The secretome of induced pluripotent stem cells reduces lung fibrosis in part by hepatocyte growth factor

Abstract

Introduction: Idiopathic pulmonary fibrosis (IPF) is a progressive and irreversible fibrotic lung disease, resulting in respiratory insufficiency and reduced survival. Pulmonary fibrosis is a result of repeated alveolar epithelial microinjuries, followed by abnormal regeneration and repair processes in the lung. Recently, stem cells and their secretome have been investigated as a novel therapeutic approach in pulmonary fibrosis. We evaluated the potential of induced pluripotent stem cells (iPSC) conditioned media (iPSC-cm) to regenerate and repair the alveolar epithelium in vitro and improve bleomycin induced lung injury in vivo.

Methods: IPSC-cm was collected from cultured iPSC derived from human foreskin fibroblasts and its biological effects on alveolar epithelial wound repair was studied in an alveolar wound healing assay in vitro. Furthermore, iPSC-cm was intratracheally instilled 7 days after bleomycin induced injury in the rat lungs and histologically and biochemically assessed 7 days after instillation.

Results: iPSC-cm increased alveolar epithelial wound repair in vitro compared with medium control. Intratracheal instillation of iPSC-cm in bleomycin-injured lungs reduced the collagen content and improved lung fibrosis in the rat lung in vivo. Profibrotic TGFbeta1 and α-smooth muscle actin (α-sma) expression were markedly reduced in the iPSC-cm treated group compared with control. Antifibrotic hepatocyte growth factor (HGF) was detected in iPSC-cm in biologically relevant levels, and specific inhibition of HGF in iPSC-cm attenuated the antifibrotic effect of iPSC-cm, indicating a central role of HGF in iPSC-cm.

Conclusion: iPSC-cm increased alveolar epithelial wound repair in vitro and attenuated bleomycin induced fibrosis in vivo, partially due to the presence of HGF and may represent a promising novel, cell free therapeutic option against lung injury and fibrosis.

Figure 1

Immunophenotypic profiles of induced pluripotent stem cell colonies and differentiation of embryoid bodies. (a) Following viral transfection of fibroblasts and culture for 2 to 3 weeks in embryonic stem cell culture medium, induced pluripotent stem cell (iPSC) colonies stained positive for the pluripotent markers OCT4, Nanog, SSEA4 and TRA-1-81 respectively. (b) Derived iPSCs have the ability to differentiate into the three germ layers in vitro as shown after in vitro differentiation of embryoid bodies: β-tubulin III (ectoderm marker), nestin (endoderm marker) and smooth muscle actin (SMA; mesoderm marker).

Figure 2

Induced pluripotent stem cell conditioned medium improves alveolar epithelial wound repair in vitro . Alveolar epithelial wound repair in vitro was increased after treatment of the wounded A549 alveolar epithelial monolayer with induced pluripotent stem cell conditioned medium (iPSC-cm) compared with fibroblast (CCD1) conditioned medium (Fibro-cm) and serum-free media as control. Data indicated as mean ± standard error of the mean. *P <0.001, **P <0.001,***P <0.0001, respectively.

Figure 3

Histological and biochemical analysis of lung fibrosis in vivo . Histological analysis of injured lungs treated with different types of conditioned media. Marked fibrosis was seen in the control groups treated with (a) media only (b) or fibroblast conditioned medium (CCD1-cm), whereas a significant decrease of fibrosis was observed in (c) the induced pluripotent stem cell conditioned medium (iPSC-cm)-treated group. Hematoxylin and eosin staining, scale bar =200 μm. (d) Semiquantitative histological analysis of lung fibrosis using the Ashcroft score showed significant reduction of fibrosis after treatment with iPSC-cm compared with media control or CCD-1-cm. Data indicated as mean ± standard error of the mean (SEM), **P <0.001. (e) In accordance with histological data, the collagen content was reduced after treatment with iPSC-cm compared with media control or CCD-1-cm. Data indicated as mean ± SEM, *P <0.01, **P <0.001.

Figure 4

Neutralizing anti-hepatocyte growth factor antibodies inhibit induced pluripotent stem cell-induced alveolar epithelial repair in vitro . In the presence of neutralizing anti-hepatocyte growth factor antibodies (HGF Ab), the induction of alveolar epithelial repair in vitro by induced pluripotent stem cell conditioned medium (iPSC-cm) was reduced in a dose-dependent manner using the concentrations of HGF Ab as indicated. Data presented as mean ± standard error of the mean, **P <0.001.

Figure 5

Neutralizing anti-hepatocyte growth factor antibody inhibit induced pluripotent stem cell conditioned medium-induced reduction of lung fibrosis in vivo . Induced pluripotent stem cell conditioned medium (iPSC-cm)-induced reduction in lung fibrosis was prevented by neutralizing anti-hepatocyte growth factor antibodies (HGF Ab) added to iPSC-cm prior to intratracheal instillation in the bleomycin-injured lung as assessed by (a) histology, (c) collagen content and (d) Ashcroft score in the lung. (b) When bleomycin-injured animals were treated with only HGF Ab there was no reduction in fibrosis, and the Ashcroft score and collagen content were significantly high. Data presented as mean ± standard error of the mean, **P <0.001).

Figure 6

Myofibroblast accumulation in vivo is reduced after treatment with induced pluripotent stem cell conditioned medium, in part by a hepatocyte growth factor-dependent mechanism. Immunohistochemistry was performed to detect myofibroblasts using alpha smooth muscle actin (αSMA) antibody. In (c) media control and (b) fibroblast (CCD1) conditioned medium-treated animals, strong αSMA staining was detected; whereas in (a) induced pluripotent stem cell conditioned medium (iPSC-cm)-treated animals, the number of αSMA-positive signals was reduced. (d) When iPSC-cm was pretreated with neutralizing anti-hepatocyte growth factor (HGF) antibody before intratracheal instillation to bleomycin-injured lungs, αSMA positivity increased compared with treatment with iPSC-cm alone. (e) Treatment with HGF neutralizing anti-HGF alone did not show any effect compared with control animals. Scale bar =500 μm.