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. 2015 Jan 23;290(4):1994-2006.
doi: 10.1074/jbc.M114.605063. Epub 2014 Dec 9.

CXCR4 receptor overexpression in mesenchymal stem cells facilitates treatment of acute lung injury in rats

Jing-Xian Yang  1 , Nan Zhang  2 , Han-Wei Wang  3 , Peng Gao  4 , Qing-Ping Yang  1 , Qing-Ping Wen  5
Affiliations

CXCR4 receptor overexpression in mesenchymal stem cells facilitates treatment of acute lung injury in rats

Jing-Xian Yang et al. J Biol Chem. .

Abstract

Novel therapeutic regimens for tissue renewal incorporate mesenchymal stem cells (MSCs) as they differentiate into a variety of cell types and are a stem cell type that is easy to harvest and to expand in vitro. However, surface chemokine receptors, such as CXCR4, which are involved in the mobilization of MSCs, are expressed only on the surface of a small proportion of MSCs, and the lack of CXCR4 expression may underlie the low efficiency of homing of MSCs toward tissue damage, which results in a poor curative effect. Here, a rat CXCR4 expressing lentiviral vector was constructed and introduced into MSCs freshly prepared from rat bone marrow. The influence of CXCR4 expression on migration, proliferation, differentiation, and paracrine effects of MSCs was examined in vitro. The in vivo properties of CXCR4-MSCs were also investigated in a model of acute lung injury in rats induced by lipopolysaccharide. Expression of CXCR4 in MSCs significantly enhanced the chemotactic and paracrine characteristics of the cells in vitro but did not affect self-renewal or differentiation into alveolar and vascular endothelial cells. In vivo, CXCR4 improved MSC homing and colonization of damaged lung tissue, and furthermore, the transplanted CXCR4-MSCs suppressed the development of acute lung injury in part by modulating levels of inflammatory molecules and the neutrophil count. These results indicated that efficient mobilization of MSCs to sites of tissue injury may be due to CXCR4, and therefore, increased expression of CXCR4 may improve their therapeutic potential in the treatment of diseases where tissue damage develops.

Keywords: Cell Migration; Lung Injury; Mesenchymal Stem Cells (MSCs); Migration; Transplantation.

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Figures

FIGURE 1.
FIGURE 1.
Generation and molecular characterization of rat MSCs from bone marrow. A, morphological characteristics of MSCs at P3 under phase-contrast microscopy. B, immunophenotype analysis of MSCs at P3 by flow cytometry. Adherent MSCs are positive to CD29, CD44, and CD90 but negative for CD34 and CD45. Data were compared with isotype-matched controls. C, immunocytochemistry to detect stromal cell markers VCAM-1 and ICAM-1 (red) on MSCs. One representative experiment of three performed is shown. DAPI+ nuclei (blue). Scale bar: 50 μm in A and C.
FIGURE 2.
FIGURE 2.
CXCR4 is highly expressed in CXCR4-transduced MSCs. A, quantitative flow cytometry for CXCR4 and CXCR7 expression in MSCs expanded in culture from P0 to P3. A small proportion of MSCs expressed CXCR4 and CXCR7, and the percentages of positive cells gradually decreased as cells were expanded in vitro. One representative experiment of three performed is shown. B, L.v.-GFP transduced MSCs in monolayer culture under fluorescence microscopy. C, quantitative analysis of the percentages of GFP+ cells for the total number of transduced MSCs (DAPI+) by image J. Symbols represent the mean ± S.D. of 4 independent experiments. D, immunocytochemistry to detect CXCR4 expression in Mock-MSCs, GFP-MSCs, and CXCR4-MSCs. All transduced MSCs expressed GFP and were visible under fluorescence microscopy within 3 days after infection, whereas CXCR4 was highly expressed only in CXCR4-transduced MSCs. One representative experiment of three performed is shown. E, comparison of percentages of CXCR4+ cells among three groups of MSCs by flow cytometric analysis. Symbols represent the mean ± S.D. (n = 3). green, GFP+; red, CXCR4+; blue, DAPI+ nuclei. Scale bar: 50 μm in B, 25 μm in D.
FIGURE 3.
FIGURE 3.
SDF-1α chemotaxis assay for CXCR4-transduced MSCs. A, MSCs were transduced with CXCR4/GFP or GFP. On day 5 cells were plated into the upper well of a 24-well Transwell plate, and different concentrations of SDF-1α were added to the lower well (50, 100 ng/ml). Assays were incubated for 5 h, and cells that migrated into the lower well were fixed, stained with crystal violet, and counted. Scale bar = 25 μm. One representative experiment of three is shown. B, quantitative analysis of percentage of migrated cells in total number of plated cells. Data represent the mean values ± S.D. of three wells from three independent experiments. The migration of CXCR4-MSCs and GFP-MSCs is compared with non-transduced cells in response to SDF-1α (50, 100 ng/ml). **, p < 0.01; CXCR4- versus GFP-MSCs at either concentration. ##, p < 0.01.
FIGURE 4.
FIGURE 4.
CXCR4 overexpression does not alter self-renewal and differentiation potential of MSCs but stimulates the cells to secrete VEGF and IL-6 in vitro. A, growth curves of MSCs. CXCR4-, GFP-, or Mock-MSCs were seeded at the same density in 24-well plates 5 days post gene-transduction. At days 1, 3, 5, 7, and 9, MSCs in each well (n = 6) were dissociated into single cells, and numbers of cells were counted with a hemocytometer and plotted as a function of time in days to reveal growth curves. B, differentiation of MSCs into lung cell types. After culture in CM for 8 days, three groups of MSCs differentiated into alveolar epithelial cells (SP-C+) and vascular endothelial cells (vWF+) as verified by immunocytochemistry. DAPI+ nuclei (blue). Scale bar: 25 μm. C, quantitative analysis of percentages of differentiated MSCs. Symbols represent the mean ± S.D. of four independent experiments. No significant differences were observed among the three groups of MSCs. E and F, levels of VEGF and IL-6 in supernatants of the three cell types were assayed by ELISA. Data represent the mean ± S.D. of three independent experiments (n = 9). Comparison between GFP-MSCs and Mock-MSCs, p > 0.05; comparison between CXCR4-MSCs and GFP-MSCs, ** p < 0.01.
FIGURE 5.
FIGURE 5.
CXCR4 overexpression enhances the ability of MSCs to modulate the inflammatory response to ALI. Rats were injected with CXCR4- or GFP-MSCs 1 h post LPS-injection, and lavage fluid from bronchial alveoli was removed 72 h later. A–C, levels of TNF-α, IL-6, and IL-10 in BAL fluid were assayed by ELISA. D, numbers of neutrophils in BAL fluid were counted. Symbols represent the mean ± S.D. of 3 independent experiments (n = 9). ALI versus control group; ##, p < 0.01. GFP- and CXCR4-MSCs versus ALI; *, p < 0.05, and **, p < 0.01, respectively. CXCR4-MSCs versus GFP-MSCs, @, p < 0.05.
FIGURE 6.
FIGURE 6.
CXCR4 overexpression enhances suppression of ALI. Lung tissue of ALI rats was histologically examined 72 h after injection of cells. A, H&E staining of lung. B, pathological score. C, lung wet/dry weight ratios. D, BAL protein concentrations. Data represent the mean ± S.D.; for each group, 10 biopsies from 5 rats were evaluated. ALI versus control animals; ##, p < 0.01. GFP- and CXCR4-MSCs versus ALI; *, p < 0.05, and **, p < 0.01 respectively. CXCR4- versus GFP-MSCs; @, p < 0.05. Scale bar: 100 μm in A.
FIGURE 7.
FIGURE 7.
Localization and migration of transplanted CXCR4-MSCs in the lungs. Lung tissue of ALI rats was harvested for immunocytochemistry with anti-CXCR4 and anti-Ki67 antibodies 2 weeks post MSC injection. A, GFP+ MSCs (green) were observed in the lung tissue in both cases, and the majority of GFP+ MSCs were Ki67-positive (red), corresponding to the proliferation potential of the cells. B, CXCR4 was strongly expressed (red) only in the lung of rats treated with CXCR4-MSCs. The white arrows highlight cells that are double-positive to CXCR4 and GFP. Nuclei were stained with DAPI (blue). Scale bar: 100 in A, 50 μm in B. C, quantitative analysis of percentages of Ki67+/GFP+/DAPI+ cells in the total number of GFP+/DAPI+ cells in lungs of ALI rats. D, quantitative analysis of the number of GFP+ MSCs (cells/mm2) in lungs of ALI rats. Symbols represent the mean ± S.D.; n = 5 rats per group, 5 sections per rat were selected, and 10 fields were photographed in each section. Cells were counted by Image J software. Comparison between groups of CXCR4-MSCs and GFP-MSCs. **, p < 0.01.
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