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. 2018 Nov 15;16(1):59-68.
doi: 10.1007/s13770-018-0163-5. eCollection 2019 Feb.

GM-CSF Enhances Mobilization of Bone Marrow Mesenchymal Stem Cells via a CXCR4-Medicated Mechanism

Jiyoung Kim #  1 , Na Kyeong Kim #  1 , So Ra Park  1 , Byung Hyune Choi  2
Affiliations

GM-CSF Enhances Mobilization of Bone Marrow Mesenchymal Stem Cells via a CXCR4-Medicated Mechanism

Jiyoung Kim et al. Tissue Eng Regen Med. .

Abstract

Background: This study was conducted to investigate the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on the mobilization of mesenchymal stem cells (MSCs) from the bone marrow (BM) into the peripheral blood (PB) in rats.

Methods: GM-CSF was administered subcutaneously to rats at 50 μg/kg body weight for 5 consecutive days. The BM and PB of rats were collected at 1, 3, and 5 days during the administration for analysis.

Results: Upon GM-CSF administration, the number of mononuclear cells increased rapidly at day 1 both in the BM and PB. This number decreased gradually over time in the BM to below the initial amount by day 5, but was maintained at a high level in the PB until day 5. The colony-forming unit-fibroblasts were increased in the PB by 10.3-fold at day 5 of GM-CSF administration, but decreased in the BM. Compared to GM-CSF, granulocyte-colony stimulating factor (G-CSF) stimulated lower levels of MSC mobilization from the BM to the PB. Immunohistochemical analysis revealed that GM-CSF induced a hypoxic and proteolytic microenvironment and increased C-X-C chemokine receptor type 4 (CXCR4) expression in the BM. GM-CSF added to BM MSCs in vitro dose-dependently increased CXCR4 expression and cell migration. G-CSF and stromal cell derived factor-1 (SDF-1) showed similar results in these in vitro assays. Know-down of CXCR4 expression with siRNA significantly abolished GM-CSF- and G-CSF-induced MSC migration in vitro, indicating the involvement of the SDF-1-CXCR4 interaction in the mechanism.

Conclusion: These results suggest that GM-CSF is a useful tool for mobilizing BM MSCs into the PB.

Keywords: Bone marrow; Granulocyte–macrophage colony-stimulating factor; Hypoxia; Mesenchymal stem cells; Mobilization.

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Conflict of interest statement

The authors have no potential conflicts of interest.All animal procedures were approved by the committee of Institutional Animal Care (INHA-IACUC Approval Number: INHA 130625-217-1).

Figures

Fig. 1
Fig. 1
Effects of GM-CSF on the colony-forming ability of stem cells in the BM and PB. SD rats were treated with PBS or 50 μg/kg body weight of GM-CSF via subcutaneous injection for 5 consecutive days. A, B The number of MNCs in the BM and PB was measured at 0, 1, 3, and 5 days after GM-CSF administration. Data show the mean values with standard deviations in BM of a femur and in 10 mL PB (n = 3/group). **p < 0.01 for day 0 versus other time points. C, D The number of CFU-F and CFU-GM in the BM and PB was determined at day 5 after GM-CSF administration. Data show the mean values with standard deviations in 106 MNCs in each group (n = 3/group). *p < 0.05 and **p < 0.01 for the PBS versus GM-CSF groups
Fig. 2
Fig. 2
Effects of GM-CSF and G-CSF on the number of MNCs and CFU-Fs in the BM and PB. SD rats were treated with PBS or 50 μg/kg body weight of GM-CSF or G-CSF via subcutaneous injection for 5 consecutive days. A The number of MNCs in the BM and PB at day 5 after administration. Data show the mean values with standard deviations in BM of a femur and in 10 mL PB (n = 3/group). B The CFU-F numbers were measured in the BM and PB at day 5 after administration. Data show the mean values with standard deviations in 106 MNCs in each group (n = 3/group). *p < 0.05 and **p < 0.01 for the PBS versus GM-CSF or G-CSF groups. #p < 0.05 for the GM-CSF versus G-CSF groups
Fig. 3
Fig. 3
Changes in the BM microenvironment caused by GM-CSF administration. SD rats were treated with PBS or 50 μg/kg body weight of GM-CSF for 5 consecutive days. A Histological sections of BM were stained with pimonidazole to observe the hypoxic state followed by immunostaining to examine HIF-1α and MMP-9 expression levels in the BM at day 5. B Histological sections of the BM were immunostained for CXCR4 and SDF-1 to examine the expression of these stem cell-related genes. Scale bars = 100 μm. The number of CXCR4- or SDF-1-positive cells was counted in the microscopic images of each group and data are presented in the graph as the mean values with standard deviations from 3 independent samples (n = 3). **p < 0.01 for the PBS versus GM-CSF groups
Fig. 4
Fig. 4
Effect of G-CSF and GM-CSF on the migration of BM MSCs in vitro. MSCs were isolated from the BM of SD rats and cultured in Transwells at passage 4 for cell migration assays. A Cells were treated with increasing concentrations of G-CSF or GM-CSF in the upper chamber as indicated. B Cells were treated with increasing concentrations of SDF-1, as indicated in the lower chamber in the absence or presence of 100 ng/mL GM-CSF in the upper chamber. In both (A) and (B), the number of cells that migrated across the Transwell membrane is expressed as the mean value with standard deviations (n = 3/group). **p < 0.01 for the untreated versus cytokine-treated groups at each concentration. #p < 0.05 and ##p < 0.01 for the SDF-1 versus SDF-1 + GM-CSF groups. C The expression of CXCR4 was examined after GM-CSF and/or SDF-1 treatment at the protein (top) and mRNA (bottom) levels. The expression of β-actin and GAPDH was examined as internal controls for each assay
Fig. 5
Fig. 5
Effect of CXCR4 siRNA on the G-CSF or GM-CSF-induced BM MSCs migration in vitro. BM MSCs from SD rats were treated at passage 4 with 100 ng/mL of G-CSF or GM-CSF with or without prior transfection of CXCR4 siRNA. A Migration of BM MSCs was examined in the untreated, G-CSF, or GM-CSF groups. Data are presented as the mean values with standard deviations (n = 3/group). **p < 0.01 for the untreated versus G-CSF or GM-CSF groups without CXCR4 siRNA. #p < 0.05 for the control and CXCR4 siRNA groups. B Decrease in CXCR4 protein and mRNA levels was confirmed by western blotting (top) and RT-PCR (bottom) as indicated
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