The only curative therapy for end-stage heart failure is orthotopic allogeneic heart transplantation. This therapy has extended the survival of patients worldwide but is limited due to the scarcity of donor organs. Potential alternative donor sources of organs for transplantation include genetically-modified (GM) large animal donors (ie, xenografts) and human organs developed in large animal hosts. These strategies utilize gene editing and somatic cell nuclear transfer technologies to engineer partially or completely humanized organs. Preclinical xenotransplantation studies of GM pig hearts into baboons have already provided an important clinical foundation, as two patients have received cardiac xenografts from GM pigs and have survived for up to 2 months. Additional issues need to be addressed in order for patients to survive more than 1 year, which would make these strategies clinically applicable. Thus, in combination with immunosuppression agents, xenogeneic and exogenic organ sources hold tremendous promise for an unlimited and transformative supply of organs for transplantation.

Myocardial cold ischemia/reperfusion (I/R) injury is an inevitable consequence of heart transplantation, significantly affecting survival rates and therapeutic outcomes. Growth Differentiation Factor 15 (GDF15) has been shown to regulate GPX4-mediated ferroptosis, playing a critical role in mitigating I/R injury. Meanwhile, verbascoside (VB), an active compound extracted from the herbaceous plant, has demonstrated myocardial protective effects. In this study, heart transplantation was performed using a modified non-suture cuff technique, with VB administered at a dose of 20 mg/kg/day via intraperitoneal injection for 3 days in vivo. In vitro, cardiomyocytes were pretreated with 50 µg/ml VB for 24 h. VB treatment significantly reduced histopathological injury, decreased myocardial injury markers, and inhibited ferroptosis and oxidative stress during myocardial cold I/R injury in vivo. In vitro experiments further demonstrated that GDF15 alleviates ferroptosis induced by hypoxic reoxygenation by upregulating GPX4. Therefore, it is concluded that VB preconditioning can effectively reduce ferroptosis induced by myocardial cold I/R after heterotopic heart transplantation, possibly through up-regulation of GDF15/GPX4/SLC7A11 pathway.

Gene-edited pig organ transplantation can solve the serious shortage of human donor organs. Currently, xenotransplantation is rapidly developing and has made significant breakthroughs. The use of GTKO (Gal knockout) pigs is an important step forward. The subsequent knockout of three genes combined with the transfer of immune-related genes effectively prolonged the survival time of non-human primate (NHP) transplantation in xenotransplantation. Due to the success of allogeneic kidney transplantation on NHP, this gene editing protocol was recently applied to clinical patients. Two patients underwent allogeneic kidney transplantation and survived for 51 days and 47 days. Exceeding the hyperacute rejection period proves that appropriate gene editing strategies and the combination of immunosuppressive agents contribute to the success of xenotransplantation. To further enhance the feasibility of pig kidney xenograft, this article mainly explores the effects of the NHP xenograft gene editing scheme and immunosuppressants on prolonging transplant survival time.

This study investigates the impact of nursing interventions, guided by behavior change theory, on the recovery of cardiac function and quality of life in patients undergoing cardiac surgery with cardiopulmonary bypass (CPB).

A total of 120 patients scheduled for CPB in the Department of Cardiology at our hospital, from February 2021 to May 2023, were enrolled. According to the study protocol, patients were randomly assigned to either a control group (n = 60) or an observation group (n = 60) post-surgery. The control group received routine nursing care, while the observation group received nursing interventions based on behavior change theory, including health education, psychological support, and behavioral incentives. Informed consent was obtained from all patients. General patient data were collected from clinical records. Cardiac function was assessed using echocardiography. The wall motion score index (WMSI) and 6-minute walk distance (6MWD) were evaluated post-care. Serum levels of inflammatory cytokines TNF-α, IL-6, and IL-10 were measured via ELISA. Quality of life was assessed using the WHOQOL-BREF questionnaire, while anxiety and depression levels were evaluated using the HAM-A and HAM-D scales, respectively.

The baseline clinical characteristics and biochemical data of both groups were comparable (P > 0.05). The observation group showed a significantly higher left ventricular ejection fraction (LVEF) compared to the control group (P < 0.05), while both left ventricular end-systolic diameter (LVESD) and left ventricular end-diastolic diameter (LVEDD) were significantly lower (P < 0.05). Additionally, the observation group had a significantly lower WMSI score and a longer 6MWD (P < 0.05). Regarding inflammatory markers, TNF-α and IL-6 levels were significantly reduced in the observation group, while IL-10 levels were significantly elevated compared to the control group (P < 0.05). In terms of quality of life, the observation group reported significantly higher scores in physical health, mental health, social relationships, and environmental factors (P < 0.05). Moreover, anxiety and depression levels were significantly lower in the observation group, as evidenced by reduced HAM-A and HAM-D scores (P < 0.05).

Nursing care guided by behavior change theory significantly improves cardiac function and overall quality of life in patients recovering from cardiac surgery with CPB. This approach enhances LVEF, reduces left intraventricular diameter, lowers inflammatory cytokine levels, and improves mental health and social functioning. These findings underscore the importance of behavior change theory-based nursing interventions in optimizing postoperative recovery.

Genetically edited pigs, modified using CRISPR-Cas9 technology, hold promise as potential sources for xenotransplantation. However, the optimal combination of genetic modifications and their expression levels for initial clinical trials remains unclear. This study investigates the generation of TKO/hCD55/hTM/hEPCR (6GE) pigs and evaluates their compatibility with human immune and coagulation systems.

The 6GE pigs were generated through iterative genome editing and F1 generation breeding. Genotyping, flow cytometry, and immunohistochemistry confirmed the knockout of GGTA1, CMAH, and B4GALNT2. Expression levels of human genes (hCD55, hTM, hEPCR) were quantified. In vitro assays using aortic endothelial cells (pAECs) from 6GE pigs assessed human serum IgM and IgG binding, complement cytotoxicity, and thrombin-antithrombin (TAT) complex levels. Blood from gene-edited pigs was used for pathophysiological analysis.

Complete knockout of GGTA1, CMAH, and B4GALNT2 was confirmed in 6GE pigs. The expression of hCD55 and hTM was approximately seven and thirteen times higher than in humans, respectively, while hEPCR levels were comparable to those in humans. In vitro, 6GE pAECs showed significantly reduced binding of human IgM and IgG compared to wild-type pAECs (IgG p<0.01, IgM p<0.0001). Similar to TKO/hCD55 pAECs, 6GE pAECs exhibited a substantial reduction in complement-mediated cytotoxicity (p<0.001) compared to TKO pAECs. Co-expression of hTM and hEPCR in 6GE pigs led to a significant decrease in thrombin-antithrombin (TAT) complex levels in co-culture with human whole blood, compared to WT (p<0.0001), TKO (p<0.01), and TKO/hCD55/hTM pigs (p<0.05). Pathophysiological analysis demonstrated excellent compatibility of 6GE pig kidneys and livers with human immune and coagulation systems. However, 6GE pigs showed increased susceptibility to infection compared to other gene-edited pigs, while TKO/hCD55 pigs were considered safe when they were all bred in a general environment.

Highly expressing hCD55, along with the co-expression of hEPCR and hTM genes, is expected to effectively reduce human complement cytotoxicity and enhance anticoagulant efficacy in genetically modified pigs. The 6GE pigs exhibited robust compatibility with human physiological and immune systems, fulfilling the criteria for clinical trials. Furthermore, it is imperative to rear donor pigs in pathogen-free (DPF) facilities to mitigate infection risks and prevent the transmission of porcine pathogens to humans.

Ventricular assist devices (VADs) invigorated the management of patients with advanced heart failure, providing a lifeline for patients awaiting transplantation or requiring long-term circulatory support. This article reviews recent advances in VAD technologies, focusing on key areas of progress to overcome existing challenges and the potential for future applications. The reduction or possible elimination of infection-prone components and the evolution to transcutaneous energy transfer systems are two main research fields to reach a new quality of life category for VADs patients. Miniaturization and enhanced biocompatibility have resulted in smaller, less invasive devices with a significantly reduced risk of complications and mortality. Advances in percutaneous ventricular assist devices have emerged, contributing to the creation of less invasive options with or without intracardiac pumps, and facilitating their use for both left and right ventricles. These devices have gained more and more features, including the use of artificial intelligence. Moreover, the possibility of long-term use of intracardiac pumps offers a potential bridge to transplantation, allowing ambulation and probably also long-term circulatory support. Despite considerable advances, challenges remain, particularly in terms of improving durability, reducing the risk of ischemic events, further refining materials, and more sophisticated control and synchronization between systems that adapt to changing physiological demands.

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 purpose of a first-in-human (FIH) clinical trial is to gather information about how the drug or device affects and interacts with the human body: its safety, side effects, and (potential) dosage. As such, the primary goal of a FIH trial is not participant benefit but to gain knowledge of drug or device efficacy, i.e., baseline human safety knowledge. Some FIH clinical trials carry significant foreseeable risk to participants with little to no foreseeable participant benefit. Participation in such trials would be a bad deal for participants, and the research is considered justifiable because of the promise of significant potential social benefit. I argue that there is an ethical tension inherent in risky FIH research and that researchers should fairly compensate risky FIH trial participants. This does not make the risk-benefit outcome more favorable for participants; rather, it amounts to a collective reckoning with the ethical tension inherent in the research.

Xenotransplantation has made significant advances recently using pigs genetically engineered to remove carbohydrate antigens, either alone or with addition of various human complement, coagulation, and anti-inflammatory ''transgenes''. Here we evaluated results associated with gene-edited (GE) pig hearts transplanted in baboons using an established costimulation-based immunosuppressive regimen and a cold-perfused graft preservation technique.

Eight baboons received heterotopic abdominal heart transplants from 3-GE (GalKO.β4GalNT2KO.hCD55, n = 3), 9-GE (GalKO.β4GalNT2KO.GHRKO.hCD46.hCD55. TBM.EPCR.hCD47. HO-1, n = 3) or 10-G (9-GE+CMAHKO, n = 2) pigs using Steen's cold continuous perfusion for ischemia minimization. Immunosuppression (IS) included induction with anti-thymocyte globulin and αCD20, ongoing αCD154, MMF, and tapered corticosteroid.

All three 3-GE grafts functioned well initially, but failed within 5 days. One 9-GE graft was lost intraoperatively due to a technical issue and another was lost at POD 13 due to antibody mediated rejection (AMR) in a baboon with a strongly positive pre-operative cross-match. One 10-GE heart failed at POD113 with combined cellular and antibody mediated rejection. One 9-GE and one 10-GE hearts had preserved graft function with normal myocardium on protocol biopsies, but exhibited slowly progressive graft hypertrophy until elective necropsy at POD393 and 243 respectively. Elevated levels of IL-6, MCP-1, C-reactive protein, and human thrombomodulin were variably associated with conditioning, the transplant procedure, and clinically significant postoperative events.

Relative to reference genetics without thrombo-regulatory and anti-inflammatory gene expression, 9- or 10-GE pig hearts exhibit promising performance in the context of a clinically applicable regimen including ischemia minimization and αCD154-based IS, justifying further evaluation in an orthotopic model.

Pleural effusions develop frequently after cardiac surgery in humans. Lung ultrasound is an essential non-invasive tool in the diagnosis and treatment of these effusions. Pleural effusions also develop regularly after preclinical cardiac xenotransplantation experiments. Unlike in the human setting, modern ultrasound devices lack pre-installed tools for calculating the volume of pleural effusions in baboons. The aim of this study was to analyze ultrasound examinations of pleural effusions after orthotopic pig-to-baboon cardiac xenotransplantation experiments in order to develop a formula for calculating the effusion volume based on ultrasound measurements.

Hearts from seven genetically modified (GGTA1-KO, hCD46/hTBM transgenic) juvenile pigs were orthotopically transplanted into male baboons. Postoperatively, the baboons were tested regularly for the development of pleural effusions using ultrasound. When thoracocentesis was required, the drained effusion volume (EV) was compared to ultrasound-derived calculations using various formulas. These calculations were based on measuring the distance between lung and diaphragm at the effusions' maximum height (Hmax). Subsequently, the most promising formula was used to describe the interobserver variability between trained and untrained staff members to predict effusion volumes based on ultrasound measurements.

Ultrasound measurement correlated very strongly with the absolute EV (r = 0.9156, p < 0.0001), with EV indexed to total body weight (r = 0.9344, p < 0.0001) and with EV indexed to body surface area (BSA) (r = 0.9394, p < 0.0001). The ratio between Hmax and EV increased with total body weight and BSA and also depended on the baboon species. The sonographic measurements taken by an experienced and an inexperienced observer showed only low interobserver variability. A Bland-Altman plot of both observers' measurements showed an overall bias of -2.39%.

Ultrasound imaging provides a simple and non-invasive tool for measuring pleural effusion quantity in baboons. This facilitates simple and efficient monitoring even in the hands of untrained personnel and may guide the decision-making to perform thoracocentesis.

In modern cardiovascular research, isolated perfused hearts have become cost-effective and highly reproducible tools to investigate the mechanisms of cardiovascular diseases (CVDs). Since they were first introduced in the nineteenth century, isolated perfused hearts have been extensively used for testing novel therapies, elucidating cardiac metabolic and electrophysiological activities, and modeling CVDs, including ischemic heart disease, arrhythmias, and hyperacute rejection. In recent years, ex vivo heart perfusion (EVHP) has shown potential in cardiac transplantation by allowing prolonged preservation and reconditioning of donor hearts. In this review, we summarize the evolution of the isolated perfused heart technique and its applications in cardiovascular research to help researchers comprehensively understand the capabilities of isolated heart models and provide guidance to use them to investigate various CVDs.

Xenotransplantation of porcine hearts has become a promising alternative to human allotransplantation, where organ demand still greatly surpasses organ availability. Before entering the clinic, however, feasibility of cardiac xenotransplantation needs to be proven, ideally in the life supporting orthotopic pig-to-nonhuman primate xenotransplantation model. In this review, we shortly outline the last three decades of research and then discuss in detail its most recent advances. These include the genetic modifications of donor pigs to overcome hyperacute rejection and coagulation dysregulation, new organ preservation methods to prevent perioperative xenograft dysfunction, experimental immunosuppressive and immunomodulatory therapies to inhibit the adaptive immune system and systemic inflammation in the recipient, growth control concepts to avoid detrimental overgrowth of the porcine hearts in nonhuman primates, and lastly, the avoidance of porcine cytomegalovirus infections in donor pigs. With these strategies, consistent survival of 6-9 months was achieved in the orthotopic xenotransplantation model, thereby fulfilling the prerequisites for the initiation of a clinical trial.

The blockade of the CD40/CD40L immune checkpoint is considered essential for cardiac xenotransplantation. However, it is still unclear which single antibody directed against CD40 or CD40L (CD154), or which combination of antibodies, is better at preventing organ rejection. For example, the high doses of antibody administered in previous experiments might not be feasible for the treatment of humans, while thrombotic side effects were described for first-generation anti-CD40L antibodies. To address these issues, we conducted six orthotopic pig-to-baboon cardiac xenotransplantation experiments, combining a chimeric anti-CD40 antibody with an investigational long-acting PASylated anti-CD40L Fab fragment. The combination therapy effectively resulted in animal survival with a rate comparable to a previous study that utilized anti-CD40 monotherapy. Importantly, no incidence of thromboembolic events associated with the administration of the anti-CD40L PAS-Fab was observed. Two experiments failed early because of technical reasons, two were terminated deliberately after 90 days with the baboons in excellent condition and two were extended to 120 and 170 days, respectively. Unexpectedly, and despite the absence of any clinical signs, histopathology revealed fungal infections in all four recipients. This study provides, for the first time, insights into a combination therapy with anti-CD40/anti-CD40L antibodies to block this immune checkpoint.

Transplantation of genetically modified porcine hearts and kidneys could become a solution to the persistent shortage of human organ donors. Progress has been made in genetic engineering of donor pigs, preservation techniques after organ harvesting and immunosuppression using co-stimulation blockade with anti-CD40/CD40L monoclonal antibodies. Progress has also been made in in the development of methods that detect pathogenic porcine viruses and prevent their transmission to the recipient. As normal land breed pig organs continue to grow in the recipient to their original size, different pig breeds (such as Auckland Island pigs) are now used which reach a final size suitable for humans. Alternatively, a knock-out of the growth hormone receptor gene has been established, e.g., in the 10GM genetically modified pigs from Revivicor/United Therapeutics, USA. The first clinical pilot studies including patients suffering from terminal heart failure are expected to start in Germany in about 2 years.

Cardiac xenotransplantation (cXT) has emerged as a solution to heart donor scarcity, prompting an exploration of its scientific, ethical, and regulatory facets. The review begins with genetic modifications enhancing pig hearts for human transplantation, navigating through immunological challenges, rejection mechanisms, and immune responses. Key areas include preclinical milestones, complement cascade roles, and genetic engineering to address hyperacute rejection. Physiological counterbalance systems, like human thrombomodulin and endothelial protein C receptor upregulation in porcine xenografts, highlight efforts for graft survival enhancement. Evaluating pig and baboon donors and challenges with non-human primates illuminates complexities in donor species selection. Ethical considerations, encompassing animal rights, welfare, and zoonotic disease risks, are critically examined in the cXT context. The review delves into immune control mechanisms with aggressive immunosuppression and clustered regularly interspaced palindromic repeats associated protein 9 (CRISPR/Cas9) technology, elucidating hyperacute rejection, complement activation, and antibody-mediated rejection intricacies. CRISPR/Cas9's role in creating pig endothelial cells expressing human inhibitor molecules is explored for rejection mitigation. Ethical and regulatory aspects emphasize the role of committees and international guidelines. A forward-looking perspective envisions precision medical genetics, artificial intelligence, and individualized heart cultivation within pigs as transformative elements in cXT's future is also explored. This comprehensive analysis offers insights for researchers, clinicians, and policymakers, addressing the current state, and future prospects of cXT.

Xenotransplant patient selection recommendations restrict clinical trial participation to seriously ill patients for whom alternative therapies are unavailable or who will likely die while waiting for an allotransplant. Despite a scholarly consensus that this is advisable, we propose to examine this restriction. We offer three lines of criticism: (1) The risk-benefit calculation may well be unfavorable for seriously ill patients and society; (2) the guidelines conflict with criteria for equitable patient selection; and (3) the selection of seriously ill patients may compromise informed consent. We conclude by highlighting how the current guidance reveals a tension between the societal values of justice and beneficence.

Inflammatory responses and coagulation disorders are a relevant challenge for successful cardiac xenotransplantation on its way to the clinic. To cope with this, an effective and clinically practicable anti-inflammatory and anti-coagulatory regimen is needed. The inflammatory and coagulatory response can be reduced by genetic engineering of the organ-source pigs. Furthermore, there are several therapeutic strategies to prevent or reduce inflammatory responses and coagulation disorders following xenotransplantation. However, it is still unclear, which combination of drugs should be used in the clinical setting. To elucidate this, we present data from pig-to-baboon orthotopic cardiac xenotransplantation experiments using a combination of several anti-inflammatory drugs.

Genetically modified piglets (GGTA1-KO, hCD46/hTBM transgenic) were used for orthotopic cardiac xenotransplantation into captive-bred baboons (n = 14). All animals received an anti-inflammatory drug therapy including a C1 esterase inhibitor, an IL-6 receptor antagonist, a TNF-α inhibitor, and an IL-1 receptor antagonist. As an additive medication, acetylsalicylic acid and unfractionated heparin were administered. The immunosuppressive regimen was based on CD40/CD40L co-stimulation blockade. During the experiments, leukocyte counts, levels of C-reactive protein (CRP) as well as systemic cytokine and chemokine levels and coagulation parameters were assessed at multiple timepoints. Four animals were excluded from further data analyses due to porcine cytomegalovirus/porcine roseolovirus (PCMV/PRV) infections (n = 2) or technical failures (n = 2).

Leukocyte counts showed a relevant perioperative decrease, CRP levels an increase. In the postoperative period, leukocyte counts remained consistently within normal ranges, CRP levels showed three further peaks after about 35, 50, and 80 postoperative days. Analyses of cytokines and chemokines revealed different patterns. Some cytokines, like IL-8, increased about 2-fold in the perioperative period, but then decreased to levels comparable to the preoperative values or even lower. Other cytokines, such as IL-12/IL-23, decreased in the perioperative period and stayed at these levels. Besides perioperative decreases, there were no relevant alterations observed in coagulation parameters. In summary, all parameters showed an unremarkable course with regard to inflammatory responses and coagulation disorders following cardiac xenotransplantation and thus showed the effectiveness of our approach.

Our preclinical experience with the anti-inflammatory drug therapy proved that controlling of inflammation and coagulation disorders in xenotransplantation is possible and well-practicable under the condition that transmission of pathogens, especially of PCMV/PRV to the recipient is prevented because PCMV/PRV also induces inflammation and coagulation disorders. Our anti-inflammatory regimen should also be applicable and effective in the clinical setting of cardiac xenotransplantation.

Cardiac xenotransplantation has seen remarkable success in recent years and is emerging as the most promising alternative to human cardiac allotransplantation. Despite these achievements, acute vascular rejection still presents a challenge for long-term xenograft acceptance and new insights into innate and adaptive immune responses as well as detailed characterizations of signaling pathways are necessary. In allotransplantation, endothelial cells and their sugar-rich surface-the endothelial glycocalyx-are known to influence organ rejection. In xenotransplantation, however, only in vitro data exist on the role of the endothelial glycocalyx so far. Thus, in the current study, we analyzed the changes of the endothelial glycocalyx components hyaluronan, heparan sulfate and syndecan-1 after pig-to-baboon cardiac xenotransplantations in the perioperative (n = 4) and postoperative (n = 5) periods. These analyses provide first insights into changes of the endothelial glycocalyx after pig-to-baboon cardiac xenotransplantation and show that damage to the endothelial glycocalyx seems to be comparable or even less pronounced than in similar human settings when current strategies of cardiac xenotransplantation are applied. At the same time, data from the experiments where current strategies, like non-ischemic preservation, growth inhibition or porcine cytomegalovirus (a porcine roseolovirus (PCMV/PRV)) elimination could not be applied indicate that damage of the endothelial glycocalyx also plays an important role in cardiac xenotransplantation.

This report comprises the contents of the presentations and following discussions of a workshop of the German Heart Transplant Centers in Martinsried, Germany on cardiac xenotransplantation. The production and current availability of genetically modified donor pigs, preservation techniques during organ harvesting, and immunosuppressive regimens in the recipient are described. Selection criteria for suitable patients and possible solutions to the problem of overgrowth of the xenotransplant are discussed. Obviously microbiological safety for the recipient and close contacts is essential, and ethical considerations to gain public acceptance for clinical applications are addressed. The first clinical trial will be regulated and supervised by the Paul-Ehrlich-Institute as the National Competent Authority for Germany, and the German Heart Transplant Centers agreed to cooperatively select the first patients for cardiac xenotransplantation.

Xenotransplantation offers the potential to meet the critical need for heart and lung transplantation presently constrained by the current human donor organ supply. Much was learned over the past decades regarding gene editing to prevent the immune activation and inflammation that cause early organ injury, and strategies for maintenance of immunosuppression to promote longer-term xenograft survival. However, many scientific questions remain regarding further requirements for genetic modification of donor organs, appropriate contexts for xenotransplantation research (including nonhuman primates, recently deceased humans, and living human recipients), and risk of xenozoonotic disease transmission. Related ethical questions include the appropriate selection of clinical trial participants, challenges with obtaining informed consent, animal rights and welfare considerations, and cost. Research involving recently deceased humans has also emerged as a potentially novel way to understand how xeno-organs will impact the human body. Clinical xenotransplantation and research involving decedents also raise ethical questions and will require consensus regarding regulatory oversight and protocol review. These considerations and the related opportunities for xenotransplantation research were discussed in a workshop sponsored by the National Heart, Lung, and Blood Institute, and are summarized in this meeting report.

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.

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.

Orthotopic cardiac xenotransplantation has seen notable improvement, leading to the first compassionate use in 2022. However, it remains challenging to define the clinical application of cardiac xenotransplantation, including the back-up strategy in case of xenograft failure. In this regard, the heterotopic thoracic technique could be an alternative to the orthotopic procedure. We present hemodynamic data of heterotopic thoracic pig-to-baboon transplantation experiments, focusing on perioperative xenograft dysfunction and xenograft overgrowth.

We used 17 genetically modified piglets as donors for heterotopic thoracic xenogeneic cardiac transplantation into captive-bred baboons. In all animals, pressure probes were implanted in the graft's left ventricle and the recipient's ascending aorta and hemodynamic data (graft pressure, aortic pressure and recipient's heart rate) were recorded continuously.

Aortic pressures and heart rates of the recipients' hearts were postoperatively stable in all experiments. After reperfusion, three grafts presented with low left ventricular pressure indicating perioperative cardiac dysfunction (PCXD). These animals recovered from PCXD within 48 h under support of the recipient's heart and there was no difference in survival compared to the other 14 ones. After 48 h, graft pressure increased up to 200 mmHg in all 17 animals with two different time-patterns. This led to a progressive gradient between graft and aortic pressure. With increasing gradient, the grafts stopped contributing to cardiac output. Grafts showed a marked weight increase from implantation to explantation.

The heterotopic thoracic cardiac xenotransplantation technique is a possible method to overcome PCXD in early clinical trials and an experimental tool to get a better understanding of PCXD. The peculiar hemodynamic situation of increasing graft pressure but missing graft's output indicates outflow tract obstruction due to cardiac overgrowth. The heterotopic thoracic technique should be successful when using current strategies of immunosuppression, organ preservation and donor pigs with smaller body and organ size.

Stem cell factor (SCF), a cytokine growth factor, is expressed in various tissues of the male and female reproductive organs, including the testis, ovary, and endometrium. Its primary function involves cell survival, differentiation, and proliferation, achieved through its binding to the c-kit receptor. This study aimed to scrutinize the effects of SCF treatment during in vitro culture (IVC) on both the developmental potential and the efficiency of establishing embryonic stem cells (ESCs) from fertilized and cloned porcine embryos. The rates of cleavage and blastocyst formation exhibited no significant differences between fertilized and cloned embryos, even with the addition of SCF. However, it's worth noting that embryos cloned with Cloud eGFP as donor cells demonstrated notably increased rates of hatched blastocysts when treated with SCF, and this increase was statistically significant (p < 0.05). Furthermore, following the complete dissection of the blastocysts, although there was no significant difference in the SCF-treated group, the area of expansion was significantly reduced (p < 0.01) in the group treated with the antagonistic blocker (ACK2) compared to both the control and SCF-treated groups. These outcomes suggest that the SCF/c-kit signaling pathway might play a pivotal role in embryo implantation. As anticipated, the efficiency of deriving ESCs was significantly higher (p < 0.01) in the group subjected to SCF treatment (12.82 ± 1.02%) compared to the control group (5.41 ± 2.25%). In conclusion, this study highlights the crucial role of SCF in enhancing the quality of porcine embryos, a vital step in obtaining high-quality ESCs.

Using pigs as organ donors has advanced xenotransplantation to the point that it is almost ready for clinical use. However, there is still a zoonotic risk associated with xenotransplantation, and the potential transmission of porcine endogenous retroviruses needs to be surveyed. Despite significant attempts to eliminate this risk, by the selection of PERV-C free pigs with low expression of PERV-A, -B, and by the genome-wide inactivation of PERV using CRISPR/Cas9, the impact of superinfection resistance (SIR) was not investigated. SIR is a viral trait that prevents reinfection (superinfection). For PERV, the underlying mechanism is unclear, whether and how cells, that harbor functional PERV, are protected. Using PERV-C(5683) as a reference virus, we investigated SIR in a newly developed in vitro model to pursue the mechanism and confirm its protective effect.

We developed three PERV-C constructs on the basis of PERV-C(5683), each of which carries a hemagglutinin tag (HA-tag) at a different position of the envelope gene (SP-HA, HA-VRA, and RPep-HA), to distinguish between primary infection and superinfection. The newly generated PERV-C(5683)-HA viruses were characterized while quantifying the viral RNA, reverse transcriptase activity, protein expression analysis, and infection studies. It was demonstrated that SP-HA and RPep-HA were comparable to PERV-C(5683), whereas HA-VRA was not replication competent. SP-HA and RPep-HA were chosen to challenge PERV-C(5683)-positive ST-IOWA cells demonstrating that PERV-C-HA viruses are not able to superinfect those cells. They do not integrate into the genome and are not expressed.

The mechanism of SIR applies to PERV-C. The production of PERV-C particles serves as a defense mechanism from superinfection with exogenous PERV-C. It was demonstrated by newly generated PERV-C(5683)-HA clones that might be used as a cutting-edge tool. The HA-tagging of PERV-C is novel, providing a blueprint for the tagging of other human tropic PERV viruses. The tagged viruses are suitable for additional in vitro and in vivo infection studies and will contribute, to basic research on viral invasion and pathogenesis. It will maintain the virus safety of XTx.

Hemocompatibility tuning was adopted to explore and refine an innovative, GA-free preparation strategy combining decellularization, riboflavin/UV crosslinking, and low-energy electron irradiation (SULEEI) procedure. A SULEEI-protocol was established to avoid GA-dependent deterioration that results in insufficient long-term aortic valve bioprosthesis durability. Final SULEEI-pericardium, intermediate steps and GA-fixed reference pericardium were exposed in vitro to fresh human whole blood to elucidate effects of preparation parameters on coagulation and inflammation activation and tissue histology. The riboflavin/UV crosslinking step showed to be less efficient in inactivating extracellular matrix (ECM) protein activity than the GA fixation, leading to tissue-factor mediated blood clotting. Intensifying the riboflavin/UV crosslinking with elevated riboflavin concentration and dextran caused an enhanced activation of the complement system. Yet activation processes induced by the previous protocol steps were quenched with the final electron beam treatment step. An optimized SULEEI protocol was developed using an intense and extended, trypsin-containing decellularization step to inactivate tissue factor and a dextran-free, low riboflavin, high UV crosslinking step. The innovative and improved GA-free SULEEI-preparation protocol results in low coagulant and low inflammatory bovine pericardium for surgical application.