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. The process of blood cell formation (hematopoiesis) has been extensively studied yet many basic mechanisms continue to be unclear. Dogma states that hematopoietic differentiation is generally stochastic in nature. In vitro stem cell studies indicate that stem cells have some ability toward directed cellular differentiation with use of specific cytokine cocktails, but lack of formation of adequate numbers of lineage specific cells currently prevents translation into the clinical realm. Prior work on cell cycle related hematopoietic stem cell (HSC) function has indicated that HSC are in a continuum, with constant reversible phenotypic variation through cycle. We have found that cell cycle position is an independent variable of the stem cell on the outcome of response to an identical stimulus. Examples of stimuli are bone marrow transplantation, progenitor expansion, and differentiation. We have found reproducible changes in the context of cell cycle position of the stem cell at the time of applying a stimulus. We have primarily studied this though initial cell cycle transit from a quiescent G0-1 state but have more recently looked at these stem cells after initial cell division and have found that this phenomenon continues to be tied to cell cycle position in a reproducible pattern. These data suggest that the functional phenotype of early marrow stem cells shifts as it traverses cell cycle. We have explored the functional ability of HSC differentiation through cell cycle, which is a reflection of time in culture and have found that there are points where differentiation toward specific lineage(s) occurs. We have found that an initial cytokine inductive signal prior to initial cell division influences the content of the cell population two weeks later. We believe that HSC have changing sensitivity to specific microenvironmental influences through cycle and this directly impacts on the HSC's progeny. We have performed experiments using purified stem/progenitors [lineage negative, rhodamine low, Hoescht low cells] and showed highly directed lineage differentiation into megakaryocytes or granulocytes at specific positions in cell cycle. The objectives of this grant are to define cell cycle related directed lineage differentiation in preclinical murine transplant models and to study human stem/progenitor cells for directed differentiation opportunities. These studies promise to further define the basic nature of the hematopoietic marrow stem cells and could lead to interesting preclinical cellular bioprocessing models for selective lineage support of various myeloablative therapy approaches or progenitor deficient states.
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