Results of Two Cases of Pig-to-Human Kidney Xenotransplantation.

BACKGROUND: Xenografts from genetically modified pigs have become one of the most promising solutions to the dearth of human organs available for transplantation. The challenge in this model has been hyperacute rejection. To avoid this, pigs have been bred with a knockout of the alpha-1,3-galactosyltransferase gene and with subcapsular autologous thymic tissue. METHODS: We transplanted kidneys from these genetically modified pigs into two brain-dead human recipients whose circulatory and respiratory activity was maintained on ventilators for the duration of the study. We performed serial biopsies and monitored the urine output and kinetic estimated glomerular filtration rate (eGFR) to assess renal function and xenograft rejection. RESULTS: The xenograft in both recipients began to make urine within moments after reperfusion. Over the 54-hour study, the kinetic eGFR increased from 23 ml per minute per 1.73 m2 of body-surface area before transplantation to 62 ml per minute per 1.73 m2 after transplantation in Recipient 1 and from 55 to 109 ml per minute per 1.73 m2 in Recipient 2. In both recipients, the creatinine level, which had been at a steady state, decreased after implantation of the xenograft, from 1.97 to 0.82 mg per deciliter in Recipient 1 and from 1.10 to 0.57 mg per deciliter in Recipient 2. The transplanted kidneys remained pink and well-perfused, continuing to make urine throughout the study. Biopsies that were performed at 6, 24, 48, and 54 hours revealed no signs of hyperacute or antibody-mediated rejection. Hourly urine output with the xenograft was more than double the output with the native kidneys. CONCLUSIONS: Genetically modified kidney xenografts from pigs remained viable and functioning in brain-dead human recipients for 54 hours, without signs of hyperacute rejection. (Funded by Lung Biotechnology.).

Immune response after pig-to-human kidney xenotransplantation: a multimodal phenotyping study.

BACKGROUND: Cross-species immunological incompatibilities have hampered pig-to-human xenotransplantation, but porcine genome engineering recently enabled the first successful experiments. However, little is known about the immune response after the transplantation of pig kidneys to human recipients. We aimed to precisely characterise the early immune responses to the xenotransplantation using a multimodal deep phenotyping approach. METHODS: We did a complete phenotyping of two pig kidney xenografts transplanted to decedent humans. We used a multimodal strategy combining morphological evaluation, immunophenotyping (IgM, IgG, C4d, CD68, CD15, NKp46, CD3, CD20, and von Willebrand factor), gene expression profiling, and whole-transcriptome digital spatial profiling and cell deconvolution. Xenografts before implantation, wild-type pig kidney autografts, as well as wild-type, non-transplanted pig kidneys with and without ischaemia-reperfusion were used as controls. FINDINGS: The data collected from xenografts suggested early signs of antibody-mediated rejection, characterised by microvascular inflammation with immune deposits, endothelial cell activation, and positive xenoreactive crossmatches. Capillary inflammation was mainly composed of intravascular CD68+ and CD15+ innate immune cells, as well as NKp46+ cells. Both xenografts showed increased expression of genes biologically related to a humoral response, including monocyte and macrophage activation, natural killer cell burden, endothelial activation, complement activation, and T-cell development. Whole-transcriptome digital spatial profiling showed that antibody-mediated injury was mainly located in the glomeruli of the xenografts, with significant enrichment of transcripts associated with monocytes, macrophages, neutrophils, and natural killer cells. This phenotype was not observed in control pig kidney autografts or in ischaemia-reperfusion models. INTERPRETATION: Despite favourable short-term outcomes and absence of hyperacute injuries, our findings suggest that antibody-mediated rejection in pig-to-human kidney xenografts might be occurring. Our results suggest specific therapeutic targets towards the humoral arm of rejection to improve xenotransplantation results. FUNDING: OrganX and MSD Avenir.

The Young Investigator Committee of the International Xenotransplantation Association-Perspective of advancements in the field in 2023.

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.

Immune response, phenotyping and molecular graft surveillance in kidney transplant recipients following severe acute respiratory syndrome coronavirus 2 vaccination.

BACKGROUND: Understanding immunogenicity and alloimmune risk following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination in kidney transplant recipients is imperative to understanding the correlates of protection and to inform clinical guidelines. METHODS: We studied 50 kidney transplant recipients following SARS-CoV-2 vaccination and quantified their anti-spike protein antibody, donor-derived cell-free DNA (dd-cfDNA), gene expression profiling (GEP), and alloantibody formation. RESULTS: Participants were stratified using nucleocapsid testing as either SARS-CoV-2-naïve or experienced prior to vaccination. One of 34 (3%) SARS-CoV-2 naïve participants developed anti-spike protein antibodies. In contrast, the odds ratio for the association of a prior history of SARS-CoV-2 infection with vaccine response was 18.3 (95% confidence interval 3.2, 105.0, p < 0.01). Pre- and post-vaccination levels did not change for median dd-cfDNA (0.23% vs. 0.21% respectively, p = 0.13), GEP scores (9.85 vs. 10.4 respectively, p = 0.45), calculated panel reactive antibody, de-novo donor specific antibody status, or estimated glomerular filtration rate. CONCLUSIONS: SARS-CoV-2 vaccines do not appear to trigger alloimmunity in kidney transplant recipients. The degree of vaccine immunogenicity was associated most strongly with a prior history of SARS-CoV-2 infection.

Caregiver exposure to hepatitis C virus following transplantation with hepatitis C viremic donor organs: A case series.

Direct-acting antiviral (DAA) therapeutics have ushered in an era in which transplanting organs from donors infected with hepatitis C virus (HCV+) into recipients without (HCV-) is an increasingly common practice. Rare but potentially life-threatening events have been reported in recipients of HCV+ organs. Since 2018 at our institution, 182 HCV- patients have received HCV+ donor organs. Here, we retrospectively reviewed cases in which recipients' family member caregivers reported sustaining needlestick exposures at home following discharge of the transplant recipient from the hospital. Caregiver needlestick exposures were passively reported in three cases of HCV+ into HCV- transplants (1.64% of such cases at our center). In all instances, the exposed individuals were aiding in diabetic management and the exposure occurred via lancets or insulin needles. In one case, the recipient viral load was undetectable at the time of the exposure but in the other two, recipients were viremic, putting their family members at risk to contract HCV infection. Surveillance for the exposed individuals was undertaken and no transmissions occurred. For centers performing HCV+ into HCV- transplants, it is important that informed consent includes discussion of potential secondary risks to family members and caregivers. Further, protocols for postexposure surveillance and for the acquisition of DAA treatment in the event of a secondary transmission should be in place.

Impact of the 2014 kidney allocation system changes on trends in A2/A2B into B kidney transplantation and organ procurement organization reporting of donor subtyping.

The current kidney allocation system (KAS) preferentially allocates kidneys from blood type A2 or A2B (A/A2B) donors to blood type B candidates. We used national data to evaluate center-level performance of A2/A2B to B transplants, and organ procurement organization (OPO) reporting of type A or AB donor subtyping, in 5-year time periods prior to (2009-2014) and following (2015-2019) KAS implementation. The number of centers performing A2/A2B to B transplants increased from 17 pre-KAS to 76 post-KAS, though this still represents only a minority of centers (7.3% pre-KAS and 32.6% post-KAS). For high-performing centers, the median net increase in A2/A2B to B transplants was 19 cases (range -2-72) per center in the 5 years post-KAS. The median net increase in total B recipient transplants was 21 cases (range -17-119) per center. Despite requirements for performance of subtyping, in 2019 subtyping was reported on only 56.4% of A/AB donors. This translates into potential missed opportunities for B recipients, and even post-KAS up to 2322 A2/A2B donor kidneys may have been allocated for transplantation as A/AB. Further progress must be made both at center and OPO levels to broaden implementation of A2/A2B to B transplants for the benefit of underserved recipients.

Characterization, biology, and expansion of regulatory T cells in the Cynomolgus macaque for preclinical studies.

Reliable in vitro expansion protocols of regulatory T cells (Tregs) are needed for clinical use. We studied the biology of Mauritian Cynomolgus macaque (MCM) Tregs and developed four in vitro Treg expansion protocols for translational studies. Tregs expanded 3000-fold when artificial antigen presenting cells (aAPCs) expressing human CD80, CD58 and CD32 were used throughout the culture. When donor peripheral blood mononuclear cells (PBMCs) were used as the single source of APCs followed by aAPCs, Tregs expanded 2000-fold. Tregs from all protocols suppressed the proliferation of anti-CD2CD3CD28 bead-stimulated autologous PBMCs albeit with different potencies, varying from 1:2-1:4 Treg:PBMC ratios, up to >1:32. Reculture of cryopreserved Tregs permitted reexpansion with improved suppressive activity. Occasionally, CD8 contamination was observed and resolved by resorting. Specificity studies showed greater suppression of stimulation by anti-CD2CD3CD28 beads of PBMCs from the same donor used for stimulation during the Treg cultures and of autologous cells than of third-party PBMC responders. Similar to humans, the Treg-specific demethylated region (TSDR) within the Foxp3 locus correlated with suppressive activity and expression of Foxp3. Contrary to humans, FoxP3 expression did not correlate with CD45RA or CD127 expression. In summary, we have characterized MCM Tregs and developed four Treg expansion protocols that can be used for preclinical applications.

Regenerative Medicine: Solution in Sight.

The retina, like other central nervous system tissues, has poor regenerative properties in humans. Therefore, diseases that cause retinal cell loss, such as Age-related macular degeneration (AMD), retinitis pigmentosa (RP), Leber congenital amaurosis, Usher syndrome, glaucoma, and diabetic retinopathy, typically result in permanent visual impairment. Stem cell technologies have revolutionized our ability to produce neural cells in abundant supply. Much stem cell research effort is focused on producing the required cell types for cell replacement, or to generate disease-in-a-dish models to elucidate novel disease mechanisms for therapeutic development. Here we review the recent advances in stem cell studies relevant to producing RPE and retinal cells, and highlight future directions.

Human Retinal Pigment Epithelium Stem Cell (RPESC).

The retinal pigment epithelium (RPE) is a pigmented cellular monolayer that supports photoreceptor cells located in the overlying neural retina. The RPE is critical for vision and its dysfunction results in numerous pathologies, several with limited available disease-altering strategies. Regeneration of the retina from RPE is robust in lower vertebrates, but is not normally exhibited in mammals. We recently found that a subpopulation of human RPE cells can be stimulated in culture to generate multipotent self-renewing cells-the RPE stem cell (RPESC). RPESC can be expanded to generate RPE progeny that are a potential source for cell replacement therapy. Alternatively, RPESC can produce mesenchymal progeny which serve as a disease model of epiretinal membrane formation. Yet another potential application of RPESCs is activation within the eye to awaken dormant endogenous repair.

Efficiency of Membrane Protein Expression Following Infection with Recombinant Adenovirus of Polarized Non-Transformed Human Retinal Pigment Epithelial Cells.

Transient expression of exogenous proteins facilitates studies of molecular mechanisms and utility for transplantation of retinal pigment epithelial (RPE) cells in culture. Here, we compared expression of the membrane protein ß5 integrin-GFP (ß5-GFP) in two recently established models of differentiated human RPE, adult RPE stem cell-derived RPE and primary fetal RPE, upon infection with recombinant adenovirus or transfection with DNA in liposomes. We varied viral titer and duration of virus incubation and examined ß5-GFP and the tight junction marker ZO-1 in manipulated cells by confocal microscopy. Fewer than 5 % of cells expressed ß5-GFP after liposome-mediated transfection. The percentage of cells with detectable ß5-GFP exceeded 90 % after adenovirus infection for as little as 1 h. Decreasing virus titer two-fold did not alter the fraction of cells expressing ß5-GFP but increased variability of ß5-GFP level among cells. In cells with low expression levels, ß5-GFP localized mostly to the apical plasma membrane like endogenous αvß5 integrin. In cells with high expression levels, ß5-GFP localized to the cytoplasm in addition to the apical surface suggesting accumulation in trafficking compartments. Altogether, adenovirus delivery yields efficient exogenous membrane protein expression of correct polarity in differentiated human RPE cells in culture.

Strong Loophole-Free Test of Local Realism.

We present a loophole-free violation of local realism using entangled photon pairs. We ensure that all relevant events in our Bell test are spacelike separated by placing the parties far enough apart and by using fast random number generators and high-speed polarization measurements. A high-quality polarization-entangled source of photons, combined with high-efficiency, low-noise, single-photon detectors, allows us to make measurements without requiring any fair-sampling assumptions. Using a hypothesis test, we compute p values as small as 5.9×10^{-9} for our Bell violation while maintaining the spacelike separation of our events. We estimate the degree to which a local realistic system could predict our measurement choices. Accounting for this predictability, our smallest adjusted p value is 2.3×10^{-7}. We therefore reject the hypothesis that local realism governs our experiment.

Cellular dynamics in pig-to-human kidney xenotransplantation.

BACKGROUND: Xenotransplantation of genetically engineered porcine organs has the potential to address the challenge of organ donor shortage. Two cases of porcine-to-human kidney xenotransplantation were performed, yet the physiological effects on the xenografts and the recipients' immune responses remain largely uncharacterized. METHODS: We performed single-cell RNA sequencing (scRNA-seq) and longitudinal RNA-seq analyses of the porcine kidneys to dissect xenotransplantation-associated cellular dynamics and xenograft-recipient interactions. We additionally performed longitudinal scRNA-seq of the peripheral blood mononuclear cells (PBMCs) to detect recipient immune responses across time. FINDINGS: Although no hyperacute rejection signals were detected, scRNA-seq analyses of the xenografts found evidence of endothelial cell and immune response activation, indicating early signs of antibody-mediated rejection. Tracing the cells' species origin, we found human immune cell infiltration in both xenografts. Human transcripts in the longitudinal bulk RNA-seq revealed that human immune cell infiltration and the activation of interferon-gamma-induced chemokine expression occurred by 12 and 48 h post-xenotransplantation, respectively. Concordantly, longitudinal scRNA-seq of PBMCs also revealed two phases of the recipients' immune responses at 12 and 48-53 h. Lastly, we observed global expression signatures of xenotransplantation-associated kidney tissue damage in the xenografts. Surprisingly, we detected a rapid increase of proliferative cells in both xenografts, indicating the activation of the porcine tissue repair program. CONCLUSIONS: Longitudinal and single-cell transcriptomic analyses of porcine kidneys and the recipient's PBMCs revealed time-resolved cellular dynamics of xenograft-recipient interactions during xenotransplantation. These cues can be leveraged for designing gene edits and immunosuppression regimens to optimize xenotransplantation outcomes. FUNDING: This work was supported by NIH RM1HG009491 and DP5OD033430.

Integrative multi-omics profiling in human decedents receiving pig heart xenografts.

In a previous study, heart xenografts from 10-gene-edited pigs transplanted into two human decedents did not show evidence of acute-onset cellular- or antibody-mediated rejection. Here, to better understand the detailed molecular landscape following xenotransplantation, we carried out bulk and single-cell transcriptomics, lipidomics, proteomics and metabolomics on blood samples obtained from the transplanted decedents every 6 h, as well as histological and transcriptomic tissue profiling. We observed substantial early immune responses in peripheral blood mononuclear cells and xenograft tissue obtained from decedent 1 (male), associated with downstream T cell and natural killer cell activity. Longitudinal analyses indicated the presence of ischemia reperfusion injury, exacerbated by inadequate immunosuppression of T cells, consistent with previous findings of perioperative cardiac xenograft dysfunction in pig-to-nonhuman primate studies. Moreover, at 42 h after transplantation, substantial alterations in cellular metabolism and liver-damage pathways occurred, correlating with profound organ-wide physiological dysfunction. By contrast, relatively minor changes in RNA, protein, lipid and metabolism profiles were observed in decedent 2 (female) as compared to decedent 1. Overall, these multi-omics analyses delineate distinct responses to cardiac xenotransplantation in the two human decedents and reveal new insights into early molecular and immune responses after xenotransplantation. These findings may aid in the development of targeted therapeutic approaches to limit ischemia reperfusion injury-related phenotypes and improve outcomes.

Hemoglobin A1c testing is associated with improved pancreas utilization for transplant.

CONTEXT: Aging, higher prevalence of diabetes, worsening obesity, and hyperglycemia among potential donors increase the likelihood that pancreata will be declined by transplant centers. Hemoglobin A1c testing, also known as glycated hemoglobin testing, identifies a donor's average blood glucose concentration for the preceding 2 to 3 months and is the standard test for identifying prolonged periods of hyperglycemia. OBJECTIVE: To compare pancreas utilization rates before and after implementation of hemoglobin A1c testing. DESIGN: A retrospective study of data from the New York Organ Donor Network was conducted. Potential donors were defined as standard criteria donors who had no history of diabetes and were not seropositive for hepatitis B or C. Criteria for "ideal" potential pancreas donors were based on age, body mass index, lipase level, and terminal creatinine level. Potential donors who did not meet the criteria for ideal donors were considered "expanded" potential pancreas donors. Pancreas utilization rate was defined as the number of pancreata transplanted divided by the number of potential pancreas donors. RESULTS: Of 779 standard criteria donors, 691 (89%) were potential pancreas donors: 251 ideal (36%) and 440 expanded (64%) donors. In 2005 and 2006, before hemoglobin A1c testing, pancreas utilization rates were 21% and 18%, respectively. In 2008, 2009, and 2010, after hemoglobin A1c testing was incorporated, utilization rates were 27%, 28%, and 32%, respectively. Utilization of ideal donors increased from 33% to 51% (P= .003), and utilization of expanded donors increased from 11% to 17% (P= .05). Pancreas utilization increased 51.0%, and pancreas discards decreased 50.8% with the implementation of hemoglobin A1c testing. CONCLUSION: Hemoglobin A1c testing may increase utilization of ideal and expanded criteria pancreata.

Staged Endovascular and Surgical Management of a Mycotic Pseudoaneurysm After Pancreas Transplant.

Mycotic pseudoaneurysms are a rare, life-threatening complication after pancreas transplant. There have been limited reports of endovascular treatment of mycotic pseudoaneurysms in pancreas transplant recipients. Herein, we report on a case of a mycotic pseudoaneurysm from Pseudomonas aeruginosa after pancreas transplant. A 53-year-old male recipient underwent an uneventful simultaneous pancreas and kidney transplant. He was readmitted 48 days posttransplant with fevers and rigors. Pan-cultures were performed and broad-spectrum antibiotics were initiated. Imaging studies demonstrated a large mycotic pseudoaneurysm arising from the right common iliac artery adjacent to the arterial Y-graft anastomosis of the transplant pancreas. Endovascular stent placement was used to exclude the pseudoaneurysm prior to transplant pancreatectomy. During pancreatectomy, the lateral wall of the common iliac artery was found to be necrotic with significant exposure of the endovascular stent. After ligation and excision of the common iliac artery, a femorofemoral bypass was performed to revascularize the lower extremity. This case report highlights the advantage of a staged endovascular and surgical management strategy for complex mycotic pseudoaneurysms after pancreas transplant.

Identifying biomarkers of heterogeneity and transplantation efficacy in retinal pigment epithelial cells.

Transplantation of retinal pigment epithelial (RPE) cells holds great promise for patients with retinal degenerative diseases, such as age-related macular degeneration. In-depth characterization of RPE cell product identity and critical quality attributes are needed to enhance efficacy and safety of replacement therapy strategies. Here, we characterized an adult RPE stem cell-derived (RPESC-RPE) cell product using bulk and single-cell RNA sequencing (scRNA-seq), assessing functional cell integration in vitro into a mature RPE monolayer and in vivo efficacy by vision rescue in the Royal College of Surgeons rats. scRNA-seq revealed several distinct subpopulations in the RPESC-RPE product, some with progenitor markers. We identified RPE clusters expressing genes associated with in vivo efficacy and increased cell integration capability. Gene expression analysis revealed lncRNA (TREX) as a predictive marker of in vivo efficacy. TREX knockdown decreased cell integration while overexpression increased integration in vitro and improved vision rescue in the RCS rats.

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

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

Identifying risk factors in renal allografts before transplant: machine-measured renal resistance and posttransplant allograft survival.

Enhancement of renal allograft function and survival in an era where expanded criteria donors are increasingly used requires validated selection criteria. The goal of this retrospective study was to evaluate the significance of pretransplant donor and allograft parameters to identify risk factors that can be used in a model to predict 1-year allograft outcomes. Donor demographic factors, donor type, and allograft parameters such as biopsy results and machine-measured renal resistance were correlated with 1-year graft outcome. The Kaplan-Meier method was used to estimate graft survival using the categorical predictors of donor type, donor age, and machine measured renal resistance at 1.5, 3, and 5 hours. The log-rank test was used to test the difference in survival curves between cohorts. The Cox regression analysis was used to estimate hazard ratios for machine-measured renal resistance, donor age, donor terminal creatinine level, donor's estimated glomerular filtration rate, cold ischemia time, and percent glomerulosclerosis. The data show that machine-measured renal resistance at 3 and 5 hours has a statistically significant inverse relationship to 1-year graft survival. All other risk factors had no correlation with 1-year graft survival. The machine-measured renal resistance at 3 hours is the earliest significant predictor of 1-year allograft outcome.

Intraoperative Verapamil Fails to Reduce Delayed Graft Function in Donation After Circulatory Death Renal Allografts.

BACKGROUND: The shortage of transplantable organs has led to increased utilization of kidneys that may be particularly vulnerable to ischemia-reperfusion injury (IRI) and delayed graft function (DGF). Kidneys from donation after circulatory death (DCD) donors have additional IRI from donor procurement that results in increased risk of DGF. Verapamil may reduce IRI in kidney allografts when given at the time of organ reperfusion. This study sought to determine if intraoperative administration of verapamil (Ver) could reduce the risk of DGF in DCD kidney transplants. METHODS: A single-center retrospective matched cohort study was performed of 93 Ver (-) kidney transplant recipients compared with 93 Ver (+) kidney transplant recipients, matched by donor age, Kidney Donor Profile Index, and DCD status. Covariates that could impact DGF risk were evaluated by univariate and multivariate logistic regression analyses. RESULTS: The Ver (-) and Ver (+) matched cohorts did not have any significant differences in the demographic covariates. There was no difference in DGF rate between the Ver cohorts in either the overall study population or within the DCD subgroup. There was a trend toward reduced DGF in the Ver (+) cohort for cold ischemia time (CIT) ≤24 h, but this failed to achieve statistical significance. On multivariate analysis, only CIT was found to be independently associated with DGF. CONCLUSIONS: Intraoperative verapamil failed to reduce DGF risk in DCD kidney allografts. Limitations to this study include nonrandomization for the intraoperative administration of verapamil and the mean CIT >24 h in the study population. Only CIT was an independent prognosticator for DGF on multivariate analysis in a cohort matched for DCD status, consistent with prior studies.

The eyeball's connected to the brain ball.

How does the human eye develop in concert with the brain to create a functioning visual system? In this issue of Cell Stem Cell, Gabriel et al. (2021) report the development of eye-like structures from forebrain organoids with light sensitivity, signal processing, and connectivity, which moves us toward answering this complex question.