This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Introduction: Improvement in bone marrow transplantation techniques can only be made with a better understanding of the hematopoietic stem cell and the regulation of hematopoiesis. Studies involving analyzing the functional ability of hematopoietic stem cell differentiation through cell cycle transit have elucidated points where dramatic differentiation toward specific lineage(s) occurs. Molecular studies confirm that stem cells have changing receptor expression through cycle; a reflection of sensitivity to specific microenvironmental influences. This may have a direct impact on the progeny of the hematopoietic stem cell. Murine studies confirmed that (lineage negative rhodamine low Hoechst low (LRH)) cells can be directed towards lineage differentiation into megakaryocytes or granulocytes at specific positions in celly cycle. A critical aspect is that these changes are not fixed differentiation steps but rather are reversible with continued cell cycle transit. This data suggests that instead of a hieracrhally defined progenitor class, the cells are in a constant state of phenotype flux. To translate these findings to human application; it is crucial to concurrently study human stem cells. This pilot project aims to parallel ongoing murine work with initiation into human studies. Methods: We will first evaluate different populations of cells and look at their cell cycle phase, cell doubling time, viability in culture and optimize the culturing conditions. We will test different populations of purified marrow aspirates and clinical samples of G-CSF primed CD34+ PBSC. Cell populations will either be sorted immediately based on cell cycle phase based on DNA content and placed in differentiation cultures, or they will be cultured with cytokines and at serial times after primary culture, sub-populations of cells will be placed in differentiation cultures such as a machrophage-granulocyte culture with G-CSF, GM-CSF and SCF. Results: An in vivo sub-lethal radiation recovery model has been developed and will allow us to test different populations of cultured cells.
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