William L. Fodor

Xenotransplantation of transgenic pig olfactory ensheathing cells promotes axonal regeneration in rat spinal cord

Here we describe transplantation of olfactory ensheathing cells (OECs) or Schwann cells derived from transgenic pigs expressing the human complement inhibitory protein, CD59 (hCD59), into transected dorsal column lesions of the spinal cord of the immunosuppressed rat to induce axonal regeneration. Non-transplanted lesion-controlled rats exhibited no impulse conduction across the transection site, whereas in animals receiving transgenic pig OECs or Schwann cells impulse conduction was restored across and beyond the lesion site for more than a centimeter. Cell labeling indicated that the donor cells migrated into the denervated host tract. Conduction velocity measurements showed that the regenerated axons conducted impulses faster than normal axons. By morphological analysis, the axons seemed thickly myelinated with a peripheral pattern of myelin expected from the donor cell type. These results indicate that xenotranplantation of myelin-forming cells from pigs genetically altered to reduce the hyperacute response in humans are able to induce elongative axonal regeneration and remyelination and restore impulse conduction across the transected spinal cord.

Production of α1,3-Galactosyltransferase-Knockout Cloned Pigs Expressing Human α1,2-Fucosylosyltransferase

Abstract The production of genetically engineered pigs as xenotransplant donors aims to solve the severe shortage of organs for transplantation in humans. The first barrier to successful xenotransplantation is hyperacute rejection (HAR). HAR is a rapid and massive humoral immune response directed against the pig carbohydrate Galα1,3-Gal epitope, which is synthesized by α1,3-galactosyltransferase (α1,3-GT). The Galα1,3-Gal antigen also contributes to subsequent acute vascular rejection events. Genetic modifications of donor pigs transgenic for human complement regulatory proteins or different glycosyltransferases to downregulate Galα1,3-Gal expression have been shown to significantly delay xenograft rejection. However, the complete removal of the Galα1,3-Gal antigen is the most attractive option. In this study, the 5′ end of the α1,3-GT gene was efficiently targeted with a nonisogenic DNA construct containing predominantly intron sequences and a Kozak translation initiation site to initiate translation of the neomycin resistance reporter gene. We developed two novel polymerase chain reaction screening methods to detect and confirm the targeted G418-resistant clones. This is the first study to use Southern blot analysis to demonstrate the disruption of the α1,3-GT gene in somatic HT-transgenic pig cells before they were used for nuclear transfer. Transgenic male pigs were produced that possess an α1,3-GT knockout allele and express a randomly inserted human α1,2-fucosylosyltransferase (HT) transgene. The generation of homozygous α1,3-GT knockout pigs with the HT-transgenic background is underway and will be unique. This approach intends to combine the α1,3-GT knockout genotype with a ubiquitously expressed fucosyltransferase transgene producing the universally tolerated H antigen. This approach may prove to be more effective than the null phenotype alone in overcoming HAR and delayed xenograft rejection.

Tissue engineering and cell based therapies, from the bench to the clinic: The potential to replace, repair and regenerate

The field of Regenerative Biology as it applies to Regenerative Medicine is an increasingly expanding area of research with hopes of providing therapeutic treatments for diseases and/or injuries that conventional medicines and even new biologic drug therapies cannot effectively treat. Extensive research in the area of Regenerative Medicine is focused on the development of cells, tissues and organs for the purpose of restoring function through transplantation. The general belief is that replacement, repair and restoration of function is best accomplished by cells, tissues or organs that can perform the appropriate physiologic/metabolic duties better than any mechanical device, recombinant protein therapeutic or chemical compound. Several strategies are currently being investigated and include, cell therapies derived from autologous primary cell isolates, cell therapies derived from established cell lines, cell therapies derived from a variety of stem cells, including bone marrow/mesenchymal stem cells, cord blood stem cells, embryonic stem cells, as well as cells tissues and organs from genetically modified animals. This mini-review is not meant to be exhaustive, but aims to highlight clinical applications for the four areas of research listed above and will address a few key advances and a few of the hurdles yet to be overcome as the technology and science improve the likelihood that Regenerative Medicine will become clinically routine.

Expression of the human α1,2-fucosyltransferase in transgenic pigs modifies the cell surface carbohydrate phenotype and confers resistance to human serum-mediated cytolysis

Hyperacute rejection (HAR) is the first critical immunological hurdle that must be addressed in order to develop xenogeneic organs for human transplantation. In the area of cell‐based xenotransplant therapies, natural antibodies (XNA) and complement have also been considered barriers to successful engraftment. Transgenic expression of human complement inhibitors in donor cells and organs has significantly prolonged the survival of xenografts. However, expression of complement inhibitors without eliminating xenogeneic natural antibody (XNA) reactivity may provide insufficient protection for clinical application. An approach designed to prevent XNA reactivity during HAR is the expression of human α1,2‐fucosyltransferase (H‐transferase, HT). H‐transferase expression modifies the cell surface carbohydrate phenotype of the xe‐nogeneic cell, resulting in the expression of the universal donor O antigen and a concomitant reduction in the expression of the antigenic Galα1,3‐Gal epitope. We have engineered various transgenic pig lines that express HT in different cells and tissues, including the vascular endothelium. We demonstrate that in two different HT transgenic lines containing two different HT promoter constructs, expression can be differentially regulated in a constitutive and cytokine‐inducible manner. The transgenic expression of HT results in a significant reduction in the expression of the Galα1,3‐Gal epitope, reduced XNA reactivity, and an increased resistance to human serum‐mediated cytolysis. Transgenic pigs that express H‐transferase promise to become key components for the development of xenogeneic cells and organs for human transplantation.—Costa, C., Zhao, L., Burton, W. V., Bondioli, K. R., Williams, B. L., Hoagland, T. A., DiTullio, P. A., Ebert, K. M., Fodor, W. L. Expression of the human α1,2‐fucosyltrans‐ferase in transgenic pigs modifies the cell surface carbohydrate phenotype and confers resistance to human serum‐mediated cytolysis. FASEB J. 13, 1762–1773 (1999)

Remyelination of the nonhuman primate spinal cord by transplantation of H-transferase transgenic adult pig olfactory ensheathing cells

Olfactory ensheathing cells (OECs) have been shown to mediate remyelination and to stimulate axonal regeneration in a number of in vivo rodent spinal cord studies. However, whether OECs display similar properties in the primate model has not been tested so far. In the present study, we thus transplanted highly‐purified OECs isolated from transgenic pigs expressing the α1,2 fucosyltransferase gene (H‐transferase or HT) gene into a demyelinated lesion of the African green monkey spinal cord. Four weeks posttransplantation, robust remyelination was found in 62.5% of the lesion sites, whereas there was virtually no remyelination in the nontransplanted controls. This together with the immunohistochemical demonstration of the grafted cells within the lesioned area confirmed that remyelination was indeed achieved by OECs. Additional in vitro assays demonstrated l) that the applied cell suspension consisted of >98% OECs, 2) that the majority of the cells expressed the transgene, and 3) that expression of the HT gene reduced complement activation more than twofold compared with the nontransgenic control. This is the first demonstration that xenotransplantation of characterized OECs into the primate spinal cord results in remyelination.

Expression of human CD59 in transgenic pig organs enhances organ survival in an ex vivo xenogeneic perfusion model

The serious shortage of available donor organs for patients with end stage organ failure who are in need of solid organ transplantation has led to a heightened interest in xenotransplantation. The major barrier to successful discordant xenotransplantation is hyperacute rejection. Hyperacute rejection results from the deposition of preformed antibodies that activate complement on the luminal surface of the vascular endothelium, leading to vessel occlusion and graft failure within minutes to hours. Endogenous membrane-associated complement inhibitors normally protect endothelial cells from autologous complement -- however, these molecules are species-restricted and therefore are ineffective at inhibiting activated xenogeneic complement. To address the pathogenesis of hyperacute rejection in the pig-to-human combination, F1 offspring were generated from a transgenic founder animal that was engineered to express the human terminal complement inhibitor hCD59. High-level cell surface expression of hCD59 was detected in the hearts and kidneys of these transgenic F1 animals, similar to expression levels in human kidney tissue. The hCD59 was expressed on both large vessel and capillary endothelium. Ex vivo perfusion experiments, using human blood as the perfusate, were performed with transgenic porcine hearts and kidneys to evaluate the ability of hCD59 to inhibit hyperacute rejection. These experiments demonstrated that transgenic organs expressing hCD69 resisted hyperacute rejection, as measured by increased organ function for both the hearts and the kidneys, as compared with control pig organs. Hearts from hCD59-expressing animals demonstrated a five-fold prolongation in function compared with controls, 109.8 +/- 20.7 min versus 21.2 +/- 2.9 min (P = 0.164). The hCD59-expressing kidneys also demonstrated significantly prolonged function at 157.8 +/- 27.0 min compared with 60.0 +/- 6.1 min for controls (P = 0.0174). Deposition of C9 neoantigen In the vasculature of porcine organs perfused with human blood was markedly reduced in organs expressing hCD59. These studies demonstrate that C5b-9 plays an important role in hyperacute rejection of a porcine organ perfused with human blood and suggest that donor pigs transgenic for hCD59 may be an integral component of successful clinical xenotransplantation.

Primary Structure and Functional Characterization of a Soluble, Alternatively Spliced Form of B7-1

Recent studies have suggested that soluble forms of B7-1 and B7-2 may exist, but transcripts that code for these molecules have not been previously described. In this study, we report the cloning and characterization of an alternatively spliced soluble form of porcine B7-1 (sB7-1) that lacks exons coding for both the transmembrane and cytoplasmic domains. Northern blot analysis of RNA from alveolar macrophages revealed an approximate 3:1 ratio of the transmembrane form of B7-1 mRNA relative to sB7-1 mRNA. Porcine B7-1 was present on the surface of both B and T cells following stimulation with PMA/ionomycin. A histidine-tagged form of porcine sB7-1 (sB7-1-His) interacted with both CD28 and CTLA-4, and effectively blocked IL-2 production from human responder cells stimulated with PHA and either porcine or human stimulator cells. In addition, sB7–1-His inhibited human T cell proliferation in response to porcine or human peripheral blood leukocytes. This study is the first report of an alternatively spliced form of B7 that codes for a soluble protein. Furthermore, these data demonstrate that porcine B7-1 interacts with the human receptors CD28 and CTLA-4, suggesting a potential role for this molecule in pig to human xenotransplantation. Possible physiological functions for the soluble form of B7-1 are discussed.

Transgenic pigs designed to express human CD59 and H-transferase to avoid humoral xenograft rejection

Abstract: Research in pig‐to‐primate xenotransplantation aims to solve the increasing shortage of organs for human allotransplantation and develop new cell‐ and tissue‐based therapies. Progress towards its clinical application has been hampered by the presence of xenoreactive natural antibodies that bind to the foreign cell surface and activate complement, causing humoral graft rejection. Genetic engineering of donor cells and animals to express human complement inhibitors such as hCD59 significantly prolonged graft survival. Strategies to decrease the deposition of natural antibodies were also developed. Expression of human α1,2‐fucosyltransferase (H transferase, HT) in pigs modifies the cell‐surface carbohydrate phenotype resulting in reduced Galα1,3‐Gal expression and decreased antibody binding. We have developed transgenic pigs that coexpress hCD59 and HT in various cells and tissues to address both natural antibody binding and complement activation. Functional studies with peripheral blood mononuclear cells and aortic endothelial cells isolated from the double transgenic pigs showed that coexpression of hCD59 and HT markedly increased their resistance to human serum‐mediated lysis. This resistance was greater than with cells transgenic for either hCD59 or HT alone. Moreover, transgene expression was enhanced and protection maintained in pig endothelial cells that were exposed for 24u2003h to pro‐inflammatory cytokines. These studies suggest that engineering donor pigs to express multiple molecules that address different humoral components of xenograft rejection represents an important step toward enhancing xenograft survival and improving the prospect of clinical xenotransplantation.

Down-regulation of Gal alpha(1,3)Gal expression by alpha1,2-fucosyltransferase: further characterization of alpha1,2-fucosyltransferase transgenic mice.

BACKGROUNDnIn pig-to-primate transplantation, antibody-mediated hyperacute rejection is the consequence of binding of natural antibodies to Gal alpha(1,3)Gal on pig endothelium. The elimination of the Gal alpha(1,3)Gal antigen from pig cells should prevent hyperacute rejection. Using in vitro techniques, we have previously reported that using the alpha1,2-fucosyltransferase gene induces the preferential expression of H substance with a concomitant reduction in the expression of Gal alpha(1,3)Gal. The aim of the present study was to examine the effect of expressing the alpha1,2-fucosyltransferase gene in vivo on Gal alpha(1,3)Gal.nnnMETHODSnThree alpha1,2-fucosyltransferase transgenic lines of mice were produced and characterized serologically and histologically.nnnRESULTSnImmunohistological studies showed heavy staining for H substance in liver, spleen, kidney, and heart, with a reduction in staining for Gal alpha(1,3)Gal. In addition, there was a reduction in the binding of human anti-Gal alpha(1,3)Gal antibody to lymphocytes from alpha1,2-fucosyltransferase transgenic mice and a substantial decrease in complement-mediated cytolysis of alpha1,2-fucosyltransferase transgenic lymphocytes when compared with that obtained with normal mice.nnnCONCLUSIONSnThe findings have important implications, in that alpha1,2-fucosyltransferase transgenic pigs could be produced as a source for humans. Such pigs should have a reduced expression of Gal alpha(1,3)Gal.

Immune parameters relevant to neural xenograft survival in the primate brain

Cicchetti F, Fodor W, Deacon TW, van Horne C, Rollins S, Burton W, Costantini LC, Isacson O. Immune parameters relevant to neural xenograft survival in the primate brain. Xenotransplantation 2003: 10: 41–49.