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The pathobiology of pig-to-primate xenotransplantation: A historical review
Abstract
The immunologic barriers to successful xenotransplantation are related to the presence of natural anti-pig antibodies in humans and non-human primates that bind to antigens expressed on the transplanted pig organ (the most important of which is galactose-α1,3-galactose [Gal]), and activate the complement cascade, which results in rapid destruction of the graft, a process known as hyperacute rejection. High levels of elicited anti-pig IgG may develop if the adaptive immune response is not prevented by adequate immunosuppressive therapy, resulting in activation and injury of the vascular endothelium. The transplantation of organs and cells from pigs that do not express the important Gal antigen (α1,3-galactosyltransferase gene-knockout [GTKO] pigs) and express one or more human complement-regulatory proteins (hCRP, e.g., CD46, CD55), when combined with an effective costimulation blockade-based immunosuppressive regimen, prevents early antibody-mediated and cellular rejection. However, low levels of anti-non-Gal antibody and innate immune cells and/or platelets may initiate the development of a thrombotic microangiopathy in the graft that may be associated with a consumptive coagulopathy in the recipient. This pathogenic process is accentuated by the dysregulation of the coagulation-anticoagulation systems between pigs and primates. The expression in GTKO/hCRP pigs of a human coagulation-regulatory protein, for example, thrombomodulin, is increasingly being associated with prolonged pig graft survival in non-human primates. Initial clinical trials of islet and corneal xenotransplantation are already underway, and trials of pig kidney or heart transplantation are anticipated within the next few years.
... H&E and DAPI staining confirmed the absence of cells after subjecting the tissue to the decellularization process ( Figure 2). Likewise, the absence of the α-Gal epitope, which is involved in the immunological rejection of xenografts, was verified [17,18]. In immunohistochemistry for α-Gal, we observed that decellularization completely eliminated this molecule located in the endothelium of blood vessels. ...
... Furthermore, the presence of the α-Gal epitope in the native and decellularized tissues was examined by indirect immunohistochemistry. The α-Gal molecule is expressed in pigs and is involved in the immunological rejection of xenotransplants [17,18]. For this, the samples were placed in a 0.6 M citrate buffer (pH 6) at 120 • C for 3 min to perform antigen retrieval, incubated in 3% hydrogen peroxide for 10 min to block endogenous peroxidase and washed with 5% albumin/1% Tween 20 at 37 • C for 1 h to block nonspecific sites and permeabilize the membranes. ...
Currently, there are no therapies that prevent the negative myocardial remodeling process that occurs after a heart attack. Injectable hydrogels are a treatment option because they may replace the damaged extracellular matrix and, in addition, can be administered minimally invasively. Reactive oxygen species generated by ischemia-reperfusion damage can limit the therapeutic efficacy of injectable hydrogels. In order to overcome this limitation, grape seed proanthocyanidins were incorporated as antioxidant compounds into a thermosensitive myocardial extracellular matrix hydrogel in this study. For the fabrication of the hydrogel, the extracellular matrix obtained by decellularization of porcine myocardium was solubilized through enzymatic digestion, and the proanthocyanidins were incorporated. After exposing this extracellular matrix solution to 37 °C, it self-assembled into a hydrogel with a porous structure. According to the physicochemical and biological evaluation, the coupling of proanthocyanidins in the hydrogel has a positive effect on the antioxidant capacity, gelation kinetics, in vitro degradation, and cardiomyocyte viability, indicating that the hydrogel coupled with this type of antioxidants represents a promising alternative for potential application in post-infarction myocardial regeneration. Furthermore, this study proposes the best concentrations of proanthocyanidins that resulted in the hydrogels for future studies in cardiac tissue engineering.
... Religious authorities from Sikhism, Buddhism, Islam, Hinduism, and Jehovah's Witnesses express disapproval of items made from pig skin when there are other alternatives [59]. Moreover, galalpha1,3 (GAL) oligosaccharide is expressed on the pig endothelium cells, and native antibodies can identify it, leading to hyper-acute rejection fail as a "white" graft [77,78]. In order to eliminate these epitopes, scientists either genetically modify pigs or add a gene for a protein called 1,2-fucosyltransferase that co-expresses with the GAL, hence reducing the frequency of the GAL's expression in pig cells [78][79][80]. ...
... Moreover, galalpha1,3 (GAL) oligosaccharide is expressed on the pig endothelium cells, and native antibodies can identify it, leading to hyper-acute rejection fail as a "white" graft [77,78]. In order to eliminate these epitopes, scientists either genetically modify pigs or add a gene for a protein called 1,2-fucosyltransferase that co-expresses with the GAL, hence reducing the frequency of the GAL's expression in pig cells [78][79][80]. It has been demonstrated that these transgenic dressings made from pig skin can decrease pain experienced by patients. ...
... Although infection of the porcine genome by porcine endogenous retrovirus (PERV) is a concern, PERV infection has not yet been observed in humans in multiple clinical studies. [8][9][10] However, innate immune reactions, acquired immune reactions, 11,12 and sustained inflammatory effects 13 occur in porcine islet xenotransplantation, and therefore administration of multiple immunosuppressants is required. 14 Accordingly, encapsulation of islets in a special capsule capable of immunoisolation, known as a bioartificial pancreas (BAP), is a promising method for protecting islets from rejection and inflammatory responses. ...
... (D) H&E images of 200 mL-0.5% CCAG device islets retrieved at 242 days after transplantation (panels 1-3). Immunostaining with anti-insulin and antiglucagon antibodies (panels 4-6); anti-IgG and anti-insulin antibodies (panels 7-10); and anti-C3 and anti-insulin antibodies (panels[11][12][13][14]. (E) H&E images of a 50 mL-0.5% ...
Islet transplantation is an effective treatment for type 1 diabetes (T1D). However, a shortage of donors and the need for immunosuppressants are major issues. The ideal solution is to develop a source of insulin-secreting cells and an immunoprotective method. No bioartificial pancreas (BAP) devices currently meet all of the functions of long-term glycemic control, islet survival, immunoprotection, discordant xenotransplantation feasibility, and biocompatibility. We developed a device in which porcine islets were encapsulated in a highly stable and permeable hydrogel and a biocompatible immunoisolation membrane. Discordant xenotransplantation of the device into diabetic mice improved glycemic control for more than 200 days. Glycemic control was also improved in new diabetic mice “relay-transplanted” with the device after its retrieval. The easily retrieved devices exhibited almost no adhesion or fibrosis and showed sustained insulin secretion even after the two xenotransplantations. This device has the potential to be a useful BAP for T1D.
... The activation of the complement ( Figure 1) plays a pivotal part in this process, which causes irreversible damage to the donor organ or even death within minutes (45). Antibody-mediated complement activation is completed within minutes and induces graft dysfunction and disruption, the pathological features of which are endothelial edema, interstitial hemorrhage, and microvascular thrombosis (46). ...
... When the HAR, AHXR, and T cell responses are prevented, coagulation dysregulation becomes more obvious following xenograft transplantation (46). Antibodies and complement cause the endothelial cells of the graft to change from an anticoagulant state to a procoagulant state, and induce immune cell infiltration and destruction of the blood vessel wall. ...
Xenotransplantation has the potential to solve the shortfall of human organ donors. Genetically modified pigs have been considered as potential animal donors for human xenotransplantation and have been widely used in preclinical research. The genetic modifications aim to prevent the major species-specific barriers, which include humoral and cellular immune responses, and physiological incompatibilities such as complement and coagulation dysfunctions. Genetically modified pigs can be created by deleting several pig genes related to the synthesis of various pig specific antigens or by inserting human complement‐ and coagulation‐regulatory transgenes. Finally, in order to reduce the risk of infection, genes related to porcine endogenous retroviruses can be knocked down. In this review, we focus on genetically modified pigs and comprehensively summarize the immunological mechanism of xenograft rejection and recent progress in preclinical and clinical studies. Overall, both genetically engineered pig-based xenografts and technological breakthroughs in the biomedical field provide a promising foundation for pig-to-human xenotransplantation in the future.
... Later, motor neuron progenitor cells derived from mouse ES cells expressing channelrhodopsin−2 were transplanted into the peripheral nerve trunk, where muscle contraction was assessed by light control [13]. Although there have been studies using mouse [26] and human iPS cells [19], this is the first study in which fetal porcine neural cells were transplanted into xenogeneic peripheral nerves. ...
... Although immunological problems and complications of the coagulation system have long been considered as problems in xenotransplantation, many issues have been addressed by the development of genetically modified pigs [26]. In addition, endogenous retroviruses cause the most problematic infectious diseases in pigs [27], but the CRISPR-Cas9 technique was developed to inactivate all retroviral genes [22]. ...
Neural cell transplantation targeting peripheral nerves is a potential treatment regime for denervated muscle atrophy. This study aimed to develop a new therapeutic technique for intractable muscle atrophy by the xenotransplantation of neural stem cells derived from pig fetuses into peripheral nerves. In this study, we created a denervation model using neurotomy in nude rats and transplanted pig-fetus-derived neural stem cells into the cut nerve stump. Three months after transplantation, the survival of neural cells, the number and area of regenerated axons, and the degree of functional recovery by electrical stimulation of peripheral nerves were compared among the gestational ages (E 22, E 27, E 45) of the pigs. Transplanted neural cells were engrafted at all ages. Functional recovery by electric stimulation was observed at age E 22 and E 27. This study shows that the xenotransplantation of fetal porcine neural stem cells can restore denervated muscle function. When combined with medical engineering, this technology can help in developing a new therapy for paralysis.
... Advances in genetic engineering technologies and immunosuppressive therapy have made significant progress in the field of xenotransplantation (1)(2)(3). Deletion of alpha-1,3galactose (Gal) xenoantigen in donor pigs by alpha-1,3galactosyltransferase gene-knockout (GTKO) genetic modification improved xenograft survival significantly in nonhuman primate (NHP) xenotransplantation models and provided a baseline platform for further genetic modification (4). Adaptation of new immune suppression (IS) regimens, including co-stimulation pathway blockades, enabled long-term survival of porcine xenografts over a year, including the heart, kidney, islets, and cornea in NHP recipients (5)(6)(7)(8). ...
... Among them, anti-Gal IgM antibodies are the most frequently and abundantly detected in humans. Accordingly, the use of GTKO pig organs prevents hyperacute rejection in NHP xenotransplantation models (4,13). However, despite the deletion of Gal antigen expression, xenograft survival is limited, especially in cases with high levels of pre-existing non-Gal antibodies or with inadequate immunosuppression that failed to prevent adaptive immune responses (14). ...
Background
Triple knockout (TKO) donor pigs lacking alpha-1,3-galactose (Gal), N-glycolylneuraminic acid (Neu5Gc), and Sd(a) expressions were developed to improve the clinical success of xenotransplantation. Neu5Gc, a sialic acid expressed on cell surfaces, recruits factor H to protect cells from attack by the complement system. Lack of Neu5Gc expression may cause unwanted complement activation, abrogating the potential benefit of gene-modified donor pigs. To investigate whether TKO porcine cells display increased susceptibility to complement activation in human serum, pathway-specific complement activation, apoptosis, and human platelet aggregation by porcine cells were compared between alpha-1,3-galactosyltransferase gene-knockout (GTKO) and TKO porcine cells.
Methods
Primary porcine peripheral blood mononuclear cells (pPBMCs) and endothelial cells (pECs) from GTKO and TKO pigs were used. Cells were incubated in human serum diluted in gelatin veronal buffer (GVB⁺⁺) or Mg⁺⁺-EGTA GVB, and C3 deposition and apoptotic changes in these cells were measured by flow cytometry. C3 deposition levels were also measured after incubating these cells in 10% human serum supplemented with human factor H. Platelet aggregation in human platelet-rich plasma containing GTKO or TKO pECs was analyzed.
Results
The C3 deposition level in GTKO pPBMCs or pECs in GVB⁺⁺ was significantly higher than that of TKO pPBMCs or pECs, respectively, but C3 deposition levels in Mg⁺⁺-EGTA-GVB were comparable between them. The addition of factor H into the porcine cell suspension in 10% serum in Mg⁺⁺ -EGTA-GVB inhibited C3 deposition in a dose-dependent manner, and the extent of inhibition by factor H was similar between GTKO and TKO porcine cells. The percentage of late apoptotic cells in porcine cell suspension in GVB⁺⁺ increased with the addition of human serum, of which the net increase was significantly less in TKO pPBMCs than in GTKO pPBMCs. Finally, the lag time of platelet aggregation in recalcified human plasma was significantly prolonged in the presence of TKO pECs compared to that in the presence of GTKO pECs.
Conclusion
TKO genetic modification protects porcine cells from serum-induced complement activation and apoptotic changes, and delays recalcification-induced human platelet aggregation. It does not hamper factor H recruitment on cell surfaces, allowing the suppression of alternative complement pathway activation.
... The immunological barriers to successful xenotransplantation relate to the presence of natural anti-pig antibodies in humans and NHPs that bind to antigens expressed on the transplanted pig organ, and activate the complement cascade, which results in rapid destruction of the graft (hyperacute rejection). The success of these techniques relies on genetically modified animals not eliciting an immune response, the introduction of novel immunosuppressive agents that target the immune system, and the development of clinically applicable methods to induce donor-specific tolerance [3,4]. ...
Background: Nonhuman primates (NHPs) play a unique role in translational science by bridging the gap between basic and clinical investigations and are often seen as a last step before clinical application. They are widely utilized in biomedical research due to their anatomical and physiological similarities to humans. Examples of commonly used species include the genera Macaca (macaques), Papio (baboons), Aotus (owl monkeys), Callithrix (marmosets), Saimiri (squirrel monkeys), and Chlorocebus (vervet monkeys). Summary: NHP models have played an instrumental role in the development of surgical techniques, each being balanced with a unique set of advantages and shortcomings. With the appropriate selection of species and anatomy, animal models can be used to provide insight into the pathophysiology of diseases, to confirm the feasibility of new surgery technologies, to assess the potential efficacy of new surgical techniques for specific clinical outcomes, and to establish reasonable safety of new techniques for specified clinical use. Robotics have augmented surgical precision for microinjections and a brain-spine robotic interface used in gait restoration, illustrating the translational potential of NHP models in human neurological research. Recent studies highlight protocols for procedures such as tubectomy and spinal cord access with minimal postoperative risk, expanding surgical possibilities. Key Messages: This review provides an overview of the recent advancements made in surgery in NHP models and the translation of these techniques to the clinical setting. Surgical refinements not only enhance animal welfare but also improve the quality of experimental outcomes. The integration of robotics, imaging, and personalized approaches signifies a transformative shift in NHP surgical models, encouraging collaboration among veterinary and research staff for continuous progress.
... However, once grafted, fresh adult pig skin can cause hyperacute rejection and hypertrophic scarring due to human-preformed antibodies against α-1,3galactose (Gal) antigens expressed on the plasma membrane of pig vessels endothelial cells. In early childhood, gut bacteria and viruses bearing the Gal antigens shared by pig endothelial cells sensitize humans, inducing anti-Gal circulating antibodies 139,149,150 . Fresh pig skin xenografts also evoked long-lasting increases in IgGs directed against porcine Gal and Neu5Gc (N-glycolylneuraminic acid) in gravely burnt patients 151,152 . ...
... Upon xenotransplantation of porcine tissues or organs into primates, the pnAbs bind to their carbohydrate antigen targets, inducing activation of the complement system, endothelial cell activation, coagulation, and antibody-dependent cellular cytotoxicity (ADCC). Therefore, wild-type porcine xenotransplants are hyperacutely rejected upon transplantation into humans and non-human primates [2]. This can be addressed by generating source pigs lacking the major carbohydrate antigens and expressing one or several human complement pathway regulatory proteins (see below). ...
Xenotransplantation of porcine organs has made remarkable progress towards clinical application. A key factor has been the generation of genetically multi-modified source pigs for xenotransplants, protected against immune rejection and coagulation dysregulation. While efficient gene editing tools and multi-cistronic expression cassettes facilitate sophisticated and complex genetic modifications with multiple gene knockouts and protective transgenes, an increasing number of independently segregating genetic units complicates the breeding of the source pigs. Therefore, an optimal combination of essential genetic modifications may be preferable to extensive editing of the source pigs. Here, we discuss the prioritization of genetic modifications to achieve long-term survival and function of xenotransplants and summarise the genotypes that have been most successful for xenogeneic heart, kidney, and islet transplantation. Specific emphasis is given to the choice of the breed/genetic background of the source pigs. Moreover, multimodal deep phenotyping of porcine organs after xenotransplantation into human decedents will be discussed as a strategy for selecting essential genetic modifications of the source pigs. In addition to germ-line gene editing, some of these modifications may also be induced during organ preservation/perfusion, as demonstrated recently by the successful knockdown of swine leukocyte antigens in porcine lungs during ex vivo perfusion.
... The porcine glycocalyx presents sugars carrying α-Gal, N-glycolylneuraminic acid (Neu5Gc), and Sd a epitopes, which can be recognized as foreign by natural antibodies circulating in human blood, and have been shown to play a significant role in the immune response to pig grafts in xenotransplantation [22,[68][69][70][71]. Antibody-mediated complement activation will lead to hyperacute rejection, a rapid destruction of the xenograft due to the complement attack, accompanied by interstitial hemorrhage, edema, and microvascular thrombosis [21,72,73]. The lack of complement and coagulation protection on the surface of ECs due to the interspecies molecular incompatibilities also leads to complement-mediated injury and coagulopathy, which are evident in various stages of xenograft rejection [74][75][76]. ...
In xenotransplantation, the vascular endothelium serves as the first point of contact between the recipient’s blood and the transplanted donor organ. The loss of the endothelium’s ability to control the plasma cascades plays a critical role in the dysregulation of the complement and coagulation systems, which greatly contribute to graft rejection and hinder long-term xenograft survival. Although it is known that an intact glycocalyx is a key feature of a resting endothelium that exhibits optimal anticoagulant and anti-inflammatory properties, the role of the endothelial glycocalyx in xenotransplantation is barely investigated so far. Here, we discuss the central role of endothelial cells and the sugar-rich endothelial glycocalyx in regulating the plasma cascades, and how the loss of these functions contributes to graft damage and rejection. We highlight the importance of preserving the regulatory functions of both endothelial cells and the glycocalyx as strategies to improve xenotransplantation outcomes.
... In fact, three xeno-antigens have been identified in porcine skin alone [36]. When the body encounters xeno-antigens within the vascular endothelial cells of a graft, an antibodymediated complement activation leads to the destruction of the transplant within hours [37]. ...
Diabetic foot ulcers (DFUs) pose a significant threat to individuals with diabetes mellitus (DM), such as lower limb amputation and severe morbidity. Bioengineered skin substitutes (BSS) are alternatives to traditional interventions for treating DFUs, but their efficacy compared to standard wound care (SWC) or other treatment types, such as allografts, remains unknown. A scoping review of human studies was conducted to identify current approaches in the treatment of DFUs using BSS as compared with other treatment options. Systematic searches in PubMed, Cochrane Library, and Web of Science were conducted to identify comparative studies that enrolled 10 or more patients and evaluated wound healing outcomes (closure, time-to-healing, and area reduction). Database searches isolated articles published from 1 December 2012 to 1 December 2022 and were conducted in accordance with PRISMA-ScR guidelines. The literature search yielded 1312 articles, 24 of which were included for the qualitative analysis. Findings in these studies demonstrated that BSS outperformed SWC in all measured outcomes, suggesting that BSS may be a superior treatment for DFUs. Of the 24 articles, 8 articles compared human amniotic membrane allografts (hAMA) to BSS. Conflicting evidence was observed when comparing BSS and hAMA treatments, highlighting the need for future research.
... HAR and AHXR were nearly removed by using α-1,3-galactosyltransferase gene-knockout (GTKO) pigs expressing human complement regulatory proteins such as CD46, CD55, and CD59, in addition to exogenous immunosuppressive therapies (Mohiuddin et al. 2014(Mohiuddin et al. , 2016. However, coagulation dysregulation is another barrier (Cooper et al. 2016), resulting in thrombotic microangiopathy (TM), systemic consumptive coagulopathy (CC), and systemic inflammatory response (Bühler et al. 2000;Cooper and Bottino 2015;Long et al. 2016). To prevent coagulation problems, various combinatorial expressions of human coagulation regulation proteins such as thrombomodulin and CD39, and anti-inflammatory protein, CD73, in pigs have been utilized (Fischer et al. 2016;Mohiuddin et al. 2016;Lee et al. 2018). ...
Cardiac xenotransplantation is the potential treatment for end-stage heart failure, but the allogenic organ supply needs to catch up to clinical demand. Therefore, genetically-modified porcine heart xenotransplantation could be a potential alternative. So far, pig-to-monkey heart xenografts have been studied using multi-transgenic pigs, indicating various survival periods. However, functional mechanisms based on survival period-related gene expression are unclear. This study aimed to identify the differential mechanisms between pig-to-monkey post-xenotransplantation long- and short-term survivals. Heterotopic abdominal transplantation was performed using a donor CD46-expressing GTKO pig and a recipient cynomolgus monkey. RNA-seq was performed using samples from POD60 XH from monkey and NH from age-matched pigs, D35 and D95. Gene-annotated DEGs for POD60 XH were compared with those for POD9 XH (Park et al. 2021). DEGs were identified by comparing gene expression levels in POD60 XH versus either D35 or D95 NH. 1,804 and 1,655 DEGs were identified in POD60 XH versus D35 NH and POD60 XH versus D95 NH, respectively. Overlapped 1,148 DEGs were annotated and compared with 1,348 DEGs for POD9 XH. Transcriptomic features for heart failure and inhibition of T cell activation were observed in both long (POD60)- and short (POD9)-term survived monkeys. Only short-term survived monkey showed heart remodeling and regeneration features, while long-term survived monkey indicated multi-organ failure by neural and hormonal signaling as well as suppression of B cell activation. Our results reveal differential heart failure development and survival at the transcriptome level and suggest candidate genes for specific signals to control adverse cardiac xenotransplantation effects.
... I), producing TKO pigs. While many humans exhibit no or minimal antibody binding to cells from TKO pigs ( Fig. 1) 46 , complement activation and coagulation pathway dysregulation may still be observed, in part associated with molecular incompatibilities between the species 47,48 . This particularly pertains to the inefficient binding of human complement and coagulation pathway proteins to pig complement-regulatory and thromboregulatory molecules, respectively. ...
In suitable patients with end-stage organ failure, the transplantation of organs from living or deceased human donors offers a much-improved quality and length of life. However, the availability of deceased human donor organs is grossly inadequate. Gene-edited pigs might provide an alternative source of organs for clinical transplantation (xenotransplantation). However, there are major immunobiological barriers to successful pig organ transplantation in human or nonhuman primate recipients. These barriers include antibody binding, activation of complement, the innate cellular response, coagulation dysregulation between pig and primate, and a systemic inflammatory response, in addition to the T cell response. These have steadily been overcome by a combination of (i) genetic engineering of the organ-source pig (aimed mainly at the innate immune response), and (ii) the administration of novel immunosuppressive agents (directed towards the adaptive immune response). The immunological barriers that remain relate to both the innate and adaptive immune responses. Pig kidney transplants have now supported immunosuppressed (anephric) nonhuman primates for periods in excess of a year and pig heart transplants for up to 9 months, although these encouraging results cannot yet be achieved consistently.
... Pig (Sus scrofa), one of the earliest domesticated animals, is estimated to have been domesticated approximately 10,000 years ago in Asia and Europe independently 44 . It serves as an indispensable source of animal protein and an important biomedical model for humans 45,46 . Currently, a total of 22 pig assemblies are publicly available on NCBI [47][48][49][50][51] , accompanied by the availability of massive high-throughput whole-genome sequences. ...
Transposable elements (TEs) are a major source of genetic polymorphisms and play a role in chromatin architecture, gene regulatory networks, and genomic evolution. However, their functional role in pigs and contributions to complex traits are largely unknown. We created a catalog of TEs (n = 3,087,929) in pigs and found that young SINEs were predominantly silenced by histone modifications, DNA methylation, and decreased accessibility. However, some transcripts from active young SINEs showed high tissue-specificity, as confirmed by analyzing 3570 RNA-seq samples. We also detected 211,067 dimorphic SINEs in 374 individuals, including 340 population-specific ones associated with local adaptation. Mapping these dimorphic SINEs to genome-wide associations of 97 complex traits in pigs, we found 54 candidate genes (e.g., ANK2 and VRTN) that might be mediated by TEs. Our findings highlight the important roles of young SINEs and provide a supplement for genotype-to-phenotype associations and modern breeding in pigs.
... Whatever serum cytotoxicity remains is largely neutralized by the expression of human complement regulatory proteins, e.g., CD46, CD55, on the pig cells. 4 There are significant molecular incompatibilities between the pig and primate coagulation systems, resulting in the development of a thrombotic microangiopathy within the pig graft and a consumptive coagulopathy in the recipient. These have largely been prevented by judicious gene editing of the pig, e.g., by the transgenic expression of human thrombomodulin (TBM) and/or endothelial cell protein C receptor (EPCR). ...
... The pathobiology of organ XTx is more complex than that of allotransplantation, with the innate immune response playing a greater role. The factors that contribute to xenograft destruction have been comprehensively reviewed previously 31 and so will only be summarized briefly here to provide context for the choices being discussed for pig design and recipient management. ...
For many patients with terminal/advanced cardiac failure, heart transplantation is the most effective, durable treatment option, and offers the best prospects for a high quality of life. The number of potentially life-saving donated human organs is far fewer than the population who could benefit from a new heart, resulting in increasing numbers of patients awaiting replacement of their failing heart, high waitlist mortality, and frequent reliance on interim mechanical support for many of those deemed among the best candidates but who are deteriorating as they wait. Currently, mechanical assist devices supporting left ventricular or biventricular heart function are the only alternative to heart transplant that is in clinical use. Unfortunately, the complication rate with mechanical assistance remains high despite advances in device design and patient selection and management, and the quality of life of the patients even with good outcomes is only moderately improved. Cardiac xenotransplantation from genetically multi-modified (GM) organ-source pigs is an emerging new option as demonstrated by consistent long-term success of heterotopic (non-life-supporting) abdominal and life-supporting orthotopic porcine heart transplantation in baboons, and by a recent 'compassionate use' transplant of the heart from a GM pig with 10 modifications into a terminally ill patient who survived for two months. In this review, we discuss pig heart xenotransplantation as a concept, including pathobiological aspects related to immune rejection, coagulation dysregulation, and detrimental overgrowth of the heart, as well as GM strategies in pigs to prevent or minimize these problems. Relevant results of heterotopic and orthotopic heart transplantation experiments in the pig-to-baboon model, microbiological and virologic safety concepts, and efficacy requirements for initiating formal clinical trials are additional topics discussed. An adequate regulatory and ethical framework as well as stringent criteria for the selection of patients will be critical for the safe clinical development of cardiac xenotransplantation, which we expect will be clinically tested during the next few years.
... 3,4 Due to this situation, xenotransplantation might become an alternative solution. 5,6 With effective encapsulation technologies, islets may be protected against graft rejection and autoimmune destruction during xenotransplantation. 7 Islets are efficiently shielded from host immune rejection through cell encapsulation at the initial stage, yet longterm therapeutic efficacy remains a challenge [8][9][10][11][12][13][14] due to foreign body response (FBR), which can cause inflammation, recruitment of leukocytes, pericapsular fibrotic overgrowth, and even ultimately graft failure. 15 Currently, islet clinical allotransplantation is performed by intraportal infusion with embolization of the islets to the liver, but this implantation site is not an ideal site, due to instant blood-mediated inflammatory reaction (IBMIR), causing a graft loss of up to 70%. ...
Objective:
Islet allotransplantation has demonstrated improved clinical outcomes using the hepatic portal vein as the standard infusion method. However, the current implantation site is not ideal due to the short-term thrombotic and long-term immune destruction. Meanwhile, the shortage of human organ donors further limits its application. To find a new strategy, we tested a new polymer combination for islet encapsulation and transplantation. Meanwhile, we explored a new site for xenogeneic islet transplantation in mice.
Method:
We synthesized a hydrogel combining alginate plus poly-ethylene-imine (Alg/PEI) for the encapsulation of rat, neonatal porcine, and human islets. Transplantation was performed into the retroperitoneal retro-colic space of diabetic mice. Control mice received free islets under the kidney capsule or encapsulated islets into the peritoneum. The biochemical indexes were measured, and the transplanted islets were harvested for immunohistochemical staining of insulin and glucagon.
Results:
Mice receiving encapsulated rat, porcine and human islets transplanted into the retroperitoneal space maintained normoglycemia for a median of 275, 145.5, and 146 days, respectively. In contrast, encapsulated xenogeneic islets transplanted into the peritoneum, maintained function for a median of 61, 95.5, and 82 days, respectively. Meanwhile, xenogeneic islets transplanted free into the kidney capsule lost their function within 3 days after transplantation. Immunohistochemical staining of encapsulated rat, porcine and human islets, retrieved from the retroperitoneal space, allowed to distinguish morphological normal insulin expressing β- and glucagon expressing α-cells at 70, 60, and 100 days post-transplant, respectively.
Conclusion:
Transplantation of Alg/PEI encapsulated xenogeneic islets into the retroperitoneal space provides a valuable new implantation strategy for the treatment of type 1 diabetes.
... Organ-disabled pig models such as the PDX1 KO/mutated (pancreas-disabled) [3][4][5][6] and the SALL1 KO (anephrogenic) [6,7] models have been generated, and some of them have been successfully complemented intraspecies using pig blastomeres, producing pancreases [3,6] and kidneys [6], respectively, the parenchymal cells of which were completely of exogenous origin, whereas the endothelial and stromal cells were formed from a mix of donor and host cells. However, ECs play a critical role in xenotransplantation rejection because they are enriched with epitopes and signaling molecules that trigger the immune response and the coagulation cascade [8]; therefore, the substitution of ECs in humanized organs is of pivotal importance, and to enable this, large-scale production of avascular pig embryos is a necessity. To date, two avascular pig models have been produced: the KDR KO [6] and the ETV2 KO [9] models. ...
Simple Summary
One of the latest goals in regenerative medicine is to use pluripotent stem cells to generate whole organs in vivo through the blastocyst complementation technique. This method consists of the microinjection of pluripotent stem cells into preimplantation embryos that have been genetically modified to ablate the development of a target organ. By taking advantage of the spatiotemporal clues present in the developing embryo, pluripotent stem cells are able to colonize the empty developmental niche and create the missing organ. Combining human pluripotent stem cells with genetically engineered pig embryos, it would be possible to obtain humanized organs that could be used for transplantation, and, therefore, solve the worldwide issue of insufficient availability of transplantable organs. As endothelial cells play a critical role in xenotransplantation rejection in all organs, in this study, we optimized a protocol to generate a vascular-disabled preimplantation pig embryo using the CRISPR/Cas9 system. This protocol could be used to generate avascular embryos for blastocyst complementation experiments and work towards the generation of rejection-free humanized organs in pigs.
Abstract
Each year, tens of thousands of people worldwide die of end-stage organ failure due to the limited availability of organs for use in transplantation. To meet this clinical demand, one of the last frontiers of regenerative medicine is the generation of humanized organs in pigs from pluripotent stem cells (PSCs) via blastocyst complementation. For this, organ-disabled pig models are needed. As endothelial cells (ECs) play a critical role in xenotransplantation rejection in every organ, we aimed to produce hematoendothelial-disabled pig embryos targeting the master transcription factor ETV2 via CRISPR-Cas9-mediated genome modification. In this study, we designed five different guide RNAs (gRNAs) against the DNA-binding domain of the porcine ETV2 gene, which were tested on porcine fibroblasts in vitro. Four out of five guides showed cleavage capacity and, subsequently, these four guides were microinjected individually as ribonucleoprotein complexes (RNPs) into one-cell-stage porcine embryos. Next, we combined the two gRNAs that showed the highest targeting efficiency and microinjected them at higher concentrations. Under these conditions, we significantly improved the rate of biallelic mutation. Hence, here, we describe an efficient one-step method for the generation of hematoendothelial-disabled pig embryos via CRISPR-Cas9 microinjection in zygotes. This model could be used in experimentation related to the in vivo generation of humanized organs.
... In recent years, the generation of bioartificial organs has gone from being an entelechy to a feasible goal, particularly achieved by some laboratories worldwide [2]. The use of transgenic xenotransplantation seeks to remedy some of these drawbacks but still faces problems that are difficult to solve [3]. There is also the generation of bioartificial organs using acellular matrices from human decellularized organs [2,[4][5][6]. ...
The excessive demand for organ transplants has promoted the development of strategies that increase the supply of immune compatible organs, such as xenotransplantation of genetically modified pig organs and the generation of bioartificial organs. We describe a method for the partial replacement of rat endothelial cells for human endothelial cells in a rat’s kidney, obtaining as a final result a rat-human bioartificial kidney. First, in order to maintain parenchymal epithelial cells and selectively eliminate rat endothelial cells, three methods were evaluated in which different solutions were perfused through the renal artery: 0.1% sodium dodecyl sulfate (SDS), 0.01% SDS, and hyperosmolar solutions of sucrose. Then, partially decellularized kidneys were recellularized with human endothelial cells and finally transplanted in an anesthetized rat. The solution of 0.1% SDS achieved the highest vascular decellularization but with high degree of damage in the parenchyma side. On the contrary, 0.01% SDS and hyperosmolar solutions achieved a partial degree of endothelial decellularization. TUNEL assays reveal that hyperosmolar solutions maintained a better epithelial cell viability contrasting with 0.01% SDS. Partially decellularized kidneys were then recellularized with human endothelial cells. Histological analysis showed endothelial cells attached in almost all the vascular bed. Recellularized kidney was transplanted in an anesthetized rat. After surgery, recellularized kidney achieved complete perfusion, and urine was produced for at least 90 min posttransplant. Histological analysis showed endothelial cells attached in almost all the vascular bed. Therefore, endothelial decellularization of grafts and recellularization with human endothelial cells derived from transplant recipients can be a feasible method with the aim to reduce the damage of the grafts.
... These genetic modifications were expected to avoid hyperacute and acute-phase graft injury. Some human genomes were inserted to avoid complement formation (hDAF, hCD46), [14][15][16][17] inhibit coagulation factor activation (hTBM, hEPCR), [18][19][20][21][22] and modulate the immune response (hCD47, hHO1). [22][23][24] In addition to human gene insertion, some pig-derived genes were deleted to avoid antibody-mediated hyperacute graft injury (pGGTA1, pβ4GalNT2, pCMAH) [25][26][27][28] and the graft overgrowth after implantation (pig growth hormone receptor, which is discussed later). ...
Pig kidney xenotransplantation is increasingly regarded as a realistic solution to the current shortage of human organ donors for patients with end-stage organ failure. Recently, the news of three pig-to-human transplantation cases has awakened public interest. Notably, the case by the Alabama team reported detailed and important findings for the xenotransplantation field. Using a genetically modified pig, two porcine kidneys were transplanted into a brain-dead recipient. They applied several approaches established in the preclinical NHP study, including gene-edited pig kidney graft and preoperative laboratory inspection such as crossmatching and infection screening. The pig-to-human kidney xenotransplantation had no unexpected events during surgery or evidence of hyperacute rejection. Unfortunately, the grafts did not work appropriately , and the study had to be terminated due to the decompensation of the recipient. While this study demonstrated the outstanding achievement in this research area, it also revealed remaining gaps to move xenotransplantation to the clinic. While brain-dead human recipients could reinforce the compatibility achievements of gene-edited pigs in NHP, their pro-inflammatory and pro-coagulant environment, in combination with short-duration of experiments will limit the assessment of kidney function, infection and rejection risk post-transplant, in particular antibody-mediated rejection. The use of successful immunosuppressive protocols of non-human primates xenotrans-plant experiments including anti-CD154 antibody will be critical to maximize the success in the first inhuman trials.
... These have only been overcome by the transgenic introduction of human coagulation-regulatory proteins into the pig. 22 Despite the complications and failures that plagued attempts at xenotransplantation for decades, Jaboulay had initiated the exploration of the pig-or goat-as a source of organs for humans. It has required solutions to numerous technological, immunological, and ethical hurdles, which over 100 years later, are now beginning to show the potential that many early proponents foresaw. ...
Mathieu Jaboulay (1860-1913) was a professor of clinical surgery in Lyon, France who is best known for his development of vascular anastomosis and for conducting the first reported renal xenotransplantation experiments on humans, using pig and goat kidneys to treat end-stage renal failure in 1906. His insights and pioneering techniques contributed significantly to allotransplantation and contemporary attempts at xenotransplantation. He is also credited with inventing several surgical instruments and novel surgical techniques that continue to influence vascular, general, and urological surgery to this day. However, this article will focus specifically on his notable contributions to xenotransplantation research and surgery.
... Combined with immunosuppressive therapy, GalTKO or the expression of one or more hCRPs dramatically reduced hyperacute rejection and prolonged the graft survival time in heterotropic nonlife supporting cardiac xenotransplantation. 613 The combination of GalTKO and hCD46 expression further prolonged nonlife-supporting xenograft survival to 236 days. 614 As the graft survival time increases, coagulation dysregulation becomes more obvious, 615 suggesting another major barrier to successful xenotransplantation. Under physiological conditions, there is a balance between coagulation and anticoagulation. ...
Chronic heart failure is the end stage of cardiac diseases. With a high prevalence and a high mortality rate worldwide, chronic heart failure is one of the heaviest health-related burdens. In addition to the standard neurohormonal blockade therapy, several medications have been developed for chronic heart failure treatment, but the population-wide improvement in chronic heart failure prognosis over time has been modest, and novel therapies are still needed. Mechanistic discovery and technical innovation are powerful driving forces for therapeutic development. On the one hand, the past decades have witnessed great progress in understanding the mechanism of chronic heart failure. It is now known that chronic heart failure is not only a matter involving cardiomyocytes. Instead, chronic heart failure involves numerous signaling pathways in noncardiomyocytes, including fibroblasts, immune cells, vascular cells, and lymphatic endothelial cells, and crosstalk among these cells. The complex regulatory network includes protein–protein, protein–RNA, and RNA–RNA interactions. These achievements in mechanistic studies provide novel insights for future therapeutic targets. On the other hand, with the development of modern biological techniques, targeting a protein pharmacologically is no longer the sole option for treating chronic heart failure. Gene therapy can directly manipulate the expression level of genes; gene editing techniques provide hope for curing hereditary cardiomyopathy; cell therapy aims to replace dysfunctional cardiomyocytes; and xenotransplantation may solve the problem of donor heart shortages. In this paper, we reviewed these two aspects in the field of failing heart signaling cascades and emerging therapeutic strategies based on modern biological techniques.
The collective efforts of scientists over multiple decades have led to advancements in molecular and cellular biology-based technologies including genetic engineering and animal cloning, that are now being harnessed to enhance the suitability of pig organs for xenotransplantation into humans. Using organs sourced from pigs with multiple gene deletions and human transgene insertions, investigators have overcome formidable immunological and physiological barriers in pig-to-non-human primate (NHP) xenotransplantation and achieved prolonged pig xenograft survival. These studies informed the design of Revivicor's (Revivicor Inc, Blacksburg, VA) genetically engineered pig with 10 genetic modifications (10 GE) (including the inactivation of 4 endogenous porcine genes and insertion of 6 human transgenes) whose hearts and kidneys have now been studied in preclinical human xenotransplantation models using brain-dead recipients. Additionally, the first two clinical cases of pig-to-human heart xenotransplantation were recently performed using hearts from this 10 GE pig at the University of Maryland. While this review focuses on xenotransplantation of hearts and kidneys, multiple organs, tissues, and cell-types from genetically engineered pigs will provide much-needed therapeutic interventions in the future.
An estimated 1.5 million Americans suffer from Type I diabetes mellitus, and its incidence is increasing worldwide. Islet allotransplantation offers a treatment, but the availability of deceased human donor pancreases is limited. The transplantation of islets from gene-edited pigs, if successful, would resolve this problem. Pigs are now available in which the expression of the three known xenoantigens against which humans have natural (preformed) antibodies has been deleted, and in which several human ‘protective’ genes have been introduced. The transplantation of neonatal pig islets has some advantages over that of adult pig islets. Transplantation into the portal vein of the recipient results in loss of many islets from the instant blood-mediated inflammatory reaction (IBMIR) and so the search for an alternative site continues. The adaptive immune response can be largely suppressed by an immunosuppressive regimen based on blockade of the CD40/CD154 T cell co-stimulation pathway, whereas conventional therapy (e.g., based on tacrolimus) is less successful. We suggest that, despite the need for effective immunosuppressive therapy, the transplantation of ‘free’ islets will prove more successful than that of encapsulated islets. There are data to suggest that, in the absence of rejection, the function of pig islets, though less efficient than human islets, will be sufficient to maintain normoglycemia in diabetic recipients. Pig islets transplanted into immunosuppressed nonhuman primates have maintained normoglycemia for periods extending more than two years, illustrating the potential of this novel form of therapy.
The pig has long been used as a research animal and has now gained importance as a potential source of organs for clinical xenotransplantation. When an organ from a wild-type (i.e., genetically unmodified) pig is transplanted into an immunosuppressed nonhuman primate, a vigorous host immune response causes hyperacute rejection (within minutes or hours). This response has been largely overcome by 1) extensive gene editing of the organ-source pig and 2) the administration to the recipient of novel immunosuppressive therapy based on blockade of the CD40/CD154 T cell costimulation pathway. Gene editing has consisted of 1) deletion of expression of the 3 known carbohydrate xenoantigens against which humans have natural (preformed) antibodies and 2) the introduction of human ‘protective’ genes. The combination of gene editing and novel immunosuppressive therapy has extended life-supporting pig kidney graft survival to greater than 1 y and of pig heart survival to up to 9 mo. This review briefly describes the techniques of gene editing, the potential risks of transfer of porcine endogenous retroviruses with the organ, and the need for breeding and housing of donor pigs under biosecure conditions.
End-stage organ failure can result from various preexisting conditions and occurs in patients of all ages, and organ transplantation remains its only treatment. In recent years, extensive research has been done to explore the possibility of transplanting animal organs into humans, a process referred to as xenotransplantation. Due to their matching organ sizes and other anatomical and physiological similarities with humans, pigs are the preferred organ donor species. Organ rejection due to host immune response and possible interspecies infectious pathogen transmission have been the biggest hurdles to xenotransplantation's success. Use of genetically engineered pigs as tissue and organ donors for xenotransplantation has helped to address these hurdles. Although several preclinical trials have been conducted in nonhuman primates, some barriers still exist and demand further efforts. This review focuses on the recent advances and remaining challenges in organ and tissue xenotransplantation.
Expected final online publication date for the Annual Review of Animal Biosciences, Volume 12 is February 2024. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
Antibody-mediated rejection (AMR) is the commonest cause of failure of a pig graft after transplantation into an immunosuppressed nonhuman primate (NHP). The incidence of AMR compared to acute cellular rejection is much higher in xenotransplantation (46% vs. 7%) than in allotransplantation (3% vs. 63%) in NHPs. Although AMR in an allograft can often be reversed, to our knowledge there is no report of its successful reversal in a pig xenograft. As there is less experience in preventing or reversing AMR in models of xenotransplantation, the results of studies in patients with allografts provide more information. These include (i) depletion or neutralization of serum anti-donor antibodies, (ii) inhibition of complement activation, (iii) therapies targeting B or plasma cells, and (iv) anti-inflammatory therapy. Depletion or neutralization of anti-pig antibody, for example, by plasmapheresis, is effective in depleting antibodies, but they recover within days. IgG-degrading enzymes do not deplete IgM. Despite the expression of human complement-regulatory proteins on the pig graft, inhibition of systemic complement activation may be necessary, particularly if AMR is to be reversed. Potential therapies include (i) inhibition of complement activation (e.g., by IVIg, C1 INH, or an anti-C5 antibody), but some complement inhibitors are not effective in NHPs, for example, eculizumab. Possible B cell-targeted therapies include (i) B cell depletion, (ii) plasma cell depletion, (iii) modulation of B cell activation, and (iv) enhancing the generation of regulatory B and/or T cells. Among anti-inflammatory agents, anti-IL6R mAb and TNF blockers are increasingly being tested in xenotransplantation models, but with no definitive evidence that they reverse AMR. Increasing attention should be directed toward testing combinations of the above therapies. We suggest that treatment with a systemic complement inhibitor is likely to be most effective, possibly combined with anti-inflammatory agents (if these are not already being administered). Ultimately, it may require further genetic engineering of the organ-source pig to resolve the problem entirely, for example, knockout or knockdown of SLA, and/or expression of PD-L1, HLA E, and/or HLA-G.
Transfusion of allogeneic human red blood cell (hRBCs) is limited by supply and compatibility between individual donors and recipients. In situations where the blood supply is constrained or when no compatible RBCs are available, patients suffer. As a result, alternatives to hRBCs that complement existing RBC transfusion strategies are needed. Pig RBCs (pRBCs) could provide an alternative because of their abundant supply, and functional similarities to hRBCs. The ability to genetically modify pigs to limit pRBC immunogenicity and augment expression of human 'protective' proteins has provided major boosts to this research and opens up new therapeutic avenues. Although deletion of expression of xenoantigens has been achieved in genetically-engineered pigs, novel genetic methods are needed to introduce human 'protective' transgenes into pRBCs at the high levels required to prevent hemolysis and extend RBC survival in vivo. This review addresses recent progress and examines future prospects for clinical xenogeneic pRBC transfusion.
Currently, the most feasible and widely practiced option for patients with end-stage organ failure is the transplantation of part of or whole organs, either from deceased or living donors. However, organ shortage has posed and is still posing a big challenge in this field. Newer options being explored are xenografts and engineered/bioengineered tissues/organs. Already small steps have been taken in this direction and sooner or later, these will become a norm in this field. However, these developments will pose different challenges for the diagnosis and management of problems as compared with traditional allografts. The approach to pathologic diagnosis of dysfunction in these settings will likely be significantly different. Thus, there is a need to increase awareness and prepare transplant diagnosticians to meet this future challenge in the field of xenotransplantation/ regenerative medicine. This review will focus on the current status of transplant pathology and how it will be changed in the future with the emerging scenario of routine xenotransplantation.
La escasez de órganos humanos para trasplante ha propiciado la búsqueda de otras alternativas, como la posibilidad de utilizar animales como donantes, lo que se conoce como xenotrasplante. El cerdo se ha convertido en la especie donante más prometedora tanto por su similitud anatómica y fisiológica con el ser humano, como por la disponibilidad de metodologías de modificación genética, que han permitido la obtención de modelos porcinos que permiten hacer frente a los riesgos del xenotrasplante. Estos riesgos están asociados con el rechazo inmunológico, la desregulación de la coagulación y la presencia de retrovirus endógenos porcinos. Aunque los resultados de los ensayos preclínicos en primates varían considerablemente, alcanzando diferentes tiempos de supervivencia en función del órgano trasplantado, la inactivación del gen que codifica el antígeno α-Gal ha resultado imprescindible para superar el rechazo hiperagudo, que es la primera y más drástica barrera inmunológica. Los hitos que se han logrado en los últimos años, incluyendo las pruebas clínicas recientes con seres humanos a los que se han realizado trasplantes de riñón o corazón de cerdos modificados genéticamente, acercan un paso más el traslado de los xenotrasplantes a la práctica clínica
Progress in pig organ xenotransplantation has been made largely through (1) genetic engineering of the organ-source pig to protect its tissues from the human innate immune response, and (2) development of an immunosuppressive regimen based on blockade of the CD40/CD154 costimulation pathway to prevent the adaptive immune response. In the 1980s, after transplantation into nonhuman primates (NHPs), wild-type (genetically unmodified) pig organs were rejected within minutes or hours. In the 1990s, organs from pigs expressing a human complement-regulatory protein (CD55) transplanted into NHPs receiving intensive conventional immunosuppressive therapy functioned for days or weeks. When costimulation blockade was introduced in 2000, the adaptive immune response was suppressed more readily. The identification of galactose-α1,3-galactose as the major antigen target for human and NHP anti-pig antibodies in 1991 allowed for deletion of expression of galactose-α1,3-galactose in 2003, extending pig graft survival for up to 6 months. Subsequent gene editing to overcome molecular incompatibilities between the pig and primate coagulation systems proved additionally beneficial. The identification of 2 further pig carbohydrate xenoantigens allowed the production of 'triple-knockout' pigs that are preferred for clinical organ transplantation. These combined advances enabled the first clinical pig heart transplant to be performed and opened the door to formal clinical trials.
Objective
The availability of clinically applied medical materials in thoracic surgery remains insufficient, especially materials for treating tracheal defects. Herein, the potential of porcine extracellular matrix (P-ECM) as a new airway reconstruction material was explored by xenotransplanting it into a canine trachea.
Methods
P-ECM was first transplanted into the buttocks of Narc Beagle dogs ( n = 3) and its overall immuno-induced effects were evaluated. Subsequently, nine dogs underwent surgery to create a tracheal defect that was 1 × 2 cm. In group A, the P-ECM was implanted parallel to the tracheal axis ( n = 3), whereas in group B the P-ECM was implanted perpendicular to the tracheal axis ( n = 6). The grafts were periodically observed by bronchoscopy and evaluated postoperatively at 1 and 3 months through macroscopic and microscopic examinations. Immunosuppressants were not administered. Statistical evaluation was performed for Bronchoscopic stenosis rate, graft epithelialization rate, shrinkage rate and ECM live-implantation rate.
Results
No sign of P-ECM rejection was observed after its implantation in the buttocks. Bronchoscopic findings showed no improvement concerning stenosis in group A until 3 months after surgery; epithelialization of the graft site was not evident, and the ECM site appeared scarred and faded. In contrast, stenosis gradually improved in group B, with continuous epithelium within the host tissues and P-ECM. Histologically, the graft site contracted longitudinally and no epithelialization was observed in group A, whereas full epithelialization was observed on the P-ECM in group B. No sign of cartilage regeneration was confirmed in both groups. No statistically significant differences were found in bronchoscopic stenosis rate, shrinkage rate and ECM live-implantation rate, but graft epithelialization rate showed a statistically significant difference (G-A; sporadic (25%) 3, vs. G-B; full covered (100%) 3; p = 0.047).
Conclusions
P-ECM can support full re-epithelialization without chondrocyte regeneration, with perpendicular implantation facilitating epithelialization of the ECM. Our results showed that our decellularized tracheal matrix holds clinical potential as a biological xenogeneic material for airway defect repair.
Introduction:
Xenografts are a now a cornerstone in the management of wound dressings. Promising results were achieved since 1960 in the application of skin substitute for skin defects.
Objective:
The objective of this study was to evaluate the efficacy of various xenografts.
Methods:
A literature research was conducted using the following query: 'Porcine skin dermatology substitute', 'bovine skin dermatology substitute', 'xenograft skin substitute dermatology', 'xenografts skin defect', 'porcine skin defect', 'bovine skin defect'.
Results:
The review yielded 35 articles pertaining to the topic. Main indications for porcine and bovine xenograft application were burn wounds and post-traumatic wounds, respectively. Mean discharge date or length of stay was at the 6th day after porcine application, and the time of graft healing was reported for 33.7% (n = 510) of patients. Promising results were seen with Matriderm and split-thickness skin graft. Most wounds achieved an excellent cosmetic result with full range of motion and a smooth contour appearance. A great variety of tissue substitutes exist, and the choice of graft application should depend on a patient's factors, product availability, wound type, size, and physician's factors.
Conclusion:
In summary, xenografts are more economic and affordable but have higher risk of infections compared to allografts.
There is a global shortage of organs for transplantation and despite many governments making significant changes to their organ donation systems, there are not enough kidneys available to meet the demand. This has led scientists and clinicians to explore alternative means of meeting this organ shortfall. One of the alternatives to human organ transplantation is xenotransplantation, which is the transplantation of organs, tissues, or cells between different species. The resurgence of interest in xenotransplantation and recent scientific breakthroughs suggest that genetically-engineered pigs may soon present a realistic alternative as sources of kidneys for clinical transplantation. It is therefore important for nurses and allied health professionals to understand what is involved in xenotransplantation and its future implications for their clinical practices. First, we explore the insufficiency of different organ donation systems to meet the kidney shortage. Second, we provide a background and a summary of the progress made so far in xenotransplantation research. Third, we discuss some of the scientific, technological, ethical, and economic issues associated with xenotransplantation. Finally, we summarise the literature on the attitudes of healthcare professionals toward xenotransplantation.
Robotic surgery is a surgical modality within the minimally invasive techniques and consists of a coupling of a robotic interface between surgeon and patient, enhancing the urologist’s skills in performing established urological procedures. It is an avant-garde modality as it allows revisiting techniques performed in open surgery, but with greater precision, privileged vision and superhuman freedom of movement. This great interest in adapting this new technology in routine procedures allowed a great development in anatomical and surgical knowledge, allowing procedures with the purpose of preserving and also reconstructing the urinary tract. One of the well-established applications in robotic surgery that allows a kidney preservation is the nephron-sparing partial nephrectomy, which avoids the complete removal of the kidney in case of localized renal tumors. Robotic surgery has added other technologies for this purpose, such as the use of 3D images and augmented reality to improve the precision and saving nephrons. Within the renal replacement therapy, robotic surgery allowed the performance of kidney transplants through the minimally invasive route, as the precision of movements allowed the creation of the anastomosis in a confined space. In addition to these applications, reconstructive urology is becoming increasingly feasible with robotic surgery, as detailed sutures can be performed with the aid of the robot. Robotic surgery continues to lead urological surgery into the future, adding technologies and allowing to follow the development of diagnostic methods and clinical therapies, always with the main objective of preserving renal function.KeywordsRobotic surgeryInnovationUrologyPartial nephrectomyKidney transplantationReconstructive urology
Due to the worldwide increase in the number of patients with end-stage kidney disease, there are concerns regarding further shortages of renal replacement therapy (RRT) supply in the future. With the discovery of induced pluripotent stem cells and the expectation that autologous pluripotent stem cells (PSCs) can be used, there has been extensive research to develop methods to artificially differentiate and produce PSCs into renal tissue such as organoids. Moreover, xenotransplantation is becoming more realistic than ever before with the pleiotropic control of rejection immunity through the combination of genome editing and new drugs. Furthermore, chimeric technologies such as blastocyst complementation and fetus organ complementation are rapidly advancing as an intermediate organ regeneration technology between technologies created from autologous stem cells alone and xenotransplantation that does not depend on stem cells. This session will present challenging strategies for the development of new RRTs.KeywordsRegenerative medicineXenotransplantationIps cellsOrganoidChimeraBlastocyst complementationFetus organ complementation
Background:
Antibody-mediated rejection has long been known to be one of the major organ failure mechanisms in xenotransplantation. In addition to the porcine α1,3-galactose (α1,3Gal) epitope, N-Glycolylneuraminic acid (Neu5Gc), a sialic acid, has been identified as an important porcine antigen against which most humans have pre-formed antibodies. Here we evaluate GalTKO.hCD46 lungs with an additional cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) gene knock-out (Neu5GcKO) in a xenogeneic ex vivo perfusion model METHODS: Eleven GalTKO.hCD46.Neu5GcKO pig lungs were perfused for up to 6 h with fresh heparinized human blood. Six of them were treated with histamine (H) blocker famotidine and 1-thromboxane synthase inhibitor Benzylimidazole (BIA) and five were left untreated. GalTKO.hCD46 lungs without Neu5GcKO (n = 18: eight untreated and 10 BIA+H treated) served as a reference. Functional parameters, blood, and tissue samples were collected at pre-defined time points throughout the perfusion RESULTS: All but one Neu5GcKO organs maintained adequate blood oxygenation and "survived" until elective termination at 6 h whereas two reference lungs failed before elective termination at 4 h. Human anti-Neu5Gc antibody serum levels decreased during the perfusion of GalTKO.hCD46 lungs by flow cytometry (∼40% IgM, 60% IgG), whereas antibody levels in Neu5GcKO lung perfusions did not fall (IgM p = .007; IgG p < .001). Thromboxane elaboration, thrombin generation, and histamine levels were significantly reduced with Neu5GcKO lungs compared to reference in the untreated groups (p = .007, .005, and .037, respectively); treatment with BIA+H masked these changes. Activation of platelets, measured as CD62P expression on circulating platelets, was lower in Neu5GcKO experiments compared to reference lungs (p = .023), whereas complement activation (as C3a rise in plasma) was not altered. MCP-1 and lactotransferin level elevations were blunted in Neu5GcKO lung perfusions (p = .007 and .032, respectively). Pulmonary vascular resistance (PVR) rise was significantly attenuated and delayed in untreated GalTKO.hCD46.Neu5GcKO lungs in comparison to the untreated GalTKO.hCD46 lungs (p = .003) CONCLUSION: Additional Neu5GcKO in GalTKO.hCD46 lungs significantly reduces parameters associated with antibody-mediated inflammation and activation of the coagulation cascade. Knock-out of the Neu5Gc sialic acid should be beneficial to reduce innate immune antigenicity of porcine lungs in future human recipients.
By 2030, diabetes will be the 7th leading cause of premature mortality worldwide, according to the World Health Organisation. The application of nanotechnology in medicine holds many possible advantages and over the past few decades, there has been huge progress in its utilisation. Nanotechnology is widely applied for cancer treatment and other diseases but, the use of it for diabetes treatment is now starting to flourish. This book presents the latest developments of nanomedicine for the treatment of different facets of diabetes and related disorders. With a multidisciplinary approach, chapters focus on previously overlooked topics in glucose sensing, insulin delivery and secretion, bioimaging and transplantation of islets. This book is suitable for researchers of nanomedicine, nanotechnology and diabetes looking into the emergence of new approaches for the treatment of this life-threatening disease.
Transplantation is often the last resort for end-stage organ failures, e.g., kidney, liver, heart, lung, and pancreas. The shortage of donor organs is the main limiting factor for successful transplantation in humans. Except living donations, other alternatives are needed, e.g., xenotransplantation of pig organs. However, immune rejection remains the major challenge to overcome in xenotransplantation. There are three different xenogeneic types of rejections, based on the responses and mechanisms involved. It includes hyperacute rejection (HAR), delayed xenograft rejection (DXR) and chronic rejection. DXR, sometimes involves acute humoral xenograft rejection (AHR) and cellular xenograft rejection (CXR), which cannot be strictly distinguished from each other in pathological process. In this review, we comprehensively discussed the mechanism of these immunological rejections and summarized the strategies for preventing them, such as generation of gene knock out donors by different genome editing tools and the use of immunosuppressive regimens. We also addressed organ-specific barriers and challenges needed to pave the way for clinical xenotransplantation. Taken together, this information will benefit the current immunological research in the field of xenotransplantation.
Purpose of review:
The field of xenotransplantation has seen remarkable progress since its inception with recent preclinical trials in human recipients pushing kidney xenotransplantation one-step closer to clinical reality. In this review, we update practicing clinicians on recent advances in kidney xenotransplantation given the proximity of clinical trials in humans.
Recent findings:
Early studies in the field established the physiologic basis of xenotransplantation and suggested that the pig kidney will support human physiology. Genetic engineering of source pigs has greatly reduced the immunogenicity of kidney grafts, and studies in nonhuman primates have demonstrated the viability of kidney xenotransplants for months after transplantation. Finally, a recent study in a novel preclinical human model demonstrated that key findings in NHP experiments are generalizable to humans, namely, the absence of hyperacute rejection.
Summary:
Overall, it appears that critical physiologic, immunologic and technical barriers to implementation of clinical trials in humans have been overcome.
To date, the wide application of cell-based biomaterials in tissue engineering and regeneration is remarkably hampered by immune rejection. Reducing the immunogenicity of cell-based biomaterials has become the latest direction in biomaterial research. Recently, genetically modified cell-based biomaterials with immunomodulatory genes have become a feasible solution to the immunogenicity problem. In this review, recent advances and future challenges of genetically modified immunomodulatory cell-based biomaterials are elaborated, including fabrication approaches, mechanisms of common immunomodulatory genes, application and, more importantly, current preclinical and clinical advances. The fabrication approaches can be categorized into commonly used (e.g., virus transfection) and newly developed approaches. The immunomodulatory mechanisms of representative genes involve complicated cell signaling pathways and metabolic activities. Wide application in curing multiple end-term diseases and replacing lifelong immunosuppressive therapy in multiple cell and organ transplantation models is demonstrated. Most significantly, practices of genetically modified organ transplantation have been conducted on brain-dead human decedent and even on living patients after a series of experiments on nonhuman primates. Nevertheless, uncertain biosecurity, nonspecific effects and overlooked personalization of current genetically modified immunomodulatory cell-based biomaterials are shortcomings that remain to be overcome.
Pig-to-human organ transplantation is a feasible solution to resolve the shortage of organ donors for patients that wait for transplantation. To overcome immunological rejection, which is the main hurdle in pig-to-human xenotransplantation, various engineered transgenic pigs have been developed. Ablation of xeno-reactive antigens, especially the 1,3-Gal epitope (GalT), which causes hyperacute rejection, and insertion of complement regulatory protein genes, such as hCD46, hCD55, and hCD59, and genes to regulate the coagulation pathway or immune cell-mediated rejection may be required for an ideal xenotransplantation model. However, the technique for stable and efficient expression of multi-transgenes has not yet been settled to develop a suitable xenotransplantation model. To develop a stable and efficient transgenic system, we knocked-in internal ribosome entry sites (IRES)-mediated transgenes into the α 1,3-galactosyltransferase (GGTA1) locus so that expression of these transgenes would be controlled by the GGTA1 endogenous promoter. We constructed an IRES-based polycistronic hCD55/hCD39 knock-in vector to target exon4 of the GGTA1 gene. The hCD55/hCD39 knock-in vector and CRISPR/Cas9 to target exon4 of the GGTA1 gene were co-transfected into white yucatan miniature pig fibroblasts. After transfection, hCD39 expressed cells were sorted by FACS. Targeted colonies were verified using targeting PCR and FACS analysis, and used as donors for somatic cell nuclear transfer. Expression of GalT, hCD55, and hCD39 was analyzed by FACS and western blotting. Human complement-mediated cytotoxicity and human antibody binding assays were conducted on peripheral blood mononuclear cells (PBMCs) and red blood cells (RBCs), and deposition of C3 by incubation with human complement serum and platelet aggregation were analyzed in GGTA1 knock-out (GTKO)/CD55/CD39 pig cells. We obtained six targeted colonies with high efficiency of targeting (42.8% of efficiency). Selected colony and transgenic pigs showed abundant expression of targeted genes (hCD55 and hCD39). Knocked-in transgenes were expressed in various cell types under the control of the GGTA1 endogenous promoter in GTKO/CD55/CD39 pig and IRES was sufficient to express downstream expression of the transgene. Human IgG and IgM binding decreased in GTKO/CD55/CD39 pig and GTKO compared to wild-type pig PBMCs and RBCs. The human complement-mediated cytotoxicity of RBCs and PBMCs decreased in GTKO/CD55/CD39 pig compared to cells from GTKO pig. C3 was also deposited less in GTKO/CD55/CD39 pig cells than wild-type pig cells. The platelet aggregation was delayed by hCD39 expression in GTKO/CD55/CD39 pig. In the current study, knock-in into the GGTA1 locus and GGTA1 endogenous promoter-mediated expression of transgenes are an appropriable strategy for effective and stable expression of multi-transgenes. The IRES-based polycistronic transgene vector system also caused sufficient expression of both hCD55 and hCD39. Furthermore, co-transfection of CRISPR/Cas9 and the knock-in vector not only increased the knock-in efficiency but also induced null for GalT by CRISPR/Cas9-mediated double-stranded break of the target site. As shown in human complement-mediated lysis and human antibody binding to GTKO/CD55/CD39 transgenic pig cells, expression of hCD55 and hCD39 with ablation of GalT prevents an effective immunological reaction in vitro. As a consequence, our technique to produce multi-transgenic pigs could improve the development of a suitable xenotransplantation model, and the GTKO/CD55/CD39 pig developed could prolong the survival of pig-to-primate xenotransplant recipients.
Type 1 diabetes (T1D) is an autoimmune disease wherein the pancreas does not produce enough insulin due to islet beta cell destruction. Despite improvements in delivering exogenous insulin to T1D patients, pancreas or islet transplantation remains the best way to regulate their glycaemia. Results from experimental islet transplantation have improved dramatically in the last 15 years, to the point where it can be comparable to pancreas transplantation, but without the accompanying morbidity associated with this procedure. As with other transplants, the limiting factor in islet allotransplantation is the relatively small number of organs made available by deceased human donors throughout the world. A strong case can be made for islet xenotransplantation to fill the gap between supply and demand; however, transplantation across species presents challenges that are unique to that setting. In the search for the most suitable animal for human xenotransplantation, the pig has many advantages that make it the likely animal of choice. Potentially one of the most beneficial advantages is the ability to genetically engineer porcine donors to be more compatible with human recipients. Several genetic manipulations have already proven useful in relation to hyperacute rejection and inflammation (instant blood mediated inflammatory reaction), with the potential of even further advancement in the near future.
We describe the incidence of early graft failure (EGF, defined as loss of function from any cause within 3 days after transplant) in a large cohort of GalTKO pig organs transplanted into baboons in three centers, and the effect of additional expression of a human complement pathway-regulatory protein, CD46 or CD55 (GalTKO.hCPRP). Baboon recipients of life-supporting GalTKO kidney (n = 7) or heterotopic heart (n = 14) grafts received either no immunosuppression (n = 4), or one of several partial or full immunosuppressive regimens (n = 17). Fourteen additional baboons received a GalTKO.hCPRP kidney (n = 5) or heart (n = 9) and similar treatment regimens. Immunologic, pathologic, and coagulation parameters were measured at frequent intervals. EGF of GalTKO organs occurred in 9/21 baboons (43%). hCPRP expression reduced the GalTKO EGF incidence to 7% (1/14; P < 0.01 vs. GalTKO alone). At 30 mins, complement deposits were more intense in organs in which EGF developed (P < 0.005). The intensity of peri-transplant platelet activation (as β-thromboglobulin release) correlated with EGF, as did the cumulative coagulation score (P < 0.01). We conclude that (i) the transgenic expression of a hCPRP on the vascular endothelium of a GalTKO pig reduces the incidence of EGF and reduces complement deposition, (ii) complement deposition and platelet activation correlate with early GalTKO organ failure, and (iii) the expression of a hCPRP reduces EGF but does not prevent systemic coagulation activation. Additional strategies will be required to control coagulation activation.
© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
Background:
With the introduction of the α1, 3-galactosyltransferase gene-knockout (GT-KO) pig and its pivotal role in preventing hyperacute rejection (HAR), coagulation remains a considerable obstacle yet to be overcome in order to provide long-term xenograft survival. Thrombomodulin (TBM) plays a critical anticoagulant and anti-inflammatory role in its part of the protein C pathway. Many studies have demonstrated the strong anticoagulant effects of TBM in xenotransplantation, but its complement regulatory effects have not been appropriately examined. Here, we investigate whether TBM can regulate complement activation as well as coagulation in response to xenogeneic stimuli.
Methods:
We transfected porcine endothelial cells (MPN-3) with adenovirus vectors containing the human TBM gene (ad-hTBM), or a control gene containing GFP (ad-GFP). The expression level of ad-hTBM was measured by flow cytometry. To confirm the anticoagulant effect of TBM, coagulation time was measured after treatment with recalcified human plasma in ad-hTBM-transfected MPN-3, and a thrombin activity assay was performed after treatment with 50% human serum in ad-hTBM-infected MPN-3.
Results:
Thrombin generation was significantly decreased in a dose-dependent manner in ad-TBM group, and coagulation time was increased in the ad-hTBM group when compared to the ad-GFP group. Complement-dependent serum toxicity assays were performed after treatment with 20% human serum or heat-inactivated human serum by LDH assay. Complement-dependent toxicity was significantly attenuated in the ad-hTBM group, but complement-independent toxicity was not attenuated in the ad-hTBM group. These results suggest that human thrombomodulin (hTBM) has complement regulatory effects as well as anticoagulant effects. To further investigate the mechanisms of complement regulation by hTBM, we deleted the EGF5, 6 domains that are involved in thrombin generation or the lectin-like domain involved in inflammation of TBM and functional tests were performed using these modified forms. We showed that the EGF5, 6 domain of TBM principally inhibits complement activation rather than the lectin domain.
Conclusion:
The EGF5, 6 domains of TBM appear to be the major domains for down-regulating the complement system rather than the lectin-like domain during xenogenic stimuli. The role of EGF5, 6 domains of hTBM may be due to inhibition of thrombin as thrombin can cleave C3a and C5a directly and hTBM may also be involved in complement regulation. Clearly then human TBM has complement regulatory effects as well as anticoagulant effects in xeno-immune response, and it is a promising target for attenuating xenograft rejection.
Xenotransplantation has the potential to alleviate the organ shortage that prevents many patients with end-stage renal disease from enjoying the benefits of kidney transplantation. Despite significant advances in other models, pig-to-primate kidney xenotransplantation has met limited success. Preformed anti-pig antibodies are an important component of the xenogeneic immune response. To address this, we screened a cohort of 34 rhesus macaques for anti-pig antibody levels. We then selected animals with both low and high titers of anti-pig antibodies to proceed with kidney transplant from galactose-α1,3-galactose knockout/CD55 transgenic pig donors. All animals received T-cell depletion followed by maintenance therapy with costimulation blockade (either anti-CD154 mAb or belatacept), mycophenolate mofetil, and steroid. The animal with the high titer of anti-pig antibody rejected the kidney xenograft within the first week. Low-titer animals treated with anti-CD154 antibody, but not belatacept exhibited prolonged kidney xenograft survival (>133 and >126 vs. 14 and 21 days, respectively). Long-term surviving animals treated with the anti-CD154-based regimen continue to have normal kidney function and preserved renal architecture without evidence of rejection on biopsies sampled at day 100. This description of the longest reported survival of pig-to-non-human primate kidney xenotransplantation, now >125 days, provides promise for further study and potential clinical translation.
© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
The fundamental problem of corneal transplantation is a severe shortage of donor tissues worldwide, resulting in approximately 1.5 million new cases of blindness annually. To explore an alternative to donor corneas, we conducted a clinical study in which implanted acellular porcine corneal stromas (APCSs) replaced the pathologic anterior corneas in 47 patients who had experienced fungal corneal infections. Subsequently, we demonstrated the safety and efficacy of APCSs in human keratoplasty for a minimum follow-up period of 6 months, during which time no recurrence of infection was observed. All corneal ulcers healed with the return of neovascularization. In addition, our results indicated that epithelialization occurred in all APCS grafts except four grafts; for these four, the grafts dissolved to varying degrees. Furthermore, most porcine grafts (n = 41) gradually became transparent without rejection, and an improvement of more than two lines in best corrected visual acuity (BCVA) was achieved in 34 eyes (∼72%). Finally, no patients showed any severe adverse reaction or any significant change in postoperative systemic safety indicators. Thus, we concluded that APCS grafts are safe and efficacious during lamellar keratoplasty in treating corneal fungal ulcers and potentially for other clinical diseases.
© Copyright 2015 The American Society of Transplantation and the American Society of Transplant Surgeons.
Previous attempts of α-1,3-galactocyltransferase knockout (GalTKO) pig bone marrow (BM) transplantation (Tx) into baboons have demonstrated a loss of macro-chimerism within 24 h in most cases. In order to achieve improved engraftment with persistence of peripheral chimerism, we have developed a new strategy of intra-bone BM (IBBM) Tx. Six baboons received GalTKO BM cells, with one-half of the cells transplanted into the bilateral tibiae directly and the remaining cells injected intravenously (IBBM/BM-Tx) with a conditioning immunosuppressive regimen. In order to assess immune responses induced by the combined IBBM/BM-Tx, three recipients received donor SLA-matched GalTKO kidneys in the peri-operative period of IBBM/BM-Tx (Group 1), and the others received kidneys 2 months after IBBM/BM-Tx (Group 2). Peripheral macro-chimerism was continuously detectable for up to 13 days (mean 7.7 days; range 3-13) post-IBBM/BM-Tx and in three animals, macro-chimerism reappeared at days 10, 14 and 21. Pig CFUs, indicating porcine progenitor cell engraftment, were detected in the host BM in four of six recipients on days 14, 15, 19 and 28. In addition, anti-pig unresponsiveness was observed by in vitro assays. GalTKO/pCMV-kidneys survived for extended periods (47 and 60 days). This strategy may provide a potent adjunct for inducing xenogeneic tolerance through BM-Tx.
© Copyright 2015 The American Society of Transplantation and the American Society of Transplant Surgeons.
Mixed chimerism approaches for induction of tolerance of solid organ transplants have been applied successfully in animal models and in the clinic. However, in xenogeneic models (pig-to-primate), host macrophages participate in the rapid clearance of porcine hematopoietic progenitor cells, hindering the ability to achieve mixed chimerism. CD47 is a cell-surface molecule that interacts in a species-specific manner with SIRPα receptors on macrophages to inhibit phagocytosis and expression of human CD47 (hCD47) on porcine cells has been shown to inhibit phagocytosis by primate macrophages. We report here the generation of hCD47 transgenic GalT-KO miniature swine that express hCD47 in all blood cell lineages. The effect of hCD47 expression on xenogeneic hematopoietic engraftment was tested in an in vivo mouse model of human hematopoietic cell engraftment. High-level porcine chimerism was observed in the bone marrow of hCD47 progenitor cell recipients and smaller but readily measurable chimerism levels were observed in the peripheral blood of these recipients. In contrast, transplantation of WT progenitor cells resulted in little or no bone marrow engraftment and no detectable peripheral chimerism. These results demonstrate a substantial protective effect of hCD47 expression on engraftment and persistence of porcine cells in this model, presumably by modulation of macrophage phagocytosis.
Background:
Recent survivals of our pig-to-baboon kidney xenotransplants have been markedly shorter than the graft survivals we previously reported. The discovery of high levels of porcine cytomegalovirus (pCMV) in one of the rejected xenografts led us to evaluate whether this reduction in graft survival might be because of the inadvertent introduction of pCMV into our α1,3-galactosyltransferase gene knockout swine herd.
Methods:
Archived frozen sections of xeno-kidney grafts over the past 10 years were analyzed for the presence of pCMV, using real-time polymerase chain reaction. Three prospective pig-to-baboon renal transplants using kidneys from swine delivered by cesarean section (C-section) and raised in isolation were likewise analyzed.
Results:
Kidney grafts, from which 8 of the 18 archived samples were derived were found to be pCMV-negative, showed a mean graft survival of 48.3 days and were from transplants performed before 2008. None showed signs of disseminated intravascular coagulopathy and were lost because of proteinuria or infectious complications. In contrast, 10 of the archived samples were pCMV positive, were from kidney transplants with a mean graft survival of 14.1 days, had been performed after 2008, and demonstrated early vascular changes and decreased platelet counts. Three prospective xenografts from swine delivered by C-section were pCMV negative and survived an average of 53.0 days.
Conclusions:
Decreased survivals of α1,3-galactosyltransferase gene knockout renal xenografts in this laboratory correlate temporally with latent pCMV in the donor animals and pCMV in the rejected xeno-kidneys. Transmission of pCMV to swine offspring may be avoided by C-section delivery and scrupulous isolation of donor animals.
Platelet activation and microthrombus formation are invariable features of xenograft rejection and the vascular injury observed when porcine organs are transplanted into primates. This pathological process could be mediated, at least in part, by aberrant interactions of von Willebrand Factor (vWF) associated with the donor vasculature with host platelets. Unlike human vWF, native porcine vWF (pvWF) interacts with human GPIb independently of shear stress or nonphysiological stimuli, eg, ristocetin. We therefore contrasted the potential of isolated human and porcine vWF–A1-domains to interact with human platelets in vitro. Both human and porcine vWF–A1-domains expressed as glycosyl phosphatidylinositol–linked FLAG fusion proteins on COS-7 cells induced GPIb-dependent aggregation and intracellular Ca++ uptake of platelets, independent of both the remainder of the vWF protein and additional modifying factors. Porcine A1-domains were more potent than human homologues, and in addition ristocetin could boost platelet aggregation only with the human A1-domain. Putative conformational changes in the porcine A1-domain could result in the heightened, ristocetin-independent interactions observed with human platelets and may be of importance for xenograft survival.
Mouse cells expressing the human complement regulatory proteins decay accelerating factor (DAF) or membrane cofactor protein (MCP) were produced both by hybridoma technology and by transfection with the appropriate cDNAs. The expression of either or both of these products protected the mouse cell from lysis by human (though not rabbit) complement in the presence of naturally occurring human anti‐mouse antibody. This effect could be abrogated by the addition of monoclonal antibody against DAF or MCP. These data suggested that the production of animals transgenic for human complement regulatory proteins should in principle be similarly protected from hyperacute xenograft rejection.
Background:
The impact that the absence of expression of NeuGc in pigs might have on pig organ or cell transplantation in humans has been studied in vitro, but only using red blood cells (pRBCs) and peripheral blood mononuclear cells (pPBMCs) as the target cells for immune assays. We have extended this work in various in vitro models and now report our initial results.
Methods:
The models we have used involve GTKO/hCD46 and GTKO/hCD46/NeuGcKO pig aortas and corneas, and pRBCs, pPBMCs, aortic endothelial cells (pAECs), corneal endothelial cells (pCECs), and isolated pancreatic islets. We have investigated the effect of the absence of NeuGc expression on (i) human IgM and IgG binding, (ii) the T-cell proliferative response, (iii) human platelet aggregation, and (iv) in an in vitro assay of the instant blood-mediated inflammatory reaction (IBMIR) following exposure of pig islets to human blood/serum.
Results:
The lack of expression of NeuGc on some pig tissues (aortas, corneas) and cells (RBCs, PBMCs, AECs) significantly reduces the extent of human antibody binding. In contrast, the absence of NeuGc expression on some pig tissues (CECs, isolated islet cells) does not reduce human antibody binding, possibly due to their relatively low NeuGc expression level. The strength of the human T-cell proliferative response may also be marginally reduced, but is already weak to GTKO/hCD46 pAECs and islet cells. We also demonstrate that the absence of NeuGc expression on GTKO/hCD46 pAECs does not reduce human platelet aggregation, and nor does it significantly modify the IBMIR to pig islets.
Conclusion:
The absence of NeuGc on some solid organs from GTKO/hCD46/NeuGcKO pigs should reduce the human antibody response after clinical transplantation when compared to GTKO/hCD46 pig organs. However, the clinical benefit of using certain tissue (e.g., cornea, islets) from GTKO/hCD46/NeuGcKO pigs is questionable.
Clinical xenotransplantation utilizing pig organs represents, in theory, a very attractive solution to the worldwide shortage of organs for human transplantation. The use of pigs as organ donors has several advantages over the choice of any other species [1]. The pig has a short gestation period, it produces large litters and its offspring grow very rapidly. Utilization of pigs would also eliminate the ethical and virological concerns associated with the use of organs from nonhuman primates [2]. However, hyperacute rejection (HAR), which occurs when transplanting between discordant xeno combinations, currently represents the major immunological barrier to the survival of pig organs into primates [3–5]. The binding of naturally occurring cytotoxic xenoreactive antibodies (XNA) mainly directed to the carbohydrate structure Galα1-3Galß1-4GlcNAc-R [6–8] on porcine endothelial cells and the activation of the recipient’s complement cascade represent the sequence of immunological events which underlie HAR [5, 9, 10].
Background:
The CD40/CD154 and CD28/B7 pathways are important in allo- and xeno-transplantation. Owing to the thrombotic complications of anti-CD154mAb, anti-CD40mAb has emerged as a promising inhibitor of costimulation. Various clones of anti-CD40mAb have been developed against primate species, e.g., clone 2C10 against rhesus monkeys. We have compared the in vitro efficacy of 2C10 to prevent a T cell response in primates and pigs.
Methods:
The binding of 2C10 to antigen-presenting cells (PBMCs [B cells]) of humans, rhesus and cynomolgus monkeys, baboons, and pigs was measured by flow cytometry, and was also tested indirectly by a blocking assay. The functional capacity of 2C10 was tested by mixed lymphocyte reaction (MLR) with polyclonal stimulation by phytohemagglutinin (PHA) and also with wild-type pig aortic endothelial cells (pAECs) as stimulators.
Results:
There was a significant reduction in binding of 2C10 to baboon PBMCs compared to rhesus, cynomolgus, and human PBMCs, and minimal binding to pig PBMCs. The blocking assay confirmed that the binding of 2C10 was significantly lower to baboon PBMCs when compared to the other primate species tested. The functional assay with PHA showed significantly reduced inhibition of PBMC proliferation in humans, cynomolgus monkeys, and baboons compared to rhesus monkeys, which was confirmed on MLR with pAECs.
Conclusions:
Since both the binding and functional activity of 2C10 in the baboon is lower than in rhesus monkeys, in vivo treatment using 2C10 in the baboon might require a higher dose or more frequent administration in comparison to rhesus monkeys. It may also be beneficial to develop species-specific clones of anti-CD40mAb.
In pig-to-baboon heart/artery patch transplantation models, adequate costimulation blockade prevents a T-cell response. After heart transplantation, coagulation dysfunction (thrombocytopenia, reduced fibrinogen, increased D-dimer) and inflammation (increased C-reactive protein [CRP]) develop. We evaluated whether coagulation dysfunction and/or inflammation can be detected following pig artery patch transplantation.
Baboons received heart (n = 8) or artery patch (n = 16) transplants from genetically engineered pigs and a costimulation blockade-based regimen. Heart grafts functioned for 15-130 days. Artery recipients were euthanized after 28-84 days. Platelet counts, fibrinogen, D-dimer, and CRP were measured.
Thrombocytopenia and reduced fibrinogen developed only in recipients of hearts not expressing a coagulation-regulatory protein (n = 4), but not in other heart or patch recipients. However, in heart recipients (n = 8), there were sustained increases in D-dimer (<0.5 to 1.9 ug/ml [P < 0.01]) and CRP (0.26-2.2 mg/dl [P < 0.01]). In recipients of artery patches, there were also sustained increases in D-dimer (<0.5 to 1.4 ug/ml [P < 0.01]) and CRP (0.26 to 1.5 mg/dl [P < 0.001]). An IL-6R antagonist suppressed the increase in CRP, but not D-dimer.
The pig artery patch model has proved valuable for determining immunosuppressive regimens that prevent sensitization to pig antigens. This model also provides information on the sustained systemic inflammation in xenograft recipients (SIXR). An IL-6R antagonist may help suppress this response.
© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
Unlabelled:
Multiple modifications of the porcine genome are required to prevent rejection after pig-to-primate xenotransplantation. Here, we produced pigs with a knockout of the α1,3-galactosyltransferase gene (GGTA1-KO) combined with transgenic expression of the human anti-apoptotic/anti-inflammatory molecules heme oxygenase-1 and A20, and investigated their xenoprotective properties.
Methods:
The GGTA1-KO/human heme oxygenase-1 (hHO-1)/human A20 (hA20) transgenic pigs were produced in a stepwise approach using zinc finger nuclease vectors targeting the GGTA1 gene and a Sleeping Beauty vector coding for hA20. Two piglets were analyzed by quantitative reverse-transcription polymerase chain reaction, flow cytometry, and sequencing. The biological function of the genetic modifications was tested in a (51)Chromium release assay and by ex vivo kidney perfusions with human blood.
Results:
Disruption of the GGTA1 gene by deletion of few basepairs was demonstrated in GGTA1-KO/hHO-1/hA20 transgenic pigs. The hHO-1 and hA20 mRNA expression was confirmed by quantitative reverse-transcription polymerase chain reaction. Ex vivo perfusion of 2 transgenic kidneys was feasible for the maximum experimental time of 240 minutes without symptoms of rejection.
Conclusions:
Results indicate that GGTA1-KO/hHO-1/hA20 transgenic pigs are a promising model to alleviate rejection and ischemia-reperfusion damage in porcine xenografts and could serve as a background for further genetic modifications toward the production of a donor pig that is clinically relevant for xenotransplantation.
Pig islet grafts have been successful in treating diabetes in animal models. One remaining question is whether neonatal pig isletlike cell clusters (NICC) are resistant to the early loss of islets from the instant blood-mediated inflammatory reaction (IBMIR).
Neonatal isletlike cell clusters were harvested from three groups of piglets-(i) wild-type (genetically unmodified), (ii) α1,3-galactosyltransferase gene-knockout (GTKO)/CD46, and (iii) GTKO/CD46/CD39. NICC samples were mixed with human blood in vitro, and the following measurements were made-antibody binding; complement activation; speed of islet-induced coagulation; C-peptide; glutamic acid decarboxylase (GAD65) release; viability.
Time to coagulation and viability were both reduced in all groups compared to freshly drawn non-anticoagulated human blood and autologous combinations, respectively. Antibody binding to the NICC occurred in all groups.
Neonatal isletlike cell clusters were subject to humoral injury with no difference associated to their genetic characteristics.
© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
Five minipig-to-baboon renal xenografts have been performed using a protocol similar to that utilized in human ABO-incompatible renal allografts. The animals survived 1, 1, 10, 13 and 22 days. Satisfactory renal function was obtained in three animals. One baboon never presented any sign of rejection and had a normal function of the xenograft at the time of death from pneumonia on the 10th postoperative day. Two baboons, who survived respectively 13 and 22 days both presented a reversible rejection crisis on the 6th postoperativey day. These results give hope for future applicability of xenografts in man.
The longest survival of a non-human primate with a life-supporting kidney graft to date has been 90 days, although graft survival > 30 days has been unusual. A baboon received a kidney graft from an α-1,3-galactosyltransferase gene-knockout pig transgenic for two human complement-regulatory proteins and three human coagulation-regulatory proteins (although only one was expressed in the kidney). Immunosuppressive therapy was with ATG+anti-CD20mAb (induction) and anti-CD40mAb+rapamycin+corticosteroids (maintenance). Anti-TNF-α and anti-IL-6R were administered. The baboon survived 136 days with a generally stable serum creatinine (0.6 to 1.6 mg/dl) until termination. No features of a consumptive coagulopathy (e.g., thrombocytopenia, decreased fibrinogen) or of a protein-losing nephropathy were observed. There was no evidence of an elicited anti-pig antibody response. Death was from septic shock (Myroides spp). Histology of a biopsy on day 103 was normal, but by day 136, the kidney showed features of glomerular enlargement, thrombi, and mesangial expansion. The combination of (i) a graft from a specific genetically engineered pig, (ii) an effective immunosuppressive regimen, and (iii) anti-inflammatory agents prevented immune injury and a protein-losing nephropathy, and delayed coagulation dysfunction. This outcome encourages us that clinical renal xenotransplantation may become a reality.
© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
Most of the genes studied in humans have homologues in other mammals; however, theα1,3galactosyltransferase (α1,3GT) gene represents a unique exception. Whereas it is highly active in nonprimate mammals, prosimians and New World monkeys (i.e., monkeys of South America), producing one of the most abundant cell surface carbohydrate epitopes, the epitope Galα1–3Galβ1α4GlcNAc-R (termed the α-gal or α-galactosyl epitope), this gene is completely inactive in Old World primates (i.e., humans, apes and Old World monkeys). As discussed below, the inactivation of α1,3GT in ancestral primates was likely to be associated with a catastrophic evolutionary event that led primates of the Old World (i.e., primates of Asia and Africa) into almost complete extinction. Because of this event, humans, apes and Old World monkeys produce very large amounts of a natural antibody against the α-gal epitope. This antibody, termed anti-Gal, constitutes 1% of circulating immunoglobulins and it prevents transplantation of organs or tissues from nonprimate mammals (e.g., pigs) into humans because it readily binds to the α-gal epitopes on such xenografts. It is impossible to determine the actual evolutionary events which have led to inactivation of the α1,3GT gene and suppression of α-gal epitope expression in ancestral Old World primates. However, information on the expression of this epitope in various species, the structure of the α1,3GT pseudogene in primates and the fossil record of primates, enable us to speculate on both the evolutionary period in which α1,3GT gene inactivation occurred, as well as possible causes for this event.
Pig islets are an alternative source for islet transplantation to treat type 1 diabetes (T1D), but reproducible curative potential in the pig-to-nonhuman primate (NHP) model has not been demonstrated. Here, we report that pig islet grafts survived and maintained normoglycemia for >6 months in four of five consecutive immunosuppressed NHPs. Pig islets were isolated from designated pathogen-free (DPF) miniature pigs and infused intraportally into streptozotocin-induced diabetic rhesus monkeys under pretreatment with cobra venom factor (CVF), anti-thymocyte globulin (ATG) induction and maintenance with anti-CD154 monoclonal antibody and low-dose sirolimus. Ex vivo expanded autologous regulatory T cells were adoptively transferred in three recipients. Blood glucose levels were promptly normalized in all five monkeys and normoglycemia (90-110 mg/dL) was maintained for >6 months in four cases, the longest currently up to 603 days. Intravenous glucose tolerance tests during the follow-up period showed excellent glucose disposal capacity and porcine C-peptide responses. Adoptive transfer of autologous regulatory T cells was likely to be associated with more stable and durable normoglycemia. Importantly, the recipients showed no serious adverse effects. Taken together, our results confirm the clinical feasibility of pig islet transplantation to treat T1D patients without the need for excessive immunosuppressive therapy.
© Copyright 2015 The American Society of Transplantation and the American Society of Transplant Surgeons.
Inflammation and immunity may be associated with endothelial cell (EC) injury and thrombus formation. We explored the mechanisms through which a humoral immune response directed against the endothelium might promote coagulation. Using the interaction of anti-EC antibodies and complement (C) with cultured EC as a model, we studied the expression and function of tissue factor, a cofactor for factor VIIa-mediated conversion of factor X to Xa. Exposure of EC to anti-EC antibodies and C in sublytic amounts stimulated the synthesis of tissue factor over a period of 16-42 h. Cell surface expression of tissue factor activity required activation of C and assembly of the membrane attack complex, because expression was inhibited by soluble CR1 and was not detected in the absence of C8. Elaboration of tissue factor messenger RNA was observed over a period of 8-30 h and required protein synthesis. Expression of tissue factor was not a direct consequence of the action of C on the EC but was a secondary response that required as an intermediate step the release of interleukin 1 alpha, an early product of the EC response to C activation. These findings suggest that, after the assembly of membrane attack complex on EC, the production of tissue factor and initiation of coagulation in a blood vessel depend on the production of interleukin 1 alpha and on its availability to stimulate affected EC.
Heparan sulfate proteoglycan associated with endothelial cells in normal blood vessels inhibits intravascular coagulation and egress of blood cells and plasma proteins, key features of hyperacute rejection. It was shown herein that exposure of cultured porcine endothelium to human serum as a source of natural antibodies and complement caused cleavage and release of 5% of endothelial cell proteoglycans within 4 min and greater than 50% within 1 h. Proteoglycan release depended on activation of the classical complement pathway and preceded irreversible cell injury. These findings suggest that loss of endothelial cell proteoglycan may be a critical step in the pathogenesis of hyperacute rejection and in diseases involving humoral injury to endothelial cells.
The cleavage of C3 is a critical step for complement (C) activation in the classical and alternative pathways. This reaction is controlled by the regulators of C activation protein family. Membrane cofactor protein (MCP) is a cofactor for the factor I-mediated inactivation of C3b and C4b. As a widely distributed membrane protein, MCP may protect host cells from inadvertent C activation. Human MCP has recently been shown to protect transfected rodent cells from human C-mediated lysis. In this report the relationship of MCP expression to C3b deposition and cytoprotection was examined using NIH/3T3 cells transfected with human MCP and exposed to human serum as a source of C and naturally occurring anti-mouse antibody. MCP inhibited C3b deposition in a dose-dependent fashion and inhibited lysis of the mouse cells expressing it. MCP did not inhibit lysis on bystander cells. These results demonstrate the protective role of MCP, at the cellular level, by an intrinsic mechanism.
Background
Three costimulation blockade-based regimens have been explored after transplantation of hearts from pigs of varying genetic backgrounds to determine whether CTLA4-Ig (abatacept) or anti-CD40mAb+CTLA4-Ig (belatacept) can successfully replace anti-CD154mAb.Methods
All pigs were on an α1,3-galactosyltransferase gene-knockout/CD46 transgenic (GTKO.CD46) background. Hearts transplanted into Group A baboons (n = 4) expressed additional CD55, and those into Group B (n = 3) expressed human thrombomodulin (TBM). Immunosuppression included anti-thymocyte globulin with anti-CD154mAb (Regimen 1: n = 2) or abatacept (Regimen 2: n = 2) or anti-CD40mAb+belatacept (Regimen 3: n = 2). Regimens 1 and 2 included induction anti-CD20mAb and continuous heparin. One further baboon in Group B (B16311) received a modified Regimen 1. Baboons were followed by clinical/laboratory monitoring of immune/coagulation parameters. At biopsy, graft failure, or euthanasia, the graft was examined by microscopy.ResultsGroup A baboons survived 15 to 33 days, whereas Group B survived 52, 99, and 130 days, respectively. Thrombocytopenia and reduction in fibrinogen occurred within 21 days in Group A, suggesting thrombotic microangiopathy (TM), confirmed by histopathology. In Group B, with follow-up for >4 m, areas of myofiber degeneration and scarring were seen in two hearts at necropsy. A T-cell response was documented only in baboons receiving Regimen 2.Conclusions
The combination of anti-CD40mAb+belatacept proved effective in preventing a T-cell response. The expression of TBM prevented thrombocytopenia and may possibly delay the development of TM and/or consumptive coagulopathy.
Acute vascular rejection (AVR), in particular microvascular thrombosis, is an important barrier to successful pig-to-primate xenotransplantation. Here, we report the generation of pigs with decreased tissue factor (TF) levels induced by small interfering (si)RNA-mediated gene silencing. Porcine fibroblasts were transfected with TF-targeting small hairpin (sh)RNA and used for somatic cell nuclear transfer. Offspring were analyzed for siRNA, TF mRNA and TF protein level. Functionality of TF downregulation was investigated by a whole blood clotting test and a flow chamber assay. TF siRNA was expressed in all twelve liveborn piglets. TF mRNA expression was reduced by 94.1 ± 4.7% in TF knockdown (TFkd) fibroblasts compared to wild-type (WT). TF protein expression in PAEC stimulated with 50 ng/mL TNF-α was significantly lower in TFkd pigs (mean fluorescence intensity TFkd: 7136 ± 136 vs. WT: 13 038 ± 1672). TF downregulation significantly increased clotting time (TFkd: 73.3 ± 8.8 min, WT: 45.8 ± 7.7 min, p < 0.0001) and significantly decreased thrombus formation compared to WT (mean thrombus coverage per viewing field in %; WT: 23.5 ± 13.0, TFkd: 2.6 ± 3.7, p < 0.0001). Our data show that a functional knockdown of TF is compatible with normal development and survival of pigs. TF knockdown could be a valuable component in the generation of multi-transgenic pigs for xenotransplantation.
© Copyright 2015 The American Society of Transplantation and the American Society of Transplant Surgeons.
Background
The lethal thrombocytopenia that accompanies liver xenotransplantation is a barrier to clinical application. Human platelets are bound by the asialoglycoprotein receptor (ASGR) on pig sinusoidal endothelial cells and phagocytosed. Inactivation of the ASGR1 gene in donor pigs may prevent xenotransplantation-induced thrombocytopenia.Methods
Transcription activator-like effector nucleases (TALENs) were targeted to the ASGR1 gene in pig liver-derived cells. ASGR1 deficient pig cells were used for somatic cell nuclear transfer (SCNT). ASGR1 knock out (ASGR1−/−) fetal fibroblasts were used to produce healthy ASGR1 knock out piglets. Human platelet uptake was measured in ASGR1+/+ and ASGR1−/− livers.ResultsTargeted disruption of the ASGR1 gene with TALENs eliminated expression of the receptor. ASGR1-/- livers phagocytosed fewer human platelets than domestic porcine livers during perfusion.Conclusions
The use of TALENs in liver-derived cells followed by SCNT enabled the production of healthy homozygous ASGR1 knock out pigs. Livers from ASGR1-/- pigs exhibit decreased human platelet uptake. Deletion of the ASGR1 gene is a viable strategy to diminish platelet destruction in pig-to-human xenotransplantation.
Background
Simultaneous inactivation of pig GGTA1 and CMAH genes eliminates carbohydrate xenoantigens recognized by human antibodies. The β4GalNT2 glycosyltransferase may also synthesize xenoantigens. To further characterize glycan-based species incompatibilities, we examined human and non-human primate antibody binding to cells derived from genetically modified pigs lacking these carbohydrate-modifying genes.Methods
The Cas9 endonuclease and gRNA were used to create pigs lacking GGTA1, GGTA1/CMAH, or GGTA1/CMAH/β4GalNT2 genes. Peripheral blood mononuclear cells were isolated from these animals and examined for binding to IgM and IgG from humans, rhesus macaques, and baboons.ResultsCells from GGTA1/CMAH/β4GalNT2 deficient pigs exhibited reduced human IgM and IgG binding compared to cells lacking both GGTA1 and CMAH. Non-human primate antibody reactivity with cells from the various pigs exhibited a slightly different pattern of reactivity than that seen in humans. Simultaneous inactivation of the GGTA1 and CMAH genes increased non-human primate antibody binding compared to cells lacking either GGTA1 only or to those deficient in GGTA1/CMAH/β4GalNT2.Conclusions
Inactivation of the β4GalNT2 gene reduces human and non-human primate antibody binding resulting in diminished porcine xenoantigenicity. The increased humoral immunity of non-human primates toward GGTA1-/CMAH-deficient cells compared to pigs lacking either GGTA1 or GGTA1/CMAH/β4GalNT2 highlights the complexities of carbohydrate xenoantigens and suggests potential limitations of the non-human primate model for examining some genetic modifications. The progressive reduction of swine xenoantigens recognized by human immunoglobulin through inactivation of pig GGTA1/CMAH/β4GalNT2 genes demonstrates that the antibody barrier to xenotransplantation can be minimized by genetic engineering.
The availability of organs and cells from deceased humans for transplantation is not meeting the demand. Xenotransplantation, specifically the transplantation of organs and cells from genetically engineered pigs, could resolve this problem. Diabetic monkeys have remained normoglycemic and insulin-independent after pig islet transplantation for >one yr, and a pig heterotopic (non-life-supporting) heart transplant recently reached the one-yr milestone in a baboon. With these encouraging results, why is it that, with some notable exceptions, research into xenotransplantation has received relatively little support by industry, government funding agencies, and medical charitable foundations? Industry appears reluctant to support research that will take more than two to three yr to come to clinical trial, and the funding agencies appear to have been "distracted" by the current appeal of stem cell technology and regenerative medicine. It has only been the willingness of living donors to provide organs that has significantly increased the number of transplants being performed worldwide. These altruistic donations are not without risk of morbidity and even mortality to the donor. Although with the best of intentions, we are therefore traversing the Hippocratic Oath of doctors to "do no harm." This should be a stimulus to fund exploration of alternative approaches, including xenotransplantation.
© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
In the pig-to-nonimmunosuppressed baboon artery patch model, a graft from an α1,3-galactosyltransferase gene-knockout pig transgenic for human CD46 (GTKO/CD46) induces a significant adaptive immune response (elicited anti-pig antibody response, increase in T cell proliferation on MLR, cellular infiltration of the graft), which is effectively prevented by anti-CD154mAb-based therapy.
As anti-CD154mAb is currently not clinically applicable, we evaluated whether it could be replaced by CD28/B7 pathway blockade or by blockade of both pathways (using belatacept+anti-CD40mAb [2C10R4]). We further investigated whether a patch from a GTKO/CD46 pig with a mutant human MHC class II transactivator (CIITA-DN) gene would allow reduction in the immunosuppressive therapy administered.
When grafts from GTKO/CD46 pigs were transplanted with blockade of both pathways, a minimal or insignificant adaptive response was documented. When a GTKO/CD46/CIITA-DN graft was transplanted, but no immunosuppressive therapy was administered, a marked adaptive response was documented. In the presence of CD28/B7 pathway blockade (abatacept or belatacept), there was a weak adaptive response that was diminished when compared with that to a GTKO/CD46 graft. Blockade of both pathways prevented an adaptive response.
Although expression of the mutant MHC CIITA-DN gene was associated with a reduced adaptive immune response when immunosuppressive therapy was inadequate, when blockade of both the CD40/CD154 and CD28/B7 pathways was present, the response even to a GTKO/CD46 graft was suppressed. This was confirmed after GTKO/CD46 heart transplantation in baboons.
Copyright © 2015. Published by Elsevier B.V.
Background:
Xenotransplantation is an appealing alternative to human allotransplantation because of a worldwide shortage of organs. One of the obstacles for xenografts is cellular rejection by the innate immune system, comprised of NK cells, monocytes, and macrophages. In this study the inhibitory function of HLA-G1, a MHC Ib molecule, on macrophage-mediated cytotoxicity was examined. Furthermore, this study also evaluates the suppressive effect of cytokine production by macrophages.
Methods:
The expression of inhibitory receptors that interact with HLA-G1, immunoglobulin-like transcript 2 (ILT2), ILT4 and KIR2DL4 (CD158d) on in vitro generated macrophages were examined by flow cytometry. Complementary DNA (cDNA) of HLA-G1, HLA-E and human β2-microglobulin (hβ2m) were prepared and transfected into swine endothelial cells (SECs). The expression of the transgenic genes was evaluated by flow cytometry, and macrophage-mediated SEC cytolysis was assessed using the macrophages.
Results:
In vitro generated macrophages expressed not only ILT2 and ILT4 but CD158d as well. The transgenic HLA-G1 on SECs indicated significant suppression in macrophage-mediated cytotoxicity, which was equivalent to that of transgenic HLA-E. Furthermore, the results on real time PCR and ELISA revealed that transgenic HLA-G1 induces the anti-inflammatory cytokines, such as IL-10 and TGF-β, and suppresses iNOS mRNA expression, indicating that transgenic HLA-G1 has suppressive effects in a broad range of transplant rejection.
Conclusion:
These results indicate that generating HLA-G1 transgenic pigs can protect porcine grafts from macrophage-mediated cytotoxicity.
Genetically modified pigs are a promising potential source of lung xenografts. Ex vivo xenoperfusion is an effective platform for testing the effect of new modifications, but typical experiments are limited by testing of a single genetic intervention and small sample sizes. The purpose of this study was to analyze the individual and aggregate effects of donor genetic modifications on porcine lung xenograft survival and injury in an extensive pig lung xenoperfusion series.
Data from 157 porcine lung xenoperfusion experiments using otherwise unmodified heparinized human blood were aggregated as either continuous or dichotomous variables. Lungs were wild type in 17 perfusions (11% of the study group), while 31 lungs (20% of the study group) had one genetic modification, 40 lungs (39%) had 2, and 47 lungs (30%) had 3 or more modifications. The primary endpoint was functional lung survival to 4 h of perfusion. Secondary analyses evaluated previously identified markers associated with known lung xenograft injury mechanisms. In addition to comparison among all xenografts grouped by survival status, a subgroup analysis was performed of lungs incorporating the GalTKO.hCD46 genotype.
Each increase in the number of genetic modifications was associated with additional prolongation of lung xenograft survival. Lungs that exhibited survival to 4 h generally had reduced platelet activation and thrombin generation. GalTKO and the expression of hCD46, HO-1, hCD55, or hEPCR were associated with improved survival. hTBM, HLA-E, and hCD39 were associated with no significant effect on the primary outcome.
This meta-analysis of an extensive lung xenotransplantation series demonstrates that increasing the number of genetic modifications targeting known xenogeneic lung injury mechanisms is associated with incremental improvements in lung survival. While more detailed mechanistic studies are needed to explore the relationship between gene expression and pathway-specific injury and explore why some genes apparently exhibit neutral (hTBM, HLA-E) or inconclusive (CD39) effects, GalTKO, hCD46, HO-1, hCD55, and hEPCR modifications were associated with significant lung xenograft protection. This analysis supports the hypothesis that multiple genetic modifications targeting different known mechanisms of xenograft injury will be required to optimize lung xenograft survival.
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
The advent of facile genome engineering using the bacterial RNA-guided CRISPR-Cas9 system in animals and plants is transforming biology. We review the history of CRISPR (clustered regularly interspaced palindromic repeat) biology from its initial discovery through the elucidation of the CRISPR-Cas9 enzyme mechanism, which has set the stage for remarkable developments using this technology to modify, regulate, or mark genomic loci in a wide variety of cells and organisms from all three domains of life. These results highlight a new era in which genomic manipulation is no longer a bottleneck to experiments, paving the way toward fundamental discoveries in biology, with applications in all branches of biotechnology, as well as strategies for human therapeutics.
Copyright © 2014, American Association for the Advancement of Science.
Background
Xenotransplantation of porcine islets can reverse diabetes in non-human primates. The remaining hurdles for clinical application include safe and effective T-cell-directed immunosuppression, but protection against the innate immune system and coagulation dysfunction may be more difficult to achieve. Islet-targeted genetic manipulation of islet-source pigs represents a powerful tool to protect against graft loss. However, whether these genetic alterations would impair islet function is unknown.Methods
On a background of α1,3-galactosyltransferase gene-knockout (GTKO)/human (h)CD46, additional genes (hCD39, human tissue factor pathway inhibitor, porcine CTLA4-Ig) were inserted in different combinations under an insulin promoter to promote expression in islets (confirmed by immunofluorescence). Seven pigs were tested for baseline and glucose/arginine-challenged levels of glucose, insulin, C-peptide, and glucagon.ResultsThis preliminary study did not show definite evidence of β-cell deficiencies, even when three transgenes were expressed under the insulin promoter. Of seven animals, all were normoglycemic at fasting, and five of seven had normal glucose disposal rates after challenge. All animals exhibited insulin, C-peptide, and glucagon responses to both glucose and arginine challenge; however, significant interindividual variation was observed.Conclusions
Multiple islet-targeted transgenic expression was not associated with an overtly detrimental effect on islet function, suggesting that complex genetic constructs designed for islet protection warrants further testing in islet xenotransplantation models.
Pigs are emerging as important large animal models for biomedical research, and they may represent a source of organs for xenotransplantation. The MHC is pivotal to the function of the immune system in health and disease, and it is particularly important in infection and transplant rejection. Pigs deficient in class I MHC could serve as important reagents to study viral immunity as well as allograft and xenograft rejection. In this study, we report the creation and characterization of class I MHC knockout pigs using the Cas9 nuclease and guide RNAs. Pig fetal fibroblasts were genetically engineered using Cas9 and guide RNAs, and class I MHC(-) cells were then used as nuclear donors for somatic cell nuclear transfer. We produced three piglets devoid of all cell surface class I proteins. Although these animals have reduced levels of CD4(-)CD8(+) T cells in peripheral blood, the pigs appear healthy and are developing normally. These pigs are a promising reagent for immunological research.
Human beings do not synthesize the glycolyl form of the sialic acid (Neu5Gc) and only express the acetylated form of the sugar, whereas a diet-based intake of Neu5Gc provokes a natural immunization and production of anti-Neu5Gc antibodies in human serum. However, Neu5Gc is expressed on mammal glycoproteins and glycolipids in most organs and cells. We review here the relevance of Neu5Gc and anti-Neu5Gc antibodies in the context of xenotransplantation and the use of animal-derived molecules and products, as well as the possible consequences of a long-term exposure to anti-Neu5Gc antibodies in recipients of xenografts. In addition, the importance of an accurate estimation of the anti-Neu5Gc response following xenotransplantation and the future contribution of knockout animals mimicking the human situation are also assessed.
The generation of pigs with genetic modifications has significantly advanced the field of xenotransplantation. New genetically engineered pigs were produced on an α1,3-galactosyltransferase gene-knockout background with ubiquitous expression of human CD46, with islet beta cell-specific expression of human tissue factor pathway inhibitor and/or human CD39 and/or porcine CTLA4-lg. Isolated islets from pigs with 3, 4 or 5 genetic modifications were transplanted intraportally into streptozotocin-diabetic, immunosuppressed cynomolgus monkeys (n = 5). Immunosuppression was based on anti-CD154 mAb costimulation blockade. Monitoring included features of early islet destruction, glycemia, exogenous insulin requirement and histopathology of the islets at necropsy. Using these modified pig islets, there was evidence of reduced islet destruction in the first hours after transplantation, compared with two series of historical controls that received identical therapy but were transplanted with islets from pigs with either no or only one genetic modification. Despite encouraging effects on early islet loss, these multi-transgenic islet grafts did not demonstrate consistency in regard to long-term success, with only two of five demonstrating function beyond 5 months.
Background
Dysregulation of coagulation is considered a major barrier against successful pig organ xenotransplantation in non-human primates. Inflammation is known to promote activation of coagulation. The role of pro-inflammatory factors as well as the relationship between inflammation and activation of coagulation in xenograft recipients is poorly understood.Methods
Baboons received kidney (n = 3), heart (n = 4), or artery patch (n = 8) xenografts from α1,3-galactosyltransferase gene-knockout (GTKO) pigs or GTKO pigs additionally transgenic for human complement-regulatory protein CD46 (GTKO/CD46). Immunosuppression (IS) was based on either CTLA4Ig or anti-CD154 costimulation blockade. Three artery patch recipients did not receive IS. Pro-inflammatory cytokines, chemokines, and coagulation parameters were evaluated in the circulation after transplantation. In artery patch recipients, monocytes and dendritic cells (DC) were monitored in peripheral blood. Expression of tissue factor (TF) and CD40 on monocytes and DC were assessed by flow cytometry. C-reactive protein (C-RP) levels in the blood and C-RP deposition in xenografts as well as native organs were evaluated. Baboon and pig C-RP mRNA in heart and kidney xenografts were evaluated.ResultsIn heart and kidney xenograft recipients, the levels of INFγ, TNF-α, IL-12, and IL-8 were not significantly higher after transplantation. However, MCP-1 and IL-6 levels were significantly higher after transplantation, particularly in kidney recipients. Elevated C-RP levels preceded activation of coagulation in heart and kidney recipients, where high levels of C-RP were maintained until the time of euthanasia in both heart and kidney recipients. In artery patch recipients, INFγ, TNF-α, IL-12, IL-8, and MCP-1 were elevated with no IS, while IL-6 was not. With IS, INFγ, TNF-α, IL-12, IL-8, and MCP-1 were reduced, but IL-6 was elevated. Elevated IL-6 levels were observed as early as 2 weeks in artery patch recipients. While IS was associated with reduced thrombin activation, fibrinogen and C-RP levels were increased when IS was given. There was a significant positive correlation between C-RP, IL-6, and fibrinogen levels. Additionally, absolute numbers of monocytes were significantly increased when IS was given, but not without IS. This was associated with increased CD40 and TF expression on CD14+ monocytes and lineageneg CD11c+ DC, with increased differentiation of the pro-inflammatory CD14+ CD11c+ monocyte population. At the time of euthanasia, C-RP deposition in kidney and heart xenografts, C-RP positive cells in artery patch xenograft and native lungs were detected. Finally, high levels of both pig and baboon C-RP mRNA were detected in heart and kidney xenografts.Conclusions
Inflammatory responses precede activation of coagulation after organ xenotransplantation. Early upregulation of C-RP and IL-6 levels may amplify activation of coagulation through upregulation of TF on innate immune cells. Prevention of systemic inflammation in xenograft recipients (SIXR) may be required to prevent dysregulation of coagulation and avoid excessive IS after xenotransplantation.