This project of the Hematopoiesis Section is focused on the basic biology of hematopoietic stem cells (HSC). HSC are a rare population of self-renewing cells that give rise to all cells in the peripheral blood. For patients with a life-threatening hematologic disease, transplantation of HSC from a healthy closely matched donor after ablation of the diseased bone marrow can be a life long cure. However, the main risk in these procedures is the transplantation of inadequate numbers of HSC. Our goal is to understand the processes that promote HSC self renewal and inhibit HSC differentiation. By manipulating the balance between self-renewal and differentiation we will be able to increase the number of stem cells and consequently increase the effectiveness of bone marrow transplantation to cure acquired or inherited hematopoietic diseases. ? Specific Aim 1: We have previously used ChIP Seq to evaluate the role of EKLF (transcription factor) binding in mouse erythroid cells. we will use this same procedure to analyze histone acetylation, CTCF and USF occupancy in human HSC (Lin- CD34+ CD38-) multipotent progenitor cells (Lin-, CD34+ CD38+) and erythroid progenitor cells (Lin-, CD71+, GlyA+). After sequencing the ChIP selected DNA, we will use newly developed programs to match each sequence tag to the genome and determine where significant enrichment has occurred. Loci at which histone acetylation changes will be analyzed to determine the pathways associated with HSC differentiation and restriction to the erythroid lineage. similar analyses will be performed for CTCF and USF-1 both of which are hypothesized to be flank regulated genes. ? Specific Aim 2: The bone marrow stromal cells are a mixed population of macrophages, endothelial and fibroblast cells that provides a matrix in which HSC become embeded. However, the maintainence of these cells in culture is not uniform and the different cytokines produced have differing effects on HSC. Near the trabecular bone where the density of the extracellular matrix is most dense, HSC are most actively undergoing self renewal. In the center of the marrow space, where the matrix is considerable softer, HSC are undergoing differentiation. To achieve a uniform enironment in which specific soluable factors can be added or subtracted we will evaluate stem cell survival and expansior or differentiation of collagen matrices of different densities designed to mimic the bone interface or the center of the marrow. We will compare the number of HSC growing on each matrix by competitive bone marrow transplantation and the ability to differentiate using in vitro colony forming assays. The goal ofthese studies is to devise an ex vivo culture condition to expand compatible donor HSC to numbers that can be safely transplanted. For enriched populations of human HSC we will perform similar experiments and follow the cell cycle status and colony forming abilities to determine whether the conditions identified for mouse can be applied to human HSC.
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