Clinical and basic laboratory studies are directed at developing efficient and safe gene transduction and ex vivo manipulation strategies for hematopoietic cells, including stem and progenitor cells and lymphocytes, and using genetic marking techniques to answer important questions about in vivo hematopoiesis. In the rhesus model, shown to be the only predictive assay for human clinical results, we have focused on optimizing gene transfer to primitive stem and progenitor cells, and using genetic marking techniques to understand stem cell behavior in vivo.We have continued to further enhance gene transfer efficiency into rhesus engrafting cells, resulting in early levels of marked cells as high as 50-80%, with stable levels of 5-35% in all lineages, a range with clinical utility. We have found that actively-cycling transduced cells have an engraftment defect that can be corrected by a short culture on a fibronectin fragment with stem cell factor alone. The high levels have allowed us to continue to track the clonal contributions to hematopoiesis for the first time in a large animal model. We have utilized a new technology that allows simultaneous assessment of multiple clonal contributions to peripheral blood populations. We have found a different population of engrafting cells that contribute for the first 1-2 months post-transplantation, that are then replaced by a very stable set of over 80 clones that contribute to all lineages now for over 3 years. We have investigated the impact of cytokine therapy, radiation, and chemotherapy on the in vivo behavior of stem cell clones, using this powerful methodology. Thus far we have shown that prolonged cytokine treatment with either G-CSF or SCF does not significantly alter the number of stem cell clones contributing to hematopoiesis, nor result in detectable clonal exhaustion or recruitment. In contrast, treatment with low dose total body irradiation results in a significant decrease in stem cell clones contributing to peripheral blood lineages. Studies with busulfan are ongoing. We have also begun to carefully investigate the lineage contributions of individual stem and progenitor cell clones, asking whether clones contribute equally to each lineage such as granulocytes, T cells, B cells, dendritic cells and mast cells. We have begun to study the contributions of these clones to other tissues, including endothelium and muscle, and have developed a rhesus macaque myocardial infarction model that has allowed us to assess whether mobilization of primitive cells via cytokines in the peri-infarct period results in improved myocardial regeneration and function post MI. Thus far, there is no evidence for a positive benefit, but we continue to study different agents and schedules for mobilization. We have discovered a novel iron oxide particle that is taken up nonspecifically and highly efficiently by all primary cell types studied, and this particle has been utilized to label and then track mesenchymal stem cells in vivo via MRI follwing intracardiac or intravenous injection in the setting of a myocardial infarction.
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