Introduction

Clinical cardiac xenotransplantation could provide a solution for end-stage heart failure patients waiting for heart transplantation who die waiting for a suitable donor or do not otherwise qualify. Two genetically engineered (GE) pig hearts, with ten gene edits (10GE), were recently transplanted into two human recipients. One survived 60 days; the other recipient survived forty1,2,3. This is a great promise to alleviate the shortage of donor organs for transplantation. Earlier, we reported the 9-month survival of a multi-gene pig heart with 7-GE in a non-human primate ((NHP) using co-stimulation blockade-(anti-CD40 antibody) based immunosuppressive (IS) (Mohiuddin Regimen)4. 7GE includes the deletion of two carbohydrate antigens (i.e., GGTA1 and β4GalNT2) along with the expression of five human genes (i.e., TBM, EPCR CD46, and CD47). In addition, three additional modifications, including the deletion of growth hormone receptors and overexpression of DAF and HO1, were made in 10GE pig hearts. Längin et al. have also reported successful life-supporting porcine cardiac xenotransplantation (XTx) in NHP5. A combination of gene modifications of donor pigs and non-ischemic cardiac preservation (NICP) improves cardiac xenograft survival significantly and prevents xenograft rejection.

The immunobiology of cardiac xenotransplantation and justification for using each gene edit have been comprehensively reviewed elsewhere but are briefly summarized below6,7.

Xenograft rejection occurs primarily due to endothelial cell activation by preformed antibodies against porcine donor antigens in the recipient8,9. Preformed and elicited antibodies also promote coagulation and activate platelets, contributing to graft loss due to thrombotic microangiopathy10. Three carbohydrate antigens (i.e., α1,3-galactose (Gal), B4GALNT2KO, and CMAHKO) required knockout based on the evidence of extensive research over the past 30 years11,12.

Studies have suggested a complex interplay between blood pressure, growth hormone receptor, and cardiac xenograft growth (or hypertrophy)13,14,15. To control the intrinsic growth of pigs and their organs (in anticipation of implantation into a non-growing chest of an adult recipient), the growth hormone receptor gene (GHR) has been knocked out4. Mini pigs, which naturally have a slow growth rate, have also been suggested for xenotransplantation16. We used the option of GHR knockout to improve our ability to size-match the donor hearts with the recipient organ4. However, in prior studies, we have shown that the cause of the growth of porcine organs after transplantation is likely multifactorial15.

Complement can activate the clotting cascade by binding complement-fixing antibodies onto endothelium. Human complement regulatory proteins like CD46, decay-accelerating factor (DAF), and CD59 have been used to prevent complement activation, with synergistic effects in combination4. In this study, we used two human complement regulatory genes (i.e., hDAF and hCD46) expression of the xenograft along a C1 Esterase inhibitor to suppress complement activation. C1 Esterase inhibitors, in contrast to the cobra venom factor used in prior studies, are already US Food and Drug Administration-approved and more readily translatable to the clinic.

Incompatibilities between primate and pig coagulation and anticoagulation factors can alter their function, contributing to coagulation dysfunction. Notable proteins include tissue factor pathway inhibitor (TFPI), thrombin, thrombomodulin (TBM), endothelial protein C receptor (EPCR), and CD39. Systemic proinflammatory cytokines (IL-6, TNF-a, etc.) upregulate or recruit recipient tissue factors (TF) on platelets and monocytes by interacting with porcine vascular endothelial cells, which can lead to coagulation through thrombin production. Therefore, donor 10-GE pigs used overexpression of two coagulation regulatory proteins (TBM and EPCR).

Several gene modifications have been incorporated to control inflammation, including CD47, which provides a “don’t eat me” signal to macrophages and anti-apoptotic genes like heme oxygenase 1 (HO1) and A207. Our 10 GE pigs expressed hCD47 and hHO1 in this study.

This study demonstrates the successful transplantation of 10-GE pig hearts with carbohydrate antigen knockout and overexpression of human genes to avoid rejection in non-human primates. These 10 GE pigs’ hearts are transplanted into humans without immediate rejections and we believe they will be suitable for providing donor organs and alleviate the donor shortage for end-stage organ failure patients who may not get human organs.

Methods

Animals

Specific pathogen-free Papio anubis baboons (Southwest National Primate Center, San Antonio, TX or MD Anderson Cancer Center, Bastrop, TX) both male and or female weighing 15–30 kg were selected based on the absence of specific pathogens of interest and used as recipients. Weight-matched GE German Landrace pigs (Revivicor Inc., Blacksburg, VA) either male or female were used as xenograft donors. Preoperative transthoracic echocardiograms (TTEs) were conducted to ensure adequate cardiac function and free of anatomic abnormalities. All animals were used in compliance with guidelines provided by the Institutional Animal Care and Use Committee (IACUC) at the University of Maryland School of Medicine. The husbandry facility was certified by both the Association for Assessment and Accreditation of Laboratory Animal Care (AALAC) and the United States Department of Agriculture (USDA) and is frequently inspected by these agencies. The social and nutritional enrichment for these animals was provided as per institutional behaviorist recommendations.

Multi-gene modifications and health status of donor pig

The pig heart used as cardiac xenograft was produced by somatic cell nuclear transfer as previously described1,4,17, which was clonally derived from fibroblasts that included ten gene edits. Gene edits included the deletion of 4 genes (i.e., a galactose-alpha-1,3-galactose (GGTA1), SDa blood group antigen (B4GALNT2), and N-glycolylneuraminic acid (CMAH) and growth hormone receptor (GHR)) and overexpression of six human genes (hCD46, hDAF, hTBM, hEPCR, CD47 HO-1). Deletion and overexpression of multi-gene were confirmed using flow cytometry western blot analysis, described previously1,18. All animals were housed in a HEPA filter-equipped clean facility and were used in compliance with guidelines provided by the Institutional Animal Care and Use Committee (IACUC) at Revivicor, Inc.

Immunosuppression (IS)

Previously reported costimulation-based IS was used4. IS regimen for all recipient baboons included induction which included Anti CD20 antibody, ATG, Berinert (C1 Esterase Inhibitor) primatized anti-CD40 (2C10R), and maintenance therapy (Mycophenolate (MMF) and primatized Anti-CD40 (2C10R), which has been summarized in Supplementary Table 1 (ST1)4,15.

Procurement of the donor heart and non-ischemic cardiac preservation (NICP)

Donor pig hearts were used from 8- to 12-week-old 10-GE GE pigs. Heart procurement was performed using blood cardioplegia with XVIVO© heart solution (XHS) from XVIVO© Perfusion, Gothenburg, Sweden19. A perfusion pressure of 20 mmHg and a physiological pH range of 7.2–7.6 was maintained in the aortic root. 10-GE pig hearts were NICP in the XVIVO© Perfusion system for at least 90 min.

Orthotopic transplantation and postoperative care

10-GE pig heart was transplanted into to baboon recipient (n = 4) in an orthotopic position using a modified Shumway procedure of atrial anastomosis technique19. Transplant without a 10-GE pig heart was not performed, however, pig hearts with different genetic modifications (3, 7, and 9-GE) were published before by us4 and others5. This is a non-human primate study and a large number of experiments could not be performed due to the scarcity of GE pigs and baboons. Therefore prior sample size calculations were not performed.

A cardiac telemetry device (Data Sciences International (DSI) St. Paul, MN) was used to monitor hemodynamics, heart rhythm, and temperature postoperatively. Baboons were extubated after hemodynamic stability was achieved on minimal ionotropic support, and adequate cardiac function was demonstrated by transesophageal echocardiography (TEE). Drug administration, ionotropic support, and other supplemental drugs (if needed) were administered through a tunneled central line tethered through the recipient’s cage. Critical care nurses and physicians provided ICU-level monitoring and management for the first 48–72 h postoperatively. As soon as deemed surgically appropriate, all baboons were placed on systemic heparin for an ACT goal of twice the baseline level. Recipient blood pressure and heart rate thresholds were not used to guide the use of any afterload reduction or chronotropic controlling agents.

Methods for evaluating xenograft function

A wireless continuous telemetry device (DSI, St. Paul, MN USA) with an implant was used to monitor the 10GE pig heart’s systolic and diastolic pressures, electrocardiogram (ECG), and the recipient’s temperature. Telemetry data from the implant was transmitted to a receiver attached and data was analyzed using Ponemah® Software (DSI, St. Paul, MN, USA)4,20.

Ultrasonography of the cardiac xenograft

TTEs were obtained on a GE Vivid I (GE Healthcare Chicago, IL) portable ultrasound machine whenever the recipient was sedated or as needed to investigate clinical status change. Color and pulse-wave Doppler were obtained across all visible native valves according to standard methods. Atrial mass was not quantified as a bi-atrial approach was utilized for transplantation. The ejection fraction was estimated visually, given the limitation in obtaining apical two-chamber views, as noted above, due to the rotation of the xenograft, preventing biplane measurements.

The American Society of Echocardiography (ASE) guidelines were used to measure the left ventricular (LV) volumetric measurements As per ASE, LV mass was calculated by using the formulas (0.8 × 1.04 [(IVS + LVID + PWT)3 – LVID3] + 0.6.16)15. IVS refers to interventricular septum thickness. LVID and PWT is left ventricular internal diameter and posterior wall thickness, respectively. Due to the nature of this calculation, we acknowledge the limitations of the calculated LV mass measurements, as even small differences in the data can lead to considerable variations in LV mass. As a result, we conducted additional calculations to account for these discrepancies.

Laboratory parameters: hematology and chemistry

Hematology, chemistry, and coagulation parameters were analyzed on peripheral blood and serum of the baboon recipients weekly for the first two months and then biweekly until the 10GE pig heart was explanted4,15.

Immune monitoring assays

Measurements of Non-Gal IgG, IgM serum antibodies, and CDC were performed as previously described to determine the antibody binding and activity with donor-specific cultured PAEC retrospectively4,21,22. Immune phenotyping for T and B cells was done by immunostaining using flow cytometry techniques. Different cell subtypes of T and B cell populations were identified using fluorochrome-labeled anti-human CD45 (Cat#563530; BD Biosciences) CD3 (Cat#552852; BD Biosciences), and CD20 (Cat#302322; Biolegend). Serum cytokine levels were measured with a Biolegend plex kit (Cat#740389; Biolegend) using flow cytometry.

Histological evaluation of biopsies and explants of xenografts

For light microscopy, paraffin sections of explanted cardiac xenografts and endomyocardial biopsies (EMBs) were stained with hematoxylin and eosin (H&E). Sections were analyzed semi-quantitatively for hemorrhage, necrosis, thrombosis, and cellular infiltrates. Immunostaining for immunoglobulins (IgG (Cat# 760-2653), IgM (Cat# 760-2654) and complement (C3d (Cat#760-4522) and C4d (Cat# 760-4436) (Roche Tissue Diagnostics Indianapolis, IN) on paraffin section were performed. An independent pathologist examined all the specimens. Aperio Digital Pathology Slide Scanner (Leica Biosystems Wetzlar, Germany) was used with 20× magnification, resulting in a field of approximately 870 microns field size. Fields with the least amount of necrosis were chosen to prevent secondary deposition of IgG and then examined for capillary staining for IgG, IgM, and C4d. The intensity was measured for C4d staining as 0: negative or equivocal staining, 1+: faint positive staining, 2+: strong positive staining per the Society of Cardiovascular Pathology (SCVP), Association for European Cardiovascular Pathology (AECVP) and the International Society of Heart and Lung Transplantation (ISHLT). Additionally, specimens were graded for microthrombi on a scale of 0–4+, where 1 + = >0 capillaries stained; 2 + = >1–5 capillaries stained; 3 + = >5–10 capillaries stained, 4 + = >10 capillaries stained, per high power field.

Statistics and reproducibility data analysis

All statistical analysis and graph tabulations, including Kaplan–Meyer curves and line graph plots, were performed on GraphPad Prism 8 (San Diego, CA).

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Results

Verification of donor pig gene expression

Genome editing was done to produce 10 gene edits pigs. Deletion of GGTA1, B4GALNT2, and CMAH was confirmed by flow cytometry of peripheral blood mononuclear cells (PBMC) stained with IB4 lectin and DBA lectin. Growth hormone receptor (GHR) gene deletion was confirmed by Next-Gen DNA sequencing (MiSeq, Illumina). Variable human gene expression of individual transgenes (i.e., hCD46, hDAF, hTBM, EPCR, hHO-1, and hCD47) was observed in non-transplanted kidney tissue samples (Supplementary Fig. S1), porcine aortic endothelial cells (PAECs) and explanted heart by flow cytometry (Supplementary Fig. S2) and by IHC (Supplementary Fig. S3), and western blot respectively (Supplementary Fig. S4)1,18. Expression of hCD47, hCD46, and hHO1 transgenes was weak in explanted xenograft while hTBM and hEPCR genes expression was increased in IHCs.

10 gene edit cardiac xenograft survival

Cardiac xenografts with ten gene edits (i.e., GGTA1KO, B4GalNT2, CMAHKO, GHRKO, hTBM, hEPCR, hCD46, hDAF, hCD47, hHO1) were successfully harvested from GE pigs (Sus scrofa) and were preserved with non-ischemic continuous perfusion cardiac preservation (NICP)in the XVIVO© Perfusion system for up to 2 h before orthotopic implantation into adult baboons (Papio anubis). Table 1 describes the exact times of cardiac perfusion in all transplanted animals. All recipient baboons (n = 4) were weaned from cardiopulmonary bypass, promptly extubated, and without the need for inotropic support after successful life-supporting orthotopic cardiac xenotransplantation (OHTx). With 10-GE donors, NHP recipient survival was 128 ± 36 days (55, 105, 127, and 225 days). This survival was less than previously reported using 7-GE xenografts (10-GE minus CMAHKO, hDAF, and HO-1) but longer than reported using 9-GE xenografts (10-GE minus GHRKO)4 (Fig. 1). Human transgene expression levels of each gene varied in 10-GE donor animals and no correlation was observed with xenograft survival.

Table 1 Summary of 10GE pig heart xenotransplantation in baboons
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Fig. 1: Cardiac xenograft survival.
Fig. 1: Cardiac xenograft survival.
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Kaplan–Meir survival curve of cardiac xenograft recipients receiving 7, 9, and 10 GE edit donor pigs.

Hematological and immune monitoring of 10 gene edit cardiac xenograft

WBC, hematocrit, and platelet counts are shown in Fig. 2. After an initial drop immediately after surgery, the platelet count recovered. Troponin release was increased as an indicator of myocardial damage for all recipients receiving 10 GE cardiac xenografts but ultimately decreased to a consistent low-level background—similar to prior studies.

Fig. 2: Hematological parameter.
Fig. 2: Hematological parameter.
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Hematology (A) White blood counts (WBC), hematocrit (HCT), platelets, and coagulation parameter of cardiac xenograft recipient receiving from 10GE donor pig.

T and B-cell lymphocytes were depleted with ATG and Rituxan and confirmed by flow cytometry. CD3+ lymphocytes were reconstituted within 48 h of transplantation (Fig. 3A). However, CD20+ lymphocyte depletion lasted for several weeks after induction and did not correlate with increased anti-pig IgG and IgM antibody titers (Fig. 3B). Flow cytometry gating strategy to determine CD3+ and CD20+ has been shown in Supplementary Methods SM1.

Fig. 3: Immunological monitoring.
Fig. 3: Immunological monitoring.
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A Immunophenotyping of T and B cells. B Non‐Gal IgG and IgM levels; anti‐pig non‐Gal IgG and IgM antibodies levels in mean fluorescence intensity (MFI) were measured in cardiac xenograft grafts baboon recipient’s serum by flow cytometry using 10GE porcine endothelial cells.

There did not appear to be any significant trends in proinflammatory and anti-inflammatory cytokines, except in the case of recipient B33500, where acute kidney injury and intra-abdominal sepsis were also present. In this specific case, there was an increase in IL-6, IFN-b, IFN-g, IL-12, IL-17, IL-23, and IL-10, with notable sparing of IL-1b (Fig. 4).

Fig. 4: Serum cytokines levels.
Fig. 4: Serum cytokines levels.
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IL-6, TNF-α, IFN-γ, IL-12p40, IL-17, IL-23, IL-1β, IFN-β, IP-10 (CXCL10) levels in the serum of cardiac xenotransplant recipients.

Ultrasonography examination of 10 gene edit cardiac xenograft

Xenograft ejection fraction was preserved similarly to prior orthotopic xenografts (Fig. 5A). Similar to other GHRKO xenografts, as previously published, LV dimensions in diastole, septal, and posterior wall thicknesses stayed relatively stable throughout the post-transplantation course (Fig. 5B). However, LV mass increased in all 10-GE xenografts over time. The most notable increases in LV mass were in those with interstitial cellular infiltrate or artery intimal thickening, similarly seen in prior publications15.

Fig. 5: 10GE cardiac xenograft measurements post-transplantation according to transthoracic echocardiogram (TTE).
Fig. 5: 10GE cardiac xenograft measurements post-transplantation according to transthoracic echocardiogram (TTE).
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A LV ejection fraction was measured longitudinally after transplantation. B Septal thickness, left ventricular (LV) dimension in end-diastole, relative wall thickness (rWT), posterior wall thickness, LV mass. LV mass percent change was calculated according to the average LV mass in the 30 days after transplantation and 30 days before euthanasia.

Postmortem histopathological and immunohistochemical evaluation of xenografts

A summary of histopathological findings after explantation is summarized in Tables 2 & 3 and Fig. 6A, B. Recipient B9314 underwent cellular rejection on POD # 55, characterized by mononuclear cellular infiltrate (Fig. 6A.i). B5317 experienced sudden v-fib arrest on POD #127 from a progressive right ventricular outflow gradient seen on echocardiography. This xenograft demonstrated patchy subendocardial necrosis and arterial intimal thickening (Fig. 6A.ii).

Table 2 Histology finding score
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Table 3 Histological and Immunohistochemistry findings
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Fig. 6: Histological and Immunohistochemistry evaluation of the 10 GE cardiac xenografts.
Fig. 6: Histological and Immunohistochemistry evaluation of the 10 GE cardiac xenografts.
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A Hematoxylin and Eosin (H&E) staining of a section from the explanted heart from the necropsy demonstrating mononuclear cell infiltrate (i), arterial intimal thickening (ii), chronic vasculopathy (iii), and microvascular thrombosis (iv). B Immunohistochemistry of right ventricle (RV) section demonstrating complement (C4d & C3) and immunoglobulin (IgG&IgM) staining.

B32651 experienced a respiratory infection, requiring euthanasia on POD #105. The xenograft was functioning normally, and the final pathology demonstrated arterial intimal thickening without necrosis (Fig. 6A.iii). B33500 was euthanized on POD #225 for acute kidney insufficiency, and on the final autopsy, there was small bowel chronic serositis with granulation tissue and expansion of the submucosa. The xenograft demonstrated patchy myodegeneration and mild microthrombi but an otherwise normal-appearing xenograft (Fig. 6A.iv).

PCMV status of donor animals and its relationship to graft survival

Porcine cytomegalovirus (PCMV) has been shown by other groups to reduce xenograft survival in non-human primates23,24,25. We have retrospectively tested 10-GE cardiac xenografts used in our study by PCR and western blot analysis18. Three out of four cardiac xenografts were found to be positive for PCMV, and their xenograft survivals were 225, 127, and 55 days. One of them (B9314) had demonstrated cellular rejection with evidence of extensive chronic mononuclear infiltrate, and immunostaining of IgG (20%), IgM (10%), and C3, C4d (<5%) antibodies in the capillaries (Fig. 6B). Micro and macrovascular thrombosis was observed in the H&E staining of the explanted xenograft (Fig. 6A). However, despite the presence of PCMV positivity, no viral inclusions were found at the time of necropsy.

The role of CMAH and GHR knockout in 10 gene graft survival

Cumulative anti-pig non-gal antibodies after transplantation in recipients receiving cardiac xenografts from 10GE were higher (Fig. 7A) than in serum from recipients with 7-GE, which do not have CMAHKO (mean normalized MFI for IgG and IgM: 3.6 vs 2.5 and 6.3 vs 3.3 respectively; Unpaired t-test p-value 0.0011 and 0.0002). However, no significant difference in cumulative anti-pig non-gal antibodies after transplantation was found when the recipient received CMAHKO xenograft from both (9-GE and 10-GE). Longitudinally, anti-pig antibodies (IgG and IgM) are shown in Supplementary Fig. S5) after cardiac xenotransplantation. We have also retrospectively analyzed the pre-existing donor-specific anti-pig IgG and IgM antibodies in the baboon recipient before transplantation (−7 days), and no correlation (r2 value 0.46 and 0.36, respectively) was found with 10 GE cardiac xenograft outcomes (Fig. 7B).

Fig. 7: Non-gal anti-pig antibodies in cardiac xenotransplantation recipients.
Fig. 7: Non-gal anti-pig antibodies in cardiac xenotransplantation recipients.
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A Cumulative mean non-gal anti-pig antibodies (IgG and IgM antibodies) after cardiac xenotransplantation in cardiac xenograft recipients receiving 7, 9, and 10GE donor pig xenograft. B Pre-existing donor-specific anti-pig IgG and IgM antibodies in the baboon recipient before transplantation and cardiac xenograft outcomes.

Discussion

Multi-gene edited donor organs from genetically engineered pigs have improved xenograft survival and have overcome hyperacute and acute xenograft rejection. Several studies, including ours, from heterotopic21,22,26,27,28,29 and life-supporting (orthotopic)4,5,29,30,31 xenotransplantations have demonstrated that iterative and progressive gene edits have yielded prolonged xenograft survival. Recently, we have reported the longest cardiac xenotransplantation from 7-GE pig donors. These included the deletion of two carbohydrate antigens (GGTA1KO, B4GalNT2), i.e., double knockout (DKO), GHR KO, and overexpression of two human anticoagulation (hTBM & hEPCR) proteins, one complement regulatory protein (hCD46), and anti-phagocytosis protein (hCD47). In contrast, 9-GE pigs contain an additional carbohydrate antigen deletion (Neu5Gc, through CMAHKO), but without GHRKO. These did not perform well as the 7-GE recipients in this limited study. Reduced xenograft survival in the recipient from the 9 GE heart may have been attributed to CMAHKO in the xenograft. Cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) gene encodes N-glycolylneuraminic acid (Neu5GcUnlike humans, Old World monkeys (OWM), including baboons, have a functional CMAH gene and so do not have antibodies that bind to anti-Neu5Gc. Exposure to a 10GE inactive CMAH reveals new epitopes, “4th xenoantigen,” and presents new targets for Nab-binding32,33,34. 10-GE also expresses six human transgenes which include complement inhibitors (CD46 & DAF), anticoagulation (EPCR &TBM), anti-phagocytosis/apoptotic (CD47), and anti-inflammatory genes (HO-1). As reported by us earlier, human multi-transgene in genetically engineered pigs appears to confer superior xenograft outcomes and reduce prevailing immediate postoperative xenotransplantation challenges35. In this study, human transgene expression of each gene varied among 10GE donor animals, and they did not correlate with xenograft survival. A larger cohort is needed to elucidate the potential effect of the expression level of each gene on xenograft survival. This investigation is not possible with a limited number of transplants reported here.

Here, we demonstrate the prolonged survival of 10-GE cardiac xenografts in baboons, up to 225 days, which is more than 9-GE but less than 7 GE xenografts. Both 9 and 10 GE xenografts employed deletion of a third carbohydrate antigen (CMAHKO), which may have contributed to the shorter survival. We and others have demonstrated that PAECs from TKO have higher IgM binding than DKO PAECs in NHP serum36,37. Yamamoto et al. have also reported that TKO PAECs have less IgM binding and lower cytotoxicity with human serum than baboons or other old-word monkey serum34.

We have also demonstrated successful cardiac xenotransplantation from 10 GE donor pigs into humans, extending human life to 60 and 40 days1. No hyperacute rejection or increased anti-pig antibodies were detected in these human patients until IVIg treatment was given to one of two patients as a rescue therapy for infections. Later on, lVIg was found to be reactive with PAECs. Porret et al. and Moazami et al. have also reported xenotransplantation of 10 GE porcine grafts to brain-dead decedent humans in separate renal and cardiac transplant experiments. A decedent human recipient who received 10 GE pig hearts survived for up to 66 hours until the termination of the experiment38. None of these studies reported a cellular or antibody-mediated rejection (AMR) assessed by conventional histology and flow cytometry, but deeper molecular analysis indicates AMR may have been present39. These studies suggest that 10 GE donor pigs have sufficient gene modifications to prevent hyperacute rejection and human xenotransplantation barriers.

The pig-to-non-human primate (NHP) model is crucial for xenotransplantation, but due to exposure to “4th xenoantigen” arising after CMAH knockout, the NHP model is less than ideal for testing 10GE xenografts. Pre-existing anti-pig antibodies in the recipient may also have contributed to the xenograft outcome. 7 GE xenografts that did not have CMAHKO and their recipients had lower anti-pig IgG and IgM antibodies and survived longer. In our first clinical case, the recipient did not show anti-pig IgG and IgM antibodies until 40 days despite CMAHKO in 10GE pig hearts. These results suggest that CMAHKO grafts may not be very suitable for NHPs.

PCMV is a DNA virus widely distributed in pigs, belongs to the roseolovirus family, and has also shown reduced xenograft survival. It is acquired early in porcine life, and its infection stays lifelong and latent. Some earlier studies have reported a short survival of xenotransplant recipients, and the cause of poor outcomes was attributed to the inadvertent reactivation of pCMV infection23,40. Morozov et al. reported a 29 and 14-day survival of a 3GE heart and GTKO kidney of PCMV-positive pigs, respectively23,41. Denner et al. reported that PCMV reactivation in orthotopic pig heart xenotransplantation was associated with reduced xenograft survival and increased levels of IL-6 and TNFα40. This limited study demonstrates that 10GE heart transplants in baboons survived long-term, irrespective of the donor’s PCMV status, and no difference in the increase of inflammatory cytokines was found, however, a larger cohort is needed to investigate the role of PCMV’s on the xenograft survival. We hypothesize that overexpression of the human inflammatory gene (HO1 and CD47) may have suppressed the effect.

Lastly, the effect of physiologic mismatch between the pig donor heart and the recipient may lead to remodeling of the myocardium (as demonstrated by hypertrophy), and how to address this in our model is controversial. Prior studies have demonstrated support for both blood pressure and heart rate control pharmacologically and by non-pharmacological means5,15,42. This study again demonstrates a lack of graft-limiting hypertrophy, but there is some hypertrophy that peaks at 2 weeks and then plateaus. Further studies need to elucidate the cause from this study and others.

This study demonstrates long-term cardiac xenotransplant survival from 10 GE donor pigs into baboons but also highlights the detrimental effect of CMAHKO on NHP models. However, an additional human gene expression (CD46, TBM, EPCR, hDAF, CD47, and HO-1) may have protected and helped prolong xenograft survival. Therefore, 10GE xenografts might be more suitable for clinical xenotransplantation than in NHPs. Therefore, further human experiments are needed to answer these questions, besides the two human compassionate use cases and decedent model experiments, for demonstrating proof-of-principle.