Hematopoietic progenitor cell mobilization by granulocyte colony-stimulating factor and erythropoietin in the absence of matrix metalloproteinase-9.

The use of mobilized hematopoietic progenitor cells (HPC) has largely replaced the use of bone marrow HPC for autologous and allogeneic transplantation; however, the mechanisms of HPC mobilization remain unclear. A better understanding of these mechanisms, may allow the development of improved (potentially more rapid and/or higher yield) HPC mobilization strategies, especially for patients who mobilize poorly using current mobilization protocols. Clinically, granulocyte colony-stimulating factor (G-CSF) is widely used to induce HPC mobilization, and evidence suggests that metalloproteinase enzymes released by activated granulocytes play an important role in the G-CSF-induced HPC mobilization. These enzymes may act to disrupt putative cell-cell and/or cell-extracellular matrix interactions within the hematopoietic microenvironment thereby releasing HPC into the blood. Matrix metalloproteinase-9 (MMP-9) appears to be important for G-CSF-induced mobilization. Using an MMP-9 knock-out (KO) mouse model, we investigated the role of MMP-9 in G-CSF and erythropoietin (EPO)-based HPC mobilization at clinically relevant cytokine doses. There were few hematologic or hematopoietic differences between the wild-type and MMP-9KO mice during steady-state hematopoiesis. When treated subcutaneously with EPO (500 U/kg per day) and G-CSF (15 microg/kg per day) for 5 days and assayed on day 6, similarly increased extramedullary hematopoiesis and numbers of HPC in the spleen and blood were observed for both the wild-type and MMP-9KO mice. These data demonstrate that MMP-9 is not required for EPO + G-CSF mobilization and that alternative mobilization mechanisms must be active at clinically relevant cytokine concentrations.

Evidence for a qualitative hierarchy within the Hoechst-33342 'side population' (SP) of murine bone marrow cells.

In vitro cobblestone area (CA)-forming cell (CAFC) and in vivo (short-term and competitive repopulation) assays demonstrate that a qualitative hierarchy exists within the Hoechst-33342-defined side population (SP) in murine bone marrow (BM). Consistent with and extending previous studies, we demonstrate that (i) hematopoietic activity found in whole BM (WBM) is concentrated within the SP, rather than the non-SP (NSP); and (ii) within the SP, those cells that more strongly efflux the dye (lower SP, LSP) are qualitatively different from those that less strongly efflux the dye (upper SP, USP). Qualitative differences are highlighted by evidence that (i) CA derived from LSP CAFC persist in culture significantly longer than CA derived from USP CAFC; (ii) short-term, multilineage repopulation of lethally irradiated mice by LSP cells is more rapid than that in mice receiving USP, NSP, whole SP (WSP), or WBM cells and (iii) LSP cells out-compete USP cells in the multilineage hematopoietic repopulation of lethally irradiated recipients. These data suggest that LSP cells are of higher quality than USP cells and potentially provide a means by which qualitative changes in primitive hematopoietic progenitors occurring naturally with aging, or clinically as a consequence of therapeutic manipulation, can be assessed.