Production of homozygous alpha-1,3-galactosyltransferase knockout pigs by breeding and somatic cell nuclear transfer

We report here our experience regarding the production of double or homozygous Gal knockout (Gal KO) pigs by breeding and somatic cell nuclear transfer (SCNT). Large White x Landrace female heterozygous Gal KO founders produced using SCNT were mated with Hampshire or Duroc males to produce a F1 generation. F1 heterozygous pigs were then bred to half-sibs to produce a F2 generation which contained Gal KO pigs. To determine the viability of mating Gal KO pigs with each other, one female F2 Gal KO pig was bred to a half-sib and subsequently a full-sib Gal KO. F1 and F2 heterozygous females were also mated to F2 Gal KO males. All three types of matings produced Gal KO pigs. To produce Gal KO pigs by SCNT, heterozygous F1s were bred together and F2 fetuses were harvested to establish primary cultures of Gal KO fetal fibroblasts. Gal KO embryos were transferred to five recipients, one of which became pregnant and had a litter of four piglets. Together our results demonstrate that Gal KO pigs can be produced by breeding with each other and by SCNT using Gal KO fetal fibroblasts.     

Knockdown of porcine endogenous retrovirus (PERV) expression by PERV-specific shRNA in transgenic pigs

Abstract: Background: Xenotransplantation using porcine cells, tissues or organs may be associated with the transmission of porcine endogenous retroviruses (PERVs). More than 50 viral copies have been identified in the pig genome and three different subtypes of PERV were released from pig cells, two of them were able to infect human cells in vitro. RNA interference is a promising option to inhibit PERV transmission. Methods: We recently selected an efficient si (small interfering) RNA corresponding to a highly conserved region in the PERV DNA, which is able to inhibit expression of all PERV subtypes in PERV‐infected human cells as well as in primary pig cells. Pig fibroblasts were transfected using a lentiviral vector expressing a corresponding sh (short hairpin) RNA and transgenic pigs were produced by somatic nuclear transfer cloning. Integration of the vector was proven by PCR, expression of shRNA and PERV was studied by in‐solution hybridization analysis and real‐time RT PCR, respectively. Results: All seven born piglets had integrated the transgene. Expression of the shRNA was found in all tissues investigated and PERV expression was significantly inhibited when compared with wild‐type control animals. Conclusion: This strategy may lead to animals compatible with PERV safe xenotransplantation.     

Buccal mucosal cell immunohistochemistry: a simple method of determining the ABH phenotype of baboons, monkeys, and pigs

BACKGROUND: Baboons and monkeys fail to express ABH antigens on red blood cells (RBCs), and the A or H antigens are expressed only weakly on the surface of pig RBCs. Baboons and monkeys have been previously blood typed by detection of ABH antigens in the saliva after administration of pilocarpine. A reliable method to ABH type pigs is by immunohistochemical staining of renal distal tubules in kidney biopsies. We describe a simple and efficient method to blood type baboons, monkeys, and pigs. METHODS: Baboons (n = 14) and cynomolgus monkeys (n = 8) were blood typed by staining of buccal mucosal smears and by determining the presence of serum anti-A or B antibodies following human type O adsorption. Pigs (n = 11) were tested for ABH type by immunohistochemistry for the presence of A, B, and H antigens using monoclonal antibodies on (i) renal biopsies, (ii) RBCs, and (iii) buccal mucosal smears, without pilocarpine administration, in addition, (iv) after adsorption on human type O RBCs to remove anti-human antibodies, the pig sera were typed by hemagglutination assay for the presence of anti-A or B antibodies using human A and B RBCs. RESULTS AND CONCLUSIONS: There was complete consistency among the results obtained using all of the above methods, except that no determination could be made from staining of RBCs in one pig. Staining of buccal mucosal cells proved to be the preferred method in all three species because: (i) expression of A or H antigen is weak on pig RBCs, making an accurate blood type determination difficult, and A, B, and H expression is non-existent on baboon and monkey RBCs, (ii) neither venepuncture nor organ biopsy is necessary, (iii) time-consuming adsorption of anti-human antibodies from the sera of the test animal is not required, and (iv) it proved a quick method of evaluation.     

Justification of specific genetic modifications in pigs for clinical organ xenotransplantation

Xenotransplantation research has made considerable progress in recent years, largely through the increasing availability of pigs with multiple genetic modifications. We suggest that a pig with nine genetic modifications (ie, currently available) will provide organs (initially kidneys and hearts) that would function for a clinically valuable period of time, for example, >12 months, after transplantation into patients with end‐stage organ failure. The national regulatory authorities, however, will likely require evidence, based on in vitro and/or in vivo experimental data, to justify the inclusion of each individual genetic modification in the pig. We provide data both from our own experience and that of others on the advantages of pigs in which (a) all three known carbohydrate xenoantigens have been deleted (triple‐knockout pigs), (b) two human complement‐regulatory proteins (CD46, CD55) and two human coagulation‐regulatory proteins (thrombomodulin, endothelial cell protein C receptor) are expressed, (c) the anti‐apoptotic and “anti‐inflammatory” molecule, human hemeoxygenase‐1 is expressed, and (d) human CD47 is expressed to suppress elements of the macrophage and T‐cell responses. Although many alternative genetic modifications could be made to an organ‐source pig, we suggest that the genetic manipulations we identify above will all contribute to the success of the initial clinical pig kidney or heart transplants, and that the beneficial contribution of each individual manipulation is supported by considerable experimental evidence.     

A line of transgenic pigs in which the expression of human decay-accelerating factor by endothelial cells is increased in the presence of inflammatory stimuli

Abstract: Aortic endothelial cell cultures (PAE) from four lines of pigs transgenic for human decay-accelerating factor (hDAF) have been used to study the response to the inflammatory stimuli bacterial lipopolysaccharide (LPS) and recombinant human TNF-α. Human umbilical vein endothelial cells (HUVEC) and PAE from normal, non-transgenic pigs were used as controls. The expression of hDAF and E-selectin on the cell surface was determined by flow cytometry. After overnight incubation, HUVEC, normal and transgenic PAE increased the relative expression of E-selectin 2–5-fold in response to LPS (25 μg/ml), and 5–40-fold in response to TNF-α(10 ng/ml). In both normal and transgenic PAE the increase in expression of E-selectin in response to TNF-α was maximal at 4 hr and significantly decreased after 20 hr. There was no significant increase in DAF expression by HUVECs in response to LPS or TNF-α, and three of the four lines of transgenic pigs studied did not increase expression of hDAF in response to either stimulus. However, endothelial cells from the transgenic line A74 exhibited a dose-dependent increase in expression of hDAF in response to LPS and TNF-α. A study of the time course of up-regulation triggered by incubation with TNF-α showed that, in contrast to the up-regulation of E-selectin, hDAF expression continued to increase for at least 3 days. This response may afford additional protection to organs from this line of transgenic pigs.     

Alterations in the Coagulation Profile in Renal Pig-to-Monkey Xenotransplantation

Five monkey recipients of a porcine renal xenograft were studied to determine the relationship between fibrin formation in acute humoral xenograft rejection (AHXR) and procoagulant and anticoagulant factor levels to establish whether changes in coagulation parameters could be used to predict AHXR and determine whether AHXR is associated with overt disseminated intravascular coagulopathy (DIC) in this model. Variable degrees of compensated consumptive coagulopathy were observed in each primate. Elevated thrombin-antithrombin (TAT), F1+2 and D-dimer levels consistent with thrombin generation and fibrin formation were recorded. There was no consumption of the main clotting inhibitors (including antithrombin) or a progressive, severe drop in fibrinogen levels and platelet counts, although grafts were left in situ. After transplantation, D-dimer levels remained persistently high, so they were of limited value in defining this coagulopathy. At post mortem, no cases of multiorgan involvement typical of overt DIC were observed. The lack of a rapid postoperative recovery of clotting inhibitor levels after transplantation was invariably associated with early poor outcome. This study shows that AHXR is associated with various degrees of compensated consumptive coagulopathy in our pig-to-primate model. No clear relationship was found between coagulation parameter levels and graft outcome.     

WZS-pig is a potential donor alternative in corneal xenotransplantation

PURPOSE: To explore the characteristics of rejection of Wuzhishan (WZS) pig-to-rhesus monkey corneal transplants and to evaluate WZS pig corneas as potential donor material for a clinical setting. METHODS: Wuzhishan pigs were used as donors and rhesus monkeys as recipients for corneal xenotransplantation. Eighteen rhesus monkeys were divided into three groups. Groups 1 and 2 underwent penetrating corneal xenotransplantation, while group 3 underwent lamellar corneal xenotransplantation. Only group 2 received subconjunctival injections with betamethasone for 3 months. All xenografts were evaluated by slit-lamp microscopy. Two recipients in each group were killed for corneal histopathological staining at 30 days after surgery. The concentrations of cytokines in the aqueous humor were detected by cytometric bead array. RESULTS: Rejection of the xenografts occurred at approximately 15 days after surgery in group 1. Rejection was delayed for more than 4 months by conjunctival injection with betamethasone (group 2). Use of lamellar corneal xenografts (group 3) maintained corneal transparency for more than 3 months. Histopathological examination in group 1 showed that the xenografts were infiltrated with inflammatory cells. The corneal endothelia were destroyed and exudative membranes were formed in the anterior chamber. However, in the betamethasone-treated group, the corneal xenografts showed only minimal edema and almost no inflammatory cell infiltrate. The corneal endothelia were intact and there was no exudative membrane formation. The concentration of interleukin (IL)4, IL5 and IL10 showed an increased shift 3 weeks after surgery in group 1 and 2, but no change of cytokine's concentration was found in group 3. CONCLUSION: Rejection of pig-rhesus xenografts occurred early in penetrating corneal transplantation, but not in lamellar corneal transplantation. The endothelium of the xenograft might be a primary target of immune attack. Corticosteroid treatment inhibited rejection of the corneal xenografts.     

Double knockout pigs deficient in N‐glycolylneuraminic acid and Galactose α‐1,3‐Galactose reduce the humoral barrier to xenotransplantation

Background

Clinical xenotransplantation is not possible because humans possess antibodies that recognize antigens on the surface of pig cells. Galα‐1,3‐Gal (Gal) and N‐glycolylneuraminic acid (Neu5Gc) are two known xenoantigens.

Methods

We report the homozygous disruption of the α1, 3‐galactosyltransferase (GGTA1) and the cytidine monophosphate‐N‐acetylneuraminic acid hydroxylase (CMAH) genes in liver‐derived female pig cells using zinc‐finger nucleases (ZFNs). Somatic cell nuclear transfer (SCNT) was used to produce healthy cloned piglets from the genetically modified liver cells. Antibody‐binding and antibody‐mediated complement‐dependent cytotoxicity assays were used to examine the immunoreactivity of pig cells deficient in Neu5Gc and Gal.

Results

This approach enabled rapid production of a pig strain deficient in multiple genes without extensive breeding protocols. Immune recognition studies showed that pigs lacking both CMAH and GGTA1 gene activities reduce the humoral barrier to xenotransplantation, further than pigs lacking only GGTA1.

Conclusions

This technology will accelerate the development of pigs for xenotransplantation research.     

Monomorphic and polymorphic carbohydrate antigens on pig tissues: implications for organ xenotransplantation in the pig-to-human model

The existence of the Gal epitope in 137 pigs belonging to 23 different breeds suggests that this antigen is either monomorphic or occurs at a high incidence in the porcine species. Its histological location at the surface of pig vascular endothelial cells makes it a target for human natural anti-Gal antibodies and complement, which may be responsible for the hyperacute vascular rejection of transplanted pig organs. The precursor carbohydrate chain (N-acetyllactosamine) and NeuAc-substituted epitopes are also exposed at the surface of pig vascular endothelium and were found in all pigs in this study. However, humans also have these two epitopes on vascular endothelium and, consequently, have not made natural antibodies against these carbohydrate antigens. Therefore, these two pig epitopes cannot be the main target of the hyperacute vascular rejection process. Three pig phenotpyes-A+(51%), A:H+(38%), and A-H-I+(11%) were identified among 37 Large-white pigs by the presence of polymorphic A, H, and I carbohydrate antigens on the brush border of the surface epithelium of small intestine. These antigens were also present in other exocrine secretions but were not detected on vascular endothelium of the same pigs, suggesting that they are not involved in the hyperacute vascular rejection, although the pig A tissue antigen can induce an immune response in 0 or B blood group recipients. Once the problem of the initial hyperacute vascular rejection directed against the Gal epitope is overcome, typing donor pigs for A, H, and I, as well as for the protein swine leukocyte antigens (SLA) and other pig antigens, may help in elucidating antigens involved in acute or chronic xenograft rejection.     

Ureteral Stenosis in HDAF Pig‐to‐Primate Renal Xenotransplantation: A Phenomenon Related to Immunological Events?

The aim of this study was to analyze the incidence of ureteral stenosis in a life-supporting human decay-accelerating factor (hDAF) transgenic pig-to-cynomolgus monkey kidney transplantation model and determine the role of possible immunological events in its pathogenesis. Thirty consecutive bi-nephrectomized cynomolgus monkeys received a kidney from hDAF transgenic pigs with or without a ureteral stent. Four monkeys were euthanized prematurely after transplantation. In the remaining 26 cases, the mean survival was 24 +/- 19 days. Except in one case, there was a close relationship between ureter and kidney in terms of type and severity of rejection. There were six ureteral stenoses; five were repaired by stent positioning and resurgery extended survival for an additional 16 +/- 10 days. The stenotic ureters showed diffuse acute humoral xenograft rejection (AHXR), while all cases with no or only focal signs of ureteral rejection never revealed ureteral obstruction. Use of a ureteral stent extends the survival of a xenografted primate, thereby helping to clarify the immunological events surrounding the onset of AHXR in kidneys in long-term xenograft recipients.     

2007 IXA Presidential Address. Progress toward an ideal source animal: opportunities and challenges in a changing world

Abstract:  Xenotransplantation holds great promise for the treatment of end-stage organ failure and tissue injury. Recent advances in our understanding of the immunological hurdles to xenotransplantation and the development of genetic modification approaches to overcome them increase the likelihood of success. Exciting advances in these areas were presented at the Joint Congress of the IXA, the Cell Transplant Society and the International Pancreas and Islet Transplant Association in Minneapolis in September, 2007. However, in order to minimize infectious risks and encourage positive public perception of xenotransplantation, clinical trials should be initiated with appropriate monitoring and oversight procedures in place and only when there is high expectation of clinical benefit.     

Genetic and functional evaluation of the level of inbreeding of the Westran pig: a herd with potential for use in xenotransplantation

BACKGROUND: The Westran pig has been purposely inbred for use in xenotransplantation. The herd originated in the wild from a limited gene pool and has been inbred by repeated full-sib matings for nine generations. METHODS: The aim of this study was to evaluate the level of inbreeding by functional assays, such as bi-directional MLR and reciprocal skin grafts between herd members, and by genetic analysis using highly polymorphic genetic markers to calculate the level of inbreeding. RESULTS: The MLR between herd members were non-reactive whereas there was a prompt response to third party pig lymphocytes, indicative of a normal immune responsiveness in Westran pigs but isogenicity of the major histocompatibility complex. Skin grafts between male siblings or female sibling skin grafts on male recipients showed prolonged survival but with few exceptions did not survive beyond 100 days suggesting that by the fifth generation the Westran herd was still mismatched at minor histocompatibility antigens. This level of functional inbreeding was confirmed by microsatellite analysis of highly polymorphic markers, which showed that 52 of 53 chromosomally dispersed markers were fixed by the ninth generation. This level of fixation was consistent with 19 to 20 generations of full-sibling inbreeding. The calculated inbreeding coefficient at generation 10 was 0.98159. CONCLUSIONS: This analysis confirms that the Westran pig is highly inbred and we propose that analysis of chromosomally dispersed highly polymorphic markers is an accurate and reproducible method for assessing the level of inbreeding of a pig herd.     

Acute cell-mediated rejection in orthotopic pig-to-mouse corneal xenotransplantation

Abstract: Background:  To investigate the role of T cells and natural killer (NK) cells in mediating corneal xenograft rejection in a pig-to-mouse model.
Methods:  Pig corneas were orthotopically transplanted into BALB/c, C57BL/6, nude, severe combined immunodeficiency (SCID), and NOD/SCID/γc^null (NOG) mice. Graft survival was clinically assessed by slit-lamp biomicroscopy and median survival times (MST) were calculated. The rejected grafts were histologically evaluated using antibodies against CD4, CD8, NK1.1, and F4/80.
Results:  The pig corneal xenografts were acutely rejected by BALB/c and C57BL/6 mice (MST 9.0 days), while nude, SCID and NOG mice rejected pig corneas in a more delayed fashion (MST 16.0, 16.4, and 16.9 days, respectively). The majority of infiltrating cells found in rejected grafts in C57BL/6 mice were macrophages and CD4⁺ T cells, while CD8⁺ T cells and NK cells were rarely found. The grafts in nude mice had markedly decreased inflammatory infiltration with small numbers of macrophages and CD4⁺ T cells. Infiltration was even more modest in grafts in SCID and NOG mice.
Conclusions:  T cells play an important role in acute rejection of pig corneal xenografts in mice, although acute rejection is not solely the result of T-cell-mediated immunity. NK cells are less likely to be involved in the rejection process.