Stem/Progenitor Cell Inversions With Cell Cycle Transit

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DESCRIPTION (provided by applicant): The process of blood cell formation (hematopoiesis) has been extensively studied but many basic mechanisms continue to be unknown. Previous studies have shown that marrow cell engraftment varies at different points in cell cycle and that these changes are reversible. In recent work we have shown that murine progenitor numbers also vary with cytokine induced cell cycle transit. Studying unseparated marrow cells we demonstrated that 7-factor responsive progenitors (HPP-CFC and CFU-c) increased markedly during the first cell cycle transit and then returned to baseline. Remarkably, these increases were tightly linked to the decreased ability of stem cells to engraft in a competitive transplant model. This phenomenon is termed stem/progenitor cell inversions. These data suggest that the functional phenotype of early marrow stem cells shifts reversibly from engraftable stem cell to progenitor and back to hematopoietic stem cell as it traverses cell cycle, and that the previously conceived stem-cell-to-progenitor unidirectional hierarchy is instead a continuum of reversible phenotypic shifts. The long term objective of this grant are to further characterize the cell cycle related inversions points as to engraftment, progenitor levels and differentiation capacity of the marrow cells studying both unseparated and highly purified stem cells in FLT-3 ligand, thrombopoietin and steel factor. We plan to assess lineage(negative) rhodamine(low) Hoechst(low) (LRH) marrow cells at different points in cell cycle as to short and long-term engraftment in a competititive transplant model, to assess 7-factor-responsive progenitors and to evaluate the capacity of purified stem cells to differentiate in the presence a 7-factor cytokine cocktail and two cocktails designed to promote granulocyte-macrophage or megakaryocyte differentiation. In the engraftment studies there will be an emphasis on G1 phase of cell cycle. The differentiation studies will be carried out in bulk Teflon cultures and on a single cell clonal basis. In order to further understand the phenotype of the stem cell at different points in cell cycle. To point ways to alternative induction studies we will characterize cytokine receptors, adhesion proteins, integrins and other epitopes on the cell surface. These studies employ high-speed cell sorting, fluorescent cell imaging and a variety of in vitro and in vivo stem/progenitor assays. They promise to further define the basic nature of the hematopoietic marrow stem cells and could lead to interesting preclinical models for selective lineage support of various myeloablative therapy approaches .