Conditioned medium from amniotic mesenchymal tissue cells reduces progression of bleomycin-induced lung fibrosis

Abstract

Background and aims: We have demonstrated recently that transplantation of placental membrane-derived cells reduces bleomycin-induced lung fibrosis in mice, despite a limited presence of transplanted cells in host lungs. Because placenta-derived cells are known to release factors with potential immunomodulatory and trophic activities, we hypothesized that transplanted cells may promote lung tissue repair via paracrine-acting molecules. To test this hypothesis, we examined whether administration of conditioned medium (CM) generated from human amniotic mesenchymal tissue cells (AMTC) was able to reduce lung fibrosis in this same animal model.

Methods: Bleomycin-challenged mice were either treated with AMTC-CM or control medium, or were left untreated (Bleo group). After 9 and 14 days, the distribution and severity of lung fibrosis were assessed histologically with a scoring system. Collagen deposition was also evaluated by quantitative image analysis.

Results: At day 14, lung fibrosis scores in AMTC-CM-treated mice were significantly lower (P < 0.05) compared with mice of the Bleo group, in terms of fibrosis distribution [1.0 (interquartile range, IQR 0.9) versus 3.0 (IQR 1.8)], fibroblast proliferation [0.8 (IQR 0.4) versus 1.6 (IQR 1.0)], collagen deposition [1.4 (IQR 0.5) versus 2.0 (IQR 1.2)] and alveolar obliteration [2.3 (IQR 0.8) versus 3.2 (IQR 0.5)]. No differences were observed between mice of the Bleo group and mice treated with control medium. Quantitative analysis of collagen deposition confirmed these findings. Importantly, AMTC-CM treatment significantly reduced the fibrosis progression between the two observation time-points.

Conclusions: This pilot study supports the notion that AMTC exert anti-fibrotic effects through release of yet unknown soluble factors.

Figure 1

Representation of fibrosis distribution and severity parameter scores at days 9 and 14 post-intratracheal bleomycin instillation. (A-D) Fibrosis distribution (A) and severity (B-D) at day 9. (E-H) Fibrosis distribution (E) and severity (F-H) at day 14. (A, E) Fibrosis distribution; (B, F) fibroblast proliferation; (C, G) collagen deposition; (D, H) alveolar obliteration. The number of mice in each group is indicated (n). Brackets represent significant differences between groups; *P<0.05; **P<0.01.

Figure 2

Digital image morphometric analysis of lung collagen deposition. (A) Microphotographs taken before (left panel) and after (right panel) the imaging analysis procedure used to quantitate the collagen staining (black areas) from the image. The identification number of each mouse is indicated on each microphotograph. (B) Median values with IQR of quantitative collagen deposition are represented as box-plots for Bleo (dark gray), Bleo + non-CM (light gray) and Bleo + AMTC-CM (white) groups. The number of mice in each group is indicated (n). Brackets represent significant differences between groups; *P<0.05.

Figure 3

Representation of overall fibrosis score at days 9 and 14 post-intratracheal bleomycin instillation. Box-plots reporting the median and IQR of values obtained from the Bleo (dark gray), Bleo + non-CM (light gray) and Bleo + AMTC-CM (white) groups are represented for each time-point. The number of mice in each group is indicated (n). Brackets represent significant differences between groups and time-points; *P<0.05; **P<0.01.