G-CSF Treatment Induces Hematopoietic Stem Cell Quiescence but Loss of Repopulating Activity

Abstract 1206

There is accumulating evidence that systemic signals, such as inflammatory cytokines, can affect hematopoietic stem cell (HSC) function. Granulocyte colony stimulating factor (G-CSF), the principal cytokine regulating granulopoiesis, is often induced in response to infection or inflammation. Additionally, G-CSF is the most commonly used agent for HSC mobilization prior to stem cell transplantation. Recently there has been a renewed interest in the use of “G-CSF primed bone marrow” for stem cell transplantation, so understanding the affect of G-CSF on bone marrow HSCs is clinically relevant. Because the G-CSF receptor is expressed on HSCs, and G-CSF creates biologically relevant modifications to the bone marrow microenvironment, we hypothesized that increased signaling through G-CSF may alter the repopulating and/or self-renewal properties of HSCs.

Due to G-CSF's role as an HSC mobilizing agent, we predicted that the number of HSCs in the bone marrow would be reduced after 7 days of G-CSF treatment. Surprisingly, we observe that stem cell numbers markedly increase, regardless of which HSC-enriched population is analyzed. C-kit⁺lineage⁻sca⁺CD34⁻ (KLS-34⁻), KLS CD41^lowCD150⁺CD48⁻ (KLS-SLAM), and KLS-SLAM CD34⁻ increase by 6.97±2.25 fold, 1.79±0.29 fold, and 2.08±0.39 fold, respectively. To assess HSC repopulating activity, we conducted competitive bone marrow transplants. Donor mice were treated with or without G-CSF for 7 days, and bone marrow was transplanted in a 1:1 ratio with marrow from untreated competitors into lethally irradiated congenic recipients. Compared to untreated HSCs, we found that G-CSF treated cells have significantly impaired long-term repopulating and self-renewal activity in transplanted mice. In fact, on a per cell basis, the long-term repopulating activity of KLS-CD34⁻ cells from G-CSF treated mice was reduced approximately 13 fold. The loss of repopulating activity per HSC was confirmed by transplanting purified HSCs. Homing experiments indicate that this loss of function is not caused by an inability to home from the peripheral blood to the bone marrow niche. As HSC quiescence has been positively associated with repopulating activity, we analyzed the cell cycle status over time of KLS-SLAM cells treated with G-CSF. This analysis revealed that after a brief period of enhanced cycling (69.8±5.0% G₀ at baseline; down to 55.9±4.1% G₀after 24 hours of G-CSF), treated cells become more quiescent (86.8±2.8% G₀) than untreated HSCs. A similar increase in HSC quiescence was seen in KLS-34⁻ cells. Thus our data show that G-CSF treatment is associated with HSC cycling alterations and function impairment.

Because G-CSF is associated with modifications to the bone marrow microenvironment, and the microenvironment is known to regulate HSCs at steady state, we asked whether the G-CSF induced repopulating defect was due to a cell intrinsic or extrinsic (secondary to alterations in the microenvironment) mechanism. To do this, we repeated the competitive transplantation experiments using chimeric mice with a mixture of wild-type and G-CSF receptor knockout (Csf3r^−/−) bone marrow cells. We find that only the repopulating activity of HSCs expressing the G-CSF receptor is affected by G-CSF, suggesting a cell-intrinsic mechanism. To identify targets of G-CSF signaling that may mediate loss of stem cell function, we performed RNA expression profiling of sorted KSL-SLAM cells from mice treated for 36 hours or seven days with or without G-CSF. The profiling data show that G-CSF treatment is associated with activation of inflammatory signaling in HSCs. Studies are in progress to test the hypothesis that activation of specific inflammatory signaling pathways mediates the inhibitory effect of G-CSF on HSC function. In summary, G-CSF signaling in HSCs, although associated with increased HSC quiescence, leads to a marked loss of long-term repopulating activity. These data suggest that long-term engraftment after transplantation of G-CSF-primed bone marrow may be reduced and requires careful follow-up.