Ex vivo cultured hematopoietic cells hold substantial promise for diverse clinical applications. For many of these applications to be realized requires that primitive pluripotent stem cells survive and expand during these cultures. Utilizing cells produced in a novel purfusion-based culture system, the investigators propose to explore key aspects of stem cell self-renewal, with particular emphasis on those questions which bear directly on the applications of ex vivo expanded stem cells to the clinic. Specifically the investigators propose to: (1) Ask whether ex vivo cultured hematopoietic cells are able to reconstitute long-term hematopoiesis in NOD/SCID mice and in fetal sheep. In these studies the investigators will compare the relative repopulating ability of ex vivo expanded human bone marrow, umbilical cord blood, and mobilized peripheral blood cells; (2) Map the 5' cis-acting sequence of the human Hoxb-4(2.6) gene, to isolate those sequences responsible for Hoxb-4 expression in hematopoietic cells. If the investigators identify cis- acting repressor sequences, they will then ask whether pharmacologic derepression at these sites results in increased self-renewal, as assayed by LTCIC expansion in vitro and increased competitive xenogeneic repopulating ability in vivo. These studies should directly address the issues for safely applying currently available stem cell preparations to diverse clinical scenarios, while paving the way for greatly increased applications for expanded stem cells in the future.
