The conceptual basis of bone marrow transplantation (BMT) rests on reconstitution of hemopoiesis after depletion of marrow from defective or undesirable stem cells. A few hundred stem cells (HSC) could be sufficient for this purpose. This requires purification/enrichment of HSC. To achieve this end a reliable assay system for measuring the in vivo repopulating ability of the human HSC is needed. Available in vitro techniques for assaying human HSC, reflect the cells differentiation potential and not its repopulating ability. Our preliminary work, using transplantation in preimmune fetal sheep suggests that this system may offer a suitable vehicle for assaying human HSC in a xenograft assay. The proposed studies use the pre-immune fetal sheep as a model to: 1) Establish an assay system for human hematopoietic stem cells (HSC) that permits the qualitative and quantitative evaluation of human HSC activity in vivo. Available in vitro clonogenic assays for hematopoietic progenitors can explore the developmental potential of human HSC, but not their in vivo reconstitutive ability. 2) Identify the in vitro clonogenic assay(s) that can best predict the in vivo reconstituting ability of human HSC by comparing the in vitro clonogenic potential of a source of HSC with its in vivo engraftment activity. 3) Assess the in vitro systems for long term culture of bone marrow (LTBMC), and the available enrichment schemes for their ability to provide expanded/purified populations of HSC capable of long-term hematopoietic reconstitution in vivo. 4) Increase the efficiency of HSC engraftment by a) the use of hematopoietic growth factors IL-3, GM-CSF, and IL-6, and b) the experimental modulation of the """"""""homing"""""""" mechanism by the use of synthetic neoglycoproteins of defined specificities, monoclonal antibodies to """"""""homing"""""""" proteins, as well as, IL-3, GM-CSF, and IL-6. It is hoped that these studies will clarify the mechanism(s) regulating HSC activity in vivo, and narrow the gap between fundamental knowledge of HSC and its application in clinical bone marrow transplantation.
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