In order to make xenotransplantation clinically feasible, methodologies to achieve specific immunological tolerance to donor antigens are likely to be needed. Donor-specific tolerance can be induced across allogeneic and closely related xenogeneic barriers by establishment of donor hematopoiesis in the recipient. However, studies in both concordant and discordant xenogeneic species combinations have revealed that non-immunologic barriers such as species differences in the growth factors and adhesive interactions which comprise the hematopoietic environment, limit the establishment and efficacy of hematopoiesis. The applicants have obtained evidence that the provision of donor species-specific cytokines can enhance donor hematopoiesis in a xenogeneic environment, and that adhesive receptor ligand interactions critical to hematopoiesis may also vary between species. The applicants intend to define the critical factors that are likely to limit porcine hematopoiesis in human recipients, with the aim of optimizing conditions to achieve tolerance in the swine to human species combination. In this application, the applicants will develop reagents to identify and isolate porcine long-term multilineage repopulating pluripotent hematopoietic stem cells. The capacity of putative stem cell enriched porcine hematopoietic cells to self renew and differentiate will be examined in vitro and in vivo stem cell transplantation studies in both homologous and xenogeneic recipients. The capacity of porcine adhesion molecules to direct relevant cell-cell interactions between swine hematopoietic progenitors and human stromal elements will be analyzed. Moreover, the role of the alpha-gal carbohydrate modification in the biology of hematopoiesis will be explored in vitro in clonogenic assays and by utilizing alpha-1,3gal knockout mice in in vivo studies. It is anticipated that the results of these studies will provide information which will guide the development of transgenic pigs whose hematopoietic cells can function in human recipients, and which can thereby reliably induce durable tolerance for swine to human xenotransplantation.
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