Allogeneic human mesenchymal stem cells for treatment of E. coli endotoxin-induced acute lung injury in the ex vivo perfused human lung

Abstract

Recent studies have suggested that bone marrow-derived multipotent mesenchymal stem cells (MSCs) may have therapeutic applications in multiple clinical disorders including myocardial infarction, diabetes, sepsis, and hepatic and acute renal failure. Here, we tested the therapeutic capacity of human MSCs to restore alveolar epithelial fluid transport and lung fluid balance from acute lung injury (ALI) in an ex vivo perfused human lung preparation injured by E. coli endotoxin. Intra-bronchial instillation of endotoxin into the distal airspaces resulted in pulmonary edema with the loss of alveolar epithelial fluid transport measured as alveolar fluid clearance. Treatment with allogeneic human MSCs or its conditioned medium given 1 h following endotoxin-induced lung injury reduced extravascular lung water, improved lung endothelial barrier permeability and restored alveolar fluid clearance. Using siRNA knockdown of potential paracrine soluble factors, secretion of keratinocyte growth factor was essential for the beneficial effect of MSCs on alveolar epithelial fluid transport, in part by restoring amiloride-dependent sodium transport. In summary, treatment with allogeneic human MSCs or the conditioned medium restores normal fluid balance in an ex vivo perfused human lung injured by E. coli endotoxin.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Schematic diagram of the ex vivo perfused human lung and experimental protocol.

Fig. 2.

Effect of endotoxin on alveolar fluid clearance and inflammation. Instillation of endotoxin into the lung lobe was associated with a significant decrease in (A) alveolar fluid clearance (AFC) and (B) an increase in inflammatory cytokine secretion. (A) The decrease in AFC in the endotoxin-injured lung lobe was dependent on the presence of fresh whole human blood in the perfusate. AFC in the endotoxin-injured lung lobe with fresh whole blood was significantly decreased compared to control AFC at 0 or 4 h measurements without endotoxin or to experiments with endotoxin without blood in the perfusate at 4 h. AFC was measured by the change in protein concentration of a 5% albumin instillate in the lung lobe over 1 h and expressed as mean AFC (%/h per 150 mL alveolar fluid) ± SD. For each condition, n = 3–6; *, P < 0.0001 vs. control AFC (0 h), †, P < 0.0013 vs. control AFC (4 h) by ANOVA (Bonferroni). (B) The addition of endotoxin into the lung lobe was associated with a significant increase in IL-1β, TNFα, and IL-8 levels in the alveolar fluid compared to the control lobe at 0 or 4 h. n = 3–6; *, P < 0.0001 vs. control (0 h), †, P < 0.0001 vs. control (4 h) for IL-1β, TNFα, and IL-8 by ANOVA (Bonferroni).

Fig. 3.

Histology sections and neutrophil counts. Human lungs exposed to endotoxin with and without MSC or its CM were fixed in 10% formalin at 4 h. Sections were stained with hematoxylin and eosin (magnification 10×). The instillation of MSC or its CM 1 h after endotoxin injury reduced the degree of edema and cellularity in the endotoxin-injured lung lobe. Total white blood cell and neutrophil counts in the alveolar fluid of each lung lobe are shown as mean ± SD, n = 3–6. *, P < 0.004 vs. control by ANOVA (Bonferroni). Measurement of WBC in MSC-treated lung lobes was confounded by the presence of the MSC themselves. Although not quite statistically significant by ANOVA (Bonferroni), P values are shown for the comparisons between LPS vs. LPS + MSCs and LPS vs. LPS + MSC-CM.

Fig. 4.

Effect of human MSCs or its CM on lung endothelial permeability to protein and Wet/Dry Ratio. Instillation of MSCs or its CM into the endotoxin injured RML or LLL 1 h later restored lung endothelial permeability to protein (A) and wet/dry (W/D) ratio (B) to control values. Data are expressed as mean % endothelial permeability or W/D ratio ± SD, n = 4–5 lungs; *, P < 0.0001 vs. control lobe, †, P < 0.0011 vs. LPS (0.1 mg/kg) injured lobe for lung endothelial permeability and *, P < 0.0014 vs. control lobe, †, P < 0.005 vs. LPS (0.1 mg/kg) injured lobe for the W/D ratio by ANOVA (Bonferroni).

Fig. 5.

Effect of human MSCs or its CM on alveolar fluid clearance (A). MSCs or its CM restored the decrease in alveolar fluid clearance in the lung lobe injured by endotoxin to control values at 4 h. n = 3–4; *, P < 0.0006 vs. control AFC; †, P < 0.0001 vs. LPS (0.1 mg/kg) AFC by ANOVA (Bonferroni). Effect of the CM of human MSCs pretreated with a KGF siRNA on alveolar fluid clearance (B). Administration of the CM of MSCs grown for 24 h pretreated with the KGF siRNA (#10818, Ambion) into the endotoxin injured lung lobe after 1 h prevented the restoration of AFC with the CM alone. The addition of recombinant KGF (100 ng) to the CM pretreated with KGF siRNA restored the decrease in AFC to control values. Data are expressed as mean AFC ± SD, n = 4–5 lungs; *, P < 0.0012 vs. control lobe by ANOVA (Bonferroni).

Fig. 6.

Effect of MSC on net fluid transport among primary cultures of human alveolar epithelial type II cells (A). Human alveolar epithelial type II cells were exposed to cytomix with and without MSC grown in the bottom chamber for 24 h. Cytomix reduced net fluid transport among the type II cells. The presence of allogeneic MSCs pretreated with a non-specific, non-targeting negative control siRNA partially restored the decrease in net fluid transport induced by cytomix. Pretreatment of MSCs with a KGF siRNA reduced the protective effect of MSCs. n = 3; *, P < 0.003 vs. control lobe by ANOVA (Bonferroni). Changes in the apical membrane and total cellular protein levels of the major sodium transport protein, αENaC, by human alveolar epithelial type II cells (B). Human alveolar epithelial type II cells exposed to cytomix with or without MSCs in the bottom chamber for 24 h. Total cellular protein or apical membrane protein levels, biotinylated using the Cell Membrane Isolation Kit (Pierce), were isolated. αENaC protein levels were measured by Western blot and expressed as the percent protein levels of control ± SD of each sample, measured in triplicates. *, P < 0.02 compared to controls for biotinylated proteins; *, P < 0.008 compared to controls for total protein by ANOVA (Bonferroni). A representative Western blot is depicted above each figure.

Fig. 7.

Effect of the addition of amiloride on the therapeutic benefit of CM on AFC. The addition of amiloride (5 × 10−4 M) to the CM (the medium of 1 × 106 cells grown for 24 h) given 1 h following endotoxin injury reduced the therapeutic effect of the MSC-CM on AFC in the lung lobe injured by endotoxin. n = 4; *, P < 0.03 vs. LPS with MSC-CM on AFC rate.