We and others have recently provided evidence that the beneficial effects exerted by placenta-derived cells after transplantation in fibrosis disease animal models, might be due to secretion of paracrine-acting factor(s) that promote endogenous tissue repair.  

In this study, we validated this hypothesis by injecting conditioned medium (CM) from cells of the mesenchymal region of human amniotic membrane (AMTCs) into mice with bleomycin-induced lung fibrosis. We also explored whether the paracrine effector(s) produced by AMTCs are specific to these cells and not produced by other cell populations, and which mechanisms underlie the anti-fibrotic effects of AMTC-derived CM (AMTC-CM).

CMs used in this study were generated from 5-day in vitro cultures of either AMTCs or other cell types (human skin fibroblasts, human peripheral blood mononuclear cells and Jurkat cells).

Fifteen minutes after bleomycin instillation, mice were intra-thoracically injected with CMs or were left untreated. After 14, 21 and 28 days, lung fibrosis was evaluated by histological analysis, by a semi-quantitative scoring system, while lung collagen was determined through a spectrophotometric technique. Lungs were also analyzed for levels of pro-inflammatory and pro-fibrotic cytokines and chemokines. Moreover, arterial blood gas analysis was performed to evaluate the effects of CMs on bleomycin-induced pulmonary dysfunctions.

Among the CM types used, AMTC-CM was the only one able to reduce lung fibrosis and ameliorate the mice impaired health status after bleomycin instillation. Indeed, AMTC-CM-treated mice: i) showed a mortality rate lower than in mice treated with the other CMs; ii) did not suffer weight loss; iii) displayed a reduction in pulmonary fibrosis and lung collagen content up to 28 days post delivery, and iv) consistent  with the presence of less severe fibrotic lesions, blood gas analyses showed that AMTC-CM-treated mice had better lung function compared to animals treated with the other CMs, with minor levels of P_CO2 and lower HCO₃^- blood concentration. In addition, 14 days after bleomycin, AMTC-CM-treated mice showed lower lung content of pro-inflammatory cytokines/chemokines such as IL-6, TNF-α, MIP-1α, MCP-1. Meanwhile, at both day 14 and 28, they showed lower levels of the pro-fibrotic factor TGF-β.

This study show that AMTC-CM displays specific anti-fibrotic properties and that may act both by reducing TGF-β levels in lung tissues through early abrogation of the pro-inflammatory cytokine network, and also by acting through a TGF-β independent mechanism, inhibiting the actions of IL-6 and MCP-1, which display direct pro-fibrotic activities.These results strongly suggest that a cell free treatment could represent a potential therapeutic strategy for pathological conditions where a reduction of inflammatory and fibrotic processes could favour reparative/regenerative actions of the endogenous cells.