Balance between Pro- and Antifibrotic Proteins in Mesenchymal Stromal Cell Secretome Fractions Revealed by Proteome and Cell Subpopulation Analysis

Abstract

Multipotent mesenchymal stromal cells (MSCs) regulate tissue repair through paracrine activity, with secreted proteins being significant contributors. Human tissue repair commonly results in fibrosis, where fibroblast differentiation into myofibroblasts is a major cellular mechanism. MSCs' paracrine activity can inhibit fibrosis development. We previously demonstrated that the separation of MSC secretome, represented by conditioned medium (CM), into subfractions enriched with extracellular vesicles (EV) or soluble factors (SF) boosts EV and SF antifibrotic effect. This effect is realized through the inhibition of fibroblast-to-myofibroblast differentiation in vitro. To unravel the mechanisms of MSC paracrine effects on fibroblast differentiation, we performed a comparative proteomic analysis of MSC secretome fractions. We found that CM was enriched in NF-κB activators and confirmed via qPCR that CM, but not EV or SF, upregulated NF-κB target genes (COX2, IL6, etc.) in human dermal fibroblasts. Furthermore, we revealed that EV and SF were enriched in TGF-β, Notch, IGF, and Wnt pathway regulators. According to scRNAseq, 11 out of 13 corresponding genes were upregulated in a minor MSC subpopulation disappearing in profibrotic conditions. Thus, protein enrichment of MSC secretome fractions and cellular subpopulation patterns shift the balance in fibroblast-to-myofibroblast differentiation, which should be considered in studies of MSC paracrine effects and the therapeutic use of MSC secretome.

Keywords: cell differentiation; cell subpopulation; extracellular vesicles; fibroblasts; fibrosis; multipotent mesenchymal stromal cells; myofibroblasts; proteomics; secretome; single cell RNA sequencing.

Figure 1

The effect of MSC secretome fractions on fibroblast-to-myofibroblast differentiation induced by TGF-β1. (a) Schematic representation of the isolation procedure for MSC secretome fractions. Subfractions efficiently preventing fibroblast-to-myofibroblast differentiation are marked in green, while fractions not preventing fibroblast-to-myofibroblast differentiation are marked pink. (b–f) Fluorescent microscopy of human dermal fibroblasts: (b) untreated (negative control); (c) exposed to TGF-β (positive control); (d) exposed to TGF-β and EV; (e) exposed to TGF-β and SF; (f) exposed to TGF-β and CM. Immunocytochemical staining for alpha-smooth muscle actin (green), F-actin staining with phalloidin (red), and nuclei staining with DAPI (blue). Stress fibers, which are characteristic of myofibroblasts, appear as yellow strands.

Figure 2

MSC secretome fractioning through ultrafiltration alters the quantitative protein content of obtained fractions. Proteins potentially mediating EV and/or SF fraction ability to prevent myofibroblast differentiation are marked green, while proteins potentially interfering with CM fraction ability to prevent myofibroblast differentiation are marked pink. (a) Venn diagram of secreted proteins identified in CM fraction compared to EV and SF subfractions. Pink—proteins identified in the CM and green—EV and SF fractions. (b) Correlation plot of the log₂ ratios of protein abundance in EV and SF subfractions to CM fraction. Green—proteins with a relative fold of enrichment ≥ 2 and pink—proteins with a relative fold of enrichment ≤ 0.5. The combined data of three independent experiments.

Figure 3

CM fraction of MSC secretome upregulates the expression of NF-κB target genes in human dermal fibroblasts. Relative gene expression compared to untreated cells measured using qPCR. Ctrl—cells treated with TGF-β1. CM, EV, and SF—cells treated with TGF-β1 and either CM fraction, EV, or SF subfraction of MSC secretome, respectively. (a) CXCL1, (b) CXCL2, (c) IL1B, (d) IL6, and (e) COX2. * indicates p < 0.05.

Figure 4

The results of scRNAseq analysis of primary human adipose-derived MSC cultures in control (a) and profibrotic (b) conditions. t-SNE plots dividing cells within cultured primary MSCs into seven clusters according to single-cell gene expression profile analysis. Notably, the MSC subpopulation (blue dots, cluster 4) with upregulated expression of genes potentially preventing myofibroblast differentiation diminishes in profibrotic conditions. (a) Control; (b) profibrotic conditions. The ellipse marks cluster 4.