Hematopoietic stem cells (HSC) are characterized by their ability to undergo self-renewal and multi-lineage differentiation. A variety of in vitro assays have been used to assess and predict the in vivo potential of HSC from different human sources for these characteristics. It is understood, however, that the in vivo long-term proliferative potential of human HSC is generally not fully addressed by in vitro assays. The investigators have developed a large animal model of human hematopoiesis in sheep which permits the engraftment and multilineage differentiation of human HSC following transplantation in utero. In this model, they have reported (a) the long-term engraftment of human fetal and purified adult HSC, (b) the development of graft versus host disease (GVHD) with light density mononuclear cells (LMNC) from human post-natal sources, and (c) the induction of donor (human)-specific tolerance in chimeric lambs. The overall aims of the studies proposed here are to use the preimmune fetal/tolerized sheep as a model to investigate and compare the in vivo potential of native and ex- vivo expanded HSC from human cord blood, bone marrow, and peripheral blood with regards to (1) the long-term (stable) engraftment, self-renewal, and differentiation in non- myeloablated and myeloablated hosts, and (2) the development of GVHD in this human/sheep xenograft model. From each source the investigators plan to compare the in vivo activity of HSC present in light density mononuclear cells (LMNC), T-depleted LMNC, CD34+-enriched, and highly purified, sorted fractions under """"""""normal"""""""" and cytokine-induced """"""""stressed"""""""" conditions. It is hoped that these studies will help identify the source(s) and HSC preparation(s) likely to provide long- term in vivo reconstitution and with low risks of GVHD and HSC exhaustion.
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