Prospects for xenotransplantation of the liver

A severe shortage of human livers for allotransplantation has sparked interest in the potential use of animals in lieu of humans as a source of livers, that is xenotransplantation. Xenotransplantation might also provide a means by which recurrence of hepatitis might be averted. Among the types of xenografts that might be undertaken are extracorporeal "xenoperfusion" or perfusion of devices containing xenogeneic hepatocytes, auxiliary liver transplants, bridge liver transplants, and hepatocyte transplants. The hurdles to xenotransplantation of the liver include the immune response of the recipient against the graft, incompatibility of the graft with complex physiologic and biochemical systems of the recipient, and the possibility of transferring infectious agent from the graft to the recipient. Recent progress in characterizing and overcoming these hurdles has encouraged some optimism regarding the ultimate application of xenotransplantation for the treatment of human disease.     

Treatment of cirrhosis and liver failure in rats by hepatocyte xenotransplantation

BACKGROUND & AIMS: Hepatocyte transplantation has been proposed as an alternative to liver transplantation for the treatment of hepatic failure. A major limitation to this form of therapy is the availability of human livers as a source of hepatocytes. The use of porcine hepatocytes might address this problem; however, xenogeneic hepatocytes are thought to be functionally incompatible across species and susceptible to irreversible rejection. METHODS: Liver cirrhosis was induced with phenobarbital and carbon tetrachloride. Only rats with decompensated liver failure that did not correct 4 weeks after the discontinuation of carbon tetrachloride were subjected to intrasplenic rat or porcine hepatocyte transplantation. The immunologic integrity of cirrhotic rats was assessed by allogeneic skin grafting, and the immune response to transplanted porcine hepatocytes was assessed by enzyme-linked immunosorbent assay. RESULTS: Porcine hepatocytes restored metabolic function and prolonged the survival of cirrhotic rats, as well as rat hepatocytes. Cirrhotic rats retained the ability to reject allogeneic skin grafts and showed an immune response to the engrafted hepatocytes. Despite this, survival of transplanted porcine hepatocytes was accepted in cirrhotic rats for a period of weeks without immunosuppression. Conventional immunosuppression with FK506 allowed successful retransplantation with hepatocytes from a second porcine donor. CONCLUSIONS: Hepatocytes transplanted between widely divergent species can function to correct liver failure in cirrhotic rats and prolong their survival. Conventional immunosuppression allows long-term functioning of xenogeneic hepatocyte retransplants and suggests that hepatocyte xenotransplantation might be useful as a bridge to liver transplantation and could potentially provide long-term hepatic support.     

Prolonged survival of porcine hepatocytes in cynomolgus monkeys

BACKGROUND & AIMS: Management of patients with liver failure can be a significant medical challenge, and transplantation of the liver is the only definitive therapy. Whole liver allotransplantation is limited by a shortage of human donors and the risks of the surgery in those most ill. Transplants consisting of xenogeneic hepatocytes might overcome these problems, and work in rodents indicates that such transplants can correct some metabolic deficiencies and can prevent the complications and mortality associated with hepatic failure. As a prelude to clinical application, we tested the feasibility of hepatocyte xenotransplantation in nonhuman primates. METHODS: One to 2 billion hepatocytes from outbred swine were transplanted into the spleens of cynomolgus monkeys using conventional immunosuppression to control rejection. Duration of graft function was determined based on assay for porcine albumin. RESULTS: Following a single infusion, xenogeneic hepatocytes functioned for more than 80 days and, following re-transplantation, for more than 253 days. Engraftment in the spleen was confirmed 40 days after transplantation by asialoglycoprotein receptor-directed nuclear scanning. The humoral immune response to the transplanted porcine cells had no discernible impact on the survival of the grafts. CONCLUSIONS: Xenotransplantation of hepatocytes should be explored as a readily available, minimally invasive form of therapy for hepatic failure.     

Mixed chimerism induces donor-specific T-cell tolerance across a highly disparate xenogeneic barrier

Induction of tolerance is likely to be essential for successful xenotransplantation because immune responses across xenogeneic barriers are vigorous. Although mixed hematopoietic chimerism leads to stable donor-specific tolerance in allogeneic and closely related xenogeneic (eg, rat-to-mouse) combinations, the ability of this approach to induce tolerance across a highly disparate xenogeneic barrier has not yet been demonstrated. In this study, we investigated the immune responses of murine T cells that developed in mice with pre-established porcine hematopoietic chimerism. Our results show for the first time that induction of porcine hematopoietic chimerism can eliminate the development of antiporcine donor responses in a highly disparate xenogeneic species. Porcine hematopoietic chimeras showed donor-specific nonresponsiveness in the mixed lymphocyte reaction, lack of antidonor IgG antibody production, and acceptance of donor skin grafts. Thus, mixed chimerism is capable of inducing tolerance in a highly disparate xenogeneic combination and may have clinical potential to prevent xenograft rejection. (Blood. 2002;99:3823-3829)     

Liver xenotransplantation: changes in lipid and lipoprotein concentration after long-term graft survival

BACKGROUND/AIMS: Today, scientists devote considerable effort to the study of mechanisms of xenograft rejection, but with liver xenotransplantation (XTx) researchers face the added problem of metabolic incompatibility between species. To date, there have been few studies of molecular xenogeneic interactions, perhaps because little progress has been made in solving immunological problems. This study is an initial analysis of lipoprotein metabolism in a hamster-to-rat hepatic xenotransplantation model. METHODS: There were 6 experimental groups (n=8): (1) male Sprague-Dawley (S.D.) rats (220-280 g); (2) male Golden Syrian hamsters (100-150 g); (3) S.D. rats, "sham" operation with immunosuppression; (4) S.D. rat-to-S.D. rat alloTx; (5) S.D. rat-to-S.D. rat alloTx with immunosuppression; (6) XTx hamster G.S-to-S.D. rat with immunosuppression. Mofetil mycophenolate (25 mg/kg/d) was administered for 14 days and FK506 (0.2 mg/kg/d) for 45 days (groups 3, 5 and 6). After 24 h fasting, animals were sacrificed (day +50 postransplantation) and a complete lipoprotein profile was determined. Serum lipoproteins were subfractioned by ultracentrifugation in density gradient. RESULTS: There was a large increase in serum lipid levels in xenografted rats compared with control rats and allografted rats. Xenografted rats presented a severely altered lipoprotein profile compared with normal rats. Surprisingly, the characterisation of lipoproteins in xenografted rats displayed the same composition as donor animals. Histological study did not show signs of alteration of the hepatic architecture. CONCLUSIONS: Since the liver is the main solid organ co-ordinator of metabolic pathways, such as lipid metabolism, hepatic xenotransplantation makes changes in lipid concentrations in the recipient and also changes in lipid compositions of lipoproteins. Hepatic xenotransplantation is not a feasible solution given the organ's metabolic complexity.     

How regenerative medicine and tissue engineering may complement the available armamentarium in gastroenterology?

The increasing shortage of donors and the adverse effects of immunosuppression have restricted the impact of solid organ transplantation. Despite the initial promising developments in xenotransplantation, roadblocks still need to be overcome and this form of organ support remains a long way from clinical practice. While hepatocyte transplantation may be effectively correct metabolic defects, it is far less effective in restoring liver function than liver transplantation. Tissue engineering, using extracellular matrix scaffolds with an intact but decellularized vascular network that is repopulated with autologous or allogeneic stem cells and/or adult cells, holds great promise for the treatment of failure of organs within gastrointestinal tract, such as end-stage liver disease, pancreatic insufficiency, bowel failure and type 1 diabetes. Particularly in the liver field, where there is a significant mortality of patients awaiting transplant, human bioengineering may offer a source of readily available organs for transplantation. The use of autologous cells will mitigate the need for long term immunosuppression thus removing a major hurdle in transplantation.     

Rapidly induced, T-cell independent xenoantibody production is mediated by marginal zone B cells and requires help from NK cells

Xenoantibody production directed at a wide variety of T lymphocyte-dependent and T lymphocyte-independent xenoantigens remains the major immunologic obstacle for successful xenotransplantation. The B lymphocyte subpopulations and their helper factors, involved in T-cell-independent xenoantibody production are only partially understood, and their identification will contribute to the clinical applicability of xenotransplantation. Here we show, using models involving T-cell-deficient athymic recipient mice, that rapidly induced, T-cell-independent xenoantibody production is mediated by marginal zone B lymphocytes and requires help from natural killer (NK) cells. This collaboration neither required NK-cell-mediated IFN-gamma production, nor NK-cell-mediated cytolytic killing of xenogeneic target cells. The T-cell-independent IgM xenoantibody response could be partially suppressed by CD40L blockade.     

Induction of human T-cell tolerance to porcine xenoantigens through mixed hematopoietic chimerism

Xenotransplantation from pigs could provide a potential solution to the severe shortage of allogeneic donor organs. Because xenogeneic tissues are subject to vigorous immune rejection, tolerance induction is likely to be essential to the success of clinical xenotransplantation. Here we explore the possibility of inducing human T-cell tolerance to porcine xenografts through mixed chimerism. We previously showed that NOD/SCID-Tg mice expressing porcine cytokine transgenes permit the induction of durable porcine hematopoietic chimerism. In this study we achieved human T-cell development in these mice by engrafting human fetal thymus/liver tissues. In porcine hematopoietic chimeras, human thymus grafts were populated with porcine class II(high) cells in addition to human cells, and human T cells were tolerant of the porcine hematopoietic donor as measured by mixed lymphocyte reaction assay and skin grafting. This study proves the principle that porcine chimerism induces tolerance of xenoreactive human T cells.     

Liver transplantation: making the best out of what we have

Since the first human liver transplantation, carried out in 1963, the procedure has become routine with an excellent outcome in terms of both quality and length of survival. One of the major challenges facing the transplant community is the shortage of donor organs. Possible approaches to overcoming this problem include changes in legislation, setting up of organizational structures, more effective use of marginal donor livers, splitting livers, and the development of living related transplants. Alternative treatments to liver transplantation have been sought, including hepatocyte transplantation, xenotransplantation, liver-directed gene therapy, extracorporeal liver support systems, tissue-engineered organs, and auxiliary partial orthotopic liver transplantation.     

Expression of a functional human complement inhibitor in a transgenic pig as a model for the prevention of xenogeneic hyperacute organ rejection.

The serious shortage of human organs available for transplantation has engendered a heightened interest in the use of animal organs (xenografts) for transplantation. However, the major barrier to successful discordant xenogeneic organ transplantation is the phenomenon of hyperacute rejection. Hyperacute rejection results from the deposition of high-titer preformed antibodies that activate serum complement on the luminal surface of the vascular endothelium, leading to vessel occlusion and graft failure within minutes to hours. Although endogenous membrane-associated complement inhibitors normally protect endothelial cells from autologous complement, they are species restricted and thus confer limited resistance to activated xenogeneic complement. To address the pathogenesis of hyperacute rejection in xenotransplantation, transgenic mice and a transgenic pig were engineered to express the human terminal complement inhibitor hCD59. High-level cell surface expression of hCD59 was achieved in a variety of murine and porcine cell types, most importantly on both large vessel and capillary endothelium. hCD59-expressing porcine cells were significantly resistant to challenge with high-titer anti-porcine antibody and human complement. These experiments demonstrate a strategy for developing a pig-to-primate xenogeneic transplantation model to test whether the expression of a human complement inhibitor in transgenic pigs could render xenogeneic organs resistant to hyperacute rejection.     

Humanized hemato-lymphoid system mice

Over the last decades, incrementally improved xenograft mouse models, supporting the engraftment and development of a human hemato-lymphoid system, have been developed and now represent an important research tool in the field. The most significant contributions made by means of humanized mice are the identification of normal and leukemic hematopoietic stem cells, the characterization of the human hematopoietic hierarchy, and their use as preclinical therapy models for malignant hematopoietic disorders. Successful xenotransplantation depends on three major factors: tolerance by the mouse host, correct spatial location, and appropriately cross-reactive support and interaction factors such as cytokines and major histocompatibility complex molecules. Each of these can be modified. Experimental approaches include the genetic modification of mice to faithfully express human support factors as non-cross-reactive cytokines, to create free niche space, the co-transplantation of human mesenchymal stem cells, the implantation of humanized ossicles or other stroma, and the implantation of human thymic tissue. Besides the source of hematopoietic cells, the conditioning regimen and the route of transplantation also significantly affect human hematopoietic development in vivo. We review here the achievements, most recent developments, and the remaining challenges in the generation of pre-clinically-predictive systems for human hematology and immunology, closely resembling the human situation in a xenogeneic mouse environment.     

Development and perspectives of bioartificial liver support

Bioartificial liver support systems containing adsorbent devices, xenogeneic whole liver perfusion, and hybrid bioartificial liver are anticipated to be effective for the treatment of severe hepatic failure. At present, whole liver perfusion and the hybrid bioartificial liver are two mainstreams in the field of the bioartificial liver, but it is still unclear whether either of them has significant beneficial effects in hepatic failure patients. We developed a new system of xenogeneic direct hemoperfusion consisting of a leukocyte adsorbent, an immunoglobulin adsorbent, and a substitute unit for hepatic function, that is, whole liver or bioreactor. Using this system, the perfusion efficiency rate and oxygen supply of the substitute unit for hepatic function were significantly increased. A further improvement of our system by incorporating a leukocytapheresis device to return the leukocyte-rich portion of the perfused blood back to the patient's body directly was undergone. Moreover, our system can be applied not only as a bioartificial liver, but also as a bioartificial kidney. To solve the problem of xenogeneic protein influx into the human body associated with extracorporeal bioartificial liver support, it is important to develop transgenic cattle in which human protein genes are transduced. Influx of porcine endogenous retrovirus into the human body has been a controversial subject. In relation to this issue, the cross-hemoperfusion method is promising in that the patient's circuit and the bioartificial liver circuit are separated by a semipermeable membrane, which can prevent any kind of virus from flowing into the patient's circuit.     

Efficacy of transplanting cryo-preserved and encapsulated xenogeneic fetal liver fragment as an auxiliary liver support in 90%-hepatectomized rats

BACKGROUND/AIMS: Xenogeneic-hepatocyte or liver-fragment transplantation could be an attractive clinical option in hepatic surgery for patients with impaired liver function if xenogeneic hepatocytes or liver fragments could be preserved for lengthy periods and if immunoisolation could be more easily achieved. METHODOLOGY: Porcine fetal and adult livers were used as xenogeneic transplants in rats. The grafts were stored frozen for more than one year in liquid nitrogen. After thawing, they were evaluated histologically and for potential function for auxiliary liver support in 90%-hepatectomized rats. The efficacy of microporous polypropylene membrane as a macrocapsule for immunoprotection was also examined. RESULTS: Frozen liver fragments could be preserved in liquid nitrogen for more than one year. Fetal fragments were better able to survive under the given conditions than the adult fragments. Macrocapsules protected the grafts from xenoantibodies. The survival rate of encapsulated fetal liver fragment-transplanted recipients on the seventh day after 90%-hepatectomy was 72%, while transplant recipients of fragments of fetal-liver, adult-liver, and encapsulated adult-liver, were 0, 0, and 0, respectively. CONCLUSIONS: Porcine fetal liver fragments survived longer in liquid nitrogen than did the adult ones. The fragments retained their capacity to provide auxiliary liver support in 90%-hepatectomized rats.     

Pig-to-Nonhuman Primates Pancreatic Islet Xenotransplantation: An Overview

The therapy of type 1 diabetes is an open challenging problem. The restoration of normoglycemia and insulin independence in immunosuppressed type 1 diabetic recipients of islet allotransplantation has shown the potential of a cell-based diabetes therapy. Even if successful, this approach poses a problem of scarce tissue supply. Xenotransplantation can be the answer to this limited donor availability and, among possible candidate tissues for xenotransplantation, porcine islets are the closest to a future clinical application. Xenotransplantation, with pigs as donors, offers the possibility of using healthy, living, and genetically modified islets from pathogen-free animals available in unlimited number of islets. Several studies in the pig-to-nonhuman primate model demonstrated the feasibility of successful preclinical islet xenotransplantation and have provided insights into the critical events and possible mechanisms of immune recognition and rejection of xenogeneic islet grafts. Particularly promising results in the achievement of prolonged insulin independence were obtained with newly developed, genetically modified pigs islets able to produce immunoregulatory products, using different implantation sites, and new immunotherapeutic strategies. Nonetheless, further efforts are needed to generate additional safety and efficacy data in nonhuman primate models to safely translate these findings into the clinic.     

Living related liver transplantation: medical and social aspects of a controversial therapy

There are more than 15,000 patients waiting for a liver transplant in the USA, with an average waiting time of 468 days and a mortality rate as high as 15-20%. Until artificial organs or xenotransplantation becomes a reality, living donor liver transplantation remains the best option for patients with end stage liver disease.     

Prospects for the temporary treatment of acute liver failure

At present, the most successful treatment of acute liver failure is orthotopic liver transplantation, with survival rates ranging from 70% to 85%. However, mortality rates for liver failure remain high because of the shortage of available donor organs. Therefore, there has been renewed interest in temporary treatment methods for patients with acute liver failure to either allow liver regeneration or await liver transplantation. It is thought that the function of the liver can only be replaced with the biological substrate, e.g. liver cells or a whole liver specimen, which requires the availability of liver tissue from xenogeneic or human sources. In this review, existing temporary liver support techniques are summarized and the potential hazards are described. These include the immunological implications of these techniques, e.g. the host versus graft reaction, which may influence the effectivity of the support system, and in the long run may sensitize the patient to subsequent allogeneic transplantation. The graft versus host reaction is also considered. At present, one of the major concerns is the threat of pig-to-human transmission of activated endogenous retrovirus present in the pig genome. An overview is given of literature concerning the transmission of retrovirus particles in vitro and in vivo. Finally, new solutions for the development of ex vivo systems for temporary treatment of patients with acute liver failure are discussed. These include the use of new immortalized human cell lines and human fetal hepatocytes, and the possibility of isolating, expanding and genetically manipulating stem cells in order to have stable differentiated and committed cells.     

Hepatocyte-based gene therapy

Hepatocyte-based gene therapy may be used to replace a missing gene product, confer proliferating ability to cultured hepatocytes, prevent allograft rejection, massively repopulate the host liver, or grow xenogeneic hepatocytes in mammalian liver. Gene transfer into isolated hepatocytes can be accomplished via nonviral or viral vectors, the viral vectors being more useful at this time. Common recombinant viruses that integrate into the host genome include murine leukemia retroviruses and lentiviruses, adenoassociated virus, and the T-antigen-deleted SV40 virus. Episomal viruses, such as adenoviruses, permit efficient gene transfer, but the transgene is lost upon proliferation of the transplanted hepatocyte in the host. Hybrid viruses that combine the high transduction efficiency of adenoviral vectors and the integrative capacity of other vectors, such as adenoassociated viruses, have been designed. Massive repopulation of the liver by transplanted hepatocytes can be achieved if a mitotic stimulus to the transplanted cells is combined with prevention of proliferation of the host hepatocytes. Treatment with a plant alkaloid or retrorsine, or preparative irradiation of the liver can be used to inhibit host hepatocellular proliferation, while partial hepatectomy, expression of Fas ligand, or thyroid hormone administration can be used as a mitotic stimulus to the transplanted cells.     

Role for CD47-SIRPalpha signaling in xenograft rejection by macrophages

We have previously proven that human macrophages can phagocytose porcine cells even in the absence of Ab or complement opsonization, indicating that macrophages present a pivotal immunological obstacle to xenotransplantation. A recent report indicates that the signal regulatory protein (SIRP)alpha is a critical immune inhibitory receptor on macrophages, and its interaction with CD47, a ligand for SIRPalpha, prevents autologous phagocytosis. Considering the limited compatibility (73%) in amino acid sequences between pig and human CD47, we hypothesized that the interspecies incompatibility of CD47 may contribute to the rejection of xenogeneic cells by macrophages. In the present study, we have demonstrated that porcine CD47 does not induce SIRPalpha tyrosine phosphorylation in human macrophage-like cell line, and soluble human CD47-Fc fusion protein inhibits the phagocytic activity of human macrophages toward porcine cells. In addition, we have verified that manipulation of porcine cells for expression of human CD47 radically reduces the susceptibility of the cells to phagocytosis by human macrophages. These results indicate that the interspecies incompatibility of CD47 significantly contributes to the rejection of xenogeneic cells by macrophages. Genetic induction of human CD47 on porcine cells could provide inhibitory signaling to SIRPalpha on human macrophages, providing a novel approach to preventing macrophage-mediated xenograft rejection.     

Immunological analysis in xenogeneic fetal porcine liver transplantation into a beagle

BACKGROUND/AIMS: While allogeneic organ transplantation has been performed safely, a major barrier in xenogeneic transplantation is how to inhibit hyperacute rejection. METHODOLOGY: We challenged xenogeneic fetal liver transplantation from pig to dog. The graft was investigated by immunohistochemical analysis on recipient's IgG, IgM and C3. RESULTS: In 1 of 4 cases, the graft escaped hyperacute rejection for about 4 hours after transplantation, however, the recipient died next day due to hemorrhage from the torn capsule of the liver due to the arterial blood pressure of the recipient. Histologically, the parenchyma showed good countenance and no congestion nor hemorrhage was shown in the vessels. On immunohistochemical analysis, canine IgG, IgM and C3 were deposited on the sinusoidal epithelium of the fetal liver more moderately than that of adult control. Fetal porcine liver showed less expression of major histocompatability complex class I antigen than that of the adult one. CONCLUSIONS: We consider that the hyperacute rejection occurred more slowly in xenogeneic fetal liver transplantation than in the adult one due to not only less expression of major histocompatability complex class I, but also lower expression of the epitope recognized by a natural antibody of the recipient.     

Bioengineered humanized livers as better three-dimensional drug testing model system

AIM To develop appropriate humanized three-dimensional ex-vivo model system for drug testing. METHODS Bioengineered humanized livers were developed in this study using human hepatic stem cells repopulation within the acellularized liver scaffolds which mimics with the natural organ anatomy and physiology. Six cytochrome P-450 probes were used to enable efficient identification of drug metabolism in bioengineered humanized livers. The drug metabolism study in bioengineered livers was evaluated to identify the absorption, distribution, metabolism, excretion and toxicity responses.RESULTS The bioengineered humanized livers showed cellular and molecular characteristics of human livers. The bioengineered liver showed three-dimensional natural architecture with intact vasculature and extra-cellular matrix. Human hepatic cells were engrafted similar to the human liver. Drug metabolism studies provided a suitable platform alternative to available ex-vivo and in vivo models for identifying cellular and molecular dynamics of pharmacological drugs.CONCLUSION The present study paves a way towards the development of suitable humanized preclinical model systems for pharmacological testing. This approach may reduce the cost and time duration of preclinical drug testing and further overcomes on the anatomical and physiological variations in xenogeneic systems.