GalT-KO Pigs Research Articles

Expression of human CD47 in pig glomeruli prevents proteinuria and prolongs graft survival following pig-to-baboon xenotransplantation.

Nephrotic syndrome is a common complication of pig-to-baboon kidney xenotransplantation (KXTx) that adversely affects outcomes. We have reported that upregulation of CD80 and down-regulation of SMPDL-3b in glomeruli have an important role in the development of proteinuria following pig-to-baboon KXTx. Recently we found induced expression of human CD47 (hCD47) on endothelial cells and podocytes isolated from hCD47 transgenic (Tg) swine markedly reduced phagocytosis by baboon and human macrophages. These observations led us to hypothesize that transplanting hCD47 Tg porcine kidneys could overcome the incompatibility of the porcine CD47-baboon SIRPα interspecies ligand-receptor interaction and prevent the development of proteinuria following KXTx. Ten baboons received pig kidneys with vascularized thymic grafts (n=8) or intra-bone bone marrow transplants (n=2). Baboons were divided into three groups (A, B, and C) based on the transgenic expression of hCD47 in GalT-KO pigs. Baboons in Group A received kidney grafts with expression of hCD47 restricted to glomerular cells (n=2). Baboons in Group B received kidney grafts with high expression of hCD47 on both glomerular and tubular cells of the kidneys (n=4). Baboons in Group C received kidney grafts with low/no glomerular expression of hCD47, and high expression of hCD47 on renal tubular cells (n=4). Consistent with this hypothesis, GalT-KO/hCD47 kidney grafts with high expression of hCD47 on glomerular cells developed minimal proteinuria. However, high hCD47 expression in all renal cells including renal tubular cells induced an apparent destructive inflammatory response associated with upregulated thrombospondin-1. This response could be avoided by a short course of weekly anti-IL6R antibody administration, resulting in prolonged survival without proteinuria (mean 170.5 days from 47.8 days). Data showed that transgenic expression of hCD47 on glomerular cells in the GalT-KO donor kidneys can prevent xenograft nephropathy, a significant barrier for therapeutic applications of xenotransplantation. The ability to prevent nephrotic syndrome following KXTx overcomes a critical barrier for future clinical applications of KXTx.

Anti-Non Gal Antibody Binding in Pig-to-Baboon and Pig-to-Human Lung Transplantation Models is Reduced by β4GalKO

Purpose Antibody-mediated xenograft rejection is inhibited by knock-out (KO) of porcine carbohydrates (CHO), including Galα1,3Gal. The β4Gal epitope was discovered using serum from xenograft-sensitized baboons. However, it remains controversial if humans and baboons have detectable titers of preformed anti-β4Gal antibody (as humans do for Neu5Gc), and whether anti-β4Gal antibody modulates lung xenograft injury. Methods In serum from 14 baboons, pre-lung transplant and post-op day1 anti-pig Ab binding against GalTKO versus GalTKO.β4GalKO (double KO (DKO)) porcine aortic endothelial cells (PAECs) was measured in animals that had received a lung from a GalTKO pig and compared to those in which the donor animal had β4GalKO (or knock down), with (TKO) or without (DKO) additional Neu5GcKO. To evaluate Ab binding to pig tissue, lung biopsies from wildtype, GalTKO, DKO and TKO pigs after 30min of ex vivo xeno-perfusion with human blood were stained for IgG and IgM binding. In one case Ab binding to GalTKO and DKO cells was also measured at 1 and 31d in a long-surviving xenolung recipient. Results IgM Ab binding in naive baboons was consistently lower on GalTKO.β4GalKO relative to GalTKO PAECs. Decline in anti-non-Gal Ab levels on post-op d1 in vivo was consistently less using GalTKO.β4GalKO lungs (31%±16%, n=3) when compared to single KO lungs (58%±16%, n=7). In a long-surviving LTX recipient animal, sensitized anti-non Gal antibody binding was almost completely abrogated when using TKO cells. Lungs with multiple CHO KOs appear to have decreased IgM tissue deposition relative to GalTKO lungs after perfusion with human blood when analyzed by Immunohistochemistry. Conclusion β4Gal is a dominant antigen after lung xenotransplantation. Binding of preformed antibody (IgM>IgG) in humans and baboons is reduced by β4GalKO. Since the β4Gal epitope presumably contributes to pig lung xenograft injury, including the β4GalKO modification in donor pigs will likely help to better control Ab-mediated xenorejection pathways and advance xenotransplantation of the lung and other organs towards the clinic.

Impact of donor and prolonged cold ischemia time of neonatal pig pancreas on neonatal pig islet transplant outcome.

Genetically modified pigs (GMP) have been developed to alleviate the shortage of donors in human islet transplantation and rejection. In this study, we characterized and compared the islets from GalTKO, GalTKO/hCD46, GalTKO/hCD46/hCD39, and wild-type (WT) neonatal pigs. Islets were isolated from GMP and WT pig pancreases that have been packaged with ice pack for at least 24hours. The difference in gene expression and function of islets were evaluated by microarray analysis and transplantation of islets under the kidney capsule of streptozotocin-induced diabetic immune-deficient mice, respectively. Blood glucose levels of these mice were monitored weekly post-transplantation for >100days, and islet grafts were collected and evaluated for the presence of endocrine cells. The genes involved in extracellular components, cell adhesion, glucose metabolism, and inflammatory response are differentially expressed between GMP and WT pig islets. Variation in the ability of pig islets in correcting the diabetic state of the mouse recipients appears to be dependent on the pig donor. In addition, prolonged cold ischemia time had a negative effect on the transplant outcome. All normoglycemic mice were able to respond well to glucose challenge despite the initial differences in the ability of islet transplants to reverse their diabetic state. Islet xenografts of normoglycemic mice contained abundant insulin- and glucagon-positive cells. The effect of GMP and WT neonatal pig islet transplants on hyperglycemia in mice appears to be dependent on the pig donor, and prolonged cold ischemia time negatively affects the neonatal pig islet transplant outcome.

MICROCAPSULE AGAINST HYPOXIA SHOWED LONGER GRAFT SURVIVAL THAN MICROCAPSULE AGAINST FIBROSIS IN DIABETIC MICE RECEIVING GALT-KO PORCINE ISLET TRANSPLANTATION

Introduction: Fibrosis is one of the most important barrier to improve encapsulated islet graft survival. Hypoxia is another major obstacle for encapsulated islet transplantation. Previously, we have developed several alginate-based microcapsules improving fibrosis (alginate-chitosan) or hypoxia (alginate-perfluorodecalin) and proved their superior effects compared with alginate microcapsule. In this study, we compared long-term graft survival of encapsulated islet with microcapsule against fibrosis or hypoxia in diabetic mice model, using 1,3-galactosyltransferase gene-knockout (GalT-KO) pigs. Materials and Methods: Islets were isolated form GalT-KO pigs and then encapsulated with alginate-chitosan or alginate-perfluorodecalin microcapsule. To magnify graft survival gap between different microcapsules, marginal doses of islets were transplanted into streoptozotocin-induced diabetic mice intraperitoneally. Blood glucose level were measured for a year. Intraperitoneal glucose tolerance test (IPGTT) was performed at 6 and 12 months after transplantation. Results: In the alginate-chitosan group, the blood glucose level of diabetic mice decreased after encapsulated islet transplantation. However, blood glucose level increased again within two months in 3 out of 4 mice. In the alginate-perfluorodecalin group, the blood glucose level of diabetic mice decreased after transplantation and maintained normoglycemia more than 6 months. Although blood glucose level tended to increase 6 months after transplantation, it remained below 300 mg/dL by one year. The area under curve (AUC) of IPGTT was similar between two groups at baseline. At 6 months after transplantation, AUC of IPGTT in alginate-perflourodecalin group lower than AUC of alginate-chitosan group. At one year after transplantation, microcapsules were retrieved. Dithizone stain was positive in most retrieved islet and fibrosis was minimal in both groups. Conclusion: In conclusion, microcapsule against hypoxia showed a longer xenograft survival than microcapsule against fibrosis in diabetic mice. This work was carried out with the support of “Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ0134532019)” Rural Development Administration, Republic of Korea.

2289-PUB: Comparison of GalT-KO Porcine Islet Xenograft Survival between Microcapsule against Hypoxia and Microcapsule against Fibrosis in Diabetic Mice Model

Hypoxia and fibrosis are one of the most important obstacles for encapsulated islet transplantation. Previously, we have developed alginate-based microcapsules improving fibrosis (alginate-chitosan) or hypoxia (alginate-perfluorodecalin) and proved their effects. In this study, we compared long-term graft survival of encapsulated islet with microcapsule against fibrosis or hypoxia in diabetic mice model, using 1,3-galactosyltransferase gene-knockout (GalT-KO) pigs. Islets were isolated form GalT-KO pigs and then encapsulated with alginate-chitosan or alginate-perfluorodecalin microcapsule. To magnify graft survival gap between different microcapsules, marginal dose of islets were transplanted into streoptozotocin-induced diabetic mice intraperitoneally. Blood glucose level were measured for a year. Intraperitoneal glucose tolerance test (IPGTT) was performed at 6 and 12 months after transplantation. In the alginate-chitosan group, the blood glucose level of diabetic mice decreased after encapsulated islet transplantation. However, blood glucose level increased again within two months in 3 out of 4 mice. In the alginate-perfluorodecalin group, the blood glucose level of diabetic mice decreased after transplantation and maintained normoglycemia more than 6 months. Although blood glucose level tended to increase 6 months after transplantation, it remained below 300 mg/dL by one year. The area under curve (AUC) of IPGTT was similar between two groups at baseline. At 6 months after transplantation, AUC of IPGTT in alginate-perflourodecalin group lower than AUC of alginate-chitosan group. At one year after transplantation, microcapsules were retrieved. Dithizone stain was positive in most retrieved islet and fibrosis was minimal in both groups. In conclusion, microcapsule against hypoxia showed a longer xenograft survival than microcapsule against fibrosis in diabetic mice. Disclosure E. Lee: None. Y. Yang: None. Y. Cho: None. S. Lee: None. J. Cho: None. K. Yoon: None. Funding Cooperative Research Program for Agriculture Science and Technology Development (PJ01345301); Rural Development Administration, Republic of Korea

Intra-bone Bone Marrow Transplantation in Pig-to-Nonhuman Primates for the Induction of Tolerance Across Xenogeneic Barriers.

Mixed chimerism and thymic tissue transplantation strategies have achieved xenogeneic tolerance in pig-to-mouse models, and both have been extended to pig-to-baboon models. A mixed chimerism strategy has shown promise toward inducing tolerance in allogeneic models in mice, pigs, nonhuman primates (NHP), humans, and a rat-to-mouse small animal xeno-model. However, even though α-1,3-galactosyltransferase gene knockout (GalTKO) pigs have been used as bone marrow (BM) donors, direct intravenous injection of porcine BM cells was detected for only up to 4days (peripheral macro-chimerism) in one case, and the rest lost chimerism within 2days.Recent data in allogeneic models demonstrated that direct injection of donor BM cells into recipient BM spaces (intra-bone bone marrow transplantation: IBBMTx) produces rapid reconstitution and a higher survival rate compared to i.v. injection. In order to minimize the loss of injected porcine BM peripherally before reaching the BM space, Yamada developed a xeno-specific regimen including IBBMTx coated with a collagen gel matrix in a preclinical pig-to-baboon model (Yamada IBBMTx). This strategy aims to achieve improved, persistent macro-chimerism as well as engraftment of BM across a xenogeneic barrier. The initial study published in 2015 demonstrated that this IBBMTx strategy leads to markedly prolonged peripheral macro-chimerism detectable for up to 23days. Furthermore, a more recent study using human CD47-transgenic (Tg) GalTKO pigs as xeno-donors achieved long-lasting macro-chimerism >60days with evidence of reduction of anti-pig natural antibodies (nAb). This is the longest macro-chimerism that has ever been achieved in a preclinical large animal xenotransplant model to date. In this chapter, we introduce a brief summary of our achievements in regard to successful tolerance induction by utilizing our novel strategy of IBBMTx as well as describe the step-by-step methodology of surgical and in vitro procedures that are required for this project.

Immortalization of porcine hepatocytes with a α-1,3-galactosyltransferase knockout background.

In vivo pig liver xenotransplantation preclinical trials appear to have poor efficiency compared to heart or kidney xenotransplantation because of xenogeneic rejection, including coagulopathy, and particularly thrombocytopenia. In contrast, ex vivo pig liver (wild type) perfusion systems have been proven to be effective in "bridging" liver failure patients until subsequent liver allotransplantation, and transgenic (human CD55/CD59) modifications have even prolonged the duration of pig liver perfusion. Despite the fact that hepatocyte cell lines have also been proposed for extracorporeal blood circulation in conditions of acute liver failure, porcine hepatocyte cell lines, and the GalT-KO background in particular, have not been developed and applied in this field. Herein, we established immortalized wild-type and GalT-KO porcine hepatocyte cell lines, which can be used for artificial liver support systems, cell transplantation, and even in vitro studies of xenotransplantation. Primary hepatocytes extracted from GalT-KO and wild-type pigs were transfected with SV40 LT lentivirus to establish immortalized GalT-KO porcine hepatocytes (GalT-KO-hep) and wild-type porcine hepatocytes (WT). Hepatocyte biomarkers and function-related genes were assessed by immunofluorescence, periodic acid-Schiff staining, indocyanine green (ICG) uptake, biochemical analysis, ELISA, and RT-PCR. Furthermore, the tumorigenicity of immortalized cells was detected. In addition, a complement-dependent cytotoxicity (CDC) assay was performed with GalT-KO-hep and WT cells. Cell death and viability rates were assessed by flow cytometry and CCK-8 assay. GalT-KO and wild-type porcine hepatocytes were successfully immortalized and maintained the characteristics of primary porcine hepatocytes, including albumin secretion, ICG uptake, urea and glycogen production, and expression of hepatocyte marker proteins and specific metabolic enzymes. GalT-KO-hep and WT cells were confirmed as having no tumorigenicity. In addition, GalT-KO-hep cells showed less apoptosis and more viability than WT cells when exposed to complement and xenogeneic serum. Two types of immortalized cell lines of porcine hepatocytes with GalT-KO and wild-type backgrounds were successfully established. GalT-KO-hep cells exhibited higher viability and injury resistance against a xenogeneic immune response.

Recent Progress in Pig to Non-Human Primate(NHP) Solid Organ Xenotransplantation in Korea

Extreme shortage of solid organ donors comparing to the transplant-needed patients is most critical problem now and only probable solution seems to be xenotransplantation. However, results of preclinical trials of solid organ xenotransplantation of pig to NHP are not sufficient to start the clinical trials yet. In Korea, although GalTKO pigs are developed already in the middle of 2000th, trials of solid organ xenotransplantation of pig to NHP are only begun from 2011. From then, 6 cases of kidney xenotransplantation and 20 cases of heart xenotransplantation have been done in our research team until now. We have used the cynomolgus monkey as recipient animal and pigs of several types of transforming just like GTKO only, GTKO.hCD46 and GTKO.hCD39 as donor animal. We have used the immunosuppressants of CD154 ab, rituximab, ATG, Tacrolimus, MMF and steroid. The longest survival of kidney and heart is 32 and 61 days for each. And among heart and kidney recipients, nearly 40% survive more than 3 weeks and longer survivals are recent cases. For the kidney transplantation, functioning graft was confirmed. There are no signs of hyper-acute rejection for the transplanted graft and even for the more than 1-month survivors, acute rejection is the cause of death. This survival results are relatively poor yet, but longest cases of survival are recent cases and so results are improving. Although solid organ xenotransplantation in Korea is just beginning and we are only team with insufficient resources and results, with the supports of governmental fund and volunteering activities of clinical experts of heart and kidney, we expect the continuing experiments and studies to make comparative good results and contribute the beginning of clinical trials of solid organ xenotransplantation Korean Rural Development Administration.

Durable Macrochimerism (>8 weeks) and Pig-Specific T and B Cell Unresponsiveness Following hCD47+ Transgenic GalTKO Pig Intra-Bone Bone Marrow Transplantation in Baboons

Background Recent data have suggested that low levels of preformed anti-non-Gal natural antibody (nAb) limit xenograft survival. Durable mixed xenogeneic chimerism resulted in tolerance of human T cell-independent B cells that produce xenoantibodies, including anti-non-Gal nAb, in mouse models. However, previous attempts at intravenous (IV) α-1,3-galactocyltransferase knockout (GalTKO) pig bone marrow (BM) transplantation (Tx) into non-human primates have been associated with very short-lived chimerism (<24 hours in most cases). In order to achieve improved engraftment with more persistent chimerism, we have recently developed an intra-bone BM (IBBM) Tx strategy in a pig-to-baboon model. We have reported continuous peripheral blood macrochimerism for up to 13 days (mean 7.7 days; range 3–13) post-IBBM/BM-Tx (n=6) and in three animals, macrochimerism reappeared at days 10, 14 and 21 (Am J Transplant 2015). Recent data has suggested that incompatibility between CD47 and SIRPα causes phagocytosis of porcine cells. In this study, we examined whether the use of BM cells from human CD47 (hCD47) transgenic pigs further improves durable chimerism and assessed anti-pig antibody responses following BM Tx,Methods: Four baboons received GalTKO/human CD47(hCD47)/hCD55 pig BM cells, and one received GalTKO/hCD55 porcine BM cells with a conditioning regimen consisting of whole body irradiation thymic, irradiation, ATG, and Rituximab followed by anti-CD40L antibody and MMF. Approximately 2/3 of BM cells were infused intraosseously and the remainder intravenously. Macrochimerism, anti-pig T cells responses, and anti-pig antibody responses were assessed following IBBM Tx. Results were compared with those from baboons that received GalTKO porcine BM cells without any other gene modification (n=6). Results As shown in the Figure (left panel), all four baboons that received hCD47+/hCD55+ porcine BM using the IBBM Tx method maintained durable macrochimerism for >50 days, and two of the 4 maintained blood levels above 1.6%, even at 8 weeks following IBBMTx (red line in the left panel of Fig). We also confirmed macrochimerism in recipients’ BM. A baboon that received hCD55+ but hCD47-negative porcine BM cells also had a slightly higher % of chimerism than GalTKO alone porcine BM cells in the first 3 weeks, but the duration was similar to GalTKO alone porcine BM cells (Blue line vs black line in the left panel of Fig). All recipients of IBBM Tx lost their anti-pig T cell responses in vitro and showed no development of elicited anti-pig ab. Notably, anti-pig nonGal nAb levels in three of 4 recipients of hCD47+/hCD55+ porcine BM cells declined markedly following IBBM Tx (right panel of Fig). Conclusion To our knowledge, this is the first demonstration of (1) durable macrochimerism beyond 8 weeks and (2) reduction of anti-non Gal nAb levels following hCD47+/hCD55+ GalTKO porcine IBBMTx in preclinical pig-to-non-human primate models. The results suggest that IBBMTx using hCD47+ porcine BM is a potent strategy to induce pig-specific T and B cell tolerance across xenogeneic barriers..NIH P01AI45897.:

Recipient T Regulatory Cells Enhance Chimerism and Prolong Galt-KO pig Skin Graft Survival in Baboons

Recipient T Regulatory Cells Enhance Chimerism and Prolong Galt-KO pig Skin Graft Survival in Baboons

Transgenic Expression of hCD47 on Vessels in Porcine GalTKO Lung Grafts in Baboons may Mitigate Acute Vascular Rejection in Baboons

Background Despite recent progress in survival times of xenografts in nonhuman primates, there are no reports of survival beyond 5 days of histologically well-aerated porcine lung grafts in baboons. Here we report our initial results of pig-to-baboon xeno lung transplantation (XLTx). Methods Study 1: Eleven baboons received genetically modified porcine left lungs as primary grafts that were histologically confirmed to be either GalT-KO alone (n=3), GalT-KO/humanCD47(hCD47)/hCD55 (n=3), GalT-KO/hD47/hCD46 (n=4) or GalTKO/hCD39/hCD55/hTBM/hEPCR (n=1) Tg swine. Study 2: Three porcine lung grafts were transplanted to baboons that had received a tolerance-inducing regimen and showed pig specific unresponsiveness in vitro by IFNg ELISPOT and FACS analysis. Two of these were hCD47+ GalTKO lungs and the third was GalTKO alone. Results Study 1: Survivals of primary lung grafts were 1, 1, 2, 3, 5, 5, 6, 7, >7, 9 and 10 days. One baboon with graft survival >7 days, whose entire lung graft remained well-aerated, was euthanized on POD7 due to malfunction of femoral catheters. No statistical difference was seen in preformed nAb cytotoxicity levels between >7 day survivors and others (20% vs 27%). hCD47 expression of donor lungs was detected on both endothelial cells (ECs) and alveoli (Alv) only in the three grafts surviving >7, 9 and 10 days. All other grafts lacked hCD47 expression in endothelial cells and were completely rejected with diffuse hemorrhagic changes and antibody/complement deposition detected in association with early graft loss. Study 2: Delayed pig lung transplant survivals were 2 days (GaTKO alone), 4 days (hCD47+/GalTKO) and 13 days (hCD47+/GalTKO). The lung graft of the 13-day survivor expressed hCD47 on both ECs and Alv while the graft of the 4-day survivor expressed hCD47 only on Alv.Conclusions To our knowledge, this is first evidence of histologically viable porcine lung grafts up to 13 days in baboons. Our results indicate that GalT-KO pig lungs are highly susceptible to acute humoral rejection. Transgenic expression of hCD47 on vascular ECs in porcine GalTKO lung grafts appeared to be important to mitigate acute vascular rejection in baboons. Lung Biotechnology PBC. NIH P01AI45897.

GalT-KO pig lungs are highly susceptible to acute vascular rejection in baboons, which may be mitigated by transgenic expression of hCD47 on porcine blood vessels.

Despite recent progress in survival times of xenografts in non-human primates, there are no reports of survival beyond 5days of histologically well-aerated porcine lung grafts in baboons. Here, we report our initial results of pig-to-baboon xeno-lung transplantation (XLTx). Eleven baboons received genetically modified porcine left lungs from either GalT-KO alone (n=3), GalT-KO/humanCD47(hCD47)/hCD55 (n=3), GalT-KO/hD47/hCD46 (n=4), or GalT-KO/hCD39/hCD46/hCD55/TBM/EPCR (n=1) swine. The first 2 XLTx procedures were performed under a non-survival protocol that allowed a 72-hour follow-up of the recipients with general anesthesia, while the remaining 9 underwent a survival protocol with the intention of weaning from ventilation. Lung graft survivals in the 2 non-survival animals were 48 and >72hours, while survivals in the other 9 were 25 and 28hours, at 5, 5, 6, 7, >7, 9, and 10days. One baboon with graft survival >7days, whose entire lung graft remained well aerated, was euthanized on POD 7 due to malfunction of femoral catheters. hCD47 expression of donor lungs was detected in both alveoli and vessels only in the 3 grafts surviving >7, 9, and 10days. All other grafts lacked hCD47 expression in endothelial cells and were completely rejected with diffuse hemorrhagic changes and antibody/complement deposition detected in association with early graft loss. To our knowledge, this is the first evidence of histologically viable porcine lung grafts beyond 7days in baboons. Our results indicate that GalT-KO pig lungs are highly susceptible to acute humoral rejection and that this may be mitigated by transgenic expression of hCD47.

Lung xenotransplantation.

This review describes the most recent progress in xeno lung transplantation (XLTx) to date. It describes the potential mechanisms of early xeno lung graft loss, as well as the latest therapeutic strategies to overcome them. Using ex-vivo perfusion models of porcine lungs with human blood, the use of genetically modified pig lungs along with novel pharmaceutical approaches has recently been studied. Strategies that have demonstrated improved lung survival include the knockout of known xenoantigens (GalTKO and N-glycolylneuraminic acid-KO), genes that regulate complement activation (hCD46 and hCD55), as well as the inflammation/coagulation cascade (human leukocyte antigen-E, human thrombomodulin, human endothelial protein C receptor, hCD47, hCD39, hCD73 and heme oxygenase-1). Furthermore, pharmacologic interventions including the depletion of pulmonary intravascular macrophages or von Willebrand factor, inhibition of thromboxane synthase and blockade of histamine receptors have also demonstrated protective effects on xeno lung grafts. Using in-vivo pig to nonhuman primate lung transplant models, these approaches have been shown to extend pulmonary xenograft survival to 5 days. The development of new multitransgenic GalTKO pigs has demonstrated prolongation of porcine xenograft survival; however, advancement in XLTx has remained frustratingly limited. Further intensive and innovative strategies including genetic manipulation of donors, as well as inflammation/coagulation dysregulation, are required to make XLTx a clinical possibility.

Generation of Induced Hepatocyte-Like Cells From Bone Marrow Mesenchymal Stem Cells Derived From an α1, 3-Galactosyltransferase Knockout Pig

Transplantation of porcine hepatocytes for the treatment of humans with acute and chronic liver diseases is considered an alternative to liver transplantation treatment. Especially, a bioartificial liver using porcine hepatocytes has the potential to facilitate liver transplantation for patients with acute-on-chronic liver failure. However, the use of porcine primary hepatocytes is limited by their low survivability under in vitro culture systems and complicated methods of isolation. To address this concern, we attempted to generate induced hepatocyte-like cells from bone marrow mesenchymal stem cells (BM-MSCs) derived from an α1, 3-Galactosyltransferase knockout (GalT KO) pig by transfecting three hepatocyte transcription factors (HNF1A, HNF4A, and FOXA3) and an additional small molecule such as A83-01, an inhibitor of TGF-ß receptor. BM-MSCs derived from the GalT KO pig could be used to minimize immune rejection in xenotransplantation in the future. The ratio of BM-MSCs and the three lentiviral vectors was maintained at MOI =1 and at one day post infection, they were cultured in a hepatocyte culture medium with or without 2 μM A83-01 for 4 weeks. Porcine induced hepatocyte-like cells (piHeps) were observed at a low frequency from 1 week onwards, regardless of A83-01; however, the presence of A83-01 increased the colony number of piHeps. Up to 4 weeks, piHeps maintained A83-01 treatment group but not without A83-01. qPCR analysis of liver-specific genes such as albumin and transferrin genes revealed that A83-01 improved the expression of these genes in the piHeps. Notably, A83-01 treatment could induce the activation of endogenic genes (pHNF1A, pHNF4A, and pFOXA3) in piHeps, as indicated by qPCR results. These results suggest that piHeps could be generated using the three mentioned factors, although at a low efficiency, and that A83-01 could improve the efficiency of piHep generation by the inhibition of epithelial-to-mesenchymal transition. These findings provide valuable information in the field of translational liver research and testing cells for the development of a bioartificial liver system and novel pharmaceuticals. This work was supported by the Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ01100203), RDA, Republic of Korea.

Molecular immunology profiles of monkeys following xenografting with the islets and heart of α-1,3-galactosyltransferase knockout pigs.

Effective immunosuppression strategies and genetically modified animals have been used to prevent hyperacute and acute xenograft rejection; however, the underlying mechanisms remain unknown. In this study, we evaluated the expression of a comprehensive set of immune system-related genes (89 genes, including five housekeeping genes) in the blood of cynomolgus monkeys (~5 yr old) used as graft recipients, before and after the xenografting of the islets and heart from single and double α-1,3-galactosyltransferase (GalT) knockout (KO) pigs (<6 weeks old). The immunosuppressive regimen included administration of cobra venom factor, anti-thymocyte globulin, rituximab, and anti-CD154 monoclonal antibodies to recipients before and after grafting. Islets were xenografted into the portal vein in type 1 diabetic monkeys, and the heart was xenografted by heterotopic abdominal heart transplantation. Genes from recipient blood were analyzed using RT(2) profiler PCR arrays and the web-based RT(2) profiler PCR array software v.3.5. Recipients treated with immunosuppressive agents without grafting showed significant downregulation of CCL5, CCR4, CCR6, CD4, CD40LG, CXCR3, FASLG, CXCR3, FOXP3, GATA3, IGNG, L10, IL23A, TRAF6, MAPK8, MIF, STAT4, TBX21, TLR3, TLR7, and TYK2 and upregulation of IFNGR1; thus, genes involved in protection against viral and bacterial infection were downregulated, confirming the risk of infection. Notably, C3-level control resulted in xenograft failure within 2 days because of a 7- to 11-fold increase in all xenotransplanted models. Islet grafting using single GalT-KO pigs resulted in upregulation of CXCL10 and MX1, early inflammation, and acute rejection-associated signals at 2 days after xenografting. We observed at least 5-fold upregulation in recipients transplanted with islets grafts from single (MX1) or double (C3, CCR8, IL6, IL13, IRF6, CXCL10, and MX1) GalT-KO pigs after 77 days; single GalT-KO incurred early losses owing to immune attacks. Our results suggest that this novel, simple, non-invasive, and time-efficient procedure (requiring only 1.5 ml blood) for evaluating graft success, minimizing immune rejection, and blocking infection.

Aberrant methylation of Meg3 in alpha1,3-galactosyltransferase knockout pig induced pluripotent stem cells

ABSTRACTPigs have been used as a good research model for xenotransplantation. Several groups have generated porcine-induced pluripotent stem cells (piPSCs) from differentiated somatic cells. Transgenic pigs with the alpha1,3-galactosyltransferase gene-knockout (GalT-KO) could successfully govern hyper acute rejection of organ transplants into primates. Thus, GalT-KO piPSCs could be a powerful cell resource for agricultural and biomedical applications. This study was performed to generate iPSCs from GalT-KO pigs and characterize their properties. We successfully generated a GalT-KO iPSC from a genetically modified pig using double alpha1,3-galactosyltransferase knockout alterations. Similar to mouse embryonic stem cells, the GalT-KO piPSCs were positive for classical pluripotency markers: POU5F1, NANOG, SOX2 and SSEA1, and were negative for: SSEA3, TRA-1-60 and TRA-1-81. Furthermore, these cells could form an embryoid body that differentiated into three germ layers in vitro, and were highly proliferative under leukemia inhibitory factor culture conditions. However, the methylation status in DMR2 of the Meg3 gene was higher in GalT-KO piPSCs than in porcine ear fibroblast. In conclusion, GalT-KO piPSCs could be successfully generated by six human factors without expression of Gal-epitopes. Although aberrant methylation observed in GalT-KO piPSCs, this cell line maintained pluripotency and had differentiation properties into all three germ layers. Therefore, GalT-KO piPSCs might be a good cell source for biomedical application and basic research.

Early graft failure of GalTKO pig organs in baboons is reduced by expression of a human complement pathway-regulatory protein.

We describe the incidence of early graft failure (EGF, defined as loss of function from any cause within 3 days after transplant) in a large cohort of GalTKO pig organs transplanted into baboons in three centers, and the effect of additional expression of a human complement pathway-regulatory protein, CD46 or CD55 (GalTKO.hCPRP). Baboon recipients of life-supporting GalTKO kidney (n = 7) or heterotopic heart (n = 14) grafts received either no immunosuppression (n = 4), or one of several partial or full immunosuppressive regimens (n = 17). Fourteen additional baboons received a GalTKO.hCPRP kidney (n = 5) or heart (n = 9) and similar treatment regimens. Immunologic, pathologic, and coagulation parameters were measured at frequent intervals. EGF of GalTKO organs occurred in 9/21 baboons (43%). hCPRP expression reduced the GalTKO EGF incidence to 7% (1/14; P < 0.01 vs. GalTKO alone). At 30 mins, complement deposits were more intense in organs in which EGF developed (P < 0.005). The intensity of peri-transplant platelet activation (as β-thromboglobulin release) correlated with EGF, as did the cumulative coagulation score (P < 0.01). We conclude that (i) the transgenic expression of a hCPRP on the vascular endothelium of a GalTKO pig reduces the incidence of EGF and reduces complement deposition, (ii) complement deposition and platelet activation correlate with early GalTKO organ failure, and (iii) the expression of a hCPRP reduces EGF but does not prevent systemic coagulation activation. Additional strategies will be required to control coagulation activation.

Results of life-supporting galactosyltransferase knockout kidneys in cynomolgus monkeys using two different sources of galactosyltransferase knockout Swine.

Various durations of survival have been observed in the xenotransplantation of life-supporting α-1,3-galactosyltransferase knockout (GalT-KO) porcine kidneys into nonhuman primates. Although others have demonstrated loss of GalT-KO-transplanted kidneys within 2 weeks, we have reported an average survival of 51 days with the cotransplantation of the kidney and vascularized thymus and an average of 29 days with the kidney alone. To determine the factors responsible for this difference in survival time, we performed xenogeneic kidney transplantations into cynomolgus monkeys with an anti-CD40L-based regimen using two different strains of GalT-KO swine, one derived from MGH miniature swine and the other obtained from Meji University. Eight cynomolgus moneys received GalT-KO kidneys. Three kidney grafts were from Massachusetts General Hospital (MGH)-Nippon Institute for Biological Science (NIBS) GalT-KO pigs and five GalT-KO grafts were from MEIJI GalT-KO swine. All cynomolgus recipients were treated identically. Recipients of kidneys from the MGH GalT-KO kidneys swine, produced by nuclear transfer in Japan, survived an average of 28.7 days, whereas recipients of MEIJI GalT-KO kidneys swine survived an average of 9.2 days. Among the differences between these two groups, one potentially revealing disparity was that the MEIJI swine were positive for porcine cytomegalovirus, whereas the MGH-derived swine were negative. This is the first study comparing renal xenotransplantation from two different sources of GalT-KO swine into nonhuman primates at a single center. The results demonstrate that porcine cytomegalovirus may be responsible for early loss of GalT-KO swine kidney xenografts.

The alpha1,3GalT knockout/alpha1,2FucT transgenic pig does not appear to have an advantage over the alpha1,3GalT knockout pig with respect to glycolipid reactivity with human serum antibodies

The human H-transferase (α2FucT) was introduced in Gal-negative pigs to produce pig organs not only free from Gal-antigens, but also in which the uncapped N-acetyllactosamine precursor had been transformed into non-xenogenic blood group H type 2 compounds. This work is the first descriptive analysis of glycolipids from the GalT-KO/FucT-TG pig. The aim was to investigate the cell membrane antigens in GalT-KO/FucT-TG tissues to explore its efficacy as an organ donor. Also, detailed knowledge on the correlation between the cellular glycosyltransferase configuration and the resulting carbohydrate phenotype expression is valuable from a basic glycobiological perspective. Neutral and acidic glycolipids from GalT-KO/FucT-TG small intestine were compared with glycolipids from two wildtype and two GalT-KO pig intestines. Glycolipid reactivity was tested on thin layer chromatography plates using chemical reagents, antibodies, lectins, and human serum. Structural characterization of neutral glycolipids was performed by LC-ESI/MS and proton NMR spectroscopy. Characterization of the glycolipid expression in GalT-KO/FucT-TG intestine showed absence of Gal antigens and decreased/unchanged levels of the N-acetyllactosamine precursor and the blood group H type 2 expression, when compared with the wildtype. The reactivity of human serum antibodies to GalT-KO/FucT-TG derived glycolipids was similar or slightly elevated when compared with GalT-KO glycolipids. Results from LC-ESI/MS and proton NMR spectroscopy revealed no established neutral xenogenic antigens in the GalT-KO/FucT-TG pig, and could thus not explain the immunologic reactivity to human serum antibodies. The antibody binding to acidic glycolipids is most likely to be explained by the abundance of N-glycolylneuraminic acid epitopes in pig tissues. Six neutral complex biantennary glycolipids with blood group H type 1, 2, Lewis(x) and Lewis(y) determinants were found, of which three were identified in this work for the first time. One of these was a nonaglycosylceramide with blood group H type 2 and lactosyl determinants linked to a lactotetraosyl core, and the other two were decaglycosylceramides with blood group H type 1 and H type 2 determinants linked to a neolactotetraosyl core, and Lewis(x) and blood group H type 1 determinants on a lactotetraosyl core, respectively. Lipid-linked carbohydrate antigens in the GalT-KO/FucT-TG pig intestine showed no or minor qualitative difference when compared with GalT-KO pigs. The GalT-KO/FucT-TG pig did not appear to have an advantage over the GalT-KO pig with respect to reactivity with human antibodies from a xenotransplantation perspective.

Increased levels of anti‐non‐Gal IgG following pig‐to‐baboon bone marrow transplantation correlate with failure of engraftment

The development of genetically modified pigs, which lack the expression of alpha 1-3 galactosyl transferase, (GalT-KO pigs) has facilitated the xenogeneic transplantation of porcine organs and tissues into primates by avoiding hyperacute rejection due to pre-existing antibodies against the Gal epitope. However, antibodies against other antigens (anti-non-Gal antibodies), are found at varying levels in the pre-transplant sera of most primates. We have previously found that baboons with high levels of pre-transplant anti-non-Gal IgG, conditioned with a non-myeloablative conditioning regimen, failed to engraft following pig-to-baboon bone marrow transplantation (Xenotransplantation, 17, 2010 and 300). Two baboons with low levels of pre-transplant anti-non-Gal IgG, conditioned with the same regimen, showed porcine bone marrow progenitors at 28days following transplantation, suggesting engraftment. These baboons also showed evidence of donor-specific hyporesponsiveness. This observation led us to investigate the hypothesis that selecting for baboon recipients with low pre-transplant anti-non-Gal IgG levels might improve engraftment levels following GalT-KO pig-to-baboon bone marrow transplantation. Five baboons, with low pre-transplant anti-non-Gal IgG levels, received transplantation of bone marrow cells (1-5×10(9) /kg of recipient weight) from GalT-KO pigs. They received a non-myeloablative conditioning regimen consisting of low-dose total body irradiation (TBI) (150cGy), thymic irradiation (700cGy), anti-thymocyte globulin (ATG), and tacrolimus. In addition, two baboons received Rituximab and Bortezomib (Velcade) treatment as well as extra-corporeal immunoadsorption using GalT-KO pig livers. Bone marrow engraftment was assessed by porcine-specific PCR on colony forming units (CFU) of day 28 bone marrow aspirates. Anti-non-Gal antibody levels were assessed by serum binding toward GalT-KO PBMC using flow cytometry (FACS). Peripheral macro-chimerism was measured by FACS using pig and baboon-specific antibodies and baboon anti-pig cellular responses were assessed by mixed lymphocyte reactions (MLR). As previously reported, two of five baboons demonstrated detectable bone marrow engraftment at 4weeks after transplantation. Engraftment was associated with lack of an increase in anti-non-Gal IgG levels as well as cellular hyporesponsiveness toward pig. Three subsequent baboons with similarly low levels of pre-existing anti-non-Gal IgG showed no engraftment and an increase in anti-non-Gal IgG antibody levels following transplantation. Peripheral macrochimerism was only seen for a few days following transplantation regardless of antibody development. Selecting for baboon recipients with low levels of pre-transplant anti-non-Gal IgG did not ensure bone marrow engraftment. Failure to engraft was associated with an increase in anti-non-Gal IgG levels following transplantation. These results suggest that anti-non-Gal-IgG is likely involved in early bone marrow rejection and that successful strategies for combating anti-non-Gal IgG development may allow better engraftment. Since engraftment was only low and transient regardless of antibody development, innate immune, or species compatibility mechanisms will likely also need to be addressed to achieve long term engraftment.