Human bone marrow-derived mesenchymal stromal cells cultured in serum-free media demonstrate enhanced antifibrotic abilities via prolonged survival and robust regulatory T cell induction in murine bleomycin-induced pulmonary fibrosis

Abstract

Background: Mesenchymal stromal cells (MSCs) are a potential therapeutic tool for pulmonary fibrosis. However, ex vivo MSC expansion using serum poses risks of harmful immune responses or unknown pathogen infections in the recipients. Therefore, MSCs cultured in serum-free media (SF-MSCs) are ideal for clinical settings; however, their efficacy in pulmonary fibrosis is unknown. Here, we investigated the effects of SF-MSCs on bleomycin-induced pulmonary inflammation and fibrosis compared to those of MSCs cultured in serum-containing media (S-MSCs). Methods: SF-MSCs and S-MSCs were characterized in vitro using RNA sequence analysis. The in vivo kinetics and efficacy of SF-MSC therapy were investigated using a murine model of bleomycin-induced pulmonary fibrosis. For normally distributed data, Student’s t test and one-way repeated measures analysis of variance followed by post hoc Tukey’s test were used for comparison between two groups and multiple groups, respectively. For non-normally distributed data, Kruskal–Wallis and Mann–Whitney U tests were used for comparison between groups, using e Bonferroni’s correction for multiple comparisons. All tests were two-sided, and P < 0.05 was considered statistically significant. Results: Serum-free media promoted human bone marrow-derived MSC expansion and improved lung engraftment of intravenously administered MSCs in recipient mice. SF-MSCs inhibited the reduction in serum transforming growth factor-β1 and the increase of interleukin-6 in both the serum and the bronchoalveolar lavage fluid during bleomycin-induced pulmonary fibrosis. SF-MSC administration increased the numbers of regulatory T cells (Tregs) in the blood and lungs more strongly than in S-MSC administration. Furthermore, SF-MSCs demonstrated enhanced antifibrotic effects on bleomycin-induced pulmonary fibrosis, which were diminished by antibody-mediated Treg depletion. Conclusions: SF-MSCs significantly suppressed BLM-induced pulmonary inflammation and fibrosis through enhanced induction of Tregs into the lungs and corrected the dysregulated cytokine balance. Therefore, SF-MSCs could be a useful tool for preventing pulmonary fibrosis progression without the demerits of serum use.