Clinical xenotransplantation has the potential to address shortages of human organs for patients with end-stage organ failure. Advances in genetic engineering, immunosuppressive regimens, and infectious disease diagnostics have improved prospects for clinical xenotransplantation. Management of the infectious risks posed by clinical xenotransplantation requires biosecure breeding and validated methods for microbiological surveillance of source animals and recipients. Novel infection control protocols may complement biosafety requirements. Infectious risks in xenotransplantation include both known human pathogens common to immunosuppressed organ recipients and from porcine organisms or xenozoonoses for which the clinical manifestations are less well defined and for which microbial assays and therapies are more limited. Some pig-specific organisms do not infect human cells but have systemic manifestations when active within the xenograft. The human risk posed by porcine endogenous retroviruses (PERV) is uncertain. There are no documented transmissions of PERV in humans and swine are available with inactivated genomic PERV loci. Metagenomic sequencing will complement more traditional diagnostic tools in the detection of any unknown pathogens in xenotransplantation recipients. Such data are required for the development of protocols for donor and recipient microbiological surveillance, infection control, and antimicrobial therapies that will enhance the safety of clinical xenotransplantation.

SUMMARYXenotransplantation, the transplantation of living organs, tissues, or cells between species, carries the potential to address the global shortage of human organs for patients with end-stage organ failure. Recent advances in genetic engineering have improved prospects for clinical xenotransplantation by reducing immune and inflammatory responses to grafts, controlling coagulation on endothelial surfaces, and modifying viral risks, including the porcine endogenous retrovirus (PERV). Management of infectious risks posed by clinical xenotransplantation requires meticulous attention to the biosecure breeding and microbiological surveillance of source animals and recipients and consideration of novel infection control requirements. Infectious risks in xenotransplantation stem from both known human pathogens in immunosuppressed transplant recipients and from porcine organisms for which the clinical manifestations, microbial assays, and therapies are generally limited. Both known and unknown zoonoses may be transmitted from pigs to humans. Some pig-specific pathogens do not infect human cells but have systemic manifestations when active within the xenograft, including porcine cytomegalovirus/porcine roseolovirus (PCMV/PRV), which contributes to graft rejection and consumptive coagulopathy. The role of porcine endogenous retrovirus (PERV) in humans remains uncertain despite the absence of documented transmissions and the availability of swine with inactivated genomic PERV. New technologies, such as metagenomic sequencing and multi-omics approaches, will be essential for detection of novel infections and for understanding interactions between the xenograft, the host's immune system, and potential pathogens. These approaches will allow development of infection control protocols, pathogen surveillance requirements, and tailored antimicrobial therapies to enhance the safety and success of clinical xenotransplantation.

Clinical xenotransplantation has the potential to address shortages of human organs for patients with end-stage organ failure. Advances in genetic engineering, immunosuppressive regimens, and infectious disease diagnostics have improved prospects for clinical xenotransplantation. Management of the infectious risks posed by clinical xenotransplantation requires biosecure breeding and validated methods for microbiological surveillance of source animals and recipients. Novel infection control protocols may complement biosafety requirements. Infectious risks in xenotransplantation include both known human pathogens common to immunosuppressed organ recipients and from porcine organisms or xenozoonoses for which the clinical manifestations are less well defined and for which microbial assays and therapies are more limited. Some pig-specific organisms do not infect human cells but have systemic manifestations when active within the xenograft. The human risk posed by porcine endogenous retroviruses (PERV) is uncertain. There are no documented transmissions of PERV in humans and swine are available with inactivated genomic PERV loci. Metagenomic sequencing will complement more traditional diagnostic tools in the detection of any unknown pathogens in xenotransplantation recipients. Such data are required for the development of protocols for donor and recipient microbiological surveillance, infection control, and antimicrobial therapies that will enhance the safety of clinical xenotransplantation.

Porcine endogenous retrovirus C (PERV-C) is a gammaretrovirus present in the genome of many, but not all, pigs. It is an ecotropic virus, able to infect only pig cells. In contrast, PERV-A and PERV-B, which are present in all pigs, can infect cells of multiple host species, including humans, thereby posing a risk for xenotransplantation when pigs are used as donor animals. Notably, PERV-C can recombine with PERV-A to produce PERV-A/C recombinants that can infect human cells and replicate to higher titers compared to the paternal PERV-A. The objective of this study is to evaluate the reliability of both existing and newly developed polymerase chain reactions (PCR) methods for detecting PERV-C, with the aim of selecting PERV-C-free pigs to be used for xenotransplantation. To detect PERV-C by PCR, specific primers targeting the region of the envelope protein gene, which differs from that of PERV-A and PERV-B due to its unique receptor binding site, must be employed. In this study, new PCR assays were developed to detect PERV-C and a total of ten PCR assays and one real-time PCR assay were evaluated for their reliability in detecting PERV-C. These assays were used to screen indigenous Greek black pigs, Auckland Island pigs, and German slaughterhouse pigs. Two of the PCR assays consistently yielded reliable results, whereas the other PCRs and the real-time PCR gave false positive results. Using the reliable assays, it was shown that one out of four indigenous Greek black pigs (using the same method in a previous publication 11 of 21 pigs were found PERV-C-negative), one out of ten German slaughterhouse pigs, the pig kidney cell line PK15, and all the Auckland Island pigs were PERV-C-negative. The reliable PCR assays will enable the screening of PERV-C-negative donor pigs to be used in xenotransplantation. Most importantly, all the Auckland Island pigs that were genetically modified in Germany for use in clinical trials were PERV-C-negative.

Allogeneic islet transplantation becomes a viable option for patients with unstable type 1 diabetes. However, considering the huge number of patients with type 1 diabetes, human donor shortage is a serious issue. To overcome the donor shortage issue, xenotransplantation is an attractive option. In fact, clinical islet xenotransplantation has been conducted since 1990s. The first clinical trial was performed using fetal pigs and demonstrated the porcine pancreatic tissue could survive in human body with immunosuppressive strategies. To scale up the islet production, Canadian group established a method for islet isolation from neonatal pigs. Their method has been used for clinical islet xenotransplantation in New Zealand, Russian, Mexico, Argentina, and China. Recently Korean group published a clinical protocol for islet xenotransplantation using adult pigs. For the next generation of islet xenotransplantation, gene-modified pigs were created. Especially "superislets" created by Belgian group demonstrated promising preclinical outcomes. With advanced donor pigs, islet xenotransplantation might become a suitable treatment for the majority of type 1 diabetic patients.

Porcine endogenous retroviruses (PERVs) are integrated into the genome of all pigs and can infect human cells in culture. However, no PERV infections have been reported in recipients following preclinical or clinical xenotransplantation or deliberate infection experiments. Detection of PERV infection in transplanted recipients is challenging due to microchimerism, such as the presence of pig cells containing PERV proviruses in the recipient. Based on our previous publications on PERV detection in xenotransplant recipients, particularly from the first clinical trials, we developed a comprehensive strategy to screen for PERV infections. Recipients can be monitored for increasing levels of viral genomic RNA and mRNA using real-time reverse transcriptase (RT)-PCR, which can indicate PERV expression and replication. To test this strategy, explanted pig hearts and organs from baboons after pig heart transplantation were analyzed. No PERV genomic RNA or mRNA was detected in these tissues, although both were found in PERV-producing human control cells. Screening for antibodies against PERV as indirect evidence of infection is the method of choice. Recombinant viral proteins were prepared for use in Western blot assays. Animal antisera generated through immunization with recombinant PERV proteins served as positive controls. No antibodies against PERV were detected in transplanted baboons, even though microchimerism was observed in many of the animals' organs. For effective antibody screening, at least two PERV proteins should be used as antigens.

Porcine endogenous retroviruses (PERVs) are integrated in the genome of all pigs. PERV-A, PERV-B and PERV-C can be released as infectious virus particles and PERV-A and PERV-B can infect human cells in culture. PERV-C does not infect human cells, but high-titer recombinant PERV-A/C can infect them. Retroviruses are able to induce immunosuppression and/or tumors in the infected host. Numerous methods have been developed to study PERV in donor pigs. No PERV infections were observed in infection experiments as well as in preclinical and clinical xenotransplantation trials. Despite this, several strategies have been developed to prevent PERV infection of the recipient. PCR-based and immunological methods are required to screen xenotransplant recipients. Since the proviruses are integrated into the pig genome, PERV infection has to be distinguished from microchimerism, e.g., the presence of pig cells in the recipient, which is common in xenotransplantation. Sensitive PCR methods using pig short interspersed nuclear elements (SINE) sequences allow to detect pig cells easily. Virus infection can also be detected by an increase of viral genomic or mRNA in human cells. The method of choice, however, is to screen for specific antibodies against PERV using different recombinant PERV proteins, purified viruses or peptides.

The world's first clinical cardiac xenotransplantation, using a genetically engineered pig heart with 10 gene modifications, prolonged the life of a 57-year-old man with no other life-saving options, by 60 days. It is foreseeable that xenotransplantation will be introduced in clinical practice in the United States. However, little clinical or regulatory progress has been made in the field of xenotransplantation in Japan in recent years. Japan seems to be heading toward a "device lag", and the over-importation of medical devices and technology in the medical field is becoming problematic. In this review, we discuss the concept of pig-heart xenotransplantation, including the pathobiological aspects related to immune rejection, coagulation dysregulation, and detrimental heart overgrowth, as well as genetic modification strategies in pigs to prevent or minimize these problems. Moreover, we summarize the necessity for and current status of xenotransplantation worldwide, and future prospects in Japan, with the aim of initiating xenotransplantation in Japan using genetically modified pigs without a global delay. It is imperative that this study prompts the initiation of preclinical xenotransplantation research using non-human primates and leads to clinical studies.

Type 1 diabetes mellitus (T1DM) is characterized by absolute insulin deficiency primarily due to autoimmune destruction of pancreatic β-cells. The prevailing treatment for T1DM involves daily subcutaneous insulin injections, but a substantial proportion of patients face challenges such as severe hypoglycemic episodes and poorly controlled hyperglycemia. For T1DM patients, a more effective therapeutic option involves the replacement of β-cells through allogeneic transplantation of either the entire pancreas or isolated pancreatic islets. Unfortunately, the scarcity of transplantable human organs has led to a growing list of patients waiting for an islet transplant. One potential alternative is xenotransplantation of porcine pancreatic islets. However, due to inter-species molecular incompatibilities, porcine tissues trigger a robust immune response in humans, leading to xenograft rejection. Several promising strategies aim to overcome this challenge and enhance the long-term survival and functionality of xenogeneic islet grafts. These strategies include the use of islets derived from genetically modified pigs, immunoisolation of islets by encapsulation in biocompatible materials, and the creation of an immunomodulatory microenvironment by co-transplanting islets with accessory cells or utilizing immunomodulatory biomaterials. This review concentrates on delineating the primary obstacles in islet xenotransplantation and elucidates the fundamental principles and recent breakthroughs aimed at addressing these challenges.

Allogeneic islet transplantation has become a standard therapy for unstable type 1 diabetes. However, considering the large number of type 1 diabetic patients, the shortage of donors is a serious issue. To address this issue, clinical islet xenotransplantation is conducted. The first clinical islet xenotransplantation was performed by a Swedish team using fetal pancreatic tissue. Thereafter, clinical trials of islet xenotransplantation were conducted in New Zealand, Russia, Mexico, Argentina, and China using neonatal pig islets. In clinical trials, fetal or neonatal pancreata are used because of the established reliable islet isolation methods. These trials demonstrate the method's safety and efficacy. Currently, the limited number of source animal facilities is a problem in terms of promoting islet xenotransplantation. This limitation is due to the high cost of source animal facilities and the uncertain future of xenotransplantation. In the United States, the first xenogeneic heart transplantation has been performed, which could promote xenotransplantation. In Japan, to enhance xenotransplantation, the 'Medical Porcine Development Association' has been established. We hope that xenogeneic transplantation will become a clinical reality, serving to address the shortage of donors.

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.

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.

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.

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.

Type 1 Diabetes (T1D) involves the autoimmune destruction of insulin-producing β-cells in the pancreas. Exogenous insulin injections are the current therapy but are user-dependent and cannot fully recapitulate physiological insulin secretion dynamics. Since the emergence of allogeneic cell therapy for T1D, the Edmonton Protocol has been the most promising immunosuppression protocol for cadaveric islet transplantation, but the lack of donor islets, poor cell engraftment, and required chronic immunosuppression have limited its application as a therapy for T1D. Encapsulation in biomaterials on the nano-, micro-, and macro-scale offers the potential to integrate islets with the host and protect them from immune responses. This method can be applied to different cell types, including cadaveric, porcine, and stem cell-derived islets, mitigating the issue of a lack of donor cells. This review covers progress in the efforts to integrate insulin-producing cells from multiple sources to T1D patients as a form of cell therapy.

One of the prerequisites for successful organ xenotransplantation is a reasonable size match between the porcine organ and the recipient's organ to be replaced. Therefore, the selection of a suitable genetic background of source pigs is important. In this study, we investigated body and organ growth, cardiac function, and genetic diversity of a colony of Auckland Island pigs established at the Center for Innovative Medical Models (CiMM), LMU Munich. Male and female Auckland Island pig kidney cells (selected to be free of porcine endogenous retrovirus C) were imported from New Zealand, and founder animals were established by somatic cell nuclear transfer (SCNT). Morphologically, Auckland Island pigs have smaller body stature compared to many domestic pig breeds, rendering their organ dimensions well-suited for human transplantation. Furthermore, echocardiography assessments of Auckland Island pig hearts indicated normal structure and functioning across various age groups throughout the study. Single nucleotide polymorphism (SNP) analysis revealed higher runs of homozygosity (ROH) in Auckland Island pigs compared to other domestic pig breeds and demonstrated that the entire locus coding the swine leukocyte antigens (SLAs) was homozygous. Based on these findings, Auckland Island pigs represent a promising genetic background for organ xenotransplantation.

Combined islet and kidney xenotransplantation for the treatment of diabetic nephropathy represents a compelling and increasingly relevant therapeutic possibility for an ever-growing number of patients who would benefit from both durable renal replacement and cure of the underlying cause of their renal insufficiency: diabetes. Here we briefly review immune barriers to islet transplantation, highlight preclinical progress in the field, and summarize our experience with combined islet and kidney xenotransplantation, including both challenges with islet-kidney composite grafts as well as our recent success with sequential kidney followed by islet xenotransplantation in a pig-to-baboon model.

The lack of self-repairability in cartilage and the formation of fibrocartilage pose significant challenges in treating knee osteoarthritis, and there is still no ideal solution. Autologous platelet lysates have been clinically applied to treat kOA and exert satisfactory cartilage-repair efficacy, but the preparation of human PL brings damage to patients and is hardly standardized.

In this study, porcine PL was developed to replace hPL, and its chondroregenerative and anti-chondrofibrosis effects were explored. Enzyme-Linked Immunosorbent Assay was applied to qualify the PL products. In vivo, partial-thickness cartilage defects were created on rats as a kOA model, and the von Frey test, histopathological observation, immunohistochemical analysis, and western blot analysis were conducted. In vitro, CCK-8 assay, real-time PCR analysis, immunofluorescence test, and WB analysis were conducted for the mechanism study of pPL.

The in vivo data showed that pPL significantly repaired the cartilage defect by improving matrix synthesis and also ameliorated the pain response in the kOA model of rats. In addition, pPL exerted an anti-fibrosis effect on cartilage by suppressing the expressions of COL1, COL3, α-SMA, VIMENTIN, SMAD2, p-SMAD2, and CTGF in cartilage. The in vitro data verified these effects and indicated that the SMAD2 pathway mediated the anti-fibrosis mechanism of pPL. Moreover, the comparable effects between pPL and rat PL indicate that there is no immune rejection from pPL.

This study firstly demonstrated the anti-kOA effects of pPL on both cartilage-repair and anti-chondrofibrosis. It developed pPL as a promising alternative to autologous PL for clinical applications.

In this clinicopathological conference, invited experts discussed a previously published case of a patient with nonischemic cardiomyopathy who underwent heart transplantation from a genetically modified pig source animal. His complex course included detection of porcine cytomegalovirus by plasma microbial cell-free DNA and eventual xenograft failure. The objectives of the session included discussion of selection of immunosuppressive regimens and prophylactic antimicrobials for human xenograft recipients, description of infectious disease risk assessment and mitigation in potential xenograft donors and understanding of screening and therapeutic strategies for potential xenograft-related infections.

Auckland Island pigs represent an inbred population of feral pigs isolated on the sub-Antarctic island for over 100 years. The animals have been maintained under pathogen-free conditions in New Zealand; they are well characterized virologically and have been used as donor sources in first clinical trials of porcine neonatal islet cell transplantation for the treatment of human diabetes patients. The animals do not carry any of the xenotransplantation-relevant viruses, and in the first clinical trials, no porcine viruses, including porcine endogenous retroviruses (PERVs) were transmitted to the human recipients. PERVs pose a special risk in xenotransplantation, since they are part of the pig genome. When the copy number of PERVs in these animals was analyzed using droplet digital PCR and primers binding to a conserved region of the polymerase gene (PERVpol), a copy number typical for Western pigs was found. This confirms previous phylogenetic analyses of microsatellites as well as mitochondrial analyses showing a closer relationship to European pigs than to Chinese pigs. When kidney cells from very young piglets were analyzed, only around 20 PERVpol copies were detected. Using these cells as donors in somatic cell nuclear transfer (SCNT), animals were born showing PERVpol copy numbers between 35 and 56. These data indicate that Auckland Island pigs have a similar copy number in comparison with other Western pig breeds and that the copy number is higher in adult animals compared with cells from young piglets. Most importantly, PERV-C-free animals were selected and the absence of an additional eight porcine viruses was demonstrated.

Microencapsulation is a promising strategy to prolong the survival and function of transplanted pancreatic islets for diabetes therapy, albeit its translation has been impeded by incoherent graft performance. The use of decellularized ECM has lately gained substantial research momentum due to its innate capacity to augment the function of cells originating from the same tissue type. In the present study, the advantages of both these approaches are leveraged in a porcine pancreatic ECM (pECM)-based microencapsulation platform, thus significantly enhancing murine pancreatic islet performance. pECM-encapsulated islets sustain high insulin secretion levels in vitro, surpassing those of islets encapsulated in conventional alginate microcapsules. Moreover, pECM-encapsulated islet cells proliferate and produce an enriched intra-islet ECM framework, displaying a distinctive structural rearrangement. The beneficial effect of pECM encapsulation is further reinforced by the temporary protection against cytokine-induced cytotoxicity. In-vivo, this platform significantly improves glucose tolerance and achieves glycemic correction in 100% of immunocompetent diabetic mice without any immunosuppression, compared to only 50% mice achieved glycemic correction by alginate encapsulation. Altogether, the results presented herein reveal that pECM-based microencapsulation offers a natural pancreatic niche that can restore the function of isolated pancreatic islets and deliver them safely, avoiding the need for immunosuppression. STATEMENT OF SIGNIFICANCE: Aiming to improve pancreatic islet transplantation outcomes in diabetic patients, we developed a microencapsulation platform based on pancreatic extracellular matrix (pECM). In these microcapsules the islets are entrapped within a pECM hydrogel that mimics the natural pancreatic microenvironment. We show that pECM encapsulation supports the islets' viability and function in culture, and provides temporal protection against cytokine-induced stress. In a diabetic mouse model, pECM encapsulation significantly improved glucose tolerance and achieved glycemic correction without any immunosuppression. These results reveal the potential of pECM encapsulation as a viable treatment for diabetes, providing a solid scientific basis for more advanced preclinical studies.

There have been high expectations in recent years of using xenotransplantation and regenerative medicine to treat humans, and pigs have been utilized as the donor model. Pigs used for these clinical applications must be microbiologically safe, that is, free of infectious pathogens, to prevent infections not only in livestock, but also in humans. Currently, however, the full spectrum of pathogens that can infect to the human host or cause disease in transplanted porcine organs/cells has not been fully defined. In the present study, we thus aimed to develop a larger panel for the detection of pathogens that could potentially infect xenotransplantation donor pigs. Our newly developed panel, which consisted of 76 highly sensitive PCR detection assays, was able to detect 41 viruses, 1 protozoa, and a broad range of bacteria (by use of universal 16S rRNA primers). The applicability of this panel was validated using blood samples from uterectomy-born piglets, and pathogens suspected to be vertically transmitted from sows to piglets were successfully detected. We estimate that, at least for viruses and bacteria, the number of target pathogens detected by the developed screening panel should suffice to meet the microbiological safety levels required worldwide for xenotransplantation and/or regenerative therapy. This panel provides greater diagnosis options to produce donor pigs so that it would render unnecessary to screen for all pathogens listed. Instead, the new panel could be utilized to detect only required pathogens within a given geographic range where the donor pigs for xenotransplantation have been and/or are being developed.

Xenotransplantation has the potential to address shortages of organs available for clinical transplantation, but concerns exist regarding potential risks posed by porcine microorganisms and parasites (MP) to the health of human recipients. In this study, a risk-based framework was developed, and expert opinion was elicited to evaluate porcine MP based on swine exposure and risk to human health. Experts identified 255 MP to include in the risk assessment. These were rated by experts for five criteria regarding potential swine exposure in the USA and human health risks. MP were subsequently categorized into three risk mitigation groups according to pre-defined rules: disqualifying porcine MP (due to their pathogenic potential, n = 130); non-disqualifying porcine MP (still relevant to consider for biosecurity or monitoring efforts, n = 40); and alert/watch list (not reported in the USA or MP not in swine, n = 85). Most disqualifying (n = 126) and non-disqualifying (n = 36) porcine MP can effectively be eliminated with high biosecurity programs. This approach supports surveillance and risk mitigation strategies for porcine MP in swine produced for xenotransplantation, such as documentation of freedom from porcine MP, or use of porcine MP screening, monitoring, or elimination options. To the authors' knowledge, this is the first effort to comprehensively identify all relevant porcine MP systematically and transparently evaluate the risk of infection of both donor animals and immunosuppressed human recipients, and the potential health impacts for immunosuppressed human recipients from infected xenotransplantation products from pigs.

Porcine cytomegalovirus (PCMV) is widely distributed in pigs and difficult to detect due to latency. PCMV infection of source pigs was associated with early graft failure after cardiac and renal xenotransplantation into nonhuman primates. Importantly, PCMV infection of the first genetically modified pig heart into a human may have contributed to the reduced survival of the patient. Sensitive and reliable assays for detection of latent PCMV infection are thus indispensable. Here, we report the development of five peptide-induced rabbit antisera specific for PCMV glycoprotein B (gB) and their validation for detection of PCMV in infected pig fallopian tube (PFT) cells by immunofluorescence and electron microscopy (EM). The anti-gB antibodies were also used for detection by Western blot analysis of PCMV purified from the supernatant of infected PFT cells. Sera of infected versus non-infected pigs have been compared. In parallel, PCMV viral load in blood samples of the animals was quantified by a novel highly sensitive nested-PCR and qPCR assay. A combination of four partly overlapping peptides from the gB C-terminus was used to establish a diagnostic ELISA for PCMV gB specific pig antibodies which is able to differentiate infected from non-infected animals and to quantify maternal antibodies in neonates. The combination of a highly sensitive nested PCR for direct virus detection with a sensitive peptide-based ELISA detecting anti-PCMV gB-antibodies, supplemented by Western blot analysis and/or immunohistochemistry for virus detection will reliably differentiate pigs with active infection, latently infected pigs, and non-infected pigs. It may significantly improve the virologic safety of xenotransplantation.

Xenotransplantation is a potential way to reduce the shortage of the needed organ grafts for the end-stage disease. Immune rejection, physiological incompatibility and bio-safety are the most critical issues.

To ensure the safety and efficacy of gene editing, second- and third-generation sequencing technologies have allowed us to obtain a clearer genetic background of donor pigs for xenotransplantation. Based on the Changsha Communiqué, the local DPF- excluded lists and DPF donor facility were established in Changsha, China. A pig-to-human islet clinical trial was conducted and overseen by the respective Chinese governmental agency.

The DPF standards for pig husbandry eliminated specific pathogens in donor pigs. We have established a PERV-C free, genetic information clean, DPF donor for xenotransplantation. A clinical trial of ten adult patients (9M:1F) with type 1 diabetes who received DPF porcine islet xenotransplantation via the portal vein were performed. Clinical accepted immunosuppressant drugs and autologous Treg were used for controlling immune rejection. No cross-species infection events occurred in this trial, and importantly, no cross-species transmission of PERV was found.

Xenotransplantation is a pioneer study and safety is the most important issue. The fundamental principles for establishing xenotransplantation donor pigs should follow the Changsha Communiqué (2008), the second WHO consultation,and the 2018 Changsha Communiqué which would finally help reducing the risks of xenotransplantation.

Despite advances in the technology of mechanical circulatory support, the need for heart transplantation continues to grow. The longevity of heart transplants continues to be superior to mechanical solutions, though the short-term differences are shrinking. In this review, we cover three timely developments and summarize the recent literature.

After stagnant rates of heart transplant activity for some years, recently, transplant volume has increased. The developments that have ignited interest have been the use of hepatitis C infected donors, which can now be safely transplanted with the advent of curative oral regimens, and the worldwide use of donors following withdrawal of life support as opposed to traditional brain death donors. In addition, the recent experience of human cardiac xenotransplantation has been very exciting, and though it is not of clinical utility yet, it holds the promise for a virtually unlimited supply of organs at some time in the future.

Much work remains to be done, but together, all three of these developments are exciting and important to be aware of in the future. Each will contribute to additional donors for human heart transplantation and hopefully will alleviate suffering and death on the waiting list.

Xenotransplantation with porcine organs has been recognized as a promising solution to alleviate the shortage of organs for human transplantation. Porcine endogenous retrovirus (PERV), whose proviral DNAs are integrated in the genome of all pig breeds, is a main microbiological risk for xenotransplantation. Over the last decades, some advances on PERVs' studies have been achieved. Here, we reviewed the current progress of PERVs including the classification, molecular structure, regulation, function in immune system, and potential risk in xenotransplantation. We also discussed the problem of insufficient study on PERVs as well as the questions need to be answered in the future work.

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 the 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 2 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. Additional topics discussed include 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. 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.

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.

Microchimerism is the presence of cells in an individual that have originated from a genetically distinct individual. The most common form of microchimerism is fetomaternal microchimerism, i.e., cells from a fetus pass through the placenta and establish cell lineages within the mother. Microchimerism was also described after the transplantation of human organs in human recipients. Consequently, microchimerism may also be expected in xenotransplantation using pig cells or organs. Indeed, microchimerism was described in patients after xenotransplantations as well as in non-human primates after the transplantation of pig organs. Here, for the first time, a comprehensive review of microchimerism in xenotransplantation is given. Since pig cells contain porcine endogenous retroviruses (PERVs) in their genome, the detection of proviral DNA in transplant recipients may be misinterpreted as an infection of the recipient with PERV. To prevent this, methods discriminating between infection and microchimerism are described. This knowledge will be important for the interpretation of screening results in forthcoming human xenotransplantations.

Islet transplantation has emerged as a curative therapy for diabetes in select patients but remains rare due to shortage of suitable donor pancreases. Islet transplantation using porcine islets has long been proposed as a solution to this organ shortage. There have already been several small clinical trials using porcine islets in humans, but results have been mixed and further trials limited by calls for more rigorous pre-clinical data. Recent progress in heart and kidney xenograft transplant, including three studies of pig-to-human xenograft transplant, have recaptured popular imagination and renewed interest in clinical islet xenotransplantation. This review outlines immunologic barriers to islet transplantation, summarizes current strategies to overcome these barriers with a particular focus on approaches to induce tolerance, and describes an innovative strategy for treatment of diabetic nephropathy with composite islet-kidney transplantation.

A major limitation of organ allotransplantation is the insufficient supply of donor organs. Consequently, thousands of patients die every year while waiting for a transplant. Progress in xenotransplantation that has permitted pig organ graft survivals of years in non-human primates has led to renewed excitement about the potential of this approach to alleviate the organ shortage. In 2022, the first pig-to-human heart transplant was performed on a compassionate use basis, and xenotransplantation experiments using pig kidneys in deceased human recipients provided encouraging data. Many advances in xenotransplantation have resulted from improvements in the ability to genetically modify pigs using CRISPR-Cas9 and other methodologies. Gene editing has the capacity to generate pig organs that more closely resemble those of humans and are hence more physiologically compatible and less prone to rejection. Despite such modifications, immune responses to xenografts remain powerful and multi-faceted, involving innate immune components that do not attack allografts. Thus, the induction of innate and adaptive immune tolerance to prevent rejection while preserving the capacity of the immune system to protect the recipient and the graft from infection is desirable to enable clinical xenotransplantation.

The practice of xenotransplantation using pig islet cells or organs is under development to alleviate the shortage of human donor islet cells or organs for the treatment of diabetes or organ failure. Multiple genetically modified pigs were generated to prevent rejection. Xenotransplantation may be associated with the transmission of potentially zoonotic porcine viruses. In order to prevent this, we developed highly sensitive PCR-based, immunologicals and other methods for the detection of numerous xenotransplantation-relevant viruses. These methods were used for the screening of donor pigs and xenotransplant recipients. Of special interest are the porcine endogenous retroviruses (PERVs) that are integrated in the genome of all pigs, which are able to infect human cells, and that cannot be eliminated by methods that other viruses can. We showed, using droplet digital PCR, that the number of PERV proviruses is different in different pigs (usually around 60). Furthermore, the copy number is different in different organs of a single pig, indicating that PERVs are active in the living animals. We showed that in the first clinical trials treating diabetic patients with pig islet cells, no porcine viruses were transmitted. However, in preclinical trials transplanting pig hearts orthotopically into baboons, porcine cytomegalovirus (PCMV), a porcine roseolovirus (PCMV/PRV), and porcine circovirus 3 (PCV3), but no PERVs, were transmitted. PCMV/PRV transmission resulted in a significant reduction of the survival time of the xenotransplant. PCMV/PRV was also transmitted in the first pig heart transplantation to a human patient and possibly contributed to the death of the patient. Transmission means that the virus was detected in the recipient, however it remains unclear whether it can infect primate cells, including human cells. We showed previously that PCMV/PRV can be eliminated from donor pigs by early weaning. PERVs were also not transmitted by inoculation of human cell-adapted PERV into small animals, rhesus monkey, baboons and cynomolgus monkeys, even when pharmaceutical immunosuppression was applied. Since PERVs were not transmitted in clinical, preclinical, or infection experiments, it remains unclear whether they should be inactivated in the pig genome by CRISPR/Cas. In summary, by using our sensitive methods, the safety of xenotransplantation can be ensured.

Intrahepatic islet transplantation is a promising β-cell replacement strategy for the treatment of type 1 diabetes. Instant blood-mediated inflammatory reactions, acute inflammatory storm, and graft revascularization delay limit islet engraftment in the peri-transplant phase, hampering the success rate of the procedure. Growing evidence has demonstrated that islet engraftment efficiency may take advantage of several bioengineering approaches aimed to recreate both vascular and endocrine compartments either ex vivo or in vivo. To this end, endocrine pancreas bioengineering is an emerging field in β-cell replacement, which might provide endocrine cells with all the building blocks (vascularization, ECM composition, or micro/macro-architecture) useful for their successful engraftment and function in vivo. Studies on reshaping either the endocrine cellular composition or the islet microenvironment have been largely performed, focusing on a single building block element, without, however, grasping that their synergistic effect is indispensable for correct endocrine function. Herein, the review focuses on the minimum building blocks that an ideal vascularized endocrine scaffold should have to resemble the endocrine niche architecture, composition, and function to foster functional connections between the vascular and endocrine compartments. Additionally, this review highlights the possibility of designing bioengineered scaffolds integrating alternative endocrine sources to overcome donor organ shortages and the possibility of combining novel immune-preserving strategies for long-term graft function.

Xenografts from genetically modified pigs have become one of the most promising solutions to the dearth of human organs available for transplantation. The challenge in this model has been hyperacute rejection. To avoid this, pigs have been bred with a knockout of the alpha-1,3-galactosyltransferase gene and with subcapsular autologous thymic tissue.

We transplanted kidneys from these genetically modified pigs into two brain-dead human recipients whose circulatory and respiratory activity was maintained on ventilators for the duration of the study. We performed serial biopsies and monitored the urine output and kinetic estimated glomerular filtration rate (eGFR) to assess renal function and xenograft rejection.

The xenograft in both recipients began to make urine within moments after reperfusion. Over the 54-hour study, the kinetic eGFR increased from 23 ml per minute per 1.73 m² of body-surface area before transplantation to 62 ml per minute per 1.73 m² after transplantation in Recipient 1 and from 55 to 109 ml per minute per 1.73 m² in Recipient 2. In both recipients, the creatinine level, which had been at a steady state, decreased after implantation of the xenograft, from 1.97 to 0.82 mg per deciliter in Recipient 1 and from 1.10 to 0.57 mg per deciliter in Recipient 2. The transplanted kidneys remained pink and well-perfused, continuing to make urine throughout the study. Biopsies that were performed at 6, 24, 48, and 54 hours revealed no signs of hyperacute or antibody-mediated rejection. Hourly urine output with the xenograft was more than double the output with the native kidneys.

Genetically modified kidney xenografts from pigs remained viable and functioning in brain-dead human recipients for 54 hours, without signs of hyperacute rejection. (Funded by Lung Biotechnology.).

Although human islet transplantation has proven to provide clinical benefits, especially the near complete amelioration of hypoglycemia, the supply of human islets is limited and insufficient to meet the needs of all people that could benefit from islet transplantation. Porcine islets, secreting insulin nearly identical to that of human insulin, have been proposed as a viable supply of unlimited islets. Further, encapsulation of the porcine islets has been shown to reduce or eliminate the use of immunosuppressive therapy that would be required to prevent rejection of the foreign islet tissue. The goal of the current study was to determine the long-term safety and efficacy of agarose encapsulated porcine islets (macrobeads) in diabetic cynomolgus macaques, in a study emulating a proposed IND trial in which daily exogenous insulin therapy would be reduced by 50% with no loss of glucose regulation. Four of six animals implanted with macrobeads demonstrated ≥ 30% reduction in insulin requirements in year 1 of follow-up. Animals were followed for 2, 3.5, and 7.4 years with no serious adverse events, mortality or evidence of pathogen transmission. This study supports the continued pursuit of encapsulated porcine islet therapy as a promising treatment option for diabetes mellitus.

A radical solution is needed for the organ supply crisis, and the domestic pig is a promising organ source. In preparation for a clinical trial of xenotransplantation, we developed an in vivo pre-clinical human model to test safety and feasibility tenets established in animal models. After performance of a novel, prospective compatible crossmatch, we performed bilateral native nephrectomies in a human brain-dead decedent and subsequently transplanted two kidneys from a pig genetically engineered for human xenotransplantation. The decedent was hemodynamically stable through reperfusion, and vascular integrity was maintained despite the exposure of the xenografts to human blood pressure. No hyperacute rejection was observed, and the kidneys remained viable until termination 74 h later. No chimerism or transmission of porcine retroviruses was detected. Longitudinal biopsies revealed thrombotic microangiopathy that did not progress in severity, without evidence of cellular rejection or deposition of antibody or complement proteins. Although the xenografts produced variable amounts of urine, creatinine clearance did not recover. Whether renal recovery was impacted by the milieu of brain death and/or microvascular injury remains unknown. In summary, our study suggests that major barriers to human xenotransplantation have been surmounted and identifies where new knowledge is needed to optimize xenotransplantation outcomes in humans.

Following the recommendations by a panel of experts gathered by the World Health Organization in 2005, an inventory was established to collect practices of human xenotransplantation worldwide (www.humanxenotransplant.org). The website was activated in October 2006, in collaboration with the International Xenotransplantation Association, the University Hospital Geneva, and the World Health Organization. A first report on the collected xenotransplantation activities was published in 2010 in the journal Transplantation. In 2020, the website was redesigned, and its hosting and management were transferred to the Sichuan Provincial People's Hospital.

We collected information from publications in scientific journals, presentations at international congresses, the internet, and declarations of International Xenotransplantation Association members on xenotransplantation procedures in humans performed over the past 10 y.

A total of 5 new applications of human xenotransplantation were identified, with pig as source animal in all applications. The procedures involved transplantation of islets of Langerhans, skin, cornea, and choroid plexus cells. The treatments were performed in China, United States, New Zealand, and Argentina. No major complications or deaths were reported.

Several clinical applications of cell or tissue xenotransplantation are ongoing around the world. Compared with the previous reported period (1995-2010, with 29 activities, mostly without governmental regulation), the recent number of clinical activities was reduced, and all were officially approved. This information should be used to inform healthcare officials, staff, and the public with the objective of encouraging good practices based on internationally harmonized guidelines driven by initiatives such as the Changsha Communiqué.

When, in 1869, Paul Langerhans detected the "islands of tissue" in the pancreas, he took the first step on a journey towards islet transplantation as a treatment for type 1 diabetes. The route has embraced developments across biosciences, surgery, gene therapy and clinical research. This review highlights major milestones along that journey involving whole pancreas transplantation, islet transplantation, the creation of surrogate insulin-secreting cells and novel islet-like structures using genetic and bio-engineering technologies. To obviate the paucity of human tissue, pluripotent stem cells and non-β-cells within the pancreas have been modified to create physiologically responsive insulin-secreting cells. Before implantation, these can be co-cultured with endothelial cells to promote vascularisation and with immune defence cells such as placental amnion cells to reduce immune rejection. Scaffolds to contain grafts and facilitate surgical placement provide further opportunities to achieve physiological insulin delivery. Alternatively, xenotransplants such as porcine islets might be reconsidered as opportunities exist to circumvent safety concerns and immune rejection. Thus, despite a long and arduous journey, the prospects for increased use of tissue transplantation to provide physiological insulin replacement are drawing ever closer.

In this minireview, we briefly outline the hallmarks of diabetes, the distinction between type 1 and type 2 diabetes, the global incidence of diabetes, and its associated comorbidities. The main goal of the review is to highlight the great potential of encapsulated pancreatic islet transplantation to provide a cure for type 1 diabetes. Following a short overview of the different approaches to islet encapsulation, we provide a summary of the merits and demerits of each approach of the encapsulation technology. We then discuss various attempts to clinical translation with each model of encapsulation as well as the factors that have mitigated the full clinical realization of the promise of the encapsulation technology, the progress that has been made and the challenges that remain to be overcome. In particular, we pay significant attention to the emerging strategies to overcome these challenges. We believe that these strategies to enhance the performance of the encapsulated islet constructs discussed herein provide good platforms for additional work to achieve successful clinical translation of the encapsulated islet technology.