Fetal Kidney Grafts and Organoids from Microminiature Pigs: Establishing a Protocol for Production and Long-Term Cryopreservation.

Fetal organs and organoids are important tools for studying organ development. Recently, porcine organs have garnered attention as potential organs for xenotransplantation because of their high degree of similarity to human organs. However, to meet the prompt demand for porcine fetal organs by patients and researchers, effective methods for producing, retrieving, and cryopreserving pig fetuses are indispensable. Therefore, in this study, to collect fetuses for kidney extraction, we employed cesarean sections to preserve the survival and fertility of the mother pig and a method for storing fetal kidneys by long-term cryopreservation. Subsequently, we evaluated the utility of these two methods. We confirmed that the kidneys of pig fetuses retrieved by cesarean section that were cryopreserved for an extended period could resume renal growth when grafted into mice and were capable of forming renal organoids. These results demonstrate the usefulness of long-term cryopreserved fetal pig organs and strongly suggest the effectiveness of our comprehensive system of pig fetus retrieval and fetal organ preservation, thereby highlighting its potential as an accelerator of xenotransplantation research and clinical innovation.

Limited availability of methods for the detection of xenotransplantation-relevant viruses in veterinary laboratories.

BACKGROUND: The German Xenotransplantation Consortium is in the process to prepare a clinical trial application (CTA) on xenotransplantation of genetically modified pig hearts. In the CTA documents to the central and national regulatory authorities, that is, the European Medicines Agency (EMA) and the Paul Ehrlich Institute (PEI), respectively, it is required to list the potential zoonotic or xenozoonotic porcine microorganisms including porcine viruses as well as to describe methods of detection in order to prevent their transmission. The donor animals should be tested using highly sensitive detection systems. I would like to define a detection system as the complex including the actual detection methods, either PCR-based, cell-based, or immunological methods and their sensitivity, as well as sample generation, sample preparation, sample origin, time of sampling, and the necessary negative and positive controls. Lessons learned from the identification of porcine cytomegalovirus/porcine roseolovirus (PCMV/PRV) in the xenotransplanted heart in the recipient in the Baltimore study underline how important such systems are. The question is whether veterinary laboratories can supply such assays. METHODS: A total of 35 veterinary laboratories in Germany were surveyed for their ability to test for selected xenotransplantation-relevant viruses, including PCMV/PRV, hepatitis E virus, and porcine endogenous retrovirus-C (PERV-C). As comparison, data from Swiss laboratories and a laboratory in the USA were analyzed. Furthermore, we assessed which viruses were screened for in clinical and preclinical trials performed until now and during screening of pig populations. RESULTS: Of the nine laboratories that provided viral diagnostics, none of these included all potential viruses of concern, indeed, the most important assays confirmed in recent human trials, antibody detection of PCMV/PRV and screening for PERV-C were not available at all. The situation was similar in Swiss and US laboratories. Different viruses have been tested for in first clinical and preclinical trials performed in various countries. CONCLUSION: Based on these results it is necessary to establish special virological laboratories able to test for all xenotransplantation-relevant viruses using validated assays, optimally in the xenotransplantation centers.

Consistent survival in consecutive cases of life-supporting porcine kidney xenotransplantation using 10GE source pigs.

Xenotransplantation represents a possible solution to the organ shortage crisis and is an imminent clinical reality with long-term xenograft survival in pig-to-nonhuman primate (NHP) heart and kidney large animal models, and short-term success in recent human decedent and clinical studies. However, concerns remain about safe clinical translation of these results, given the inconsistency in published survival as well as key differences between preclinical procurement and immunosuppression and clinical standards-of-care. Notably, no studies of solid organ pig-to-NHP transplantation have achieved xenograft survival longer than one month without CD40/CD154 costimulatory blockade, which is not currently an FDA-approved immunosuppression strategy. We now present consistent survival in consecutive cases of pig-to-NHP kidney xenotransplantation, including long-term survival after >3 hours of xenograft cold preservation time as well as long-term survival using FDA-approved immunosuppression. These data provide critical supporting evidence for the safety and feasibility of clinical kidney xenotransplantation. Moreover, long-term survival without CD40/CD154 costimulatory blockade may provide important insights for immunosuppression regimens to be considered for first-in-human clinical trials.

Novel factors potentially initiating acute antibody-mediated rejection in pig kidney xenografts despite an efficient immunosuppressive regimen.

Antibody-mediated rejection (AMR) is a common cause of graft failure after pig-to-nonhuman primate organ transplantation, even when the graft is from a pig with multiple genetic modifications. The specific factors that initiate AMR are often uncertain. We report two cases of pig kidney transplantation into immunosuppressed baboons in which we identify novel factors associated with the initiation of AMR. In the first, membranous nephropathy was the initiating factor that was then associated with the apparent loss of the therapeutic anti-CD154 monoclonal antibody in the urine when severe proteinuria was present. This observation suggests that proteinuria may be associated with the loss of any therapeutic monoclonal antibody, for example, anti-CD154 or eculizumab, in the urine, resulting in xenograft rejection. In the second case, the sequence of events and histopathology tentatively suggested that pyelonephritis may have initiated acute-onset AMR. The association of a urinary infection with graft rejection has been well-documented in ABO-incompatible kidney allotransplantation based on the expression of an antigen on the invading microorganism shared with the kidney graft, generating an immune response to the graft. To our knowledge, these potential initiating factors of AMR in pig xenografts have not been highlighted previously.

A brief review of the current status of pig islet xenotransplantation.

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.

A Comparison Between GalT-/-;hCD39;hCD55 and GalT-/-;hCD39;hCD46;hCD55;TBM Pig Kidneys Transplanted in Nonhuman Primates.

Because there is a shortage of donor kidneys, researchers are exploring the possibility of using genetically modified pig kidneys for transplantation. Approaches involving knockout of carbohydrate genes or knockin of protective proteins have been attempted to determine the best gene modifications. In this study, we utilized GalT-/-;hCD39;hCD55 and GalT-/-;hCD39;hCD46;hCD55;thrombomodulin (TBM) pigs for transplantation in nonhuman primates (NHPs). The NHPs survived for 4 weeks after kidney transplantation (4 WAT) from the GalT-/-;hCD39;hCD55 pig and for 6 WAT from the GalT-/-;hCD39;hCD46;hCD55;TBM pig. However, messenger RNA (mRNA) sequencing and immunohistochemistry analysis revealed that the 6 WAT kidney exhibited more severe apoptosis, inflammation, loss of renal function, and renal fibrosis than the 4 WAT kidney. These results indicate that additional knockin of complement regulator (hCD46) and coagulation regulator (TBM) is not enough to prevent renal damage, suggesting that improved immune suppression is needed for more prolonged survival.

Development and characterization of islet-derived mesenchymal stem cells from clinical grade neonatal porcine cryopreserved islets.

BACKGROUND: Porcine tissues display a great potential as donor tissues in xenotransplantation, including cell therapy. Cryopreserving clinical grade porcine tissue and using it as a source for establishing therapeutic cells should be advantageous for transportation and scheduled manufacturing of MSCs. Of note, we previously performed encapsulated porcine islet transplantation for the treatment of unstable type 1 diabetes mellitus in the clinical setting. It has been reported that co-transplantation of islets and Mesenchymal stem cells (MSCs) enhanced efficacy. We assume that co-transplantation of porcine islets and porcine islet-derived MSCs could improve the efficacy of clinical islet xenotransplantation. METHODS: MSCs were established from fresh and cryopreserved non-clinical grade neonatal porcine islets and bone marrow (termed non-clinical grade npISLET-MSCs and npBM-MSCs, respectively), as well as from cryopreserved clinical grade neonatal porcine islets (termed clinical grade npISLET-MSCs). Subsequently, the cell proliferation rate and diameter, surface marker expression, adipogenesis, osteogenesis, and colony-forming efficiency of the MSCs were assessed. RESULTS: Cell proliferation rate and diameter did not differ between clinical grade and non-clinical grade npISLET-MSCs. However, non-clinical grade npBM-MSCs were significantly shorter and smaller than both npISLET-MSCs (p < 0.05). MSC markers (CD29, CD44, and CD90) were strongly expressed in clinical grade npISLET-MSCs and non-clinical grade npISLET-MSCs and npBM-MSCs. The expression of MSC-negative markers CD31, CD34, and SLA-DR was low in all MSCs. Clinical grade npISLET-MSCs derived from adipose and osteoid tissues were positive for Oil Red and alkaline phosphatase staining. The results of colony-forming assay were not significantly different between clinical grade npISLET-MSCs and non-clinical grade npBM-MSCs. CONCLUSION: The method described herein was successful in of developing clinical grade npISLET-MSCs from cryopreserved islets. Cryopreserved clinical grade porcine islets could be an excellent stable source of MSCs for cell therapy.

Xenotransplantation experiments in brain-dead human subjects-A critical appraisal.

Brain-dead human subjects (decedents) were recently introduced as a potential preclinical experimental model in xenotransplantation. Brain death is associated with major pathophysiological changes, eg, structural injury and cell infiltration in vital organs, and major hormonal, metabolic, inflammatory, and hemodynamic changes. In 2 of the 3 initial experiments, the design of the experiments resulted in little or no new information becoming available. In the third, the experiment was unfortunately unsuccessful as neither of the 2 pig kidneys transplanted into the decedent functioned adequately. Failure may well have been associated with the effects of brain death, but an immune/inflammatory response to the xenograft could not be excluded. Subsequently, 2 further pig kidney transplants and 2 pig heart transplants have been carried out in human decedents, but again the data obtained do not add much to what is already known. In view of the profound changes that take place during and after brain death, it may prove difficult to determine whether graft failure or dysfunction results from the effects of brain death or from an immune/inflammatory response to the xenograft. A major concern is that, if the results are confusing, they may impact decisions relating to the introduction of clinical xenotransplantation.

Xenoreactive antibodies in α-granules of human platelets bind pig liver endothelial cells.

Pig liver xenotransplantation is limited by a thrombocytopenic coagulopathy that occurs immediately following graft reperfusion. In vitro and ex vivo studies from our lab suggested that the thrombocytopenia may be the result of a species incompatibility in platelet glycosylation. Realization that platelet α-granules contain antibodies caused us to reevaluate whether the thrombocytopenia in liver xenotransplantation could occur because IgM and IgG from inside platelet α-granules bound to pig liver sinusoidal endothelial cells (LSECs). Our in vitro analysis of IgM and IgG from inside α-granules showed that platelets do carry xenoreactive antibodies that can bind to known xenoantigens. This study suggests that thrombocytopenia occurring following liver xenotransplantation could occur because of xenoreactive antibodies tethering human platelets to the pig LSEC enabling the platelet to be phagocytosed. These results suggest genetic engineering strategies aimed at reducing xenoantigens on the surface of pig LSEC will be effective in eliminating the thrombocytopenia that limits survival in liver xenotransplantation.

Meeting report: Xenotransplantation Development Conference in Neijiang, China.

A conference on progress in the development of xenotransplantation in China was held in Neijiang, Sichuan, in May 2023, and was attended by approximately 100 established researchers and trainees. Progress in xenotransplantation research was reviewed by both Chinese and foreign experts. The topics discussed ranged from genetic engineering of pigs and the results of pig-to-nonhuman primate organ transplantation to the requirements for designated pathogen-free (DPF) pig facilities and regulation of xenotransplantation. This conference served as an opportunity to collectively advance the development of xenotransplantation in China and pave the way for its clinical application.

Design and testing of a humanized porcine donor for xenotransplantation.

Recent human decedent model studies1,2 and compassionate xenograft use3 have explored the promise of porcine organs for human transplantation. To proceed to human studies, a clinically ready porcine donor must be engineered and its xenograft successfully tested in nonhuman primates. Here we describe the design, creation and long-term life-supporting function of kidney grafts from a genetically engineered porcine donor transplanted into a cynomolgus monkey model. The porcine donor was engineered to carry 69 genomic edits, eliminating glycan antigens, overexpressing human transgenes and inactivating porcine endogenous retroviruses. In vitro functional analyses showed that the edited kidney endothelial cells modulated inflammation to an extent that was indistinguishable from that of human endothelial cells, suggesting that these edited cells acquired a high level of human immune compatibility. When transplanted into cynomolgus monkeys, the kidneys with three glycan antigen knockouts alone experienced poor graft survival, whereas those with glycan antigen knockouts and human transgene expression demonstrated significantly longer survival time, suggesting the benefit of human transgene expression in vivo. These results show that preclinical studies of renal xenotransplantation could be successfully conducted in nonhuman primates and bring us closer to clinical trials of genetically engineered porcine renal grafts.

Short-term function and immune-protection of microencapsulated adult porcine islets with alginate incorporating CXCL12 in healthy and diabetic non-human primates without systemic immune suppression: A pilot study.

Replacement of insulin-producing pancreatic beta-cells by islet transplantation offers a functional cure for type-1 diabetes (T1D). We recently demonstrated that a clinical grade alginate micro-encapsulant incorporating the immune-repellent chemokine and pro-survival factor CXCL12 could protect and sustain the integrity and function of autologous islets in healthy non-human primates (NHPs) without systemic immune suppression. In this pilot study, we examined the impact of the CXCL12 micro encapsulant on the function and inflammatory and immune responses of xenogeneic islets transplanted into the omental tissue bilayer sac (OB; n = 4) and diabetic (n = 1) NHPs. Changes in the expression of cytokines after implantation were limited to 2-6-fold changes in blood, most of which did not persist over the first 4 weeks after implantation. Flow cytometry of PBMCs following transplantation showed minimal changes in IFNγ or TNFα expression on xenoantigen-specific CD4+  or CD8+  T cells compared to unstimulated cells, and these occurred mainly in the first 4 weeks. Microbeads were readily retrievable for assessment at day 90 and day 180 and at retrieval were without microscopic signs of degradation or foreign body responses (FBR). In vitro and immunohistochemistry studies of explanted microbeads indicated the presence of functional xenogeneic islets at day 30 post transplantation in all biopsied NHPs. These results from a small pilot study revealed that CXCL12-microencapsulated xenogeneic islets abrogate inflammatory and adaptive immune responses to the xenograft. This work paves the way toward future larger scale studies of the transplantation of alginate microbeads with CXCL12 and porcine or human stem cell-derived beta cells or allogeneic islets into diabetic NHPs without systemic immunosuppression.

Xenografts Show Signs of Concentric Hypertrophy and Dynamic Left Ventricular Outflow Tract Obstruction After Orthotopic Pig-to-baboon Heart Transplantation.

BACKGROUND: Orthotopic cardiac xenotransplantation has seen substantial advancement in the last years and the initiation of a clinical pilot study is close. However, donor organ overgrowth has been a major hurdle for preclinical experiments, resulting in loss of function and the decease of the recipient. A better understanding of the pathogenesis of organ overgrowth after xenotransplantation is necessary before clinical application. METHODS: Hearts from genetically modified ( GGTA1-KO , hCD46/hTBM transgenic) juvenile pigs were orthotopically transplanted into male baboons. Group I (control, n = 3) received immunosuppression based on costimulation blockade, group II (growth inhibition, n = 9) was additionally treated with mechanistic target of rapamycin inhibitor, antihypertensive medication, and fast corticoid tapering. Thyroid hormones and insulin-like growth factor 1 were measured before transplantation and before euthanasia, left ventricular (LV) growth was assessed by echocardiography, and hemodynamic data were recorded via a wireless implant. RESULTS: Insulin-like growth factor 1 was higher in baboons than in donor piglets but dropped to porcine levels at the end of the experiments in group I. LV mass increase was 10-fold faster in group I than in group II. This increase was caused by nonphysiological LV wall enlargement. Additionally, pressure gradients between LV and the ascending aorta developed, and signs of dynamic left ventricular outflow tract (LVOT) obstruction appeared. CONCLUSIONS: After orthotopic xenotransplantation in baboon recipients, untreated porcine hearts showed rapidly progressing concentric hypertrophy with dynamic LVOT obstruction, mimicking hypertrophic obstructive cardiomyopathy in humans. Antihypertensive and antiproliferative drugs reduced growth rate and inhibited LVOT obstruction, thereby preventing loss of function.

Auxiliary liver xenotransplantation technique in a transgenic pig-to-non-human primate model: A surgical approach to prolong survival.

Xenotransplantation using pigs' liver offers a potentially alternative method to overcome worldwide donor shortage, or more importantly as a bridge to allotransplantation. However, it has been challenged by profound thrombocytopenia and fatal coagulopathy in non-human primate models. Here we suggest that a left auxiliary technique can be a useful method to achieve extended survival of the xenograft. Fifteen consecutive liver xenotransplants were carried out in a pig-to-cynomolgus model. Right auxiliary technique was implemented in two cases, orthotopic in eight cases, and left auxiliary in five cases. None of the right auxiliary recipients survived after surgery due to hemorrhage during complex dissection between the primate's right lobe and inferior vena cava. Orthotopic recipients survived less than 7 days secondary to profound thrombocytopenia and coagulopathy. Two out of five left auxiliary xenotransplants survived more than 3 weeks without uncontrolled thrombocytopenia or anemia, with one of them surviving 34 days, the longest graft survival reported to date. Left auxiliary xenotransplant is a feasible approach in non-human primate experiments, and the feared risk of thrombocytopenia and coagulopathy can be minimized. This may allow for longer evaluation of the xenograft and help better understand histopathological and immunological changes that occur following liver xenotransplantation.

Pig-to-human heart xenotransplantation in two recently deceased human recipients.

Genetically modified xenografts are one of the most promising solutions to the discrepancy between the numbers of available human organs for transplantation and potential recipients. To date, a porcine heart has been implanted into only one human recipient. Here, using 10-gene-edited pigs, we transplanted porcine hearts into two brain-dead human recipients and monitored xenograft function, hemodynamics and systemic responses over the course of 66 hours. Although both xenografts demonstrated excellent cardiac function immediately after transplantation and continued to function for the duration of the study, cardiac function declined postoperatively in one case, attributed to a size mismatch between the donor pig and the recipient. For both hearts, we confirmed transgene expression and found no evidence of cellular or antibody-mediated rejection, as assessed using histology, flow cytometry and a cytotoxic crossmatch assay. Moreover, we found no evidence of zoonotic transmission from the donor pigs to the human recipients. While substantial additional work will be needed to advance this technology to human trials, these results indicate that pig-to-human heart xenotransplantation can be performed successfully without hyperacute rejection or zoonosis.

Partial heart xenotransplantation: A research protocol in non-human primates.

Partial heart transplantation is a new type of transplant that delivers growing heart valve replacements for babies. Partial heart transplantation differs from orthotopic heart transplantation because only the part of the heart containing the heart valve is transplanted. It also differs from homograft valve replacement because viability of the graft is preserved by tissue matching, minimizing donor ischemia times, and recipient immunosuppression. This preserves partial heart transplant viability and allows the grafts to fulfill biological functions such as growth and self-repair. These advantages over conventional heart valve prostheses are balanced by similar disadvantages as other organ transplants, most importantly limitations in donor graft availability. Prodigious progress in xenotransplantation promises to solve this problem by providing an unlimited source of donor grafts. In order to study partial heart xenotransplantation, a suitable large animal model is important. Here we describe our research protocol for partial heart xenotransplantation in nonhuman primates.

Genetic knockout of porcine GGTA1 or CMAH/GGTA1 is associated with the emergence of neo-glycans.

BACKGROUND: Pig-derived tissues could overcome the shortage of human donor organs in transplantation. However, the glycans with terminal α-Gal and Neu5Gc, which are synthesized by enzymes, encoded by the genes GGTA1 and CMAH, are known to play a major role in immunogenicity of porcine tissue, ultimately leading to xenograft rejection. METHODS: The N-glycome and glycosphingolipidome of native and decellularized porcine pericardia from wildtype (WT), GGTA1-KO and GGTA1/CMAH-KO pigs were analyzed by multiplexed capillary gel electrophoresis coupled to laser-induced fluorescence detection. RESULTS: We identified biantennary and core-fucosylated N-glycans terminating with immunogenic α-Gal- and α-Gal-/Neu5Gc-epitopes on pericardium of WT pigs that were absent in GGTA1 and GGTA1/CMAH-KO pigs, respectively. Levels of N-glycans terminating with galactose bound in ß(1-4)-linkage to N-acetylglucosamine and their derivatives elongated by Neu5Ac were increased in both KO groups. N-glycans capped with Neu5Gc were increased in GGTA1-KO pigs compared to WT, but were not detected in GGTA1/CMAH-KO pigs. Similarly, the ganglioside Neu5Gc-GM3 was found in WT and GGTA1-KO but not in GGTA1/CMAH-KO pigs. The applied detergent based decellularization efficiently removed GSL glycans. CONCLUSION: Genetic deletion of GGTA1 or GGTA1/CMAH removes specific epitopes providing a more human-like glycosylation pattern, but at the same time changes distribution and levels of other porcine glycans that are potentially immunogenic.

Xenotransplantation literature update March 2021-March 2023.

After a significant hiatus imposed by the COVID-19 pandemic, we hereby restart Xenotransplantation literature updates. With the recently performed clinical xenotransplantation cases and the much-heightened interest in the field, we have determined that this is an optimal time to reinstate this section. There has been an invigorated focus on unique challenges posed by pig-to-human xenotransplantation, and specific attention will be given to this aspect. In this issue, we aimed to cover the gap and compiled the most relevant publications from March 2021 to March 2023.