Organ availability currently poses one of the biggest obstacles to clinical transplantation. Xenotransplantation, or the use of organs from non-human donors, may provide a solution to this problem. Although attempts at clinical xenotransplantation using organs from nonhuman primates have met with some success, their use is restricted by limited availability, legal and ethical considerations, and the potential for transmitting dangerous viral diseases. Miniature swine provide a very attractive alternative in these regards. This laboratory has demonstrated the efficacy of bone marrow transplantation (BMT) for inducing transplantation tolerance across xenogeneic barriers using both concordant rat/mouse and discordant swine/mouse models, and studies are now in progress to apply this approach to the discordant large animal model of swine to cynomolgus monkey. These studies have, however, demonstrated the difficulty in achieving lasting engraftment of donor hematopoietic stem cells (HSC), presumably due in part to species differences in cytokine networks and stromal microenvironments responsible for HSC regulation. As an initial step toward the use of xenogeneic BMT for the induction of tolerance in humans toward organs from swine, the aims of this proposal are to use bone marrow culture systems to study the interactions of swine HSC and human bone marrow stroma with the goals of dissecting the regulatory mechanisms associated with HSC expansion and swine bone marrow engraftment in a human bone marrow stromal environment, and to test chimerism-enhancing strategies in a swine/cynomolgus BMT model. For this purpose, the ability of human bone marrow stroma to support the expansion of swine HSC will first be measured using previously established long-term bone marrow culture (LTBMC) techniques and myeloid progenitor colony assays. These systems will then be used to determine whether HSC from swine and human bone marrow compete for functional hematopoietic """"""""niches"""""""" via a competitive dilution analysis, as well as to determine whether a competitive advantage can be gained using HSC from swine fetal liver or cord blood. These experimental systems will also be used to determine whether swine HSC can be preferentially expanded using recombinant swine hematopoietic cytokines. As a preclinical model, these latter studies will be followed up in vivo by determining whether the indicated cytokines can enhance mixed chimerism in partially myeloablated cynomolgus monkeys transplanted with swine bone marrow. Finally, antibody blocking studies will be performed using the mixed LTBMC assay in order to identify human adhesion molecules and cytokine receptors which participate in the expansion of human HSC in a species-specific fashion. Candidate genes will be identified for use in subsequent studies to genetically engineer, or """"""""humanize"""""""", swine HSC. It is anticipated that these studies will provide both a critical step toward the goal of using swine BMT as a tolerizing regimen for clinical xenotransplantation, and a unique model for studying requirements for HSC expansion and engraftment.
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