Fig 1.
Schematic of the experimental model.
Mice received a single endotracheal (e.t.) injection of 1.5 U/kg body weight of bleomycin sulfate to induce lung injury (day 0). Intravenous (i.v.) injection of human mesenchymal stromal cells obtained from whole umbilical cord (hUC-MSC) (2.5 × 105 cells) into the tail vein was performed 24 hours (day 1) and 7 days (day 7) after bleomycin administration. Mice groups were sacrificed at days 8, 14 and 21 after bleomycin administration (i.e., 24 hours, 7 days and 14 days after second hUC-MSC infusion, respectively).
Fig 2.
hUC-MSC down-regulate bleomycin-induced lung inflammation.
Histology of mouse lungs 8 days (A-D), 14 days (E-H) and 21 days (I-L) after endotracheal injection of sterile saline (saline) or bleomycin (bleomycin), the latter also followed by intravenous infusion of hUC-MSC (bleomycin+hUC-MSC) or sterile saline (bleomycin+saline). Lung sections obtained from C57BL/6 mice (n = 8 per group) were stained with H&E. Controls (A,E,I) demonstrated normal lung architecture. 8 days post bleomycin injury, peribronchial and perivascular inflammatory infiltrates were observed (B). Alveolar and interstitial infiltrates progressively increased from day 8 to 21, with progressive distortion of lung architecture and formation of fibrotic foci (F,J). At each time point, bleomycin-induced alterations were significantly attenuated by hUC-MSC treatment (D,H,L), but not by saline (C,G,K). Representative microscopic images (10× magnification) of three independent experiments are shown. (M) The histopathological inflammatory score of lung sections obtained from C57BL/6 mice (n = 8 per group) that received the aforementioned treatments was calculated. Results are mean ± SD (n = 8 per group) and are representative of three independent experiments. * = P < 0.05 compared to bleomycin treated mice.
Fig 3.
hUC-MSC down-regulate bleomycin-induced lung fibrosis.
Collagen content in mouse lungs 8 days (A-D), 14 days (E-H) and 21 days (I-L) after endotracheal injection of sterile saline (saline) or bleomycin (bleomycin), the latter also followed by intravenous infusion of hUC-MSC (bleomycin+hUC-MSC) or sterile saline (bleomycin+saline). Lung sections obtained from C57BL/6 mice (n = 8 per group) were stained with Picrosirius Red. Controls (A,E,I) demonstrated normal lung architecture. 8 days post bleomycin injury, initial thickening of the alveoli and septa was observed (B). Collagen deposition progressively increased from day 8 to 21, with progressive distortion of lung architecture and formation of fibrotic foci (F,J). At each time point, bleomycin-induced alterations were significantly attenuated by hUC-MSC treatment (D,H,L), but not by saline (C,G,K). Representative microscopic images (10× magnification) of three independent experiments are shown. (M) The Ashcroft fibrosis score of lung sections obtained from C57BL/6 mice (n = 8 per group) that received the aforementioned treatments was calculated. Results are mean ± SD (n = 8 per group) and are representative of three independent experiments. * = P < 0.05, ** = P < 0.01, compared to bleomycin treated mice. (N) Hydroxyproline content in C57BL/6 mouse lungs that received the aforementioned treatments. Results are mean ± SD (n = 8 per group) expressed as a percent of the value obtained from endotracheal saline treated mice and are representative of three independent experiments. * = P < 0.05, ** = P < 0.01 compared to bleomycin treated mice.
Fig 4.
Time course of cytokines and matrix components in lung tissue assessed by quantitative real-time PCR.
Gene expression analysis of Col1A1 (A), TGFβ (B), α-SMA (C), IL-1β (D), IL-2 (E), IL-6 (F) and IL-10 (G) in whole lung mRNA obtained at days 8, 14 and 21 after endotracheal injection of sterile saline (saline) or bleomycin (bleomycin), the latter also followed by intravenous infusion of hUC-MSC (bleomycin+hUC-MSC) or sterile saline (bleomycin+saline). Results are expressed as mean ± SD (n = 5 per group) and are representative of three independent experiments performed in triplicate. * = P < 0.05, ** = P < 0.01 compared to bleomycin treated mice.
Fig 5.
Protein expression of cytokines and matrix components in lung tissue assessed by Western blotting.
Collagen Col1A1 (A), TGFβ (B), IL-1β (C), IL-2 (D), IL-6 (E) and IL-10 (F) protein levels in whole lung tissue lysates obtained from C57BL/6 mice 21 days after endotracheal injection of sterile saline (saline) or bleomycin (bleo), the latter also followed by intravenous infusion of hUC-MSC (bleo+hUC-MSC) or sterile saline (bleo+saline). Results are representative of three independent experiments. Densitometric analysis of protein bands is normalized to actin and expressed as mean ± SD (n = 5 per group). ** = P < 0.01, *** = P < 0.001 compared to bleomycin treated mice.
Fig 6.
Immunohistochemical determination of α-SMA in lung tissue.
Peribronchial and perivascular deposition of α-SMA in C57BL/6 mouse lungs at day 21 after injection of endotracheal sterile saline only (A), endotracheal bleomycin only (B), endotracheal bleomycin followed by intravenous sterile saline (C) or endotracheal bleomycin followed by intravenous hUC-MSC (D). Lung sections were immunostained with anti-α-SMA antibody. Representative microscopic images (40× magnification) of three independent experiments are shown.
Fig 7.
Decrease of galectin-3 positive cells in bleomycin-injured lung tissue upon administration of hUC-MSC.
Macrophage infiltration in mouse lungs at days 8 (A-D) and 21 (E-H) after endotracheal injection of sterile saline (saline) (A, E) or bleomycin (bleomycin) (B, F), the latter also followed by intravenous infusion of hUC-MSC (bleomycin+hUC-MSC) (D, H) or sterile saline (bleomycin+saline) (C, G). Lung sections obtained from C57BL/6 mice (n = 8 per group) were immunostained with anti-galectin-3 antibody. At each time point, the number of immunoreactive macrophages infiltrating the lungs are significantly decreased by hUC-MSC infusion in comparison to bleomycin-treated mice. Representative microscopic images (40× magnification) of three independent experiments are shown. (I) Cell count of galectin-3 positive macrophages in C57BL/6 mouse lung sections, and (J) quantitative real-time PCR analysis of galectin-3 gene expression in whole lung mRNA, obtained at days 8 and 21. Results are expressed as mean ± SD of positively immunostained cell count per sample (n = 5 per group) and are representative of three independent experiments. * = P < 0.05, ** = P < 0.01, *** = P < 0.001 compared to bleomycin treated mice.
Fig 8.
Decrease of arginase-I positive cells in bleomycin-injured lung tissue upon administration of hUC-MSC.
Macrophage infiltration in mouse lungs at days 8 (A-D) and 21 (E-H) after endotracheal injection of sterile saline (saline) (A, E) or bleomycin (bleomycin) (B, F), the latter also followed by intravenous infusion of hUC-MSC (bleomycin+hUC-MSC) (D, H) or sterile saline (bleomycin+saline) (C, G). Lung sections obtained from C57BL/6 mice (n = 8 per group) were immunostained with anti-Arginase I antibody. At each time point, the number of immunoreactive macrophages infiltrating the lungs is significantly diminished by hUC-MSC infusion in comparison to bleomycin-treated mice. Representative microscopic images (40× magnification) of three independent experiments are shown. (I) Cell count of arginase I positive macrophages in C57BL/6 mouse lung sections. Results are mean ± SD of positive immunostained cell count per sample (n = 8 per group) and are representative of three independent experiments. ** = P < 0.01 compared to bleomycin treated mice.
Fig 9.
Intravenous administration of human fibroblasts does not prevent the development of bleomycin-induced lung injury.
Histology (A-B), collagen content (C-D) and macrophage infiltration (E-H) of mouse lungs 21 days after bleomycin endotracheal injection followed by human fibroblasts intravenous infusion. Lung sections obtained from C57BL/6 mice (n = 8 per group) that received endotracheal bleomycin only (bleomycin) or endotracheal bleomycin followed by intravenous human fibroblasts (bleomycin+fibroblasts) were stained with H&E (A-B), Picrosirius Red (C-D), anti-galectin-3 (E-F) or anti-arginase I (G-H) antibodies. Representative microscopic images (10× or 40× magnification) of three independent experiments are shown. Quantitative real-time PCR gene expression analysis of Col1A1, TGFβ and αSMA (I), and IL-1β, IL-2, IL-6 and IL-10 (J) in whole lung mRNA obtained at day 21 from C57BL/6 mice receiving the aforementioned treatments. Results are expressed as mean ± SD (n = 5 per group) and are representative of three independent experiments performed in triplicate. (K) Quantification of galectin-3 and arginase-I positive macrophages in C57BL/6 mouse lungs 21 days after bleomycin injection with or without subsequent human fibroblast infusion. Results are mean ± SD of positive immunostained cell count per sample (n = 8 per group) and are representative of three independent experiments.
Fig 10.
hUC-MSC do not induce any biological effects on control mice treated with endotracheal saline.
Histology (A-B), collagen content (C-D) and macrophage infiltration (E-H) of mouse lungs 21 days after sterile saline endotracheal injection followed by hUC-MSC intravenous infusion. Lung sections obtained from C57BL/6 mice (n = 8 per group) that received endotracheal sterile saline only (saline) or endotracheal sterile saline followed by intravenous hUC-MSC (saline+hUC-MSC) were stained with H&E (A-B), Picrosirius Red (C-D), anti- galectin-3 (E-F) and anti-arginase I (G-H) antibodies. Representative microscopic images (10× or 40× magnification) of three independent experiments are shown. Quantitative real-time PCR gene expression analysis of Col1A1, TGFβ and αSMA (I), and IL-1β, IL-2, IL-6 and IL-10 (J) in whole lung mRNA obtained at day 21 from C57BL/6 mice receiving the aforementioned treatments. Results are expressed as mean ± SD (n = 5 per group) and are representative of three independent experiments performed in triplicate. (K) Quantification of galectin-3 and arginase I positive macrophages in C57BL/6 mouse lungs 21 days after saline injection with or without subsequent hUC-MSC infusion. Results are mean ± SD of positive immunostained cell count per sample (n = 8 per group) and are representative of three independent experiments.
Fig 11.
Detection of hUC-MSC by quantitative real-time PCR assay for human GAPDH.
Human GAPDH assessed in RNA extracted from cultured hUC-MSC prior to infusion or from lung tissue of C57BL/6 mice (n = 5 per group) receiving endotracheal bleomycin followed by intravenous hUC-MSC (bleomycin+hUC-MSC). Days 8, 14, 21 refer to bleomycin administration, and correspond, respectively, to 1, 7 and 14 days after the second hUC-MSC infusion. Results are expressed as mean ± SD (n = 5 per group) and are representative of three independent experiments performed in triplicate.
Fig 12.
Detection of hUC-MSC in lung tissue by vimentin IHC.
Lung sections obtained from C57BL/6 mice (n = 8 per group) receiving endotracheal bleomycin only (A, B, E) or endotracheal bleomycin followed by intravenous hUC-MSC (C, D, F) were immunostained with anti-human vimentin antibody. (C,D) Lung sections from hUC-MSC-treated mice show at day 8 (i.e. one day after second hUC-MSC infusion) numerous immunoreactive cells, distributed mainly in the septa and in the peribronchial tissue (200X), that exhibit at higher magnification (400X) a polygonal or stellate shape and enlarged nuclei (arrows). (A,B) In lung sections from mice treated with bleomycin only, the anti-human vimentin staining is extracellular and unspecific; no cells with polygonal or stellate shape reactive for human vimentin are evident (200X and 400X). (F) Lung sections from hUC-MSC-treated mice show at day 21 (i.e. 14 days after second hUC-MSC infusion) normal alveolar architecture with no fibrosis or inflammation, which are evident in the untreated mice (E), and only few vimentin-positive cells (arrows).
Fig 13.
Detection of hUC-MSC in lung tissue by HLA-1 and CD105 IHC.
Lung sections obtained from C57BL/6 mice receiving endotracheal bleomycin followed by intravenous hUC-MSC were immunostained with anti-HLA-1 (A-B) or anti-CD105 (C-D) antibodies. Representative microscopic images (200X and 400X) of three independent experiments are shown,. Lung sections from hUC-MSC-treated mice show at day 21 (i.e. 14 days after second hUC-MSC infusion) normal alveolar architecture with no fibrosis or inflammation, and only few positive cells (arrows, 400X).