HGF as a Strong Mobilizer of Hematopoietic Stem Cells to Peripheral Blood.

How hematopoietic stem cells (HSC) in bone marrow (BM) contribute to liver regeneration remains to be resolved. The mechanisms which mobilize HSC from BM to peripheral blood (PB) and govern their homing to the liver are unknown. Hepatocyte growth factor (HGF) is locally and generally increased following liver injury, suggesting that it promotes proliferation, adhesion, and survival of hepatocytes. PB stem cells mobilized by cytokines are widely used for clinical transplantation. However, it is not known if HGF can mobilize HSC to PB or if it has the capability to differentiate HSC into hepatocytes. In this study we examined whether HGF can mobilize HSC from BM to PB using the murine stem cell transplantation model.

First, we found that HGF transgenic mice, which have high serum levels of HGF concentration, had many colony-forming cells in PB, suggesting HGF increases circulation of HSC and progenitor cells. After determining through RT-PCR analysis that lineage (Lin) ⁻ BM cells express the tyrosine kinase receptor c-MET, we speculated that the c-MET-HGF axis modulates the recruitment of HSC from BM to PB. We also examined the effects of exogenous HGF in mobilizing stem cells from BM to PB. To determine whether HGF can mobilize HSC to PB, we investigated the expression of CD34 using flow cytometric analysis. The CD34⁺ cells in PB mobilized by HGF increased in a dose-dependent pattern and reached a plateau at 0.1mg/kg of recombinant HGF administration. Significant increases in CD34⁺ cells in PB were noted at 3h after HGF infusion. The continual administration of HGF every 24h increased the CD34⁺ cells in PB to maximum levels at 4 days. Finally, the absolute number of CD34⁺ cells in PB after HGF administration was as much as the number of those cells after administration at 12-hour intervals subcutaneously with 0.125mg/kg of recombinant human granulocyte-colony stimulating factor (G-CSF) for 4 consecutive days. To investigate engraftment of the mobilized cells to BM, 0.1mg/kg HGF was injected into Ly-5.1 mice every 24h for 4 days. Lin⁻ cells in PB were collected 3h 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 of mice transplanted with Lin⁻ cells from HGF-treated mice was 1.8%, whereas that of the mice transplanted with untreated PB cells was 0%. Multilineage engraftment was confirmed by the presence of the Thy-1⁺ cells, B220⁺ cells, and Mac-1/Gr-1⁺ cells. When we tested the CD34 expression of the stem cells transplanted, the majority of the cells expressed CD34. Then we tracked single Lin⁻, Sca-1⁺, c-kit⁺, CD34⁺ PB cells from G-CSF-treated transgenic-enhanced green fluorescent protein (EGFP) mice that were injected into spleen of the CCl₄-induced liver-injured B6 mice along with 500 Lin⁻, Sca-1⁺, c-kit⁺, CD34⁺ PB cells from G-CSF-treated normal B6 mice. Two months later, donor-derived GFP⁺ cells were identified among recipient hepatocytes in liver-injured mice using immunohistochemistry for GFP.

These findings demonstrate that stem cells with long-term engraftment capabilities can be mobilized by HGF, and that HSC in PB mobilized by HGF are capable of differentiating into hepatocytes, suggesting HGF contributes to liver regeneration partly by mobilizing HSC to PB.