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Xenotransplantation – State of the Art
Acta Veterinaria Scandinavica volume 45, Article number: S7 (2004)
Xenotransplantation is defined as the transplantation of tissues or cells between two zoologically different species. Due to the shortage of human organs for allotransplantation, i.e. transplantation between humans, essential efforts aimed at development of a donor for animal-human xenotransplantation have been performed over the last decade.
Rejections caused by xenotransplantation
Transplantation between two different species may give rise to two types of graft rejection. Between so-called discordant species a hyperacute rejection (HAR) occurs within minutes or hours. In so-called concordant species this type of rejection does not occur, but over days a delayed xenograft rejection (DXG)  will occur. In all transplanted organs, xeno- as well as allo-transplanted, chronic graft rejection (CRG) will occur after years.
Man is concordant with old world primates, but due to several reasons, such as breeding difficulties, risk of retroviral epidemics, concerns for the use of endangered species, etc., the discordant pig is considered the donor of choice.
HAR is primarily caused by initiation of the complement cascade. The antigen known to be activating the classical pathway is the alpha-gal epitope  (Figure 1); the epitope alternative to the primate AB0 blood group system in most non-primate mammals. In species, in which the antigen is absent, circulating antibodies are present. In humans IgG against the alpha-gal epitope counts for up to a total of 1% of circulating IgG . These antibodies probably derive from a reaction to members of the intestinal flora, especially Enterobacteriaceae spp., but also other types of infectious agents possess the alpha-gal epitope as a structural element in their cell walls .
Strategies to prevent rejection
Acute rejections after allotransplantation are generally prevented by immunosuppressive treatment, but although HAR may be prevented by cobra snake venom  and certain complement regulatory immunocomponents , it seems unlikely that complement cascade activation can be prevented by the same means without seriously interfering with the health and well-being of the recipient. Therefore, the donor or source animal needs to be modified to prevent recognition of complement-activating antigens. Transgenic techniques are the tools for this.
Insertion of genes may be done by the pronucleus microinjection method , which functions well in a range of animal species, including pigs. Three types of genes may be attractive to insert in a xenograft donor. The H-transferase, which fucosylates N-acetyl-lactosamine in competition with the gal-transferase (Figure 1), has been inserted in both mice [5, 28] and pigs [20, 28]. Cells of H-transferase transgenic animals show higher resistance towards human sera [20, 28], but in the mice produced by Chen and coworkers , it was found that although the alpha-gal epitope was only eliminated in those cells, in which it was also low in non-transgenic mice. Furthermore, it has been impossible to produce homozygotic H-transferase animals , i.e. too high an expression of the H-transferase in gal transferase producing animals seems to be toxic. The mechanisms behind this are not fully known, but lectin binding studies show that the structure of the cell walls of H-transferase transgenic animals is changed far beyond the sole deficit of the alpha-gal epitope and presence of the H antigen. Some crypt antigen, Tn and Forssman, may even become exposed on the cellular wall, which may increase the risk of DXG of organs from a H-transferase transgenic donor . This reduces the likelihood of such a transgenic animal becoming a future xenograft donor. Another approach attempted in mice is to insert genes coding for the enzyme, alpha-1,3-galatosidase, which resynthesises the alpha-gal epitope into a compound not attacked by natural antibodies produced by humans. These mice tended to secrete more proteins in the urine than the wild type. Furthermore, low body weights, partial damage to hair growth and early death occurred more frequently . Therefore, neither does this approach seem to be applicable for the production of a xenograft source animal.
A third and more successful approach may be to insert genes, which identify the tissue as homologue to the complement system. Therefore, it binds parts of the proteins involved in the complement cascade, thereby disabling both the classical and the alternative pathway, so-called complement-regulatory factors (CRF) (Figure 2). The most important ones in xenotransplantation research are human decay accelerating factor (H-DAF), membrane cofactor protein (MCP) and CD 59. Both mice and pigs transgenic for CRF's have been produced, and the transgenic animals have been combined with one another and with animals changed on alpha-gal related genes.
To knock-out loci for the upstart of a transgenic source animal colony demands the use of the embryonic stem cell method for transfection ; a method, which so far is only available in mice. However, pig cloning and nuclear transfer techniques  represent a reasonable alternative. Two groups reported the successful production of alpha-gal knock out mice in the mid-nineties , and around New Year 2002/2003 two other groups reported the successful production of alpha-gal knock out pigs by cloning and nuclear transfer [8, 21]. Although those pigs appearing in these two publications were hemizygotic, one of these groups has, by cloning of cells from these pigs, successfully produced homozygotic alpha-gal knock out pigs, which seem to be fully viable .
Transplantation experiments from pigs to primates
The baboon, and to a lesser extent the rhesus monkey, are the animals of choice for modelling pig to human xenotransplantation. Transplantation of non-transgenic pig organs to these primates results in HAR within hours . Transplantation from CRF transgenic pigs to baboons has been attempted, primarily with the heart  and kidney  and it is evident that CRF's prevent HAR, while DXG occurs within one or two weeks [1, 4]. Grafts become infiltrated by macrophages, T cells and B cells and the recipients develop thrombocytopenia and abnormalities in coagulation . Supplementary supportive treatments of recipients with antithymocyte serum , C1 inhibitor or cyclophosphamide may prolong graft survival up to 40 days . Extracorporal perfusion using CRF transgenic pig liver cells seem to offer an improvement compared to perfusion using non-transgenic pig cells . Results of transplantation of organs from alpha-gal knock out pigs to primates have not yet been published.
The term xenozoonosis covers infections that may spread from the transplanted organ to the recipient. One might fear that ordinary zoonotic infections known to be present in pigs, such as encephalymyocarditis virus  and rotaviruses  may pose a risk for the recipients, but these infections are easy to handle during the animal production phase and would in any case mostly be a risk to the individual; a risk, that probably would be outweighed by the benefits from xenotransplantation.
Much more concern has been related to the risk of activating porcine endogenous retroviruses (PERV) in the recipients, which hereafter may spread the infection to relatives and, thereby, cause a world-wide retroviral epidemic. The discussion was initiated by the experience that retrovirus produced by the PK-15 kidney cell line (PERV-PK) infected human kidney 293 cell lines and co-cultivation of X-irradiated PK-15 cells with human cells resulted in a broader range of human cell infection, including human diploid fibroblasts and B- and T-cell lines . Host range analyses have shown that type A and B PERV Env's have wider host ranges including several human cell lines, compared with type C PERV, which infect only one human cell line . However, in a retrospective study 15 immunosuppressed baboons were tested for a specific immune response against PERV after transplantation of porcine endothelial cells, mononuclear blood cells, and lungs. Anti-PERV antibody expression was analyzed with peptide-based, enzyme-linked immunosorbent assays and highly sensitive Western Blot assay showing no evidence of PERV specific humoral immune response . Retrospective studies of patients exposed to living pig tissues have neither been able to show such cross species transmission .
Functional problems related to xenotransplanted organs
Physiological and anatomical differences between man and pig may be speculated to be obstructive for successful xenotransplantation in various ways. E.g. man is the only animal living in an upright position, and therefore gravity may have a different impact on the lung, heart, liver, and kidney. Differences on the humoral and enzymatic basis may be even more pronounced. Hormones and enzymes are regulated by complicated and often species-specific mechanisms, e.g. growth hormones may stimulate the xenografts to unrestricted growth. If enzymes are not removed by the liver they may reach a level not compatible with life. Products like albumin are carriers for other molecules and need to be compatible for binding sites. Essential porcine and human proteins differ significantly, e.g. albumin have an amino acid identity of less than 65%, erythropoetin (EPO) of less than 82%, and complement of less than 70% . Therefore, especially the liver at present seems to be an unlikely candidate for xenotransplantation.
Future perspectives of xenotransplantation
The production of the alpha-gal knock out pig has been a major step-forward in xenotransplantation research. Although the road towards xenotransplantation still seems long, especially for complicated organs such as the liver, the fact that research in the future may be more directed against DXG, which may be handled by other means than transgenesis, e.g. immunosuppression, gives rise to optimism. Also, xenotransplantation with transgenic organs may in the future offer a higher quality of transplantation if the organs could be modified in such a way, that all types of rejection could be omitted, thus avoiding the life-long treatment with immunosuppressives,
Adams DH, Kadner A, Chen RH, Farivar RS: Human membrane cofactor protein (MCP, CD 46) protects transgenic pig hearts from hyperacute rejection in primates. Xenotransplantation. 2001, 8: 36-40. 10.1046/j.0908-665X.2000.00085.x.
World's first cloned double knock-out pigs lack both copies of gene involved in hyperacute rejection in humans. Roslin, Edinburgh. 2002
Brewer LA, Lwamba HCM, Murtaugh MP, Palmenberg AC, Brown C, Njenga MK: Porcine encephalomyocarditis virus persists in pig myocardium and infects human myocardial cells. J Virol. 2001, 75: 11621-11629. 10.1128/JVI.75.23.11621-11629.2001.
Buhler L, Yamada K, Kitamura H, Alwayn IPJ, Basker M, Appel JZ, Colvin RB, White-Scharf ME, Sachs DH, Robson SC, Awwad M, Cooper DKC: Pig kidney transplantation in baboons: Anti-Gal alpha 1-3Gal IgM alone is associated with acute humoral xenograft rejection and disseminated intravascular coagulation. Transplantation. 2001, 72: 1743-1752. 10.1097/00007890-200112150-00007.
Chen CG, Fisicaro N, Shinkel TA, Aitken V, Katerelos M, vanDenderen BJW, Tange MJ, Crawford RJ, Robins AJ, Pearse MJ, DApice AJF: Reduction in Gal-alpha 1,3-Gal epitope expression in transgenic mice expressing human H-transferase. Xenotransplantation. 1996, 3: 69-75. 10.1111/j.1399-3089.1996.tb00121.x.
Chen RH, Kadner A, Mitchell RN, Adams DH: Mechanism of delayed rejection in transgenic pig-to-primate cardiac xenotransplantation. J Surg Res. 2000, 90: 119-125. 10.1006/jsre.2000.5864.
Cowan PJ, Aminian A, Barlow H, Brown AA, Chen CG, Fisicaro N, Francis DMA, Goodman DJ, Han WR, Kurek M, Nottle MB, Pearse MJ, Salvaris E, Shinkel TA, Stainsby GV, Stewart AB, d'Apice AJF: Renal xenografts from triple-transgenic pigs are not hyperacutely rejected but cause coagulopathy in non-immunosuppressed baboons. Transplantation. 2000, 69: 2504-2515. 10.1097/00007890-200006270-00008.
Dai YF, Vaught TD, Boone J, Chen SH, Phelps CJ, Ball S, Monahan JA, Jobst PM, McCreath KJ, Lamborn AE, Cowell-Lucero JL, Wells KD, Colman A, Polejaeva IA, Ayares DL: Targeted disruption of the alpha 1,3-galactosyltransferase gene in cloned pigs. Nature Biotechnol. 2002, 20: 251-255. 10.1038/nbt0302-251.
Eyestone WH, Campbell KH: Nuclear transfer from somatic cells: applications in farm animal species. J Reprod Fertil Suppl. 1999, 54: 489-497.
Galili U, Mandrell RE, Hamadeh RM, Shohet SB, Griffiss JM: Interaction between human natural anti-alpha-galactosyl immunoglobulin G and bacteria of the human flora. Infect Immun. 1988, 56: 1730-1737.
Galili U, Rachmilewitz EA, Peleg A, Flechner I: A unique natural human IgG antibody with anti-alpha-galactosyl specificity. J exp Med. 1984, 160: 1519-1531. 10.1084/jem.160.5.1519.
Gewurz H, Clark DS, Cooper MD, Varco RL, Good RA: Effect of cobra venom-induced inhibition of complement activity on allograft and xenograft rejection reactions. Transplantation. 1967, 5: 1296-1303. 10.1097/00007890-196709000-00008.
Ghanekar A, Lajoie G, Luo YG, Yang HJ, Choi J, Garcia B, Cole EH, Greig PD, Cattral MS, Phillips MJ, Cardella CJ, Levy GA, Zhong R, Grant DR: Improvement in rejection of human decay accelerating factor transgenic pig-to-primate renal xenografts with administration of rabbit antithymocyte serum. Transplantation. 2002, 74: 28-35. 10.1097/00007890-200207150-00006.
Gordon JW, Scangos GA, Plotkin DJ, Barbosa JA, Ruddle FH: Genetic transformation of mouse embryos by microinjection of purified DNA. Proc nat Acad Sci USA. 1980, 77: 7380-7384. 10.1073/pnas.77.12.7380.
Gossler A, Doetschman T, Korn R, Serfling E, Kemler R: Transgenesis by means of blastocyst-derived embryonic stem cell lines. Proc nat Acad Sci USA. 1986, 83: 9065-9069. 10.1073/pnas.83.23.9065.
Hammer C: Physiological obstacles after xenotransplantation. Xenotransplantation. 1998, 862: 19-27.
Hansen AK, Farlov H, Bollen P: Microbiological monitoring of laboratory pigs. Lab Anim. 1997, 31: 193-200. 10.1258/002367797780596248.
Herring C, Cunningham DA, Whittam AJ, Fernandez-Suarez XM, Langford GA: Monitoring xenotransplant recipients for infection by PERV. Clin Biochem. 2001, 34: 23-27. 10.1016/S0009-9120(00)00187-9.
Ikematsu S, Kaname T, Ozawa M, Yonezawa S, Sato E, Uehara F, Obama H, Yamamura K, Muramatsu T: Transgenic mouse lines with ectopic expression of alpha-1,3-galactosyltransferase – production and characteristics. Glycobiology. 1993, 3: 575-580. 10.1093/glycob/3.6.575.
Koike C, Kannagi R, Takuma Y, Akutsu F, Hayashi S, Hiraiwa N, Kadomatsu K, Muramatsu T, Yamakawa H, Nagai T, Kobayashi S, Okada H, Nakashima I, Uchida K, Yokoyama I, Takagi H: Introduction of alpha(1,2)-fucosyltransferase and its effect on alpha-Gal epitopes in transgenic pig. Xenotransplantation. 1996, 3: 81-86. 10.1111/j.1399-3089.1996.tb00123.x.
Lai LX, Kolber-Simonds D, Park KW, Cheong HT, Greenstein JL, Im GS, Samuel M, Bonk A, Rieke A, Day BN, Murphy CN, Carter DB, Hawley RJ, Prather RS: Production of alpha-1,3-galactosyltransferase knockout pigs by nuclear transfer coning. Science. 2002, 295: 1089-1092. 10.1126/science.1068228.
Martin U, Steinhoff G: Porcine endogenous Retroviruses (PERV): In vitro artifact or big problem for xenotransplantation?. Dtsch tierärztl Wchschr. 1999, 106: 146-149.
Martin U, Tacke SJ, Simon AR, Schroder C, Wiebe K, Lapin B, Haverich A, Denner J, Steinhoff G: Absence of PERV specific humoral immune response in baboons after transplantation of porcine cells or organs. Transplant Intern. 2002, 15: 361-368. 10.1111/j.1432-2277.2002.tb00179.x.
Mollnes TE, Fiane AE: Role of complement in xenotransplantation. Allergy. 2002, 57: 75-78. 10.1034/j.1398-9995.57.s72.10.x.
Patience C, Takeuchi Y, Weiss RA: Infection of human cells by an endogenous retrovirus of pigs. Nature Med. 1997, 3: 282-286. 10.1038/nm0397-282.
Rees MA, Butler AJ, Chavez-Cartaya G, Wight DGD, Casey ND, Alexander G, Khuder SA, White DJG, Friend PJ: Prolonged function of extracorporeal hdaf transgenic pig livers perfused with human blood. Transplantation. 2002, 73: 1194-1202. 10.1097/00007890-200204270-00003.
Salerno CT, Kulick DM, Yeh CG, Guzman-Paz M, Higgins PJ, Benson BA, Park SJ, Shumway SJ, Bolman RM, Dalmasso AP: A soluble chimeric inhibitor of C3 and C5 convertases, complement activation blocker-2, prolongs graft survival in pig-to-rhesus monkey heart transplantation. Xenotransplantation. 2002, 9: 125-134. 10.1034/j.1399-3089.2002.1o042.x.
Sharma A, Okabe J, Birch P, McClellan SB, Martin MJ, Platt JL, Logan JS: Reduction in the level of Gal(alpha 1,3)Gal in transgenic mice and pigs by the expression of an alpha(1,2)fucosyltransferase. Proc nat Acad Sci USA. 1996, 93: 7190-7195. 10.1073/pnas.93.14.7190.
Takeuchi Y, Patience C, Magre S, Weiss RA, Banerjee PT, Le Tissier P, Stoye JP: Host range and interference studies of three classes of pig endogenous retrovirus. J Virol. 1998, 72: 9986-9991.
Tearle RG, Tange MJ, Zannettino ZL, Katerelos M, Shinkel TA, vanDenderen BJW, Lonie AJ, Lyons I, Nottle MB, Cox T, Becker C, Peura AM, Wigley PL, Crawford RJ, Robins AJ, Pearse MJ, DApice AJF: The alpha-1,3-galactosyltransferase knockout mouse – Implications for xenotransplantation. Transplantation. 1996, 61: 13-19. 10.1097/00007890-199601150-00004.
Vangerow B, Hecker JM, Lorenz R, Loss M, Przemeck M, Appiah R, Schmidtko J, Jalali A, Rueckoldt H, Winkler M: C1-inhibitor for treatment of acute vascular xenograft rejection in cynomolgus recipients of h-DAF transgenic porcine kidneys. Xenotransplantation. 2001, 8: 266-272. 10.1034/j.1399-3089.2001.00130.x.
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Hansen, A.K., Dahl, K., Sørensen, D.B. et al. Xenotransplantation – State of the Art. Acta Vet Scand 45, S7 (2004). https://doi.org/10.1186/1751-0147-45-S1-S7
- Complement Cascade
- Membrane Cofactor Protein
- Porcine Endothelial Cell
- Specific Humoral Immune Response
- Human Cell Infection