Xenotransplantation Research Research Articles

Aortobrachial bypass for acute vascular insufficiency due to tumor embolization

Department of Thoracic and Cardiovascular Surgery, Sejong General Hospital, Sejong Heart Institute, Bucheon, Republic of Korea Department of Thoracic and Cardiovascular Surgery, Seoul National University College of Medicine, Seoul National University Children’s Hospital, Republic of Korea Department of Thoracic and Cardiovascular Surgery, Seoul National University Children’s Hospital, Xenotransplantation Research Center, Seoul National University Hospital Clinical Research Institute, 28 Yongon-dong, Jongro-gu, Seoul 110-744, Republic of Korea Department of Surgery, Seoul National University College of Medicine, Seoul National University Children’s Hospital, Republic of Korea

224 DERIVATION AND IMMUNOLOGICAL CHARACTERISTICS OF PORCINE EMBRYONIC STEM-LIKE CELLS

Pluripotent cells from porcine blastocysts can be used to serve as an in vitro model as well as an unlimited cell source for therapeutic xenotransplantation. In xenotransplantation, foreign organs and tissues are rejected because of the immune responses in the recipient. This rejection is determined by the naturally occurring cell surface antigen, major histocompatibility antigen (MHC), knowledge of which is essential to advance our understanding of the processes implicated in immune responses. In mouse and human embryonic stem (ES) cells, MHC expression patterns are changed upon their differentiation in vitro. Therefore, immunological characteristics of undifferentiated and/or differentiated pluripotent ES-like cells derived from porcine blastocysts could provide valuable information to xenotransplantation research. For such purposes, swine leukocyte antigen (SLA) isotype and its expression pattern were determined in porcine ES-like cells. Porcine inner cell masses from Day 7 in vivo-produced blastocysts were isolated by immunosurgery and seeded on a feeder layer. Isolated cells were cultured in a mixture of DMEM : HF-10 (1 : 1) with 15% FBS, 1.7 mM l-glutamine, 1% penicillin/streptomycin, 0.1 mM β-mercaptoethanol, 1000 U mL-1 leukemia inhibitory factor, 40 ng mL-1 stem cell factor, and 20 ng mL-1 basic fibroblast growth factor in humidified environment of 5% CO2 in air at 38�C. Porcine ES-like cells showing typical morphology were subpassaged by physical separation. Embryoid body (EB) formation was induced by culturing small clumps of cells by the hanging drop method. Total RNA was extracted from porcine ES-like cell and EBs. PCR amplification was performed by using locus-specific primer pairs for swine leukocyte antigen (SLA) 1 and 2, followed by sequencing of amplified DNA fragments. These sequences were compared with known SLA sequences, and their SLA allele types were determined. Porcine ES-like cells showed the typical morphology of ES cells, closer to that of human than mouse cells. They showed typical expression of pluripotent stem cell markers, determined by immunochemistry and PCR analysis. When porcine ES-like cells formed EBs, differentiation specific markers of all 3 germ layers, �%-fetoprotein (endoderm marker), neurofilament (ectoderm marker), and cardiac troponin1 (mesoderm), were detected in the EBs. In porcine ES-like cells, two new alleles (SLA-1*06ck01 and *06ck02, and SLA-2*06ck01 and *06ck02) were identified. Therefore, our porcine ES-like cell line showed heterozygous allele isotypes in SLA-1 and SLA-2. EBs derived from porcine ES-like cells showed the same sequencing results, indicating no alteration of SLA-1 and SLA-2 isotypes in porcine ES-like cells and their differentiated derivatives. Further experiments for determining SLA-3 allele isotype in porcine ES-like cells and EBs and cell surface expression of the SLA antigens in these cells will be performed. In conclusion, porcine ES-like cells were established from in vivo-derived blastocysts and their SLA isotypes were consistent regardless of their differentiation.

77 PRODUCTION OF BAN MINIPIG EMBRYO BY SOMATIC CELL NUCLEAR TRANSFER

Ban pig is a one of the wild miniature pig breeds of the Vietnamese highland characterized by small body size and adult weight from 40 to 50 kg. SCNT has been applied to this breed as a tool for its conservation to maintain biodiversity as well as to multiply the minipigs as models for research and application in human xenotransplantation. In this paper we present the results of experiments on the production of Ban minipig embryos by transfer of their somatic cell nuclei into enucleated oocytes prepared from domestic pig (Landrace). Skin explants were collected from a young Ban male. After disinfection, the samples were cut into small pieces and cultured for 7-10 days in DMEM medium supplemented with 10% heat treated fetal bovine serum at 39�C under 5% CO2. The cultures resulting in cell confluence were used for further passage and cryopreservation by rapid freezing in medium containing 10% DMSO and 0.1 M sucrose. The ovaries collected from prepubertal Landrace gilts from a slaughterhouse were transported to the laboratory in PBS at 30�C. Cumulus-oocyte complexes were aspirated from follicles of 3-6 mm in diameter using an 18-gauge needle. They were washed in Hepes-buffered TCM-199, in vitro matured (IVM) in a modified North Carolina State University-37 medium (NCSU-37) containing 10% (v/v) porcine follicular fluid, 0.6 mM cysteine, 1 mM dibutytyl cyclic AMP (dbcAMP), 10 IU/mL eCG, and 10 IU/mL hCG for 22 h, and then held for an additional 14 h in the modified NCSU-37 without dbcAMP, eCG, and hCG. The mature oocytes were separated from cumulus cells by vortexing in 0.1% hyaluronidase in TCM-199. Metaphase II oocytes showing a clear first polar body (PB) were selected for enucleation in the presence of cytochalasin B. For nuclear transfer, fibroblasts were induced to quiescent state by culture in a medium with a low fetal bovine serum (FBS) concentration, and a single cell was transferred into each enucleated oocyte. Fusion was induced with needle electrodes and two direct current (DC) pulses of 30 V for 25 �s in Zimmerman medium with 0.3 M mannitol. The fused oocytes were activated with 7% ethanol in HEPES-TCM-199 for 5 min and cultured in the medium containing cyclohexamide for 6 h before further culture in vitro. The percentage of reconstructed oocytes fused, cleaved, and developed to the morula and blastocyst stages was assessed under a stereomicroscope at Days 2, 3, and Day 6-7, after nuclear transfer. The results showed that the rate of oocytes with a clear PB, obtained after IVM for 36 h, was 52.8% (n = 500). From 294 reconstructed oocytes, 192 (72.7%) fused; a total of 156 (81.2%) cleaved to the 4-8-cell stage, 96 (50%) developed to the morula stage, and 24 (12.5%) developed to the blastocyst stage at Days 2-3, 5-6, and 7 after nuclear transfer, respectively. A karyotype study showed a normal distribution of chromosome number (2n = 38) for the cell population after passage and for reconstructed embryos. These results suggest that IVM oocytes prepared from domestic pig can be successfully used for the production of the SCNT BAN minipig embryos. This work was supported by grant from the JSPS-VAST Joint Research Project for FY 2004 and by Project 82.

208 CYTOLYTIC ANALYSIS AND NUCLEAR TRANSFER OF hCD46-TRANSGENIC PORCINE EMBRYONIC GERM CELLS TO DEVELOP AND IN VITRO MODEL OF XENOTRANSPLANTATION

Pigs are considered the most likely source of organs for xenotransplantation due to their anatomical and physiological similarities to humans. Production of transgenic pigs including addition of human complement-regulatory protein genes and deletion of alpha-1,3-galactosyl transferase gene may overcome hyperacute rejection (HAR), the first and currently the most critical immunological hurdle in the development of xenogeneic organs for human transplantation. However, even after resolving HAR in pig-to-human xenotransplantation, a series of other transgenic pigs may be required to alleviate subsequent acute and chronic rejection and incompatibility of porcine proteins to human counterparts. The production of transgenic pigs is not only labor-intensive, time-consuming, and costly, but also the usefulness of such pigs in transplantation to humans is unpredictable. For these reasons, development of a reliable in vitro procedure to pre-evaluate effectiveness of the transgenic approach would be beneficial. This study was preformed to establish an in vitro model of xenotransplantation using porcine embryonic germ (EG) cells, undifferentiated stem cells derived from culture of primordial germ cells. Porcine EG cells were maintained in feeder-free state in DMEM containing 15% (v/v) fetal bovine serum and 1000 units/mL leukemia inhibitory factor. Human complement down-regulator hCD46 (also known as MCP, membrane cofactor protein) gene under the regulation of cytomegalovirus promoter was introduced into porcine EG cells. Transfected cells were selected by antibiotic treatment and confirmed by PCR. To test the resistance of hCD46-transgenic EG cells to human xenoreactive natural antibody and complement, EG cells were cultured for 1.5 days in DMEM containing 15% (v/v) normal human serum. The treatment with human serum did not affect the survival of hCD46-transgenic EG cells, whereas with the same treatment approximately one half of non-transfected EG cells failed to survive (P < 0.01). Transgenic EG cells presumably capable of overcoming HAR were used as nuclear donors for subsequent transfer of nuclei into enucleated oocytes. Among 110 reconstituted oocytes, 19 (17.3%) developed to the blastocyst stage. Analysis of individual nuclear transfer embryos by PCR indicated that 89.5% (17/19) of embryos contained transgene hCD46. The PCR-negative embryos might be due to an incomplete antibiotic selection of cells after transfection. Overall, the results of the present study demonstrate that the cell culture-based model of xenotransplantation may validate the usefulness of particular transgenic pigs prior to actual production. Further experiments on differentiation of transgenic EG cells into various cell types, cytolytic analysis of such cells to assess efficiency of xenotransplantation, and subsequent production and transfer of transgenic clone embryos to recipients may provide a useful new procedure to accelerate xenotransplantation research.

Westran — highly inbred pigs for xenotransplantation research

This paper reviews the history, breeding and biological characterisation of a highly inbred line of pigs, the Westran, developed by Westmead Hospital and the Veterinary Faculty of the University of Sydney, as an experimental large animal model for transplantation research. The review ranges from the historical origins of Westran pigs, from a pair of animals released on Kangaroo Island in 1803, to the context and relevance of the line in xenotransplantation (i.e. the transplantation of organs, tissues and cells across species boundaries).

Screening and analysis of porcine endogenous retrovirus in Chinese banna minipig inbred line

Pigs have been the most likely animal as the source of cells, tissues, and organs for xenotransplantation. But the use of pigs in xenotransplantation is associated with the risk of porcine endogenous retrovirus (PERV) transmission. Previous studies have identified that the proviruses are integrated into the genome of normal pigs and that virus particles released from the porcine cells can infect human cells in vitro. As a unique inbred pig, Banna minipig inbred (BMI) has a huge potential value for xenotransplantation and medical research. It has been the focal experimental animal for pig-to-human xenotransplantation in China, due to its clear genetic background and tiny individual differences. To evaluate whether the potential risk of PERV exists in inbred pigs, a series of screening experiments were performed herein. The results of PCR with primers specific for gag, pol, and env showed that proviruses existed in the genome of BMI, and the PERV subtypes were PERV-A and PERV-B. PERV mRNA was expressed functionally in BMI. Positive results of an RT assay identified that PERV in BMI had potential infectivity, but the concentration of PERV reverse transcriptase in BMI was almost 20 times lower than that of HIV. These results suggested that gag, pol and env genes of PERV were not lost during inbreeding, which created favorable conditions to produce viral particles that could possibly infect human cells in xenotransplantation.

Report of the Workshop on Xenotransplantation

Although the basic concept and medical promise of xenotransplantation appears simple, clinical and scientific, realisation has proven elusive, with success unlikely in the near future. Speakers in this workshop discussed the current European perspective on xenotransplantation; the range of clinical protocols under consideration and the health problems they are designed to address; alternative approaches with an emphasis on reducing the need for transplantation, while increasing the supply and utilisation of human organs; and animal welfare aspects of xenotransplantation research and potential clinical applications.

Piscine islet xenotransplantation.

Tilapia, a teleost fish species with large anatomically discrete islet organs (Brockmann bodies; BBs) that can be easily harvested without expensive and fickle islet isolation procedures, make an excellent donor species for experimental islet xenotransplantation research. When transplanted into streptozotocin-diabetic nude or severe combined immunodeficient mice, BBs provide long-term normoglycemia and mammalian-like glucose tolerance profiles. However, when transplanted into euthymic recipients, the mechanism of islet xenograft rejection appears very similar to that of islets from "large animal" donor species such as the very popular fetal/neonatal porcine islet cell clusters (ICCs). Tilapia islets are more versatile than ICCs and can be transplanted (1) into the renal subcapsular space, the cryptorchid or noncryptorchid testis, or intraportally as neovascularized cell transplants; (2) as directly vascularized organ transplants; or (3) intraperitoneally after microencapsulation. Unlike the popular porcine ICCs, BBs function immediately after transplantation; thus, their rejection can be assessed on the basis of loss of function as well as other parameters. We have also shown that transplantation of tilapia BBs into nude mice can be used to study the possible implications of cross-species physiological incompatibilities in xenotransplantation. Unfortunately, tilapia BBs might be unsuitable for clinical islet xenotransplantation because tilapia insulin differs from human insulin by 17 amino acids and, thus, would be immunogenic and less biologically active in humans. Therefore, we have produced transgenic tilapia that express a "humanized" tilapia insulin gene. Future improvements on these transgenic fish may allow tilapia to play an important role in clinical islet xenotransplantation.

Advances in pig genomics and functional gene discovery

Advances in pig gene identification, mapping and functional analysis have continued to make rapid progress. The porcine genetic linkage map now has nearly 3000 loci, including several hundred genes, and is likely to expand considerably in the next few years, with many more genes and amplified fragment length polymorphism (AFLP) markers being added to the map. The physical genetic map is also growing rapidly and has over 3000 genes and markers. Several recent quantitative trait loci (QTL) scans and candidate gene analyses have identified important chromosomal regions and individual genes associated with traits of economic interest. The commercial pig industry is actively using this information and traditional performance information to improve pig production by marker-assisted selection (MAS). Research to study the co-expression of thousands of genes is now advancing and methods to combine these approaches to aid in gene discovery are under way. The pig's role in xenotransplantation and biomedical research makes the study of its genome important for the study of human disease. This review will briefly describe advances made, directions for future research and the implications for both the pig industry and human health.

Cultural and ethical analysis of problem orientation in xenotransplantation development

THROUGHOUT THE WORLD xenotransplantation researchers are confronted not only with medical and technological difficulties but also with cultural and ethical ambiguities. Within the research program “Treatment of Diabetes by Transplantation of Porcine Islets” at Karolinska Institute/Huddinge University Hospital in Stockholm, Sweden, an interview study has been performed concerning the cultural and ethical issues related to xenotransplantation research and development. The purpose of the study is to evaluate, from cultural and ethical viewpoints, the way in which the xenotransplantation approach is perceived among individuals who either professionally or by personal plight are engaged in the scientific development of treating diabetes by transplantation of porcine islets.

Efficient production by sperm-mediated gene transfer of human decay accelerating factor (hDAF) transgenic pigs for xenotransplantation.

A large number of hDAF transgenic pigs to be used for xenotransplantation research were generated by using sperm-mediated gene transfer (SMGT). The efficiency of transgenesis obtained with SMGT was much greater than with any other method. In the experiments reported, up to 80% of pigs had the transgene integrated into the genome. Most of the pigs carrying the hDAF gene transcribed it in a stable manner (64%). The great majority of pigs that transcribed the gene expressed the protein (83%). The hDAF gene was transmitted to progeny. Expression was stable and found in caveolae as it is in human cells. The expressed gene was functional based on in vitro experiments performed on peripheral blood mononuclear cells. These results show that our SMGT approach to transgenesis provides an efficient procedure for studies involving large animal models.

Role of complement in xenotransplantation.

The xenotransplantation research is driven by the increasing gap between the number of patients with end-stage organ failure on waiting lists for transplantation and the supply of allografts. The lack of success in developing suitable artificial organs for permanent treatment of organ failure has further strengthened the need for xenotransplantation research. Pigs are now generally accepted to be the source animal of choice. Transplantation of pig organs to humans faces several barriers which have to be overcome before it comes to clinical application: (1) anatomical and physiological conditions; (2) immunological rejection mechanisms; (3) molecular compatibility between signal molecules of the two species; (4) risk of transmission of microorganisms, particularly pig endogenous retroviruses; and (5) legal and ethical aspects both with respect to the animal and the recipient. Here we will focus on the role of the complement system in the rejection of immediately vascularized pig-to-primate xenografts. The hyperacute rejection occurring within minutes after transplantation is mediated by binding of natural antibodies to the Galalpha(l-3)Gal epitope on the endothelial cells with subsequent complement activation. Whereas inhibition of complement activation protects against hyperacute rejection, the role of complement in the later rejection phases is less clarified.

Biliary atresia.

Biliary atresia.

Lectin interactions with alpha-galactosylated xenoantigens.

alpha-Galactosylated xenoantigens (Galalpha1-3Galbeta1-4GlcNAcbeta1 and Galalpha1-3Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc) are often detected with the alpha-Gal specific lectin Griffonia simplicifolia 1 isolectin B4 (GS1 B4). However, this lectin exhibits a broad and variable specificity for carbohydrates terminating in alpha-Gal. Thus, both false positive and false negative results may occur when GS1 B4 is used to determine natural antigens in xeno (pig-to-primate) transplantation research. To refine the tools for detecting alpha-galactosylated antigens we have studied the binding of various alpha-galactophilic lectins to alpha-galactosylated neoglycoproteins. The lectins were: Euonymus europaeus agglutinin (EEA), Griffonia simplicifolia 1 isolectin B4 (GS1 B4), Maclura pomifera agglutinin (MPA) and Pseudomonas aeruginosa agglutinin (PA-IL). Although both GS1 B4 and MPA strongly bound glycoconjugates terminating in Gal there seems to be some differentiation in their sugar binding preferences. MPA was the only lectin that showed high affinity for the pentasaccharide Galalpha1-3Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc and for the Galalpha-glycans on non-primate thyroglobulin. The length of the xenoantigenic carbohydrate chain may influence the nature of the inhibition when a simple sugar is used to inhibit GS1 B4 binding to the xenoantigen. Inhibition studies of MPA GS1 B4 interaction further suggest that both lectins attach to the same site of the carbohydrate antigen and that GS1 B4 in addition binds to at least one other site that has no affinity for MPA. When lectins are used for recognition and investigation of natural Galalpha-antigens, we propose that GS1 B4 and MPA should accompany each other.

Porcine livers perfused with human blood mount a graft-versus-"host" reaction.

A major focus of xenotransplantation research is the interaction between human immune effector mechanisms and porcine tissues. We present evidence that a transplanted porcine organ might also mount a significant immune response to a human recipient. Isolated porcine livers were perfused with fresh human blood. Plasma samples were analyzed for complement production by reverse CH50 analysis. ELISA was used to determine the amount and class of porcine immunoglobulin in human blood after xenoperfusion. Flow cytometry was used to determine the specificity and class of porcine immunoglobulin in human blood after xenoperfusion and to determine whether porcine immunoglobulins had bound to the human lymphocytes in the blood perfusing the porcine livers. Electron microscopy was used to evaluate the interaction of porcine Kupffer cells and human erythrocytes. Over the course of 72 hr of extracorporeal perfusion of porcine livers with human blood, the hematocrit fell progressively to as low as 2.5% of starting values, a phenomenon not seen in experiments using porcine blood. We have demonstrated both porcine complement and immunoglobulin in the human blood after xenoperfusion. The porcine antibodies in the human blood have specificity for human lymphocyte antigens. In fact, with increasing duration of perfusion, 40% of the xenoperfusions showed increasing titers of porcine antibodies with specificity for human lymphocyte antigens suggesting a response by primed porcine lymphocytes. However, the majority of erythrocytes are extracted directly by Kupffer cells in the liver. These data demonstrate the ability of porcine livers to generate both a humoral and cellular graft versus host response to human cells.

Isolectins I-A and I-B of Griffonia(Bandeiraea) simplicifolia: CRYSTAL STRUCTURE OF METAL-FREE GS I-B4 AND MOLECULAR BASIS FOR METAL BINDING AND MONOSACCHARIDE SPECIFICITY

Seeds from the African legume shrub Griffonia simplicifolia contain several lectins. Among them the tetrameric lectin GS I-B(4) has strict specificity for terminal alpha Gal residues, whereas the closely related lectin GS I-A(4) can also bind to alpha GalNAc. These two lectins are commonly used as markers in histology or for research in xenotransplantation. To elucidate the basis for the fine difference in specificity, the amino acid sequences of both lectins have been determined and show 89% identity. The crystal structure of GS I-B(4), determined at 2.5-A resolution, reveals a new quaternary structure that has never been observed in other legume lectins. An unexpected loss of both Ca(2+) and Mn(2+) ions, which are necessary for carbohydrate binding in legume lectins, may be related to a particular amino acid sequence Pro-Glu-Pro in the metal binding loop. Comparison with demetallized concanavalin A reveals a different process for the loss of metal ions and for the subsequent loss of carbohydrate binding activity. The GS I-A x alpha GalNAc and GS I-B x alpha Gal complexes were constructed using homology modeling and docking approaches. The unusual presence of an aromatic amino acid at position 47 (Tyr in I-A and Trp in I-B) explains the strong preference for alpha-anomeric sugars in both isolectins. Alteration at one amino acid position, Ala(106) in I-A versus Glu(106) in I-B, is the basis for the observed specificities toward alpha GalNAc and alpha Gal.

21st Workshop of the Study Group on Artificial Insulin Delivery, Pancreas and Islet Transplantation (AIDPIT) of the European Association for the Study of Diabetes (EASD)

THROUGHOUT THE WORLD xenotransplantation researchers are confronted not only with medical and technological difficulties but also with cultural and ethical ambiguities. Within the research program “Treatment of Diabetes by Transplantation of Porcine Islets” at Karolinska Institute/Huddinge University Hospital in Stockholm, Sweden, an interview study has been performed concerning the cultural and ethical issues related to xenotransplantation research and development. The purpose of the study is to evaluate, from cultural and ethical viewpoints, the way in which the xenotransplantation approach is perceived among individuals who either professionally or by personal plight are engaged in the scientific development of treating diabetes by transplantation of porcine islets.

Progress and potential for gene-based medicines.

During the past decade researchers have explored the potential of gene-based medicines to extend current treatments employing chemical entities and proteins. However, progress has been slower than was originally predicted due to our limited knowledge of the genetic components of major diseases, the complexity of developing active biological agents as therapies, and the stringent and time-consuming tests necessary to ensure safety prior to introduction of these novel modalities in the clinic. In spite of the present technology challenges and clinical setbacks in gene therapy it is anticipated that gene-based medicines will find their niche in disease prevention and management strategies in the coming decade, extending the repertoire of medicines available to satisfy key unmet medical needs. Additionally, progress in xenotransplantation research is creating the opportunity to use gene-modified porcine organs for human transplantation. This innovative approach aims to address the current insufficiency of human donor organs for clinical transplantation.

Neural tissue xenotransplantation: what is needed prior to clinical trials in Parkinson's disease? Neural Tissue Xenografting Project.

Embryonic allografted human tissue in patients with Parkinson's disease has been shown to survive and ameliorate many of the symptoms of this disease. Despite this success, the practical problems of using this tissue coupled to the ethical restrictions of using aborted human fetal tissue have lead to an exploration for alternative sources of suitable material for grafting, including xenogeneic embryonic dopaminergic-rich neural tissue. Nevertheless, xenografted neural tissue itself generates a number of practical, ethical, safety, and immunological issues that have to be addressed prior to any clinical xenotransplant program. In this article we review these critical issues and set out the criteria that we consider need to be met in the development of our clinical xenotransplantation research programs. We advocate that these, or similar, criteria should be adopted and made explicit by other centers contemplating similar clinical trials.

REPORT FROM THE SWISS SCIENCE COUNCIL PROGRAMME ON TECHNOLOGY ASSESSMENT

Xenotransplantation research, as a part of biomedical research, investigates the question of whether it is possible to take living organs, tissues, or cells from animals and transplant them into humans. It is hoped that the xenotransplantation of organs will close the widening gap between the growing demand for organs and the limited supply of donor organs; cellular xenotransplantation aims at providing first or improved therapies for a variety of diseases.