Secretome of Mesenchymal Stromal Cells Prevents Myofibroblasts Differentiation by Transferring Fibrosis-Associated microRNAs within Extracellular Vesicles

Abstract

Fibroblasts differentiation into myofibroblasts is a central event of tissue fibrosis. Multipotent mesenchymal stromal cells (MSCs) secretome can interfere with fibrosis development; despite precise underlying mechanisms remain unclear. In this study, we tested the hypothesis that MSC secretome can affect fibroblast' differentiation into myofibroblasts by delivering regulatory RNAs, including microRNAs to these cells. Using the model of transforming growth factor-beta (TGFbeta)-induced fibroblast differentiation into myofibroblasts, we tested the activity of human MSC secretome components, specifically extracellular vesicles (MSC-EV). We showed that MSC-EV down-regulated secretion of extracellular matrix proteins by fibroblasts as well as suppressed their contractility resulting in prevention as well as reversion of fibroblasts differentiation to myofibroblasts. High-throughput sequencing of RNAs extracted from MSC-EV has revealed many fibrosis-associated microRNAs. Fibroblast treatment with MSC-EV led to direct transfer of microRNAs, which resulted in the elevation of most prominent fibrosis-associated microRNAs, including microRNA-21 and microRNA-29c. Using MSC-EV transfection by antagomirs to these microRNAs we demonstrated their involvement in the suppression of fibroblast differentiation in our model. Taken together, MSC secretome can suppress fibrosis by prevention of fibroblast differentiation into myofibroblasts as well as induce de-differentiation of the latter by direct transfer of specific microRNAs.

Keywords: extracellular vesicles; fibrosis; mesenchymal stem/stromal cells; microRNA; myofibroblasts; secretome.

Figure 1

Mesenchymal stem/stromal cells conditioned medium (MSC-CM) fractions added simultaneously with transforming growth factor-beta (TGFbeta) prevented the TGFbeta-induced increase of aSMA expression in fibroblasts. Analysis of aSMA expression in serum-free cultured control fibroblasts (− TGFbeta), in fibroblasts after exposure to TGFbeta (+ TGFbeta) and TGFbeta with the components of MSC-CM (+ TGFbeta + MSC-EV; + TGFbeta + MSC-SF). (A) RT-PCR (n = 9). (B) Western blotting (n = 3).

Figure 2

MSC-CM fractions added simultaneously with TGFbeta prevented the TGFbeta-induced aSMA redistribution into stress fibers in fibroblasts. Immunofluorescent analysis (aSMA (green), phalloidin (red), DAPI (blue)) of the content of aSMA in serum-free cultured control fibroblasts (− TGFbeta), in fibroblasts after exposure to TGFbeta (+ TGFbeta) and TGFbeta with the components of MSC-CM (+ TGFbeta + MSC-EV; + TGFbeta + MSC-SF). Scale bar: 100 μm.

Figure 3

MSC-CM fractions added simultaneously with TGFbeta prevented the TGFbeta-induced intracellular redistribution of vinculin from perinuclear zone to the focal adhesion contact sites in fibroblasts. Immunofluorescent analysis (vinculin (green), phalloidin (red), DAPI (blue)) of the content of vinculin in serum-free cultured control fibroblasts (− TGFbeta), in fibroblasts after exposure to TGFbeta (+ TGFbeta) and TGFbeta with the components of MSC-CM (+ TGFbeta + MSC-EV; + TGFbeta + MSC-SF). Scale bar: 100 μm.

Figure 4

MSC-CM fractions added simultaneously with TGFbeta prevented the TGFbeta-induced increase of the collagen type I production and contractile activity of treated fibroblasts. Changes in the functional activity of serum-free cultured control fibroblasts (− TGFbeta), fibroblasts after exposure to TGFbeta (+ TGFbeta) and TGFbeta with the components of MSC-CM (+ TGFbeta + MSC-EV; + TGFbeta + MSC-SF). (A) RT-PCR on collagen type I (n = 9). (B) Collagen contraction assay (n = 3) B.1 Graph. B.2 Macro photos.

Figure 5

Human dermal fibroblasts conditioned medium (HDF-CM) fractions added simultaneously with TGFbeta were not able to prevent the TGFbeta-induced aSMA redistribution into stress fibers and contractile activity of fibroblasts. The expression of aSMA in serum-free cultured control fibroblasts (− TGFbeta), in fibroblasts after exposure to TGFbeta (+ TGFbeta), and TGFbeta with the components of HDF-CM (+ TGFbeta + HDF-EV; + TGFbeta + HDF-SF). (A) Immunofluorescent analysis (aSMA (green), phalloidin (red), DAPI (blue)). Scale bar: 100 μm. (B) Collagen contraction assay.

Figure 6

MSC-CM fractions added after the TGFbeta treatment reversed the TGFbeta-induced increase of aSMA expression and its redistribution into stress fibers in fibroblasts. Analysis of aSMA expression in the fibroblasts after exposure to TGFbeta (+ TGFbeta) and the subsequent replacement of growth medium by the components of MSC-CM (+ TGFbeta –> MSC-EV; + TGFbeta –> MSC-SF) or control serum-free DMEM (+ TGF b –> DMEM). (A) Immunofluorescence analysis (aSMA (green), phalloidin (red), DAPI (blue)). Scale bar: 100 μm. (B) Western blotting. * p < 0.05 (compared to TGFbeta –> DMEM group).

Figure 7

MSC-CM fractions added after the TGFbeta treatment reversed the TGFbeta-stimulated contractile activity of fibroblasts. Changes in the functional activity of the fibroblasts after exposure to TGFbeta (+ TGFbeta) and the subsequent replacement of growth medium by the components of MSC-CM (+ TGFbeta –> MSC-EV; + TGFbeta –> MSC-SF) or control serum-free DMEM (+ TGFbeta –> DMEM). Collagen contraction assay. * p < 0.05 (compared to TGFbeta –> DMEM group).

Figure 8

Characterization of extracellular vesicles (EVs) released from mesenchymal stromal cell (MSC) after 48 h of conditioning. (A,B) Particle size distributions of EV preparations (red) and control aliquots of MSC-CM after 30 min of conditioning (grey) for non-concentrated (A) and 5-fold concentrated (B) samples measured by nanoparticle tracking analysis (NTA). (C) MSC-EV imaging by the transmission electron microscopy (EVs are indicated by arrows).

Figure 9

Analysis of RNA profile in MSC-EV by RNA-seq. (A) Different types of RNAs are represented in MSC-EV. Cluster of microRNAs marked by red oval. (B) Variability of microRNA representation in donor adipose tissue-derived MSC-EV by Venn diagrams. (C) The most abundant microRNAs in MSC-EV (top-10). (D) Fibrosis-associated microRNA representation in MSC-EV based on their predicted gene target analysis.

Figure 10

MSC-EV transfer microRNAs from MSC to fibroblasts within MSC-EV. (A–D) Uptake of MSC-EV transfected with FAM-labeled (green) oligos by fibroblasts after 48 h of incubation. (A,B) wide-field microscopy, (C) confocal microscopy, (D) confocal microscopy, sagittal section. Immunofluorescent analysis (FAM (green), PKH26 (red), DAPI (blue)). (E,F) Expression of miR-21 (E) and miR-29c (F) in cultured control fibroblasts (− TGFbeta) and in fibroblast after exposure to TGFbeta (+ TGFbeta) incubated for 96 h in the presence of MSC-EV transfected with specific microRNA mimics or antagomirs. Real-time PCR was used for the analysis. The results were normalized to the untreated control fibroblasts (− TGFbeta). (G) Expression of miR-21 and miR-29c target gene (collagen IV) in cultured control fibroblasts (− TGFbeta) and in fibroblast after exposure to TGFbeta (+ TGFbeta) incubated for 96 h in the presence of MSC-EV transfected with specific microRNA mimics or anti-miRs. Real-time PCR was used for the analysis. EV: MSC-EV without transfection, NC: MSC-EV transfected by negative control oligos (control for miR mimics), IC: MSC-EV transfected by inhibitory control oligos (control for anti-miRs), mimic miR-21: MSC-EV transfected by mimic miR-21, mimic miR-29: MSC-EV transfected by mimic miR-29, anti-miR-21: MSC-EV transfected by anti-miR-21, anti-miR-29: MSC-EV transfected by anti-miR-29 * p < 0.05 (compared to the correspondent control group).

Figure 11

Inhibition of selected microRNAs (miR-21 and miR-29c) by antagomiRs attenuates EV-mediated MSC ability to prevent TGFbeta-induced differentiation of fibroblasts into myofibroblasts evaluated by collagen type I production (A) and aSMA expression (B), Western blotting (n = 3). EV: MSC-EV without transfection, IC: MSC-EV transfected by inhibitory control oligos (control for anti-miRs). anti-miR-21: MSC-EV transfected by anti-miR-21, anti-miR-29: MSC-EV transfected by anti-miR-29 * p < 0.05 (compared to control groups). (C) Common targets of miR-21 and miR-29c predicted using MirNet service. (D) Impact of miR-21 transferred by MSC-EV into the regulation of selected pro-fibrotic targets in fibroblasts evaluated by Western blotting (n = 1).