounter fewer social objections regarding their utilization, based on their current use as a source of food. They can also be raised under specific pathogen-free (SPF) housing conditions. Additionally, their organs are similar in size to those of humans, and there is a reasonable genetic similarity between pig and human^[19]. Genetic engineering techniques have facilitated the development of genetically modified pigs with reduced immunogenicity and enhanced organ function, addressing some of the key challenges in xenotransplantation. Despite these advantages, concerns about the risk of cross-species transmission of infectious diseases, known as xenozoonosis, and the potential for immune rejection still warrant careful consideration^[20].

Historical clinical experience of liver xenotransplantation

Liver xenotransplantation has evolved over the past several decades through a series of evolving strategies. Initial efforts involved extracorporeal liver perfusion (ECLP) as a temporary support measure, followed by auxiliary (heterotopic) and orthotopic transplantation trials in both humans and NHPs. While early clinical attempts were limited by technical and immunological challenges, these studies provided critical insights into organ viability, graft function, and species-specific barriers, laying the groundwork for modern genetically engineered porcine liver xenotransplantation.

Extracorporeal liver hemoperfusion

ECLP involves directing a patient’s blood through a whole liver, from either a human or an animal source^[21] and was first described in 1958 by Otto as an experimental model in dogs^[22,23]. This technique was developed to offer temporary support for those experiencing reversible hepatic failure, especially those at risk of death before a suitable human donor liver becomes available or who are ineligible for liver allotransplantation^[24]. It was intended to facilitate the recovery of native liver function during the regenerative process or serve as a bridge to eventual allotransplantation.

In 1964, Sen and colleagues conducted one of the earliest clinical trials involving ECLP using human cadaveric livers to treat four patients with hepatic coma. The perfusions led to biochemical improvements in all patients; however, survival was not prolonged^[25]. In 1965, however, Eiseman et al^[26] pioneered ECLP using pig livers in eight patients who were unresponsive to other treatments and deemed to have a terminal prognosis. These patients underwent liver perfusion for durations ranging from 1 to 6.5 hours. Clinical outcomes varied, with some patients exhibiting minor to significant neurological improvements; however, none of the patients achieved long-term survival. ECLP was then used sporadically over the last four decades due to advancements toward orthotopic liver transplantation (OLT).

Subsequent studies documented that the first use of transgenic porcine livers as a bridge to transplantation was in 2000^[27]. This small study involved two patients with acute liver failure who lacked available human donors. The patients underwent ECLP for over 16 hours, allowing sufficient time to locate and transport a human liver to the transplant center. Both patients subsequently received successful human liver transplants, resulting in excellent graft function and recovery.

Although the sample size is limited, studies on ECLP using pig livers in patients with acute liver failure have suggested a potential survival benefit^[24]. During the short support periods evaluated, pig livers provided life-sustaining support comparable to human livers.

These trials also yielded important immunological insights. Early observations indicated that pig livers might undergo relatively attenuated rejection compared with other porcine organs, possibly related to reduced complement activity in liver failure^[28]. However, subsequent evidence has challenged this assumption, underscoring the complexity of liver-specific immune responses in xenotransplantation^[29].

Clinically, pig liver support may be particularly valuable for patients with acute liver failure, such as acetaminophen toxicity, who retain regenerative potential but require temporary hepatic support during recovery^[24].

Auxiliary liver xenotransplantation

Dr. Starzl, a pioneer in clinical liver xenotransplantation, first reported the use of an auxiliary or heterotopic liver transplantation as a short-term viable alternative for managing hepatic failure^[30–32]. This approach has been documented in seven cases^[33], with the first reported by Starzl et al, who used a chimpanzee liver as temporary hepatic support for a 7-year-old patient, connecting it to femoral vessels^[31]. These grafts functioned briefly, producing bile and clearing bilirubin, but synthetic function remained limited, and thrombosis rapidly compromised the graft. The heterograft was removed 25 hours after revascularization; it became discolored and turgid due to multiple thrombi in the hepatic artery^[31]. Subsequent attempts with baboon donors (1969–1971) achieved survival of less than 3 days^[28,33].

In 1995, Makowka et al reported the first pig-to-human clinical liver xenotransplantation in a 26-year-old woman with long-standing autoimmune hepatitis and hepatitis C^[34]. The procedure was undertaken as a bridge therapy to provide temporary metabolic support while awaiting a human donor organ. Pre-transplant plasmapheresis and ex vivo perfusion strategies were employed to reduce circulating antipig antibodies. The wild-type (WT) pig liver initially demonstrated functional activity (active bile production), with transient stabilization of key biochemical parameters (platelets, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate). However, antibody depletion was incomplete, and rapid rebound of antibodies and complement activation led to acute rejection, evident within 3 hours post-transplantation. The patient died 34 hours after transplantation as a result of irreversible cerebral injury. At autopsy, the xenograft exhibited extensive thrombosis and ischemic necrosis^[35]. Despite the lack of long-term survival, this trial demonstrated that pig livers can function in humans, albeit transiently, and highlighted the need for immunomodulation, antibody management, and thrombosis prevention.

Orthotopic liver xenotransplantation

The first orthotopic liver xenotransplantation was reported in 1969 by Starzl using chimpanzees as donors. These initial attempts achieved limited success, with patient survival times ranging from 2 to 9 days^[36]. Five years later, in 1974, Starzl’s team reported modestly improved survival of up to 14 days. Histopathological analysis of the transplanted livers revealed ischemic injury and centrilobular cholestasis, with little evidence of classic lymphocyte-mediated cellular rejection^[37].

In 1992 and 1993, baboon-to-human OLT was conducted using FK 506 and cyclophosphamide as immunosuppressants. These protocols resulted in further improvements in survival, extending to 26 and 70 days, respectively, reflecting enhanced control of rejection^[38]. The immunosuppressive regimen was effective in limiting both cellular and antibody rejection; however, the recipient ultimately died from invasive aspergillosis, attributed to excessive immunosuppression used to overcome rejection, which remained a major barrier at the time^[39]. Overall, the clinical outcomes were insufficient to justify continuation of these experimental trials^[7].

Preclinical xenotransplantation experience in NHPs

In 1968, British surgeon Sir Roy Calne from Cambridge, UK, conducted the first study pioneering pig-to-NHPs liver xenotransplantation (Fig. 1). Seven orthotopic liver xenografts were reported, with four baboons dying from uncontrollable hemorrhage within 6–30 hours. Centrilobular necrosis and poor liver function were prominent features. The longest survival was 3.5 days in one animal that received steroids and azathioprine as part of the immunosuppression regimen^[40].

Figure 1.

Timeline of key milestones in genetically engineered pig liver xenotransplantation (1968–2024).

Two years later, additional studies were conducted involving pig-to-primate liver xenografts, using rhesus monkeys and chimpanzees. These studies had worse outcomes, as none of the subjects survived more than 12 hours. The biological incompatibility was evident, with intravascular coagulation as the prominent feature and centrilobular necrosis observed on postmortem histopathology^[41]. This early work set a precedent, establishing a standard model for preclinical studies in liver xenotransplantation^[42].

Over two decades later, in 1994, Powelson and colleagues performed orthotopic liver transplants using pigs as donors for cynomolgus monkeys and baboons^[43]. The survival times in these experiments varied from 2 to 75 hours, during which they, like in previous studies, observed hyperacute rejection (HAR), indicating that significant rejection issues persisted. In 1998, Luo et al^[44] also reported poor outcomes, with no pig liver surviving more than 5.5 hours. There was an immediate severe vascular response after reperfusion, typical of HAR (congestion and hemorrhage), even with immunosuppressive administration.

Collectively, historical clinical and preclinical liver xenotransplantation studies (as shown in Table 1) reveal recurring barriers, including immunological rejection, thrombocytopenia, coagulation dysregulation, and limited graft viability. Early extracorporeal and auxiliary approaches provided proof-of-concept by demonstrating transient metabolic support, whereas orthotopic transplantation exposed the critical impact of species-specific physiological and immunological incompatibilities. Together, these milestones offer both validation and caution, showing that liver xenotransplantation is feasible in principle but only with deliberate mitigation of immune, coagulation, and developmental barriers. These insights now guide contemporary pig-to-NHPs studies and planned human trials, reinforcing the need for advanced genetic engineering, targeted immunomodulation, and rigorous preclinical validation to enable clinical translation.

Table 1.

Experimental experience of xenotransplantation in non-human primates.

Reference	Donor genotype	Recipient species	Procedure type	Sample size	Survival (max)
Calne et al, 1968[40]	WT	Baboon	OLT	7	3 days
Calne et al, 1970[45]	WT	Chimpanzee	OLT	-	<1 day
Calne et al, 1970[41]	WT	Rhesus/chimpanzee	OLT	4	12 hours
Powelson et al, 1994[43]	WT	Cynomolgus	OLT	4	<1–3 days
Luo et al, 1998[44]	WT	Rhesus/baboon	OLT/HLT	6; 2	5.5 hours
Hayashi et al, 1998[32]	WT	Baboon	OLT	-	<1 day

GTKO, α−1,3-galacto-syltransferase gene-knockout; HLT, heterotopic liver transplantation; HT, H-transferase; OLT, orthotopic liver transplantation; WT, wild-type (genetically unmodified).

Key challenges in pig liver xenotransplantation

Immunological rejection

Xenotransplantation stands as the leading near-term solution to address the current organ shortage. However, its clinical translation is fundamentally constrained by immunological barriers that are both quantitatively stronger and mechanistically distinct from those encountered in allotransplantation (Fig. 2). Unlike allotransplantation, in which rejection is primarily mediated by adaptive immune responses that are often controllable with conventional calcineurin inhibitor (CNI)-based regimens, xenotransplantation triggers an immediate, hierarchical, and temporally ordered immunopathological cascade. In this cascade, preformed natural antibodies and complement activation initiate endothelial injury, innate immune pathways amplify vascular inflammation and coagulation disturbances, and adaptive T-and B-cell responses subsequently sustain and propagate graft damage^[46–48].

Figure 2.

Key challenges in pig liver xenotransplantation. The figure is designed using BioRender software (https://BioRender.Com/h40u885).

Xenograft rejection can be broadly classified into HAR, acute humoral xenograft rejection (AHXR), and acute cellular rejection, representing distinct yet interrelated immunological stages that evolve from rapid antibody-mediated injury to subsequent adaptive immune responses.

HAR and acute humoral xenograft rejection

HAR represents the primary and dominant initiating mechanism, occurring within minutes to hours after reperfusion and is initiated by pre-existing xenoreactive antibodies present in human and NHP sera. These antibodies predominantly recognize carbohydrate antigens expressed on WT porcine endothelial cells, leading to rapid activation of the classical and alternative complement pathways, endothelial disruption, platelet activation, and widespread intravascular thrombosis, culminating in immediate graft failure^[46,49,50]. Among these antigens, α-galactose-1,3-galactose (α-Gal) is the most immunodominant. Anti-Gal antibodies constitute approximately 1–4% of circulating human immunoglobulins and arise early in life following exposure to Gal-positive commensal bacteria, as humans and Old-World primates lack α-Gal expression due to an inactivating mutation in the α−1,3-galactosyltransferase gene^[47,51].

AHXR, also referred to as acute vascular rejection or delayed xenograft rejection, typically develops days to weeks after transplantation and is driven by elicited antibodies targeting non-Gal antigens, most notably N-glycolylneuraminic acid (Neu5Gc) and the SDa blood group antigen. Once the initial α-Gal-mediated HAR is overcome, these antibodies induce complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity (ADCC), resulting in progressive endothelial activation, microvascular inflammation, and graft dysfunction^[52]. This sequence establishes a clear antigenic hierarchy, with α-Gal dominating early rejection and non-Gal antigens shaping longer-term graft outcomes.

Genetic deletion of α-Gal markedly reduces HAR, but it does not prevent AHXR or ensure durable graft survival, underscoring the necessity for multi-gene-edited donor pigs, such as triple knockout (TKO) animals lacking α-Gal, Neu5Gc, and SDa antigens^[52–55]

To overcome the hyperacute and acute rejections, advances in donor genetic engineering (as illustrated in Figure 3) and perioperative management have been done with improved short-term survival in liver xenotransplantation models. Early strategies incorporated expression of human complement regulatory proteins, such as CD55 (also known as decay-accelerating factor, or DAF). Pigs modified with h-DAF transgenes demonstrated significantly improved survival times, with graft function lasting up to 8 days without HAR and normal coagulation. This marked a substantial improvement over previous attempts with WT pig livers, where survival was less than 12 hours, consistent with earlier studies^[56]. Subsequent generations of genetically engineered pigs have been systematically designed to target xenoantigens, complement pathways, the coagulation cascade, and additional immune-relevant genes, culminating in the generation of donor pigs harboring nine or more genetic modifications (Table 2)^[21]. Polytransgenic livers, which express complement regulatory proteins, prevent HAR and maintain hemostasis when transplanted into baboons^[57–62].

Figure 3.

The process of creating genetically engineered pigs for liver xenotransplantation. Key genetic modifications include knocking out genes like GGTA1, B4GALNT2, and CMAH to reduce immunogenicity, while inserting genes such as CD46, CD55, and CD59 for complement regulation, HO-1 and A20 for reducing inflammation, and EPCR and TBM for coagulation control. Genetically modified pig cells undergo nuclear transfer into enucleated oocytes, developing into embryos that are raised in biosecure farms. These genetically engineered pigs serve as organ donors, and immunosuppressive strategies are used during transplantation to ensure the recipient’s acceptance of the organ. The figure is designed using Biorender software (https://BioRender.Com/sa1sm31).

Table 2.

Overview of gene-edited pig liver xenotransplantation in humans and NHPs.

Study	Recipient	Procedure type	Genetic modifications	Immunosuppressive agents	Survival/outcome
Ramirez et al, 2000[56]	Baboon	OLT	hCD55	CyP, CsA, and Cs	≤8 days; Baboons transplanted with livers from unmodified pigs survived less than 12 hours, while those with h-DAF transgenic livers survived 4 and 8 days without hyperacute rejection.
Ramirez et al, 2005[58]	Baboon	OLT	hCD55 + hCD59 + H-transferase	CyP, CsA, Cs, Rituximab, Daclizumab, and MMF	≤24 hours; HAR prevented, and the livers produced bile and kept prothrombin activity above 35%.
Esker et al, 2010[59]	Baboon	OLT	GTKO or GTKO + hCD46	Cs, MMF, ATG, Tacrolimus, and CVF	≤7 days; Profound thrombocytopenia developed within hours of reperfusion, leading to spontaneous bleeding at multiple sites.
Esker et al, 2010[65]	Baboon	OLT	GTKO or GTKO + hCD46	Cs, CyP, MMF, ATG, Tacrolimus, and CVF	≤7 days; Baboons died from bleeding due to thrombocytopenia; liver function and coagulation stayed near-normal, with pig proteins and coagulation factors produced from day 1 and functioning.
Esker et al, 2012[66]	Baboon	OLT	GTKO/hCD46	Cs, CyP, MMF, ATG, Tacrolimus, CVF, and CL	≤7 days; livers showed hemorrhagic necrosis, platelet aggregation, platelet-fibrin thrombi, monocyte/macrophage margination mainly in sinusoids, and endothelial cell hypertrophy.
Kim et al, 2012[57]	Baboon	OLT	GTKO	ATG, LoCd2b, CVF, anti-CD154, Azathioprine, Tacrolimus, and Cs	≤9 days; Amicar maintained platelet counts above 40 000/μl and prolonged survival to 8–9 days, but animals died from bleeding and sepsis despite no rejection, indicating more coagulation and infection controls are needed.
Yeh et al, 2014[67]	Baboon	HLT	GTKO	ATG, CVF, Cs, and Tacrolimus	≤15 days; heterotopic liver xenografts developed severe thrombocytopenia but no bleeding for up to 8 days; deaths were due to graft thrombosis or sepsis, suggesting primate clotting support may prevent bleeding but not graft thrombosis.
Ji et al, 2015[68]	Tibetan macaques	HLT	GTKO	ATG, CVF, MMF, anti-CD154, Tacrolimus, and Cs	≤13.5 days; recipient tissue factor activation disrupts coagulation after pig-to-primate liver transplantation due to tissue factor pathway inhibitor incompatibility, which may be improved by pig bone marrow stem cells expressing human tissue factor pathway inhibitor.
Navarro-Alvarez et al, 2016[69]	Baboon	OLT	GTKO	ATG, CVF, Cs, anti-CD154, LoCD2b, and Tacrolimus	≤7 days; continuous infusion of recombinant factor VIIa stabilized platelets, reduced transfusions, and prevented hepatic thrombotic microangiopathy, whereas bolus human prothrombin complex concentrate caused thrombosis and graft failure.
Shah et al, 2016[70]	Baboon	OLT	GTKO	ATG, CVF, Cs, Tacrolimus, and belatacept	25 days; continuous infusion of human coagulation factors with infection control and costimulation blockade controlled coagulopathy, preserved platelets, prevented thrombotic microangiopathy, and extended the survival.
Shah et al, 2017[60]	Baboon	OLT	GTKO	ATG, CVF, Cs, Tacrolimus, and belatacept	≤29 days; continuous human prothrombin concentrate infusion, and costimulation blockade achieved up to 29 days of rejection-free survival.
Zhang et al, 2017[71]	Tibetan macaques	HLT	GTKO/CD47	ATG, CVF, Cs, Tacrolimus, and MMF	≤14 days; liver xenografts showed mild platelet loss without severe coagulation issues or rejection, but early graft damage correlated with sharp rises in inflammatory cytokines, likely driving leukocyte infiltration.
UPenn[72]	Brain-dead human patient	ECLP	Not specified (gene-edited porcine liver)	Not disclosed	3 days: stable perfusion with no rejection observed.
Tao et al, 2025[73]	Brain-dead human patient with liver cancer	HLT	6-gene edited GGTA1, β 4GalNT2, CMAH; hCD46, hCD55, and hTBM	ATG, Ravulizumab, Etanercept, MMF, Tacrolimus, and Rituximab	10 days; pig liver functional for; bile and albumin produced; no rejection
Zhang et al, 2025[74]	Living human (71 years) with liver cancer	OLT auxiliary	10 gene edits GGTA1, CMAH, β 4GalNT2, CD46, CD55, CD59, CD39, TBM, EPCR, and CD47	Rituximab, basiliximab, Cs, tacrolimus, sirolimus, and MMF	38 days; patient survived 171 days total; the xenograft liver was removed after 38 days due to complications
Kunming[75]	Brain-dead human patient	OLT auxiliary	8 genes edited (GGTA1, CMAH, β 4GalNT2 KO; hCD46, hCD55, hCD59, hTBM, and hCD39)	Not disclosed	Not mentioned; good early graft perfusion; liver and kidney functional for days

ATG, anti-thymocyte globulin; CL, clodronate liposomes; Cs, corticosteroids; CsA, cyclosporine; CVF, cobra venom factor; CyP, cyclophosphamide; GTKO, α−1,3-galactosyltransferase gene-knockout; HLT, heterotopic liver transplantation; LoCD2b, rat anti-primate CD2 IgG; MMF, mycophenolate mofetil; OLT, orthotopic liver transplantation.

Additionally, HAR and AHXR can be prevented by using plasmapheresis and pigs genetically engineered to lack α-Gal and two other non-Gal antigens (triple knockout or TKO pigs)^[49]. Although the knockout of α-Gal antigens reduces the T cell response, it is not sufficient for the long-term survival of a xenograft^[63]. Shah et al evaluated pig-to-primate liver xenotransplantation using α−1,3-galactosyltransferase knockout pigs, human prothrombin concentrate infusion, and an immunosuppression regimen that included anti-thymocyte globulin (ATG), FK-506, methylprednisolone, and co-stimulation blockade (with either belatacept or anti-CD40 mAb)^[42]. Four baboons underwent transplantation, with two surviving nearly 1 month (25 and 29 days) rejection free. The extended survival was attributed to the infusion of functional clotting factors, which prevented bleeding and thrombocytopenia, issues that have historically limited success in xenotransplantation. Despite stable liver function, euthanasia was required due to complications such as cholestasis, plantar ulcers, seizures, and cytomegalovirus infection. The findings demonstrate that porcine livers can sustain primates for several weeks under specific conditions, underscoring the potential of liver xenotransplantation. However, the study also highlights the need for optimization of immunosuppressive regimens and management strategies to address complications such as infections and other transplant-related issues to ensure long-term success^[64].

Cellular xenograft rejection

Cellular rejection remains a significant obstacle even in extensively genetically modified xenografts. Human T-cell proliferation in response to porcine antigens has been shown to be comparable to responses elicited by allogeneic human cells in vitro^[76–78]. Importantly, xenogeneic stimulation induces a qualitatively distinct inflammatory phenotype characterized by enhanced innate immune activation.

Specifically, exposure to pig cells, but not allogeneic human cells or concordant xenogeneic monkey cells, induces selective upregulation of NCF4, a transcriptionally regulated component of the NADPH oxidase complex that is closely associated with reactive oxygen species (ROS) generation and inflammasome activation. This finding highlights a xenogeneic-specific innate sensing pathway rather than a generic response to foreign tissue. In parallel, xenogeneic stimulation increases High Mobility Group Box-1 (HMGB1) release, a damage-associated molecular pattern that enhances antigen presentation, promotes dendritic cell maturation, and lowers the activation threshold for adaptive immunity.

Through ROS-dependent signaling, NCF4 upregulation indirectly promotes inflammasome activation, leading to downstream production of pro-inflammatory cytokines and endothelial activation. Furthermore, porcine cell lysates, but not intact cells, markedly increase interleukin-26 expression, implicating DNA-binding cytokine signaling pathways in xenogeneic immune amplification^[79,80]. Importantly, these immune pathways do not operate independently; rather, inflammasome activation, HMGB1 release, and ROS signaling function as upstream amplifiers that lower the threshold for antibody-mediated complement injury, NK-cell cytotoxicity, and subsequent adaptive immune escalation. This crosstalk helps explain why suppression of T-cell responses alone is insufficient for durable xenograft protection.

Beyond cell-intrinsic innate sensing pathways, systemic inflammatory responses also contribute significantly to xenograft injury. Xenotransplantation studies have highlighted interleukin-6 (IL-6)-driven acute phase responses as a hallmark of innate immune activation, with early work demonstrating elevated IL-6 and downstream C-reactive protein (CRP) production in xenograft recipients. Given that the liver is the primary source of CRP, pig liver xenotransplantation represents a unique context in which recipient IL-6 may directly drive graft-derived acute phase responses^[81]. Although CRP was not specifically measured in early pig liver xenotransplantation studies, activation of the IL-6–CRP axis may contribute to endothelial activation and amplification of humoral and cellular rejection pathways.

Natural killer (NK) cells constitute a major innate immune barrier to successful xenotransplantation by recognizing “missing-self” signals arising from interspecies MHC class I incompatibility. Consequently, donor genetic engineering strategies designed to reconstitute inhibitory NK signaling have received increasing attention^[49,82]. In this context, transgenic expression of the non-classical human MHC class I molecule HLA-E in combination with human β2-microglobulin on porcine cells has been demonstrated to significantly attenuate human NK cell-mediated cytotoxicity in vitro through engagement of inhibitory CD94/NKG2A receptors, thereby conferring partial protection against NK-driven xenogeneic cellular injury^[82]. In parallel, the use of regulatory T-cells (Tregs) has emerged as a promising adjunctive approach capable of suppressing effector T-cell responses and promoting donor-specific tolerance in xenotransplantation models^[83–85].

While T cell- and NK cell-mediated mechanisms dominate cellular rejection, humoral immune responses driven by B cells and plasma cells represent a central determinant of graft survival. Host B cells differentiate into plasma cells that secrete high-affinity xenoreactive antibodies targeting major porcine antigens, including αGal, Neu5Gc, and SDa, which contribute significantly to both HAR and AHXR through complement activation and ADCC^[86,87]. Importantly, terminally differentiated plasma cells lack CD20 expression and therefore evade conventional B cell depletion strategies such as rituximab, resulting in persistent antibody production and ongoing graft injury^[88]. To address this, agents targeting plasma cells directly, such as the proteasome inhibitor bortezomib or anti-CD38 monoclonal antibodies, have shown promise in allotransplant and emerging xenotransplant models by reducing donor-specific antibody levels, lowering C4d deposition, and improving graft histology^[89,90]. However, its use in xenotransplantation remains limited by toxicity and the need for further species-specific studies^[90].

Beyond total antibody levels, the subclass distribution of IgG plays a critical role in mediating effector responses. In primates, IgG1 and IgG3 are the most potent activators of complement and Fcγ receptor-mediated cytotoxicity, leading to ADCC and endothelial injury^[91,92]. For instance, human anti-porcine IgG3 antibodies have been shown to strongly induce complement deposition and endothelial activation^[92]. In contrast, IgG2 and IgG4 exhibit limited complement-fixing capabilities, with IgG4 often associated with non-inflammatory or regulatory responses^[91]. These findings suggest that profiling IgG subclass responses may provide prognostic value in assessing rejection risk and may eventually offer a target for immunomodulation in xenotransplantation^[93].

While B-cell depletion combined with plasma cell-targeted therapy has extended graft survival in preclinical models, next-generation approaches, including IgG subclass silencing, Fc gamma receptor blockade, and plasma cell-specific gene targeting such as BCMA-directed CAR T cells, are under investigation to further blunt pathogenic humoral responses^[94]. Overall, these innate, humoral, and cellular mechanisms function in a stepwise and interconnected manner, forming the causal framework that drives early graft injury, adaptive immune escalation, and ultimately xenograft failure. Together with advances in genetic engineering to eliminate major xenoantigens, these humoral-targeted strategies may help achieve durable graft survival and reduce the risk of antibody-mediated liver xenograft failure.

Coagulation dysfunction

Studies in pigs with Gal, Neu5Gc, and SDa knockouts have shown that transplant-induced coagulopathy remains a major biological barrier to successful liver xenotransplantation^[95,96]. These coagulation abnormalities occur in a defined sequence, beginning with rapid endothelial activation upon reperfusion, progressing to dysregulated thrombin generation, and culminating in platelet consumption, fibrin deposition, and microvascular injury. Direct interaction between human blood and porcine endothelial cells induces a markedly procoagulant phenotype, with clotting times shortened by more than 850 seconds, reflecting profound activation of the coagulation cascade^[97]. This early shift toward hypercoagulability is driven by molecular incompatibilities in key endothelial anticoagulant pathways, including thrombomodulin (TBM), tissue factor pathway inhibitor (TFPI), endothelial protein C receptor (EPCR), and CD39, all of which normally function in a coordinated manner to suppress thrombin generation and maintain vascular quiescence^[51].

In the xenogeneic setting, impaired cross-species functionality of these regulatory proteins disrupts anticoagulant signaling at the endothelial surface, favoring unchecked thrombin formation immediately after graft reperfusion. Porcine TBM interacts inefficiently with human thrombin, resulting in reduced activation of the protein C pathway and amplification of coagulation signaling, which promotes vascular injury and inflammatory cell recruitment^[95]. This defect is further compounded by early tissue factor exposure and inadequate TFPI activity, collectively accelerating thrombin generation during the critical early post-transplant period. In parallel, coagulation and complement systems are tightly interconnected, and insufficient regulation of complement activation exacerbates endothelial injury and prothrombotic signaling, underscoring the importance of complement regulatory proteins such as CD46, CD55, and CD59 in xenograft protection^[51].

Another major contributor to coagulation dysregulation is the species-specific incompatibility between porcine von Willebrand factor (pvWF) and primate platelet glycoprotein Ib (GPIb). Unlike the shear-dependent interaction observed in allotransplantation, pvWF binds primate GPIb with high affinity even under low-shear or static conditions, triggering spontaneous platelet adhesion, activation, and aggregation^[52]. This aberrant interaction accelerates microvascular thrombosis and platelet consumption within the graft, linking endothelial activation directly to downstream consumptive coagulopathy. Collectively, these findings support a model in which endothelial dysfunction, defective anticoagulant signaling, complement–coagulation crosstalk, and vWF–platelet mismatches converge to drive the characteristic coagulation abnormalities observed in liver xenotransplantation.

To mitigate these barriers, genetic modification strategies incorporating human coagulation regulatory proteins, including TBM, TFPI, EPCR, and CD39, into donor pigs have been pursued with encouraging results^[52,98]. By restoring key anticoagulant pathways and dampening thrombin generation, these approaches aim to stabilize the xenogeneic endothelium during the vulnerable reperfusion phase and reduce early graft-associated coagulopathy. However, despite these advances, coagulation dysfunction remains incompletely controlled, highlighting the need for integrated genetic and pharmacological strategies to achieve durable hemostatic balance in liver xenotransplantation.

Thrombocytopenia

Platelet activation is common in pig xenograft recipients and significantly contributes to thrombotic microangiopathy and consumptive coagulopathy. This excessive platelet activation initiates thrombotic microangiopathy, which subsequently propagates the systemic consumptive coagulopathy and hemorrhagic complications observed in the recipient. The severity and kinetics of this process vary by organ type, with liver xenografts uniquely inducing profound and immediate thrombocytopenia compared with pig heart or kidney xenotransplants performed under similar experimental conditions^[29,99]. This organ-specific vulnerability reflects the distinctive hepatic sinusoidal environment, which exposes circulating platelets to pvWF–GPIb interactions and asialoglycoprotein receptor (ASGR)-mediated clearance, unlike heart or kidney grafts.

Esker et al^[59] utilized genetically modified GTKO and GTKO/CD46 pig liver and demonstrated preserved hepatic function and recipient survival for up to 7–9 days; however, survival was consistently limited by severe thrombocytopenia, coagulopathy, and bleeding despite the absence of overt rejection. Notably, thrombocytopenia often develops within the first hour following reperfusion, indicating that platelet loss is driven by intrinsic molecular incompatibilities rather than immune-mediated graft injury. One key mechanism involves the enhanced binding affinity of pvWF for human and NHPs GPIb, which leads to exaggerated platelet adhesion, activation, and downstream amplification and aggregation in vitro and in vivo^[100].

Similarly, genetically modified (GTKO ± hCD46) liver xenografts could sustain life for at least 9 days in immunosuppressed baboons. However, even without signs of rejection, survival has been reduced due to severe thrombocytopenia, coagulopathy, and bleeding. This is attributed to incompatibility between the pig ASGR and its human/NHP ligand, leading to platelet sequestration by pig liver cells^[35,101]. The mismatch between porcine ASGR and human ligands results in aberrant platelet internalization by hepatocytes, explaining the profound and immediate platelet loss uniquely associated with liver xenotransplantation.

Recently, pigs lacking ASGR have been developed, and their livers showed significantly reduced human platelet uptake in an ex vivo perfusion model by addressing aberrant phagocytosis^[51]. The dual silencing with Cas9 endonuclease and gRNA of GGTA1 and cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) genes has shown superior results in reducing human platelet uptake compared to GGTA1^−/− and ASGR1^−/− livers^[102,103]. These findings suggest that simultaneous correction of carbohydrate- and receptor-mediated incompatibilities may be necessary to effectively prevent thrombocytopenia.

Potential zoonotic risks

Xenotransplantation carries the risk of zoonotic pathogens being transmitted from the donor animal, such as pigs, to the immunocompromised human recipient^[104]. The specific pathogens of concern vary by animal species, with swine being the primary focus of research and clinical applications (Fig. 2). To mitigate this risk, exclusion lists have been compiled to identify pathogens that might be transmitted from pigs to humans. Research has primarily investigated the risk of porcine endogenous retrovirus (PERV) transmission in pig cell and tissue transplantation to various species, including NHPs, minks, rats, guinea pigs, and severe combined immunodeficiency mice^[105]. Aside from one documented case in a guinea pig, no evidence of transmission has been observed in immunosuppressed humans. Additionally, pig-derived pathogens have not been identified in immunosuppressed humans except for the hepatitis E virus^[106]. Since predicting which organisms will present a health risk is virtually impossible before clinical trials, the current strategy for preventing infectious disease transmission in pig-to-human xenotransplantation involves the careful selection of “pathogen-free” pigs. This is achieved through strict herd isolation, ongoing monitoring of source animals, detailed breeding records, microbiological testing, and routine veterinary care to maintain the health of the donor pigs. Breeding swine under strict biosecure conditions is a critical strategy to minimize pathogen exposure^[107,108]. The groundbreaking cardiac xenotransplantation from a genetically modified pig to a human on 7 January 2022 marked a significant advancement and offered a unique opportunity to evaluate potential infectious risks in a true clinical pig-to-human transplantation setting^[109].

A significant concern for xenotransplantation is the zoonotic viral transmission from swine, particularly the transmission of PERVs, which are integrated into the pig genome and cannot be eradicated by SPF breeding. Early in vitro studies demonstrated that replication-competent PERV-A and PERV-B can infect certain human cell lines, including human embryonic kidney (HEK-293) cells, whereas PERV-C shows limited infectivity unless recombination or phenotypic mixing occurs^[110]. These observations established a theoretical biological basis for cross-species transmission and raised concerns regarding possible oncogenic or immunosuppressive effects.

However, these theoretical risks have not been substantiated in vivo. Extensive preclinical studies involving NHPs (monkey and baboon) recipients have consistently failed to demonstrate PERV transmission, even under conditions of intense immunosuppression and long-term molecular surveillance^[111]. Although the clinical effect of PERV is unknown; however, like other retroviruses, PERV theoretically predisposes to the risks of tumors, leukemia, and neurodegeneration. Concerns about PERV infection inducing immunosuppression arise because PERV is related to viruses known to cause immunodeficiency in animals such as murine and feline leukemia viruses. Additionally, there are worries about potential human-to-human transmission, incorporation of PERV into the human genome, and potential recombination with human endogenous retroviruses^[112–114]. However, clinical xenotransplantation studies involving porcine islets and other tissues have not detected PERV infection in human recipients using sensitive molecular assays. To date, no productive PERV infection or PERV-associated disease has been documented in humans or in swine.

In parallel with these observations, multiple strategies have been developed to further mitigate residual PERV risk. These include the selection of donor animals lacking specific PERV subtypes, the use of antiviral agents and RNA-interference approaches, and most notably genome editing to inactivate PERV proviruses^[115]. CRISPR-Cas9-mediated inactivation of all PERV copies has been achieved in porcine cell lines and used to generate viable PERV-inactivated pigs, effectively abolishing in vitro infectivity for human cells^[20,116,117]. Although issues such as off-target effects and large-scale breeding remain under evaluation, this genetic approach represents a decisive safety enhancement. From a regulatory perspective, PERV is now regarded as a manageable theoretical risk rather than a demonstrated clinical barrier. Nevertheless, regulatory agencies, including the Food and Drug Administration (FDA), continue to recommend repeated post-transplant testing and long-term surveillance of recipients, even when PERV-inactivated donor animals are used, reflecting a precautionary framework appropriate for first-in-human xenotransplantation^[118].

Other porcine viruses are also considered in xenotransplantation biosafety. Porcine cytomegalovirus (PCMV) is another significant concern in xenotransplantation due to its association with graft failure. PCMV, a member of the Herpesviridae family, is endemic in pigs and can be transmitted via nasal secretions and in utero^[119,120]. Its reactivation in xenograft recipients can lead to systemic inflammation and immune activation, contributing to graft rejection. PCMV has been directly implicated in the failure of a pig-to-human heart transplant in 2022, highlighting the importance of thorough screening and elimination from donor pigs and raising concerns about the adequacy of screening processes for this virus^[121].

On the other hand, porcine circovirus (PCV) is a distinct virus, with two distinct strains of PCV, namely PCV1 and PCV2, that have been characterized. While PCV1 was isolated from pig kidney cell culture (PK15 cells)^[122], recent studies have shown that PCV2 can infect human cells in vitro^[123]. Researchers have been unable to identify PCV1 and PCV2 in genetically engineered pigs, though two other subtypes, PCV3a and PCV3b, were found in the spleen, liver, lung, kidney, and hearts explanted from recipient baboons following genetically engineered cardiac xenografts. While the clinical significance of PCV remains unclear, the presence of PCV3 was notably higher in long-term survivors^[124]. Through early weaning, rigorous biosecurity, and screening, PCMV and PCV can be effectively managed in breeding herds, reducing their risks in xenotransplantation^[125,126].

Immunosuppressive approaches and adjunctive therapies in rejection management

The success of liver xenotransplantation using genetically engineered porcine donors is critically dependent on the capacity to control immune rejection mechanisms that are fundamentally distinct from those encountered in liver allotransplantation. Unlike allografts, where adaptive T cell-driven responses predominate and are often adequately controlled by CNI-based regimens, xenografts elicit an immediate, antibody-dominated and innate-amplified immune response^[127,128]. This qualitative difference explains why conventional immunosuppressive protocols are insufficient to prevent early graft injury and why xenotransplantation requires intensified, multi-layered, and mechanism-specific immunomodulatory strategies.

Immunosuppressive protocols

In xenotransplantation, the immune response is markedly strong due to the substantial genetic differences between species, resulting in both hyperacute and acute vascular rejection. The immunosuppressive regimens traditionally used in allotransplantation are often insufficient in xenotransplantation settings, thus requiring a combination of novel immunosuppressive agents and strategies.

Xenotransplantation provokes stronger and more rapid humoral and cellular immune responses than liver allotransplantation, driven by preformed xenoreactive antibodies, complement activation, and innate immune effector pathways. Consequently, standard triple-drug regimens comprising a CNI, mycophenolate mofetil (MMF), and corticosteroids, while effective in allotransplantation, are insufficient to prevent hyperacute or acute vascular rejection in pig-to-primate liver xenotransplantation. Contemporary xenotransplant protocols therefore retain this backbone but apply it at “desensitization” intensity, with greater emphasis on controlling antibody-mediated rejection^[86,129]. Induction therapy typically includes T cell-depleting agents such as ATG or alemtuzumab, followed by maintenance therapy with CNIs, mycophenolate, and corticosteroids^[129]. Crucially, these regimens are augmented with costimulation blockade, reflecting the central role of CD40–CD154 and CD28–CD80/86 pathways in xenogeneic T cell activation and proliferation in different models^[130–133]. Anti-CD154 monoclonal antibodies were highly effective in prolonging graft survival in pig-to-primate models but were limited clinically by thromboembolic complications^[8,134,135]. Additionally, blockade of the CD28 pathway alone using CTLA4Ig has been insufficient to prevent the development of anti-pig antibodies and cellular infiltration of the graft, even when combined with ATG and MMF in pig-to-baboon xenotransplantation^[136]. Consequently, current strategies favor anti-CD40 monoclonal antibodies combined with CTLA4Ig or belatacept-based CD28 blockade. These agents now constitute a core component of several successful multi-gene-edited pig heart and kidney xenotransplant models and are increasingly being adapted for liver xenotransplantation.

Because antibody-mediated rejection remains a dominant cause of xenograft loss, there is increasing emphasis on B-cell-and plasma cell-directed therapies. As described earlier, B cells generate high-affinity xenoreactive antibodies against αGal, Neu5Gc, SDa, and additional porcine targets, while terminally differentiated plasma cells, lacking CD20 expression, escape conventional B cell depletion strategies^[86–90]. In this context, rituximab is used to reduce circulating CD20⁺ B cells, whereas proteasome inhibitors and anti-CD38 monoclonal antibodies have been employed in selected preclinical models to deplete plasma cells, reduce donor-specific antibody levels, and diminish C4d deposition within the graft^[89,90].

As major carbohydrate xenoantigens are eliminated in TKO donor pigs, residual antibody binding to so-called “new” glycan and protein antigens becomes increasingly apparent. Several groups have reported persistent anti-pig antibody reactivity directed against targets that may be analogous to swine leukocyte antigens^[49,137,138]. Notably, in a recent study by Wang et al, it was reported that patients with end-stage liver disease and liver transplant recipients exhibited higher IgG reactivity against TKO pig cells than healthy controls, and that both SARS-CoV-2 infection and COVID-19 vaccination were associated with increased anti-TKO pig antibody levels^[139]. Although no xenografts were implanted, these findings suggest that intercurrent viral infection and vaccination can modulate xenoreactive antibody repertoires and may influence future xenograft outcomes. For prospective liver xenotransplant recipients, these data support careful pre-transplant antibody profiling, post-infection or post-vaccination monitoring of anti-pig titers, and judicious adjustment of B-cell- and plasma cell-targeted therapy, while maintaining recommended vaccination because of its clear benefit in preventing severe disease.

Adjunctive therapies

Adjunctive therapies act at defined points along the rejection cascade and complement primary immunosuppressive regimens. These approaches can be broadly categorized into antibody and complement-directed strategies, coagulation and platelet support, and anti-inflammatory and innate immune modulation.

Plasmapheresis and immunoadsorption are routinely used to reduce pre-existing xenoreactive IgG and IgM, particularly in sensitized recipients or those with high baseline anti-pig titers, and were already applied in early liver xenotransplantation attempts^{[35,49,64,86,87]}. Intravenous immunoglobulin is sometimes administered following antibody removal to modulate Fc receptor engagement and complement activation, although most supporting evidence derives from allotransplantation^[87]. Complement inhibitors, including C1 esterase inhibitor and cobra venom factor, have reduced endothelial injury and HAR in pig-to-primate models, particularly when combined with donor pigs expressing human complement regulatory proteins. While clinical experience with pharmacologic complement blockade in liver xenotransplantation remains limited, proposed pilot protocols include its use in recipients with a high xenoreactive antibody burden^[137,140].

A second category of adjunctive therapies addresses the profound coagulation disturbances characteristic of pig liver xenografts. Early thrombocytopenia, thrombotic microangiopathy, and consumptive coagulopathy arise from species-specific incompatibilities in TBM, von Willebrand factor, and related pathways as described earlier^{[52,64,95,141]}. In pig-to-baboon liver studies, infusion of human clotting factors, including prothrombin complex concentrates, fibrinogen, and antithrombin, combined with carefully titrated anticoagulation, has been essential to prolong survival and to complement donor expression of human coagulation regulators^[42,52,69].

A third, predominantly preclinical category includes anti-inflammatory and innate immune modulators. High-dose corticosteroids are widely administered at reperfusion and during suspected rejection^[48,95,142], while IL-6 blockade and Janus kinase inhibitors are being explored in pig-to-primate models to attenuate cytokine release, endothelial activation, and HMGB1-associated inflammatory signatures characteristic of xenogeneic immune stimulation^[79,80,143].

Regulatory T cells and tolerance induction

The induction of tolerance is a key goal in xenotransplantation, aiming to achieve long-term graft acceptance with minimal reliance on immunosuppressive drugs. Tregs have the unique ability to suppress alloreactive and xenoreactive immune responses, promoting an immunological environment conducive to graft tolerance^[138]. However, despite their potential, a major challenge remains the instability and loss of function of ex vivo expanded Tregs after therapeutic infusion. Notably, while preclinical studies suggest a role for Tregs in promoting graft survival, the in vivo therapeutic efficacy of ex vivo expanded Tregs in human allotransplant patients is yet to be demonstrated^[144–151]. Furthermore, co-stimulation blockade with CTLA4Ig has been associated with a reduced frequency of donor-reactive Tregs in some models, raising additional concerns regarding its long-term effectiveness in tolerance induction^[152,153].

Human–pig chimeric liver production via blastocyst complementation

Genetically engineered pigs can serve as a platform for producing human-compatible liver organs through a strategy known as blastocyst complementation, as illustrated in Figure 4. In this technique, human pluripotent stem cells (hPSCs) are injected into genetically modified pig embryos at the blastocyst stage, where key liver-developmental master genes, such as hematopoietically expressed homeobox (HHEX) or prospero-Related Homeobox 1 (PROX1), have been knocked out. This targeted gene deletion creates a vacant developmental niche that the injected human cells can occupy, enabling them to colonize the embryo, differentiate, and generate functional, humanized liver tissue as the pig develops^[154]. To date, however, published studies have consistently demonstrated partial and heterogeneous human hepatocyte engraftment, rather than complete organ-scale humanization, underscoring the experimental nature of this approach.

Figure 4.

Embryonic development and creation of a chimeric embryo through somatic cell nuclear transfer (SCNT) and blastocyst complementation. (A) Embryonic development in days: The normal progression of embryonic development is shown, starting from fertilization on day 1, leading to a two-cell stage embryo by day 2, then a morula on day 3. By day 4, the embryo reaches the early blastocyst stage, and by day 5, it develops into a late blastocyst with a defined inner cell mass. (B) Cloning by SCNT: The steps for generating a chimeric embryo using SCNT are depicted. A donor cell, specifically a FAH^−/− fibroblast, has its nucleus transferred into an enucleated recipient oocyte. Mature oocytes are isolated and matured in vitro, followed by the removal of their nuclei. After nuclear transfer, the newly reconstituted oocyte undergoes embryonic development, forming a two-cell stage embryo (day 2), a morula (day 3), and then an early blastocyst (day 4). By day 5, a late blastocyst with an inner cell mass is formed. FAH^−/− (host) and FAH⁺ (donor) blastocysts are combined to create a chimeric embryo, which contains cells from both the host and donor inner cell masses, offering potential for targeted organ generation in the host pig.

This approach leverages pigs’ anatomical and physiological similarities to humans, making them suitable for this purpose. Several strategies have been explored to enhance donor cell survival and hepatic contribution, including optimization of hPSCs pluripotent states and host genetic modifications designed to favor donor cell competitiveness^[155,156]. A notable example of this approach is the use of fumarylacetoacetate hydrolase (FAH)-deficient pigs, as shown in Figure 4. FAH deficiency leads to toxic metabolite accumulation and progressive hepatocyte loss, thereby providing a selective growth advantage to FAH-positive donor hepatocytes^[157]. Administration of NTBC [2-(2-nitro-4-trifluoromethyl-benzoyl)-cyclohexane-1,3-dione] during gestation prevents toxic buildup, supporting the survival of FAH-deficient embryos until the human cells can fully repopulate the liver niche^[158]. This example demonstrates the potential of FAH-deficient pigs as a platform for developing humanized liver tissues, however importantly, even in this optimized context, human cell contribution remains incomplete and variable, and the resulting livers retain a substantial porcine cellular and vascular component.

While gene knockout strategies targeting FAH, HHEX, or PROX1 can enhance niche permissiveness, recent analyses indicate that refining the specificity of host genetic modifications may be necessary to better control donor cell contribution and reduce unintended chimerism^[159]. At the same time, increasing the extent of host genetic manipulation has been associated with impaired embryonic development and reduced viability in interspecies models, underscoring a practical balance between niche engineering and developmental competence. Collectively, these observations highlight that current blastocyst complementation approaches are limited not only by technical constraints but also by intrinsic interspecies developmental incompatibilities.

Additional barriers include immune incompatibility, competition between residual host cells and donor human cells, and pronounced developmental asynchrony between human pluripotent cells and porcine embryonic tissues. Differences in cell-cycle dynamics, species-specific signaling environments, and epigenetic regulation collectively limit sustained human cell integration. Beyond these biological constraints, the potential contribution of human cells to non-target tissues, particularly the brain, raises significant ethical concerns and has prompted calls for stricter control of donor cell fate and tissue specificity in interspecies chimerism models. Wang et al demonstrated that optimization of naïve hPSCs culture conditions can improve chimeric competency, with notable success in kidney models; however, achieving comparably high levels of human contribution in the liver has thus far remained elusive^[160]. These observations suggest that hepatic chimerism may pose distinct biological constraints compared with other organ systems.

Recent critical analyses reinforce that, despite technical advances, human–pig liver chimerism has not yet achieved the efficiency or functional integration required for clinical relevance and should currently be regarded as an exploratory platform rather than a translational pathway^[98,161]. Future progress will likely depend on coordinated advances in generating chimera-competent hPSCs lines, fine-tuning donor–host developmental timing, and engineering liver-specific niches that enhance human cell contribution while minimizing off-target integration and ethical concerns. Addressing these challenges is essential before human–pig chimeric liver generation can be realistically considered as a solution to the persistent shortage of transplantable livers.

Ex situ liver perfusion (OPOC) in xenotransplantation

Ex situ liver perfusion, also referred to as organ perfusion and organ conditioning (OPOC), is emerging as an important platform for advancing liver xenotransplantation research. In this approach, a porcine liver is maintained outside the body and continuously perfused with oxygenated, nutrient-rich perfusate under normothermic or subnormothermic conditions^[162].

Ex situ perfusion serves two critical purposes: functional and immunological assessment of the genetically engineered liver. It allows researchers to evaluate the metabolic, synthetic, and detoxification capacity of genetically engineered pig livers in a controlled setting before transplantation into NHPs or human recipients. This helps verify that the organ can produce bile, synthesize albumin, and maintain stable hemodynamics under physiological flow conditions^[163].

Additionally, OPOC provides a unique ex vivo model to study the early immune responses triggered when human blood or plasma contacts porcine endothelial surfaces. This includes observing HAR phenomena, platelet activation, and coagulation dysregulation; major barriers to successful pig-to-human liver transplantation^[164,165].

Recent preclinical protocols have demonstrated that ex situ perfusion can sustain porcine livers for extended periods while maintaining liver-specific functions and allowing for therapeutic interventions. For example, immunosuppressive agents, gene therapies, or targeted complement inhibitors can be administered during perfusion to test their effectiveness in mitigating early rejection events before actual in vivo transplantation^{[164,166–168]}.

While many of these applications reflect foundational goals of organ perfusion research, a growing body of work now leverages ex situ perfusion as a translational decision platform. In this context, OPOC is not only a preservation tool but also a dynamic testing environment where viability thresholds, immune-modulatory strategies, and perfusion-based therapeutics can be iteratively evaluated under physiological flow and temperature, conditions that cannot be reproduced by static cold storage and isolated in vitro systems cannot replicate. This paradigm shift mirrors developments in clinical perfusion research, where ex situ platforms have begun to support viability assessment and targeted interventions in human donor livers for days rather than hours, enabling recovery, functional testing, and potential therapeutic modification prior to transplant decisions^[169].

By bridging in vitro experiments and in vivo xenotransplantation models, OPOC has become a valuable tool to refine graft preparation, optimize genetic modifications, and improve safety profiles. Rather than simply recapitulating established concepts of normothermic machine perfusion, this technology is increasingly positioned as an enabling step in the translational pipeline, reducing biological risk, guiding immunomodulatory regimens, and providing comparative performance data that can inform both preclinical study designs and early clinical xenoperfusion strategies^[170]. Even with these advances, challenges remain, including maintaining stable long-term perfusion without ischemia-reperfusion injury, limiting endothelial dysfunction during human blood exposure, and achieving reproducibility across perfusion platforms, highlighting the need for standardized, physiologically predictive xenoperfusion models.

Recent xenotransplantation trials using genetically engineered pig liver

In January 2022, a landmark procedure was performed at the University of Maryland Medical Center, marking the first successful transplantation of a genetically modified pig heart into a human patient, David Bennett. Bennett, a 57-year-old man with terminal heart disease, received a pig heart that had undergone 10 specific genetic modifications to prevent HAR and the addition of human genes to improve compatibility with the human immune system^[109]. The transplantation aimed to provide the patient with an alternative to conventional heart transplantation, for which he was not eligible. Although the initial outcomes were promising, and Bennett showed signs of recovery, he passed away 2 months later, with preliminary findings suggesting complications related to the transplant^[171]. This trial opened the door for using different genetically engineered organs, including the liver and kidney, for xenotransplantation. Therefore, 2024 has been pivotal in the field of liver xenotransplantation.

University of Pennsylvania genetically engineered pig liver trial

In recent trials, researchers at the University of Pennsylvania in Philadelphia, PA, USA, successfully utilized an external, genetically engineered pig liver to sustain a brain-dead patient for 72 hours without encountering HAR. One of the key goals has been to ensure that the pig liver can perform essential detoxification and metabolic functions in the human body. This represents the longest survival of such a graft to date. These trials involve extensive genetic modifications to address immune rejection and blood clotting issues. Preclinical studies have demonstrated that pig livers can support human patients for several days, showing promise in addressing critical shortages of human liver donors. While the full details of this experiment are not yet available, it represents a significant milestone for using porcine liver as a bridge for patients with acute liver failure and for potential liver xenotransplantation (Unpublished; based on institutional press release, University of Pennsylvania, March 2024)^[72].

Breakthroughs in pig liver xenotransplantation in China

In March 2024, a team led by Dou Kefeng and Tao Kaishan from Xijing Hospital of Air Force Medical University successfully transplanted a genetically modified pig liver heterotopically into a brain-dead individual^[73]. The study was conducted under strict national regulatory oversight, with approval from institutional ethics committees and in accordance with Chinese guidelines governing clinical xenotransplantation research. The donor organ was sourced from a designated pathogen-free facility (Chengdu Clonorgan Biotechnology Co., Ltd.), and comprehensive biosafety screening was performed, including surveillance for PERVs and PCMV.

The donor liver underwent six targeted genetic modifications, including deletion of the three major carbohydrate xenoantigens (GGTA1, β4GalNT2, and CMAH) to reduce hyperacute and acute humoral rejection, as well as insertion of human complement regulatory proteins (hCD46 and hCD55) and hTBM to mitigate complement activation and coagulation dysregulation. The transplant was performed heterotopically as an auxiliary graft, allowing continuous monitoring of graft-specific function without immediate life-sustaining dependence.

Postoperatively, the recipient was managed under a predefined immunosuppressive and monitoring protocol, including T-cell depletion with ATG and B-cell suppression with rituximab. Continuous biochemical monitoring demonstrated stable hepatic enzyme levels, preserved hepatic blood flow, and active graft function, with progressive bile production reaching approximately 66.5 ml by postoperative day 10. Porcine albumin synthesis was detectable in circulation, confirming metabolic activity of the xenograft. Although early thrombocytopenia was observed, platelet counts recovered over time. Serial histological analyses revealed graft regeneration without fibrogenesis or evidence of hyperacute, acute humoral, or cellular rejection. Ultrastructural analyses provided further mechanistic insight into graft integrity. Scanning electron microscopy demonstrated that liver sinusoidal endothelial cells retained well-differentiated fenestrae, indicating preservation of microcirculatory architecture following transplantation. Consistently, transmission electron microscopy showed no significant differences in hepatocyte ultrastructure between postoperative day 0 and day 10, and no viral particles were detected, supporting both functional stability and biosafety during the observation period.

Notably, the study was electively terminated on postoperative day 10 at the explicit request of the patient’s family, underscoring the central role of informed consent and surrogate decision-making in first-in-human xenotransplantation studies. While the limited duration precluded assessment of long-term survival, and it is unlikely that bile and porcine albumin production alone would be sufficient to support human physiology over extended periods, this landmark trial nevertheless provided unprecedented functional, immunological, ultrastructural, and biosafety insights that cannot be obtained from NHP models alone.

In May 2024, Sun Beicheng and colleagues at the First Affiliated Hospital of Anhui Medical University, in collaboration with Wei Hong-Jiang (Yunnan Agricultural University), reported an orthotopic auxiliary transplantation of a genetically engineered pig liver into a 71-year-old patient with advanced hepatocellular carcinoma who was not eligible for curative resection due to extensive tumor burden and compromised native liver volume^[74]. This procedure represented a major translational step in porcine liver xenotransplantation, moving beyond brain-dead recipient models to a living human patient and was conducted with comprehensive informed consent from the patient and his daughter obtained after detailed discussions of risks, experimental nature, and lack of alternative curative options. The donor pig was a Diannan miniature pig and carried 10 defined genetic modifications, including knockout of the three major carbohydrate xenoantigens (GGTA1, CMAH, and β4GalNT2), overexpression of three human complement regulatory proteins (CD46, CD55, and CD59), overexpression of three human coagulation-regulatory molecules (CD39, TBM, and EPCR), and expression of human CD47 to inhibit macrophage-mediated phagocytosis. The patient received induction immunosuppression with rituximab, basiliximab, and steroids, followed by maintenance therapy with tacrolimus, sirolimus, MMF, and steroids, with additional post-transplant doses of rituximab and basiliximab.

In the first 31 days post-transplant, the xenograft demonstrated sustained metabolic activity, with continued bile production, synthesis of coagulation factors, and stable serum markers of liver function, without evidence of hyperacute or acute rejection or clinically overt infection. However, on postoperative day 31, features consistent with xenotransplantation-associated thrombotic microangiopathy (xTMA) emerged, characterized by laboratory and clinical evidence of microvascular injury. Despite intervention with targeted therapies, including complement inhibition (eculizumab) and plasma exchange, this complication necessitated the removal of the porcine graft on day 38. Following graft explantation, resolution of xTMA was achieved, and the patient’s native liver demonstrated sufficient regenerative capacity to maintain metabolic needs. During the later clinical course, the patient experienced episodes of upper gastrointestinal hemorrhage, a recognized serious complication in the setting of complex liver pathology and coagulation perturbations, which ultimately contributed to the patient’s death on postoperative day 171. Throughout the follow-up period, there were no documented episodes of clinically confirmed cross-species infection, and serial clinical assessments did not reveal evidence of zoonotic transmission, aligning with prior biosafety expectations.

Additionally, in May 2024, a team led by Dong Jiahong and Zeng Zhong reported the world’s first combined transplantation of a gene-edited pig liver and kidney into a brain-dead recipient in Kunming, China (unpublished)^[75]. This unprecedented procedure marked a new frontier in xenotransplantation, demonstrating its feasibility and potential for future clinical applications.

Lessons learned from recent xenotransplantation trials

Recent clinical experiences in cardiac and hepatic xenotransplantation demonstrate that successful translation critically depends on precise genetic engineering of donor pigs. Elimination of major carbohydrate xenoantigens and incorporation of human complement-, coagulation-, and immune-regulatory transgenes are essential for preventing HAR and enabling early graft function. Nevertheless, variability in outcomes raises an important question: what is the optimal combination, number, and tissue-specific expression of transgenes required to achieve durable graft function, and how should donor pig design be further refined to meet these requirements?

Beyond classical immunological rejection, recent liver xenotransplantation trials have identified physiological and hemato-logical incompatibilities, particularly platelet consumption, coagulation imbalance, and xTMA, as dominant barriers to sustained graft function. These findings suggest that improved control of cross-species coagulation, individualized anticoagulation strategies, and enhanced microvascular monitoring will be central to future progress.

Current evidence supports liver xenotransplantation primarily as a temporary bridging strategy rather than a destination therapy. Although porcine livers can transiently provide bile production, protein synthesis, and coagulation support, their functional reserve appears limited, and long-term physiological sufficiency remains uncertain. A clear definition of clinically meaningful endpoints, such as duration of metabolic support, prevention of liver failure complications, and facilitation of native liver recovery or transition to allotransplantation, will be critical for future trials.

Auxiliary xenograft models also introduce interpretative challenges, as residual native liver tissue may confound assessment of xenograft-specific function and regeneration. How can graft-derived function be reliably distinguished from native liver recovery, and which porcine-specific biomarkers or imaging strategies should be standardized to enable accurate functional attribution? Standardized use of porcine-specific biomarkers, functional assays, and longitudinal imaging will be necessary to accurately delineate graft contribution and regenerative interactions.

While current immunosuppressive regimens effectively prevent early rejection, their intensity raises concerns regarding long-term toxicity, infection risk, and oncological safety. Future strategies should emphasize immune profiling to guide personalized immunosuppression and clarify the dynamics of humoral and cellular xenoimmune responses. In parallel, does cross-species cellular migration or microchimerism occur, and if so, what are its immunological and clinical consequences?

Finally, as liver xenotransplantation advances from brain-dead models to living patients, ethical considerations, including patient selection, informed consent, and appropriate clinical indications, must remain central. At present, xenotransplantation is best positioned for carefully selected patients with otherwise untreatable liver failure, where the goal is short-term physiological support rather than cure.

In conclusion, recent liver xenotransplantation trials establish a compelling proof of concept that genetically engineered pig livers can engraft and function in humans under rigorous clinical and biosafety conditions. At the same time, they underscore substantial unresolved biological, hematological, and ethical challenges. Addressing these issues will determine whether liver xenotransplantation emerges as a viable clinical bridge or remains an experimental milestone shaping the future of hepatology.

Ethical and regulatory considerations

The tremendous potential of xenotransplantation to alleviate the global shortage of human donor organs is accompanied by complex ethical, societal, and regulatory challenges that demand careful and transparent management to ensure responsible clinical translation. Among the foremost ethical concerns are the risks of xenozoonotic infection, the crossing of species boundaries, donor animal welfare, fully informed patient consent, and robust lifelong monitoring of recipients and their contacts^[172–175].

Animal welfare considerations lie at the core of ethical xenotransplantation practice. Genetically modified pigs, the primary source animals, must be raised under stringent welfare standards, guided by the 3Rs principle – Replacement, Reduction, and Refinement – to minimize animal use, enhance animal well-being, and ensure humane treatment throughout breeding, genetic modification, and organ procurement^[176,177]. Source pigs are typically housed in highly controlled, biosecure facilities that provide superior care compared to standard commercial farming, and all procedures must comply with institutional and national animal ethics legislation and oversight by ethics committees. Recent frameworks emphasize that genetic modifications should not compromise donor animal health or welfare and that researchers have a moral obligation to uphold a “minimum moral standing” that recognizes pigs as sentient beings capable of experiencing pain and stress, thereby mandating exceptional standards of husbandry and veterinary oversight^[178–181]

Crossing species boundaries raises philosophical and cultural questions about the moral status of animals used for human benefit among bioethicists, policymakers, and the public. Some bioethicists argue for a more biocentric perspective, weighing the intrinsic value of animal life alongside human health needs. Balancing these perspectives with the overwhelming benefit to patients, often facing terminal disease and long waiting lists, remains an ongoing societal discussion^[179].

From a regulatory perspective, xenotransplantation is governed by a complex, multi-jurisdictional framework of national and international guidance rather than a unified global statute. The WHO has long been engaged in coordinating international regulatory principles for xenotransplantation, issuing a series of expert consultations and communiqués (e.g., the Changsha Communiqués) that articulate core principles for infectious disease surveillance, donor biosecurity, and ethical conduct of clinical trials. These documents advocate that xenotransplantation should only proceed when effective national regulatory control and surveillance mechanisms overseen by competent authorities are in place^[182].

In the United States, the FDA regulates xenotransplantation products as biological and combination products subject to the Public Health Service Act and the Federal Food, Drug, and Cosmetic Act. FDA guidance, such as the PHS Guideline on Infectious Disease Issues in Xenotransplantation and the Source Animal, Product, Preclinical, and Clinical Issues guidance, requires robust source animal screening, detailed preclinical evidence (including NHPs data), investigational New Drug submissions, and lifelong monitoring protocols for xenograft recipients to reduce zoonotic risks and protect public health^[118,183].

Within the European regulatory landscape, xenotransplantation products may fall under the framework of advanced therapy medicinal products and relevant European Medicines Agency (EMA) scientific guidelines for xenogeneic cell-based products. However, there has been no recent specific EU law exclusively regulating solid organ xenotransplantation, and current frameworks rely on broader medicinal product and biologics statutes, with additional requirements on quality, safety, and clinical trial oversight performed at both national competent authority and EMA levels^[184].

China has also prioritized xenotransplantation, guided by expert consensus and draft regulatory structures influenced by the WHO global consultations held in Changsha. National guidelines and expert consensus documents classify organ xenotransplantation technologies as high-risk medical technologies requiring stringent ethical review, infectious disease control, biosecurity measures, and structured preclinical and clinical study design. Although formal legislative frameworks continue to evolve, China’s National Health Commission and other bodies have emphasized the importance of pathogen-free breeding programs, thorough preclinical evaluation, and ethics committee approval in xenotransplantation research^[185,186].

Across these jurisdictions, key ethical standards such as comprehensive informed consent remain paramount. Prospective recipients must fully understand the unique risks associated with xenotransplantation, including potential latent porcine pathogens and lifelong surveillance requirements. Clear and transparent consent processes reinforce patient autonomy while fostering trust and upholding ethical integrity in emerging xenotransplantation programs^[187].

Public perception and social license are also vital. Organizations such as the International Xenotransplantation Association, the WHO, and national regulators stress ongoing public dialogue, engagement with religious and community leaders, and transparent sharing of trial results to ensure broad societal support^[188]. Harmonization of all international regulations is an urgent priority to streamline approvals, avoid redundant barriers, and uphold global standards for patient safety and ethical practice. Together, robust animal welfare protections, informed consent processes, long-term surveillance, and up-to-date regulatory frameworks are essential for the safe and ethical advancement of xenotransplantation. Balancing these safeguards with the potential to transform outcomes for thousands of patients suffering from end-stage organ failure remains the field’s central ethical mandate.

Despite evolving national and international guidance, current regulatory frameworks remain insufficiently adapted to the complexities of next-generation genetically engineered xenografts. Existing rules often lack explicit criteria for assessing the long-term genomic stability and safety of extensively edited donor animals, standardized thresholds for acceptable degrees of multigenic modification, and harmonized definitions of preclinical evidence required for first-in-human xenotransplantation studies. Importantly, Hurst and Cooper highlighted unresolved issues that are incompletely addressed by current WHO, FDA, EMA, and national guidelines, including consistent patient selection criteria for early clinical use, comprehensive mitigation of xenozoonotic risks to recipients and bystanders, and the absence of structured public engagement mechanisms to inform regulatory decision-making^[189]. Together, these gaps, exacerbated by divergent regional regulatory pathways, underscore the need for dynamic, adaptive oversight models that integrate advanced genetic engineering realities with robust ethical safeguards to support safe and responsible clinical translation.

Conclusion and future perspectives

Xenotransplantation stands poised to transform organ transplantation by addressing the global shortage of human donor organs and providing life-saving options for patients with end-stage organ failure. Advances in genetic engineering, organ preservation, and immunomodulation have significantly improved graft survival and function in preclinical models, demonstrating the translational potential of carefully engineered porcine organs.

Despite these advances, critical challenges remain. Immunological and coagulation barriers, graft viability, interspecies compatibility, and economic considerations continue to limit progress toward clinical application. Integrated strategies that combine genetic refinement, optimized immunosuppressive and coagulation management, and robust organ preservation methods will be essential. Continued investment in research and development is crucial to overcoming these scientific and technical challenges, including refining genetic engineering approaches, improving immunosuppressive therapies, and ensuring the long-term safety and functionality of transplanted organs. Standardized preclinical models and evaluation platforms, such as ex situ perfusion, are vital for testing interventions and ensuring safety prior to human trials.

To achieve clinical implementation, the field must also address ethical, regulatory, and societal considerations, including public acceptance, zoonotic risk, and animal welfare. Global collaboration, knowledge sharing, and harmonized guidelines will accelerate progress and facilitate safe clinical translation. By focusing on these priorities, scientific, translational, economic, and ethical, xenotransplantation can move steadily toward clinical reality, offering a sustainable, life-saving solution for patients worldwide.

HIGHLIGHTS.

• Genetically engineered pigs emerge as key xenograft donors.

• Historical overview of liver xenotransplantation trials and experimental studies.

• Immunological rejection remains a critical obstacle in xenotransplantation.

• Genetic modifications and therapies tackle coagulation and infection risks.

• Insights from recent clinical trials advance xenotransplantation knowledge.

• Ethical and regulatory considerations are necessary for clinical translation.

• Future strategies focus on overcoming barriers for clinical applications.

Data availability statement

The data presented in this article are publicly available.