Design and testing of a humanized porcine donor for xenotransplantation

In the paper

“Over the years, various genetically engineered porcine donors have been created and their kidneys transplanted into Old World monkeys (OWMs)4–6.”

What the sources say

• Supported

  The Materials and Methods (Animals, “Pig-to-Rhesus Renal Transplantation”) describe two genetically engineered pig donor types and state that their kidneys were transplanted into rhesus macaques, which are Old World monkeys. This supports the claim attributed to the paper. (LLM, conf=0.94)

  cites [4] Adams, A. B. et al. Anti-C5 antibody tesidolumab reduces early antibody-mediated rejection and prolongs survival in renal xenotransplantation. Ann. Surg. 274, 473–480 (2021). source
• Supported

  The Methods (Animals) describe two genetically engineered pig donor types, with six or three genetic modifications, whose kidneys were transplanted into baboons. The Introduction also reports earlier transplantation of kidneys from genetically engineered pigs into rhesus monkeys. Together, these passages support the cited background statement. (LLM, conf=0.92)

  cites [5] Iwase, H. et al. Immunological and physiological observations in baboons with life-supporting genetically engineered pig kidney grafts. Xenotransplantation 24, e12293 (2017). source
• Supported

  The Introduction describes several genetic modifications to porcine donors and discusses pig-to-monkey kidney transplantation. Methods > Animals documents this study’s transplantation of kidneys from αGal-knockout/CD55-transgenic pigs into rhesus macaques; the References also identify genetically engineered pig-kidney transplants into baboons. No page numbers are available in our copy. (LLM, conf=0.91)

  cites [6] Kim, S. C. et al. Long‐term survival of pig‐to‐rhesus macaque renal xenografts is dependent on CD4 T cell depletion. Am. J. Transplant. 19, 2174–2185 (2019). source

In the paper

“Although renal grafts derived from the porcine donors lacking these three glycans and carrying various human transgenes have been tested in OWMs, graft survival was short8 or not all human transgenes were expressed10.”

What the sources say

• Partly supported

  The Results (In vivo study; page unavailable) report that TKO pig kidney grafts were tested in baboons and that recipient survival was 1, 4, and 61 days, significantly shorter than in the GTKO group. The Materials and Methods table lists six human transgenes for each TKO donor, while the Discussion says transgene expression was uniformly good in the great majority of cases. The paper does not establish that some of those transgenes were unexpressed in the TKO grafts. (LLM, conf=0.85)

  cites [8] Yamamoto, T. et al. Old World monkeys are less than ideal transplantation models for testing pig organs lacking three carbohydrate antigens (triple-knockout). Sci. Rep. 10, 9771 (2020). sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites [10] Ma, D. et al. Successful long-term TMA- and rejection-free survival of a kidney xenograft with triple xenoantigen knockout plus insertion of multiple human transgenes. Transplantation https://doi.org/10.1097/01.tp.0000698660.82982.ca (2020). source

In the paper

“Xenotransplantation may offer a transformative solution to the worldwide organ shortage crisis1–3.”

What the sources say

• Supported

  The paper’s opening passage (section E; page not provided) describes a shortage of transplant organs and says xenotransplantation could provide an unlimited, renewable source of lifesaving organs. That supports the citing sentence’s hedged description of a potential solution to organ shortages. (LLM, conf=0.90)

  cites [1] Montgomery, R. A. et al. Results of two cases of pig-to-human kidney xenotransplantation. N. Engl. J. Med. 386, 1889–1898 (2022). source
• Supported

  The Discussion calls xenotransplantation 'arguably the most pragmatic solution to the organ shortage crisis.' The Abstract likewise describes pigs as a promising organ source while acknowledging that further work is needed. These passages support the citing sentence’s qualified statement that xenotransplantation may offer a solution. (LLM, conf=0.92)

  cites [2] Porrett, P. M. et al. First clinical‐grade porcine kidney xenotransplant using a human decedent model. Am. J. Transplant. 22, 1037–1053 (2022). source
• Partly supported

  The Abstract reports that a patient ineligible for a human heart transplant received a pig heart that functioned normally for several weeks, supporting xenotransplantation as a possible alternative. The paper does not address the worldwide organ shortage or establish that xenotransplantation could solve it; the graft failed on day 49. (LLM, conf=0.78)

  cites [3] Griffith, B. P. et al. Genetically modified porcine-to-human cardiac xenotransplantation. N. Engl. J. Med. 387, 35–44 (2022). sourceExperimental — accuracy not yet measured

In the paper

“In humans, GGTA1 (ref. 18) and CMAH19 evolved into pseudogenes and the α-Gal and the Neu5Gc epitopes are not expressed (Fig. 1a).”

What the sources say

• ?Not checked — no full text of the sourcecites [18] Shaper, N. L., Lin, S. P., Joziasse, D. H., Kim, D. Y. & Yang-Feng, T. L. Assignment of two human alpha-1,3-galactosyltransferase gene sequences (GGTA1 and GGTA1P) to chromosomes 9q33-q34 and 12q14-q15. Genomics 12, 613–615 (1992). source
• Supported

  The citation to this paper is attached to CMAH, not GGTA1. The Abstract reports that a deletion in the human CMP-NeuAc hydroxylase gene makes its protein inactive and accounts for the absence of NeuGc in human glycoconjugates. It does not use the word “pseudogene,” but the reported loss of gene function supports that paraphrase. (LLM, conf=0.85)

  cites [19] Irie, A., Koyama, S., Kozutsumi, Y., Kawasaki, T. & Suzuki, A. The molecular basis for the absence of N-glycolylneuraminic acid in humans. J. Biol. Chem. 273, 15866–15871 (1998). source

In the paper

“Although elimination of growth hormone receptor gene expression could reduce organ sizes2,3, it comes with other undesired biological consequences7.”

What the sources say

• Partly supported

  Section 2.3.1 reports that growth hormone receptor knockout was accompanied by reduced serum IGF-1 levels and body weight, establishing a biological effect beyond organ size. It does not characterize the lower IGF-1 level as undesired. The Discussion attributes altered renal structural integrity to the pigs’ genetic modifications collectively, not specifically to growth hormone receptor knockout. (LLM, conf=0.75)

  cites [2] Porrett, P. M. et al. First clinical‐grade porcine kidney xenotransplant using a human decedent model. Am. J. Transplant. 22, 1037–1053 (2022). sourceExperimental — accuracy not yet measured
• ?Could not verify

  Citation 7 is attached to the claim that eliminating growth hormone receptor expression has undesired biological consequences. The Methods section says the receptor was knocked out to reduce xenograft growth, and the Results section reports later graft injury, but neither attributes that injury or another adverse consequence to the knockout. The Results say the mechanism of the injury was still under investigation. (LLM, conf=0.72)

  cites [3] Griffith, B. P. et al. Genetically modified porcine-to-human cardiac xenotransplantation. N. Engl. J. Med. 387, 35–44 (2022). sourceExperimental — accuracy not yet measured
• Supported

  Citation 7 is attached to the undesired biological consequences. The Results sections report transient juvenile hypoglycemia and increased body fat with a reduced muscle-to-fat ratio in GHR-KO pigs. The Results section on organ growth also reports smaller organs in these pigs. Page numbers are not supplied in our copy. (LLM, conf=0.91)

  cites [7] Hinrichs, A. et al. Growth hormone receptor-deficient pigs resemble the pathophysiology of human Laron syndrome and reveal altered activation of signaling cascades in the liver. Mol. Metab. 11, 113–128 (2018). source

In the paper

“Although the human B4GALNT2 gene is functional22, its expression levels vary and naturally occurring mutations have been identified that correlate with the Sd(a-) phenotype23.”

What the sources say

• Supported

  The paper’s opening summary reports that the human gene is expressed and that its expressed catalytic domain produces an enzyme that forms the Sd(a)/Cad antigen structure. It also reports varying expression across the cell types and tissues studied. The separate citation 23 is attached to the mutation–phenotype claim, so that claim is not attributed to this source. Section and page labels are unavailable in our copy. (LLM, conf=0.92)

  cites [22] Montiel, M.-D., Krzewinski-Recchi, M.-A., Delannoy, P. & Harduin-Lepers, A. Molecular cloning, gene organization and expression of the human UDP-GalNAc:Neu5Acalpha2-3Galbeta-R beta1,4-N-acetylgalactosaminyltransferase responsible for the biosynthesis of the blood group Sda/Cad antigen: evidence for an unusual extended cytoplasmic domain. Biochem. J. 373, 369–379 (2003). source
• Supported

  Citation 23 is attached most directly to the mutation claim. The Results and discussion section identifies naturally occurring B4GALNT2 missense and splice-site variants in individuals with the Sd(a−) phenotype. The paper also analyzes B4GALNT2 expression patterns across cell lines; the separate claim that the gene is functional is attached to citation 22. (LLM, conf=0.90)

  cites [23] Stenfelt, L. et al. Missense mutations in the C-terminal portion of the B4GALNT2-encoded glycosyltransferase underlying the Sd(a−) phenotype. Biochem. Biophys. Rep. 19, 100659 (2019). source

In the paper

“Third, the donors carried porcine endogenous retrovirus (PERV) sequences in their genome, which present a zoonotic risk, as PERV transmission to human cells in culture and their integration into the human genome have been demonstrated11,12.”

What the sources say

• Supported

  The passage labeled Abstract states that PERVs occur in the genome of all pig strains and can infect and integrate into human cells in culture. It describes transmission as a potential xenotransplantation risk, while noting that transmission to humans in clinical settings has not been shown. (LLM, conf=0.92)

  cites [11] Niu, D. et al. Inactivation of porcine endogenous retrovirus in pigs using CRISPR-Cas9. Science 357, 1303–1307 (2017). source
• ?Not checked — no full text of the sourcecites [12] Patience, C., Takeuchi, Y. & Weiss, R. A. Infection of human cells by an endogenous retrovirus of pigs. Nat. Med. 3, 282–286 (1997). source

In the paper

“Although overlapping in their anti-apoptotic activities, TNFAIP3 and HMOX1 achieve this outcome by their unique biological functions and dampen inflammation under distinct circumstances.”

What the sources say

• ?Not checked — no full text of the sourcecites [38] Lee, E. G. et al. Failure to regulate TNF-induced NF-κB and cell death responses in A20-deficient mice. Science 289, 2350–2354 (2000). source
• Partly supported

  The Discussion (page not specified in our copy) describes HO-1’s anti-inflammatory effects and identifies heme degradation products, including carbon monoxide, as contributors to cytoprotection and anti-apoptotic activity. The paper does not address TNFAIP3 or compare the two proteins’ functions or the circumstances in which they dampen inflammation. (LLM, conf=0.87)

  cites [39] Kapturczak, M. H. et al. Heme oxygenase-1 modulates early inflammatory responses. Am. J. Pathol. 165, 1045–1053 (2004). sourceExperimental — accuracy not yet measured

In the paper

“Xenograft haemotoxin and eosin and periodic acid–Schiff-stained samples were scored by current Banff criteria including C4d deposition42.”

What the sources say

• Supported

  The BANFF LESION SCORES section defines the Banff scoring criteria, including a C4d lesion score. DIAGNOSTIC WORK-UP OF BIOPSIES specifies hematoxylin-eosin and periodic acid–Schiff stains and calls C4d staining indispensable. The citation identifies the scoring criteria the citing authors applied to their xenograft samples; it does not attribute those samples to the guide. (LLM, conf=0.91)

  cites [42] Roufosse, C. et al. A 2018 reference guide to the Banff Classification of Renal Allograft Pathology. Transplantation 102, 1795–1814 (2018). source

In the paper

“We included both TNFAIP3 and HMOX1 in our donors, as both transgenes have been shown to provide benefit in vitro and in vivo40,41.”

What the sources say

• ?Not checked — no full text of the sourcecites [40] Oropeza, M. et al. Transgenic expression of the human A20 gene in cloned pigs provides protection against apoptotic and inflammatory stimuli. Xenotransplantation 16, 522–534 (2009). source
• Partly supported

  The Results report that HMOX1-transgenic endothelial cells were protected against apoptosis in vitro, and the Discussion reports resistance to rejection during ex vivo kidney perfusion. That is not an in vivo test of the transgenic pig kidneys, so this source does not establish the claimed in vivo benefit for the HMOX1 transgene. The separate TNFAIP3 citation is not assessed here. (LLM, conf=0.88)

  cites [41] Petersen, B. et al. Transgenic expression of human heme oxygenase-1 in pigs confers resistance against xenograft rejection during ex vivo perfusion of porcine kidneys. Xenotransplantation 18, 355–368 (2011). sourceExperimental — accuracy not yet measured

In the paper

“TNFAIP3 is a deubiquitinating enzyme and inhibits TNF-mediated apoptosis38, whereas HMOX1 is involved in the initial step of haem degradation and has been shown to have antioxidant and anti-inflammatory effects38,39.”

What the sources say

• ?Not checked — no full text of the sourcecites [38] Lee, E. G. et al. Failure to regulate TNF-induced NF-κB and cell death responses in A20-deficient mice. Science 289, 2350–2354 (2000). source
• Supported

  The Discussion states that HO-1 catalyzes the rate-limiting step in heme degradation. It also describes antioxidant mechanisms of HO-1 induction and evidence for its anti-inflammatory effects. Citation 39 is attached to the HMOX1 portion of the sentence, not the TNFAIP3 portion. (LLM, conf=0.93)

  cites [39] Kapturczak, M. H. et al. Heme oxygenase-1 modulates early inflammatory responses. Am. J. Pathol. 165, 1045–1053 (2004). source

In the paper

“Rejection was assessed by histopathological examination, based on the Banff classification of renal allograft pathology42, with modifications for xenotransplantation (Table 1 and Extended Data Table 1).”

What the sources say

• Supported

  The Abstract identifies this paper as a reference guide to the Banff Classification of Kidney Allograft Pathology. The BANFF LESION SCORES section says the classification assesses histopathological changes in renal transplant biopsies, particularly features of rejection. The citation is attached to the Banff classification; the xenotransplantation modifications are described as the citing authors’ own methods. (LLM, conf=0.92)

  cites [42] Roufosse, C. et al. A 2018 reference guide to the Banff Classification of Renal Allograft Pathology. Transplantation 102, 1795–1814 (2018). source

In the paper

“Although a porcine donor carrying a CMAH knockout is thought not to survive in OWMs considering complication from a CMAH knockout8,9, renal grafts derived from the 3KO.7TG ± RI porcine donor supported life long-term in cynomolgus monkeys, up to 758 days.”

What the sources say

• Partly supported

  The Discussion (page not provided) says that loss of Neu5Gc in triple-knockout pigs exposes an antigen recognized by Old World monkeys, creating a greater barrier to graft survival. The Results (page not provided) report shorter survival for triple-knockout kidneys in baboons than for GTKO kidneys, but do not establish that a graft with a CMAH knockout cannot survive in an Old World monkey: one triple-knockout recipient survived 61 days. The study tested triple-knockout organs, not the effect of CMAH knockout alone. (LLM, conf=0.86)

  cites [8] Yamamoto, T. et al. Old World monkeys are less than ideal transplantation models for testing pig organs lacking three carbohydrate antigens (triple-knockout). Sci. Rep. 10, 9771 (2020). sourceExperimental — accuracy not yet measured
• Partly supported

  The Discussion (page not available) reports that deleting CMAH on a GGTA1-knockout background increased baboon and rhesus antibody binding, a potential complication for pig-to-primate transplantation. The paper measured antibody binding, not graft survival, and does not say that CMAH-knockout porcine grafts are unlikely to survive in Old World monkeys. (LLM, conf=0.86)

  cites [9] Estrada, J. L. et al. Evaluation of human and non-human primate antibody binding to pig cells lacking GGTA1/CMAH/β4GalNT2 genes. Xenotransplantation 22, 194–202 (2015). sourceExperimental — accuracy not yet measured

In the paper

“In addition, data suggest that CMAH inactivation may produce a novel antigen xenogenic to the OWMs, which may be a target of preformed antibody binding, leading to complement activation in OWM serum8,9.”

What the sources say

• Supported

  The Discussion (page not provided) says that loss of Neu5Gc expression in triple-knockout pigs apparently reveals a ‘4th xenoantigen’ recognized by antibodies in Old World monkeys. The Results (page not provided) report that OWM IgM binding correlates with complement-dependent cytotoxicity against those pig cells. This supports the claim’s tentative wording. (LLM, conf=0.91)

  cites [8] Yamamoto, T. et al. Old World monkeys are less than ideal transplantation models for testing pig organs lacking three carbohydrate antigens (triple-knockout). Sci. Rep. 10, 9771 (2020). source
• Partly supported

  The Results and Discussion (page not available in our copy) report that pig cells lacking GGTA1 and CMAH bound more baboon and rhesus antibodies than cells lacking GGTA1 alone. This supports the suggested increase in antibody binding after CMAH inactivation, but the paper does not establish that a novel antigen causes it or report complement activation in Old World monkey serum. (LLM, conf=0.88)

  cites [9] Estrada, J. L. et al. Evaluation of human and non-human primate antibody binding to pig cells lacking GGTA1/CMAH/β4GalNT2 genes. Xenotransplantation 22, 194–202 (2015). sourceExperimental — accuracy not yet measured

In the paper

“Three KEC types were identified, including endothelial cells (PECAM1+PLVAP−EHD3−GATA5−), fenestrated endothelial cells (PECAM1+PLVAP+) and glomerular endothelial cells (PECAM1+EHD3+GATA5+) (Fig. 2d).”

In the paper

“WT KECs showed C3b deposition and 3KO KECs showed less deposition when incubated in the cynomolgus monkey serum.”

In the paper

“Among the three units, expression of the CAG and ssUBC units were higher, whereas expression of the ssEEF1A1 unit was at a lower level.”

In the paper

“When incubated with human serum, human umbilical vein endothelial cells do not show C3b deposition (Fig. 1d), suggesting no antibody binding and/or complete mitigation of complement activation.”

In the paper

“Binding by OWM serum IgG and IgM was similar, although substantial residual antibody binding (especially IgM binding) was detected with the 3KO KECs (Fig. 1b).”

In the paper

“As expected, the 3KO KECs lacked α-Gal, Neu5Gc and Sd(a) (Extended Data Fig. 3c) and the 3KO.7TG ± RI KECs expressed human transgenes, as analysed by flow cytometry (Extended Data Fig. 4a, CD46, CD55, EPCR and thrombomodulin; Extended Data Fig. 5a, CD47) and by western blot (Extended Data Fig. 5c, TNFAIP3).”

In the paper

“Althoughthe 3KO KECs showed significant reduction in C3b deposition compared with the WT KECs, they retained substantial residual C3b deposition (Fig. 1d), suggesting that porcine complement regulators are less effective in mitigating human complement activation.”

In the paper

“In comparison, OWMs express the Neu5Gc antigen.”

In the paper

“Furthermore, when subjected to fluid challenge studies, both groups responded to the challenges similarly, providing further evidence that the 3KO.7TG kidneys functioned normally (Extended Data Fig. 1d).”

In the paper

“For RNA-seq, contralateral, biopsy and necropsy samples were analysed and showed that all human transgenes were expressed and, again, those under the CAG and ssUBC promoters were at a higher level, whereas those under the ssEEF1A1 promoter were at a lower level (Extended Data Fig. 2a).”

In the paper

“All seven transgenic proteins were detected in the contralateral kidney, in both the glomeruli and the tubular cells, and expression was maintained in the renal graft at necropsy on post-transplantation day 176.”

In the paper

“These edits disrupted the three glycan synthesis genes (eight alleles; Extended Data Fig. 1b) (3KO), had a transgenic construct (referred to as Payload 15S (PL15S)) inserted hemizygously into the AAVS1 site (Extended Data Fig. 1a) (7TG), and inactivated the PERV elements (59 copies) (RI) (Extended Data Fig. 1c) carried in the Yucatan female cells Yuc25F.”

In the paper

“Enhanced clotting of human whole blood ex vivo was observed, measured as thrombin–antithrombin (TAT) complex formation, probably due to the inability of the porcine cells to generate aPC (Fig. 1g).”

In the paper

“Transcripts from the CAG and ssUBC cassettes were readily detected in the three endothelial cell types, whereas ssEEF1A1 cassette transcripts were found in endothelial cells and fenestrated endothelial cells, but not in glomerular endothelial cells.”

In the paper

“Nonetheless, both the human TNFAIP3 and HMOX1 proteins were detected in kidneys by IHC (Fig. 2c), and TNFAIP3 was detected by western blot (Extended Data Fig. 5c).”

In the paper

“When WT and 3KO porcine KECs were incubated with cynomolgus monkey serum, similar results were obtained, although higher residual C3b levels on 3KO porcine cells were observed, compared with those in human serum (Fig. 1e).”

In the paper

“In an antibody-binding assay, the porcine wild-type (WT) kidney endothelial cells (KECs) bound a high level of human IgG and IgM and binding was significantly reduced when the three xenoantigens were eliminated (3KO KECs) (Fig. 1b).”

In the paper

“The IHC photomicrographs from all completed NHP studies were scanned and quantified (Extended Data Fig. 2b–e), which showed that all transgenic proteins were detected.”

In the paper

“PL15S carries seven human genes (Supplementary Table 3), including CD46 and CD55 from the complement cascade, THBD and PROCR from the coagulation pathway, CD47, which is involved in innate immunity, and TNFAIP3 and HMOX1, which dampen ischaemia–reperfusion injury, apoptosis and inflammation.”

In the paper

“IHC confirmed that the three glycan antigens are expressed in the WT porcine kidney, but have been eliminated from the 3KO.7TG (donor ID 21077) and the 3KO.7TG.RI (donor ID A9161) kidneys, a pattern similar to that of the human kidney.”

In the paper

“WT porcine KECs bound human and NHP preformed antibodies, whereas 3KO porcine KECs showed markedly reduced antibody binding.”

In the paper

“Porcine KECs (WT or 3KO) did not produce aPC, whereas HUVECs readily produced aPC.”

In the paper

“Here we present data produced from the porcine donor, A9161, carrying the 3KO.7TG.RI genotype, whose kidney was transplanted into NHP recipient M6521 and achieved a graft survival time of 176 days.”

In the paper

“In addition, antibody-mediated rejection (AMR)/thrombotic microangiopathy (TMA) (M12121), TMA (CY1061, CY1062 and MB1027) and T cell-mediated rejection (TCMR) (M11521) were also observed.”

In the paper

“Other parameters, including serum albumin, serum potassium and blood platelet counts, generally remained within normal range, except when associated with renal failure (serum albumin and potassium) (Supplementary Fig. 5).”

In the paper

“Five out of the six 3KO ± RI kidneys showed evidence of acute tubular injury (ATI), a feature not included in the Banff criteria but representing general kidney injury.”

In the paper

“Using a SIRPα reporter cell line, we found that 3KO.7TG ± RI KECs expressing transgenic human CD47 (Extended Data Fig. 5a) activated the SIRPα signalling pathway, and pre-incubation with the anti-human CD47 antibody blocked SIRPα signalling in a dose-dependent manner (Fig. 3h).”

In the paper

“Although 3KO substantially reduced the level of pre-formed anti-porcine antibody binding, minor xenoantigens remain and contributed to residual antibody binding (Fig. 1b).”

In the paper

“When the incubation time was extended to 15 h, the 3KO modification was insufficient to fully protect the cells even from cell death by human serum, whereas 3KO.7TG ± RI KECs were protected against both human and cynomolgus monkey serum cytotoxicity, beyond the protection afforded by 3KO (Extended Data Fig. 4b).”

In the paper

“When incubated with human or cynomolgus monkey serum for 45 min, WT cells were lysed, whereas the 3KO modifications alone almost completely abolished complement-dependent cytotoxicity of human serum, but not cynomolgus monkey serum (Fig. 3c and Extended Data Fig. 4b,c).”

In the paper

“Unlike WT or 3KO porcine cells, 3KO.7TG ± RI KECs readily produced aPC (Fig. 3f), reduced TAT formation (Fig. 3g) and regulated coagulation of human whole blood as efficiently as human control cells (Extended Data Fig. 4h).”

In the paper

“In 3KO.7TG ± RI kidney recipients, those who developed renal failure exhibited TMA with or without AMR at time of euthanasia and only two showed evidence of ATI.”

In the paper

“In addition, 3KO KECs overexpressing EPCR, thrombomodulin or both were engineered and showed that both EPCR and thrombomodulin contributed to aPC production, with thrombomodulin having a more significant effect in this experimental system (Extended Data Fig. 4g).”

In the paper

“Among the long-term survivors, simple appearing, benign cysts in the kidney were observed.”

In the paper

“Using blocking antibodies (Extended Data Fig. 4f), we showed that both EPCR and thrombomodulin contributed to aPC production (Fig. 3f), with thrombomodulin having a more prominent effect.”

In the paper

“Unlike allotransplantation, T cell-mediated rejection was not a major pathology in xenotransplantation, with only one graft loss meeting Banff criteria for TCMR.”

In the paper

“IHC analyses of 3KO were performed for all contralateral kidney samples included in this study and 3KO phenotypes confirmed for all.”

In the paper

“Similar to humans, OWMs carry preformed antibodies to α-Gal and Sd(a), but unlike humans, they lack preformed antibodies to Neu5Gc, as they possess a functional CMAH gene25 (Fig. 1a).”

What the sources say

• ?Not checked — no full text of the sourcecites [25] Morozumi, K. et al. Significance of histochemical expression of hanganutziu–deicher antigens in pig, baboon and human tissues. Transplant. Proc. 31, 942–944 (1999). source

In the paper

“RNA-seq and immunohistochemistry (IHC) were performed for all completed 3KO.7TG ± RI renal transplants, except for donor 21405 whose contralateral kidney biopsy sample was not available (n = 11).”

In the paper

“Given the synergistic effect of the two proteins, we reasoned that coagulation may be better regulated when both are provided.”

In the paper

“The variable graft survival time may be inherent to kidney xenotransplantation and/or unique to the OWM model.”

In the paper

“In addition, the study was not blind for all involved.”

In the paper

“The pairing of a porcine donor with an NHP recipient was dictated by availability.”

In the paper

“Finally, activation of innate immune cells in the xenograft induces inflammation and apoptosis of endothelial cells13.”

What the sources say

• ?Not checked — no full text of the sourcecites [13] Robson, S. C., Am Esch, J. S. & Bach, F. H. Factors in xenograft rejection. Ann. N. Y. Acad. Sci. 875, 261–276 (1999). source

In the paper

“Thrombomodulin binds to thrombin and functions as a cofactor in the thrombin-induced activation of protein C, whereas EPCR binds to protein C and presents it to the thrombomodulin–thrombin activation complex.”

What the sources say

• ?Not checked — no full text of the sourcecites [36] Esmon, C. T. The protein C pathway. Chest 124, 26S–32S (2003). source

In the paper

“Therefore, the number of NHP transplants per porcine donor genotype group was empirically determined, based on historical data reported in literature and what could be reasonably achieved.”

In the paper

“In allotransplantation, TMA usually reflects an antibody-mediated or drug-related process, whereas in xenotransplantation, substantial species coagulation or complement regulatory protein incompatibility may contribute to TMA, even in the absence of de novo donor-specific antibody (dnDSA).”

In the paper

“Given the similar performance of KECs isolated from 3KO.7TG or 3KO.7TG.RI donors in functional assays (Fig. 3 and Extended Data Figs. 3–5), we analysed transplants performed with 3KO kidneys with and without RI as one group and 3KO.7TG and 3KO.7TG.RI kidneys as a second group.”

In the paper

“With these considerations, pathology samples were assessed with a ‘xenotransplantation-adapted’ criteria (Extended Data Table 1 and Supplementary Table 6).”

In the paper

“Although both thrombomodulin and EPCR contribute to aPC production, their mechanisms of action differ36.”

What the sources say

• ?Not checked — no full text of the sourcecites [36] Esmon, C. T. The protein C pathway. Chest 124, 26S–32S (2003). source

In the paper

“Monkeys were screened for the presence of anti-porcine IgG and IgM (described above), and animals with low anti-porcine IgG and anti-IgM were selected as recipients.”