Xenotransplantation has been iteratively improved over the last decade in pre-clinical models, with first-in-human clinical trials underway. The 2025 IXA Congress in Geneva was held in parallel with meetings involving World Health Organization leaders to support the development of new guidance on xenotransplantation in this rapidly evolving field. Key scientific themes of the meeting included the introduction of multiple-gene edited pigs for xenotransplantation research and clinical trials, better characterization of innate and adaptive immune responses and xenograft preservation that optimizes ramifications of ischemia-reperfusion injury during implantation. This comes at a time when gene-edited pig organs are being used for human xenotransplantation, a development that demands safety, reproducibility, durability, and a clear mechanistic understanding of rejection and tolerance.

A 4-h preservation time threshold for cardiac allografts is the current standard in heart transplantation, but novel technologies are proposed to decrease the morbidity associated with prolonged allograft storage. This study examined adult heart transplant recipients from 2000-2015 and 2020-2023 in the United States, stratified into an early (2000-2015) and modern era (2020-2023), then into standard (≤ 4 h) and prolonged (≥ 5 h) preservation time groups within each era. This study reinforced the 4-h threshold in the early era, where prolonged preservation significantly increased one-year mortality (HR 1.60, 95% CI 1.36-1.90). However, this association was no longer significant in the modern era (HR 1.14, 95% CI 0.85-1.50). A sub-analysis showed that using machine perfusion devices for allograft storage was not associated with one-year mortality (HR 1.15, 95% CI 0.79-1.70). Spline analysis demonstrated possible inflection points between 4 and 5 h and 8-9 h in the modern era, and further analysis found that 5-8 h of preservation did not increase one-year mortality (HR 1.09, 95% CI 0.80-1.47) relative to the ≤ 4 h group. In conclusion, the association between cardiac allograft preservation duration and morbidity has decreased in the modern era. Today, a 4-h preservation duration threshold may be too restrictive.

OBJECTIVE:The safety of using teenage donor hearts for adult recipients is unclear. Moreover, the appropriateness of predicted heart mass ratio as a size metric for teenage donors is unknown. METHODS:We used the United Network for Organ Sharing database to perform a retrospective analysis of heart-only adult transplant recipients (>18 years). Donors were stratified as teenage (age 12-17 years, N = 5772) or adult donors (age ≥18 years, N = 57,922). Predicted heart mass ratio was computed using age, sex, height, and weight. Predicted heart mass ratio was categorized into septiles, after excluding outliers 2 Z-scores above or below the mean. RESULTS:The mean ages of teenage and adult donors were 16 ± 1.4 years and 34 ± 11 years, respectively (P < .001). Average predicted heart mass ratio was higher for recipients of teenage (1.29 ± 0.15) compared with adult (1.12 ± 0.14) donors (P < .001). Cox proportional hazards model adjusting for confounders showed increased survival among recipients of teenage donor hearts compared with those with adult donors at 1 year (hazard ratio, 0.96, 95% CI, 0.75-0.99) and 5 years (hazard ratio, 0.84, 95% CI, 0.76-0.93). Adjusted analysis within the teenage cohort found that predicted heart mass ratio septiles were not associated with 1- and 5-year survivals. CONCLUSIONS:Teenage donor hearts appear to be safe for transplantation in adult recipients. However, teenage donor hearts used for transplantation tend to be oversized with limited data available on undersized teenage donor hearts. Further assessment of the best sizing metrics is needed.

Improvement in gene modifications of donor pigs has led to the prevention of early cardiac xenograft rejection and significantly prolonged cardiac xenograft survival in both heterotopic and orthotopic preclinical non-human primate (NHP) models. This progress formed the basis for FDA approval for compassionate use transplants in two patients. Based on our earlier report of 9-month survival of seven gene-edited (7-GE) hearts transplanted (life-supporting orthotopic) in baboons, we transplanted 10 gene-edited pig hearts into baboons (n = 4) using non-ischemic continuous perfusion preservation (NICP) and immunosuppression regimen based on co-stimulation blockade by anti-CD40 monoclonal antibody. This pivotal study expands on the 7-GE backbone, with 3 additional gene edits, using 10-GE pigs as donors to baboon recipients. 10 GE cardiac xenografts provide life-supporting function up to 225 days (mean 128 ± 36 days) in a non-human primate model. Undetectable or latent porcine cytomegalovirus (PCMV) does not influence cardiac xenograft survival in this study but still needs more exploration with a larger cohort. Xenograft histology demonstrates adipose (Fat) deposition (n = 1), chronic vasculopathy (n = 1), micro and macro thrombosis, and acute cellular rejection (n = 1). These data demonstrate that 10 GE cardiac xenografts have variable cardiac xenograft survival in NHP due to perhaps presence of 4th antigen and require further study. However, these 10GE organs may be suitable for clinical cardiac xenotransplantation and have already been utilized in two human cases. There is a shortage of organs donated for use in transplantation. Instead, animal organs could potentially be used for people with end-stage organ failure. We modified pig hearts to make them more like human organs and transplanted them into non-human primates. The pig hearts functioned in the non-human primates for up to 225 days. These hearts could also potentially be used in people with heart failure. Singh, Goerlich et al. transplant 10 gene modified pig hearts into non-human primates. Life-supporting function occurred for up to 225 days but there was evidence of adipose deposition, chronic vasculopathy, micro and macro thrombosis, and acute cellular rejection.

Background. Efforts to expand the heart donor pool have included the use of extended criteria donors (e.g., advanced age or comorbid conditions). Another potential avenue is donor hearts with pre‐existing coronary artery disease (CAD). Methods. A retrospective cohort study was performed using the Organ Procurement and Transplantation Network database between 10/1/1987 and 12/3/2020. Postoperative complications, graft failure, and survival were examined between recipients of donor hearts with abnormal (CAD donor) versus normal (non‐CAD donor) coronary angiogram. Analysis was performed in unmatched cohorts as well as after propensity score matching. Results. A total of 12,230 heart transplant recipients were identified, of which 940 (7.6%) had an abnormal donor coronary angiogram. There were no differences between CAD and non‐CAD donor groups in acute rejection, stroke, or dialysis prior to discharge or treatment for rejection <1 year. However, the CAD donor group had a greater pacemaker incidence (5.2% vs. 3.6%, P = 0.02). After matching, 785 patients were in each group and there were no differences in perioperative outcomes or treatment for rejection <1 year. There was no difference in 10‐year freedom from graft failure (53.1% vs. 54.6%, log rank = 1.03, and P = 0.31) or cumulative survival (54.7% vs. 55.8%, log rank = 0.63, and P = 0.43) between the matched CAD and non‐CAD donor groups. Conclusion. The presence of CAD may not be a contraindication to transplantation. Carefully selected donor hearts with CAD may have equivalent postoperative and long‐term outcomes to donor hearts without CAD. Further study in this area may expand the pool of donors.

The 2023 IXA conference, hosted in San Diego, CA, brimmed with excitement against the backdrop of recent innovations in both the pre-clinical and clinical realms with several first-in-human applications of xenotransplantation. The theme, "Pigs are flying," alluded to the adage that xenotransplantation would only become a clinical reality "when pigs fly," suggesting a day that might never come. The event witnessed significant attendance, with 600 participants-the highest in the history of an IXA-IPITA joint congress. Among the attendees were members of the Food and Drug Administration (FDA), the National Institutes of Health (NIH), and corporate sponsors deeply engaged in the field. We summarize the latest topics from the congress, ranging from the pros/cons of decedent models of xenotransplantation and genetic engineering of porcine heart valves, solid organs, and cells for clinical translation and their regulatory and ethical landscape.

Objective: Genetically engineered pigs are thought to be an alternative organ source for patients in end-stage heart failure unable to receive a timely allograft. However, cardiac xenografts exhibit growth and diastolic heart failure within 1 month after transplantation. Grafts function for up to 6 months, but only after administration of temsirolimus and afterload-reducing agents to reduce this growth. In this study we investigated the growth and hemodynamics of growth hor-mone receptor (GHR) knockout xenografts, without the use of adjuncts to prevent intrinsic graft growth after transplantation.Methods: Genetically engineered pig hearts were transplanted orthotopically into weight-matched baboons between 15 and 30 kg, using continuous perfusion pres-ervation before implantation (n = 5). Xenografts included knockout of carbohy-drate antigens and knockin of human transgenes for thromboregulation, complement regulation, and inflammation reduction (grafts with intact growth hor-mone, n = 2). Three grafts contained the additional knockout of GHR (GHR knockout grafts; n = 3). Transthoracic echocardiograms were obtained twice monthly and comprehensively analyzed by a blinded cardiologist. Hemodynamics were measured longitudinally after transplantation.Results: All xenografts demonstrated life-supporting function after transplantation. There was no difference in intrinsic growth, measured using septal and posterior wall thickness and left ventricular mass, on transthoracic echocardiogram out to 1 month in either GHR knockout or GHR intact grafts. However, hypertrophy of the septal and posterior wall was markedly elevated by 2 months post transplanta-tion. There was minimal hypertrophy out to 6 months in GHR knockout grafts. Phys-iologic mismatch was present in all grafts after transplantation, which is largely independent of growth.Conclusions: Xenografts with GHR knockout show reduced post-transplantation xenograft growth using echocardiography >6 months after transplantation, without the need for other adjuncts. (J Thorac Cardiovasc Surg 2023;165:e69-81)

Veno-arterial extracorporeal membrane oxygenation (VA-ECMO) for end-stage heart failure is a powerful tool to reverse acute shock and bridge a patient either to recovery, durable ventricular assist device (VAD), transplantation, or to extend a time for ongoing reevaluation of a patient’s clinical status. Patient selection is paramount. Patients without a reasonable chance for recovery or who are otherwise not candidates for advanced surgical therapy can have VA-ECMO initiation as a bridge to nowhere without an exit strategy. In this chapter, careful consideration is given to patients with heart failure requiring VA-ECMO in terms of patient selection, indications, management and auxiliary mechanical circulatory support (MCS) devices while on VA-ECMO.

Background: Our institution performed the first genetically modified porcine cardiac xenotransplantation in a human. Due to limited data on monitoring xenografts, our laboratory conducted longitudinal noninvasive imaging for surveillance and clinical evaluation. Methods: We performed weekly transthoracic echocardiograms (TTE) with global longitudinal strain (GLS) over 2 months with endomyocardial biopsy (EMBx) correlation. Results: LV ejection fraction (LVEF) remained normal or hyperdynamic throughout the patient’s clinical course. However, myocardial thickness and GLS varied post-transplant. Initial EMBx, LVEF, and GLS were normal. Despite little change in LVEF, the patient experienced a persistent deterioration in hemodynamic status which correlated with a dramatic increase in LV myocardial thickness (1.1 to 1.6 cm) with echogenicity and change in GLS (-18.2 to -11.5%) (Figure 1). EMBx revealed diffuse capillary damage with extravasated erythrocytes and edema without evidence of antibody or acute cellular rejection. The abrupt increase in myocardial thickness and more positive GLS suggested restriction from myocardial edema and capillary damage as a possible mechanism for xenograft failure. Conclusions: Echocardiography provided real-time clinical and prognostic value in the first transgenic cardiac xenotransplant. Given the tremendous interest and hope for cardiac xenotransplantation, investigation with longitudinal TTEs with GLS will be important for future management.

Introduction: Our medical center performed the first genetically modified cardiac xenotransplant in human and performed longitudinal invasive and noninvasive hemodynamic measurements throughout the recipient’s hospitalization. We hypothesized that echocardiographic derived hemodynamic assessments would correlate with invasive measurements within the cardiac xenotransplant. Methods: Transthoracic echocardiography (TTE) and right heart catheterizations were performed in the cardiac xenotransplant. Retrospective analysis of TTE and invasive hemodynamics were analyzed to assess correlation between various parameters. The following invasive hemodynamics were measured or calculated: right atrial pressure (RAP), pulmonary artery systolic and diastolic pressures (PASP, PADP), pulmonary vascular resistance (PVR), pulmonary artery pulsatility index (PAPi), cardiac output (CO), cardiac index (CI), right ventricular stroke work index (RVSWI) and cardiac power output (CPO). TTE measurements and calculations included: RAP based on inferior vena cava size and collapsibility, PASP calculated via tricuspid regurgitation velocity (TRV), PADP based on end diastolic pressure gradient, right ventricular outflow time-velocity integral (TVI RVOT), PVR (TRV/TVI RVOT x 10 +0.16) PAPi, CO (using left ventricular outflow tract (LVOT) velocity-time integral and LVOT area), CI (from TTE-derived CO), CPO and RVCPI {4x(TRV) 2 } x TAPSE as a surrogate of RVSWI. Results: Pearson correlation for RVSWI and RVCPI using invasive measurements obtained at the time of TTE was -0.095 and at time of mixed venous blood gas (mVBG) was 0.963. Mean absolute difference in time between mVBG and TTE was 4h 59 min. Pearson correlation for catheter-based continuous CO (CCO) monitoring with TTE was 0.965. Conclusions: In a retrospective analysis of porcine cardiac xenotransplant function, there was a strong correlation between directly measured hemodynamics with TTE, particularly when mVBG was obtained.

Extracorporeal membrane oxygenation (ECMO) provides innumerous benefits in the preoperative and postoperative/critical care phases of patient care. Typically, ECMO is used in emergency situations after standard medical practices have failed, or a life-threatening complication during the periprocedural time of a percutaneous intervention. This inherent morbidity and mortality with utilization of ECMO is from vascular and neurologic complications. However, recent evidence suggests the use of prophylactic ECMO can reduce morbidity and mortality associated with high-risk patients undergoing challenging percutaneous cardiac interventions. Prophylactic ECMO has been utilized for several years in challenging percutaneous coronary interventions (PCIs) with tremendous success. In addition to PCI, prophylactic ECMO has been utilized in both transcatheter aortic valve implantation, ventricular ablations, and percutaneous mitral valve repair. While ECMO will continue to remain a bailout strategy for disastrous intraoperative complications, careful preoperative planning and patient selection allow prophylactic ECMO to be utilized with tremendous percutaneous operative success.

Introduction: Humans have natural antibodies against some of the pig antigens (e.g., 1,3 Galactosyltranserase (GT), beta-1,4-N-Acetyl-Galactosaminyltransferase 2 (b-Gal), and N-Glycolylneuraminic acid (Neu5Gc)) which may trigger hyperacute rejection. A heart from a genetically engineered (GE) pig that lacks GTKO, b-GalKO, and cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAHKO) along with overexpression of human gene (CD46, DAF, TBM, EPCR, CD47, and HO-1) was transplanted in a human patient with heart failure after expanded access authorization from FDA. We report the recipient’s anti-pig antibody levels at various time points after cardiac xenotransplantation (xTX). Methods: Serum from the recipient was collected before and after cardiac xTX at multiple time points and tested for anti-pig non-gal antibodies (IgG and IgM) using donor-specific porcine aortic endothelial cells (PAECs) from 10 GE pigs, TKO and GTKO by flow cytometry. Antibodies binding with PAECs for IgG and IgM were determined by calculating normalized mean fluorescence intensity (MFI, i.e., Geometric mean) with reference to positive control. Results: The first GE pig heart-to-human transplant recipient survived for 60 days. Anti-pig non-gal antibodies were dropped after xTX and remained low until 47 days, but IgG antibodies sharply increased. The rise in anti-non-gal antibodies coincided with the increase in troponin release. This increase in IgG antibodies also occurred after intravenous IgG (IVIG) administration. Later, IVIG was also tested for binding with PAECs which was also found to have high binding to donor PAECs. Conclusion: Our study demonstrates that human recipients had a very low titer of non-gal or anti-pig IgG and IgM antibodies during the first 47 days when the graft function was excellent. The antibody levels increased after 47 days coinciding with xenograft dysfunction. Early detection of these antibodies is a powerful tool and may allow a window for proper interventions to prevent antibody-mediated rejection.

Porcine cytomegalovirus (PCMV) infection is an important concern in xenotransplantation (xTx). It has been shown that PCMV can induce endothelial cell activation, leading to a pro-coagulant state, which can cause consumptive coagulopathy and thrombotic microangiopathy in xenograft recipients. In this study, life-supporting cardiac xenotransplantation in non-human primates (NHPs, Baboons) was performed without knowing PCMV status from genetically engineered (GE) donor pigs with 7 (n=2), 9 (n=2), and 10 (n=8) gene modifications. All the recipients received anti-CD40 co-stimulation blockade-based immunosuppression. The cardiac xenograft survival in NHPs ranged from days 14 to 264. Retrospectively, GE donor pigs were tested for PCMV by sensitive PCR. Six (50%) of twelve donor pigs were positive for latent PCMV. No statistically significant difference was found in xenograft or baboon survival among PCMV-positive and negative groups. Two of six PCMV-positive cardiac xenografts were rejected and demonstrated acute cellular rejection. One of them had excessive adipose (Fat) deposition and micro and macro thrombosis, and the longest survival of the baboon was 225 days. Whereas three of six PCMV-negative cardiac xenografts were rejected and had chronic vasculopathy, the baboon’s longest survival was 264 days. These results suggest that the presence or absence of PCMV in the xenograft does not affect their survival.

Background A genetically engineered pig cardiac xenotransplantation was done on Jan 7, 2022, in a non-ambulatory male patient, aged 57 years, with end-stage heart failure, and on veno-arterial extracorporeal membrane oxygenation support, who was ineligible for an allograft. This report details our current understanding of factors important to the xenotransplantation outcome. Methods Physiological and biochemical parameters critical for the care of all heart transplant recipients were collected in extensive clinical monitoring in an intensive care unit. To ascertain the cause of xenograft dysfunction, we did extensive immunological and histopathological studies, including electron microscopy and quantification of porcine cytomegalovirus or porcine roseolovirus (PCMV/PRV) in the xenograft, recipient cells, and tissue by DNA PCR and RNA transcription. We performed intravenous immunoglobulin (IVIG) binding to donor cells and single-cell RNA sequencing of peripheral blood mononuclear cells. Findings After successful xenotransplantation, the graft functioned well on echocardiography and sustained cardiovascular and other organ systems functions until postoperative day 47 when diastolic heart failure occurred. At postoperative day 50, the endomyocardial biopsy revealed damaged capillaries with interstitial oedema, red cell extravasation, rare thrombotic microangiopathy, and complement deposition. Increased anti-pig xenoantibodies, mainly IgG, were detected after IVIG administration for hypogammaglobulinaemia and during the first plasma exchange. Endomyocardial biopsy on postoperative day 56 showed fibrotic changes consistent with progressive myocardial stiffness. Microbial cell-free DNA testing indicated increasing titres of PCMV/PRV cell-free DNA. Post-mortem single-cell RNA sequencing showed overlapping causes. Interpretation Hyperacute rejection was avoided. We identified potential mediators of the observed endothelial injury. First, widespread endothelial injury indicates antibody-mediated rejection. Second, IVIG bound strongly to donor endothelium, possibly causing immune activation. Finally, reactivation and replication of latent PCMV/PRV in the xenograft possibly initiated a damaging inflammatory response. The findings point to specific measures to improve xenotransplant outcomes in the future. Funding The University of Maryland School of Medicine, and the University of Maryland Medical Center.

Introduction: Our center performed the first cardiac xenotransplant of a genetically modified porcine heart into a human. We sought to identify whether left ventricular global longitudinal strain (LV-GLS) correlates with invasive hemodynamics in this novel clinical scenario. Methods: Transthoracic echo (TTE) with and without contrast and right heart catheterization were performed after cardiac xenotransplantation. Retrospective analyses of TTE and hemodynamic data were performed to assess correlation between various parameters. The following invasive hemodynamic parameters were obtained: mean pulmonary artery pressure (mPAP) and thermodilution cardiac index (TDCI). Standard Doppler indices and LV-GLS with and without contrast was measured. A strain based index, Mitral E velocity/LV-GLS and Mitral E velocity/contrast enhanced (CE) LV-GLS was calculated. Results: There was a moderate positive correlation of LV-GLS and CE LV-GLS with mPAP and moderate negative correlation of LV-GLS with TDCI. There were weaker positive correlations with E/LV-GLS and E/CELV- GLS with mPAP and a weaker negative correlation of E/LV-GLS with TDCI. See Table. Conclusion: Our data shows that LV-GLS correlates with invasive hemodynamics in a porcine to human xenotransplant. It highlights the limitations of Doppler derived indices due to impact of donor and recipient atrial mechanics, loading conditions and LV compliance. LV-GLS complements invasive hemodynamics in the management of a cardiac xenotransplant.

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

Introduction: The first genetically engineered (GE) pig-to-human cardiac xenotransplantation (xTX) was performed after expanded access authorization from FDA at our institution. We report the immune monitoring status in the recipient after cardiac xTX. Methods: Peripheral blood mononuclear cells (PBMCs) of the recipient were immunostained for T and B cells and analyzed with flow cytometry. Intracellular cytokine-secreting cells and serum cytokine were analyzed. Anti-pig non-gal antibodies (IgG and IgM) were tested using donor-specific porcine aortic endothelial cells (PAECs) from the 10 GE pigs. Results: The 10 GE pig heart recipients received modified immunosuppression (IS) which was significantly less than previously reported successful IS for cardiac xTX in non-human primates due to pancytopenia. Induction included ATG (after xTX), Rituxan (one dose only), C1 esterase, and costimulation blockade with humanized anti-CD40 (KPL404) antibody. KPL404, MMF, and an anti-viral drug were used for maintenance. The recipient survived for 60 days. Initially, peripheral T and B cells were depleted. T cells repopulated 10 days after Tx, but B cells remained low throughout and re-emerged after POD 47. The ratio of CD4/CD8 began increasing around day 19, peaked at day 21, and then started to decline before increasing again around POD 50. There was no significant difference in peripheral intracellular cytokine-secreting cells (IFN-g, TNFa, and IL-17) after xTX compared to pre-xTX levels. Anti-pig antibody levels in the patient’s serum dropped with induction therapy and remained low until POD 47. However, a sharp increase of IgG and IgM was observed to a lesser extent, coinciding with the increase in serum troponin and xenograft failure. Conclusion: Our study demonstrates that immune monitoring accurately reflects the immune response status of the recipient after cardiac xTX and continuous monitoring will aid in the early detection of rejection even and allow effective intervention.