Mobilized peripheral blood stem and progenitor cells (HSPC) are the preferred cell source for hematopoietic transplantation;however our understanding of the mobilization process is incomplete. Work by us and others using cellular and genetic models have identified a "trans" acting component to mobilization by G-CSF and GRObeta, and the demonstration of a role for mature polymorphonuclear neutrophils (PMN) in mobilization. These studies have led to the hypothesis that release of proteases alters adhesive and intercellular interactions that result in peripheralization of HSPC. Although not proven, what is emerging is that mobilization is a complex process with broad interdependencies that can be stimulated by multiple mechanisms. We hypothesize that what appears to be multiple mechanisms, may in fact reflect multiple points of intervention along a central mobilization axis that includes adhesion co-receptors, chemokines and their receptors and interdependent inter and intracellular signaling components. We have identified a novel mobilization strategy utilizing the CXCR2 ligand GRObeta in combination with the CXCR4 antagonist AMD3100 that results in unexpected synergistic mobilization of HSPC with enhanced stem cell properties, and additional synergy when used in combination with G-CSF. We hypothesize that these cells may represent better hematopoietic grafts for transplant and gene therapy. Mechanism of mobilization studies lead us to a second hypothesis that synergistic mobilization results from unexpected receptor crosstalk between the CXCR4 and CXCR2 receptors that results in elevated and sustained PMN release of proteases, particularly MMP-9, that facilitates cell egress. We propose in this application to characterize the HSPC populations mobilized by GRObeta plus AMD3100 alone and with G-CSF, determine the mechanism of action of mobilization relative to PMN and MMP-9, and evaluate the utility of GRObeta plus AMD3100 mobilized HSPC as an improved hematopoietic cellular graft for hematopoietic transplant and gene therapy application. Project Relevance: Identifying regulatory pathways involved in stem cell mobilization will greatly enhance our knowledge of the stem cell niche and reward us with optimized procedures to collect populations of stem cells with enhanced properties that will positively impact stem cell transplantation and gene therapy.