Fig. 3
Comparison of the anti-fibrotic effect of hiPS-EVs derived from different oxygen conditions on human cardiac fibroblasts (hCFs). A Schematic illustration of the experimental pipeline. B Fluorescence-based microscopic analysis of the efficiency of fibroblasts-to-myofibroblasts transition (FMT) in hCFs. α-SMA-positive fibers stained in green. Representative images are shown. C Analysis of the percentage of myofibroblasts in hCF populations (% ± SD, n = 9). D Detection of α-SMA protein in hCFs by Western blot in relation to GAPDH level. E Densitometric analysis of α-SMA protein level in hCFs in arbitrary units [AU] (n = 3–9). F Measurement of the expression level of pro-fibrotic genes (ACTA2, COL1A1, COL3A1) by real-time qPCR method (n = 3–6). Abbreviations: CTRL, control hCFs, not treated with TGFβ; TGFβ, hCFs treated with 1 ng/ml of TGFβ; EV-DF, hCFs treated with EVs released by human dermal fibroblasts (hDFs); EV-N, hCFs treated with EVs collected in normoxia (21% O2); EV-H5, hCFs treated with EVs collected in hypoxia 5% O2; EV-H3, hCFs treated with EVs collected in hypoxia 3% O2. All data are presented as the mean ± SD. Statistical significance was tested using the Kruskal–Wallis test with Dunn’s post hoc test (C, E, F: COL1A1) and one-way ANOVA and Tukey’s post hoc test (F: ACTA2, COL3A1). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001