Abstract
The mechanisms of mobilization of hematopoietic stem cell (HSC) from bone marrow (BM) to peripheral blood (PB) by cytokines are poorly understood. The number of circulating HSC can be dramatically increased, or mobilized, by a wide variety of stimuli including hematopoietic growth factors, chemotherapy, and chemokines. Recent studies suggest that hematopoietic proteases, such as matrix metalloproteinase-9 (MMP-9), enable BM repopulating cells to translocate to circulating pool. In this study we investigated the mechanism of stem cell mobilization by the hepatocyte growth factor (HGF).
First, we examined the effects of exogenous HGF on mobilizing HSC from BM to PB. To investigate engraftment of the mobilized cells from BM, 0.1mg/kg HGF was injected into Ly-5.1 mice every 24 hours for 4 days. Lin− cells in PB were collected 3 hours after the last injection of HGF and then injected into lethally-irradiated Ly-5.2 C57BL/6 mice. Two months after transplantation, the level of engraftment was assessed by analysis of donor (Ly-5.1) cells in the nucleated cells of the PB of recipient mice. The mean percentage of donor cells in mice transplanted was 1.8%, whereas that in the mice transplanted with untreated PB cells was 0%.
Second, we investigated the mechanisms of HSC-mobilization by HGF from BM to PB. After determining through RT-PCR analysis that Lin− BM cells express the tyrosine kinase receptor c-met and that Lin− CD34− c-kit+ Sca-1+ BM cells does not express c-met, we speculated that the HGF/c-Met involved in HSC mobilization, focusing on the stromal cells. The murine stromal cells were cultured and stimulated with HGF under a serum-free medium condition. Phosphorylation of c-Met protein and activation of stem cell factor (SCF) in stromal cells were induced after HGF treatment. However, MMP-9 was not simultaneously activated after the HGF stimulation. The levels of SCF in supernatant began to increase 1 hour after HGF-stimulation. These in vitro data suggest that HGF can effectively promote the release of SCF from stromal cells, resulting in the mobilization effect of HGF.
Third, to investigate engraftment of the mobilized cells by HGF, G-CSF, or PBS in male MMP-9 −/− mice, PB mononuclear cells were collected from HGF-, G-CSF-, or PBS-treated male MMP-9−/− mice, and then injected into lethally-irradiated female wild-type mice. Two months after transplantation, the presence of Sry gene in BM in transplanted female mice was confirmed by PCR analysis, showing the existence of the chimera. We found the Sry gene in the mice transplanted with PB cells from HGF- and G-CSF-treated mice, but not in the mice transplanted with PBS-treated PB cells. HGF and G-CSF can mobilize HSC from BM to PB in MMP-9−/− mice.
These findings demonstrate that stem cells with long-term engraftment capabilities can be mobilized by HGF, and that the activation of SCF induced by HGF/c-Met from stromal cells is involved in the mobilization mechanisms of HGF but not MMP-9.
Generation of murine stromal cell lines supporting hematopoietic stem cell proliferation by use of recombinant retrovirus vectors encoding simian virus 40 large T antigen.
