Bone marrow-derived mesenchymal stem cells in repair of the injured lung

Abstract

We sought to determine whether an intact bone marrow is essential to lung repair following bleomycin-induced lung injury in mice, and the mechanisms of any protective effects conferred by bone marrow-derived mesenchymal stem cell (BMDMSC) transfer. We found that myelosupression increased susceptibility to bleomycin injury and that BMDMSC transfer was protective. Protection was associated with the differentiation of engrafted BMDMSC into specific and distinct lung cell phenotypes, with an increase in circulating levels of G-CSF and GM-CSF (known for their ability to promote the mobilization of endogenous stem cells) and with a decrease in inflammatory cytokines. In vitro, cells from injured, but not from normal, mouse lung produced soluble factors that caused BMDMSC to proliferate and migrate toward the injured lung. We conclude that bone marrow stem cells are important in the repair of bleomycin-injured lung and that transfer of mesenchymal stem cells protects against the injury. BMDMSC localize to the injured lung and assume lung cell phenotypes, but protection from injury and fibrosis also involves suppression of inflammation and triggering production of reparative growth factors.

Figure 1.

(a) Infusion of BMDMSC prevented mortality in mice myelosuppressed with busulfan before bleomycin. Sixty-six percent of myelosuppressed mice (n = 6) given bleomycin died without BMDMSC transfer. There was no mortality in any of the other experimental groups (n = 5 animals per group). Histologic sections of lungs obtained from: (b, c) normal C57BL/6 mice; (d, e) 14 d after 4 U/kg busulfan (lungs appear normal); (f, g) 14 d after bleomycin (increased cellularity and fibrosis typical of bleomycin injury); (h, i) 14 d after bleomycin following busulfan myelosuppression (apparently more extensive increased cellularity and fibrosis compared with f and g); (j, k) 14 d after bleomycin followed by BMDMSC transfer (minimal alterations in lung architecture compared with f and g); (l, m) 14 d after bleomycin followed by BMDMSC transfer in myelosuppressed mice showing apparent protection (compare with h and i), but more abnormalities than present in the BMDMSC transplanted mice without myelosuppression (compare with j and k).

Figure 2.

Infusion of BMDMSC reduces lung damage induce by bleomycin. Morphometric analysis of histologic sections of total left lung was done to determine the percentage of the lung that was affected. Pictures (magnification: ×2) were analyzed using AxioVision 4.2 software (Carl Zeiss, Thornwood, NY). The graphic represents the average from 5–9 histologic lung sections. Bleomycin caused substantial injury and BMDMSC infusion reduced bleomycin-induced injury (*P < 0.05, bleomycin-BMDMSC versus bleomycin, versus busulfan-bleomycin, and versus busulfan-bleomycin-BMDMSC). In myelosuppressed animals, this measure of injury was on average worse than in nonmyelosuppressed animals and on average less severe when animals received BMDMSC. Since only the lungs of the one-third of the myelosuppressed animals that received BMDMSC survived to 14 d were analyzed, the histologic measurements likely underestimate the degree of injury in the entire group.

Figure 3.

Bleomycin injury induces localization of BMDMSC to injured lung. Histologic analysis by indirect immunofluorescence assay (IIFA) with anti-GFP antibodies (green), of the lungs from animals: (a) 14 d after bleomycin without BMDMSC infusion; (b) normal mice infused with BMDMSC (no GFP-positive cells); (c) 14 d after bleomycin followed by BMDMSC (a modest number of green fluorescing cells are present); (d) 14 d after bleomycin followed by BMDMSC in a busulfan myelosuppressed animal (numerous green cells are present); (e) Busulfan treated mice infused with BMDMSC (no localization of donor cells to the lungs); (f) Morphometric analysis of the intensity of GFP staining from the different samples. All microphotographs were taking at ×40 magnification.

Figure 4.

Donor BMDMSC localizing to injured lung assume lung cell phenotypes. Sections were analyzed in double-stained IIFA with anti-GFP (green) and antibodies to specific cell type markers (red); co-localization in each case appears yellow (arrows point to double positive cells). (a–d) Anti-vimentin (fibroblast). (a) Normal control; (b) 14 d after bleomycin; (c) 14 d after busulfan followed by BMDMSC (no lung injury); (d) 14 d after bleomycin followed by BMDMSC in a busulfan myelosuppressed animal. (e, f) Anti-aquaporin (type I alveolar epithelium). (e) Fourteen days after busulfan followed by BMDMSC (no lung injury); (f) 14 d after bleomycin followed by BMDMSC in busulfan myelosuppressed animal. (g, h) Anti-pro–surfactant protein C (type II alveolar epithelium). (g) Fourteen days after busulfan followed by BMDMSC (no lung injury); (h) 14 d after bleomycin followed by BMDMSC in busulfan myelosuppressed animal. (i, j) Anti–smooth muscle actin (SMA-1, myofibroblasts). (i) Fourteen days after busulfan followed by BMDMSC (no lung injury); (j) 14 d after bleomycin followed by BMDMSC in busulfan myelosuppressed animal. (k) Percentage of GFP-positive cells that express lung cell phenotype markers in myelosupressed mice treated with bleomycin and infused with BMDMSC. All microphotographs were taken at ×40 magnification.

Figure 5.

BMDMSC infusion attenuates bleomycin-induced increases in lung expression of immune-related cytokines and increases circulating concentrations of G-CSF and GM-CSF. (a) Serum concentrations of G-CSF and GM-CSF in control animals (n = 4) and 14 d after bleomycin with (n = 4) or without (n = 5) BMDMSC transfer. BMDMSC transfer resulted in significantly elevated serum concentrations of both factors. The differences were significant (P < 0.05) after one-way ANOVA and repeated-measures ANOVA between animals treated with BMDMSC and the other two groups (**). (b) Expression of cytokines in lung tissue determined by real-time PCR in control mice, (n = 3) and mice given bleomycin with (n = 3) or without (n = 3) BMDMSC transfer 14 d after bleomycin. Bleomycin treatment caused prolonged increased expression of these immune-related cytokines and transfer BMDMSC tended to suppress this response.

Figure 6.

Cells from injured lungs produce humoral factor(s) that cause BMDMSC proliferation and chemotaxis in vitro. BMDMSC obtained from GFP-positive mice were grown for 7 d, and equally distributed in the lower well of a 6-well Transwell plate. Cell preparations from lungs of normal mice or lungs from mice 14 d after bleomycin were placed in the upper well on a 1.5-inch filter with 3-μm pores. The co-cultures were maintained for 5 d and then analyzed for GFP-positive cells. (a) Photomicrographs of the upper and lower chambers of a Transwell in which the same number of BMDMSC were placed in the lower well and the same number of cells from either normal or bleomycin-injured lung was placed in the upper well. In the presence of injured lung, there were many more GFP-positive cells in the lower chamber. With normal lung, there was no evidence of migration of the GFP-positive cells to the upper chamber, but in the presence of injured lung there was marked migration and the cells appeared in clumps. (b) Quantitative assessment of GFP-positive cells in both upper and lower chambers by fluorescence intensity.