Extracellular Matrix Synthesis and Remodeling by Mesenchymal Stromal Cells Is Context-Sensitive

Abstract

Matrix remodeling could be an important mode of action of multipotent mesenchymal stromal cells (MSC) in extracellular matrix (ECM) disease, but knowledge is limited in this respect. As MSC are well-known to adapt their behavior to their environment, we aimed to investigate if their mode of action would change in response to healthy versus pathologically altered ECM. Human MSC-derived ECM was produced under different culture conditions, including standard culture, culture on Matrigel-coated dishes, and stimulation with the pro-fibrotic transforming growth factor-β1 (TGFβ1). The MSC-ECM was decellularized, characterized by histochemistry, and used as MSC culture substrate reflecting different ECM conditions. MSC were cultured on the different ECM substrates or in control conditions for 2 days. Culture on ECM increased the presence of surface molecules with ECM receptor function in the MSC, demonstrating an interaction between MSC and ECM. In MSC cultured on Matrigel-ECM and TGFβ1-ECM, which displayed a fibrosis-like morphology, gene expression of collagens and decorin, as well as total matrix metalloproteinase (MMP) activity in the supernatant were decreased as compared with control conditions. These results demonstrated that MSC adapt to their ECM environment, which may include pathological adaptations that could compromise therapeutic efficacy.

Keywords: MSC-derived ECM; collagen; extracellular matrix (ECM); matrisome; matrix metalloproteinases (MMP); matrix remodeling; mesenchymal stromal cells (MSC); mode of action; tissue-inhibitors-of-matrix-metalloproteinases (TIMP).

Figure 1

Decellularized MSC-derived ECM obtained after culturing the MSC for 2 days on standard tissue culture plates (ECM), on Matrigel-coated tissue culture plates (Matrigel-ECM), or on standard tissue culture plates with TGFβ1-supplemented medium (TGFβ1-induced ECM). Alcian blue staining was weak, suggesting low glycosaminoglycan content of the MSC-derived ECM, while picrosirius red staining was more distinct, demonstrating the presence of collagens. Note the fibrous structure of the TGFβ1-induced ECM.

Figure 2

Metabolic activity and microscopy. (A) Metabolic activity of MSC after culture on the different MSC-derived ECM substrates (ECM, Matrigel-ECM, TGFβ1-ECM) or in the respective control conditions without MSC-derived ECM (tissue culture plastic (TCP), Matrigel), as determined by MTS-assay (n = 5 independent experiments). (B) Representative images of surface molecule (CD44, CD29) and actin cytoskeleton (phalloidin) staining of MSC cultured on the different substrates. Note the higher intensity of the stainings in MSC cultured on ECM, indicating that the surface molecules interacting with the ECM are more present and the cytoskeleton formation is more pronounced.

Figure 3

Relative gene expression of the extracellular matrix components collagen 1A2 (COL1A2), collagen 3A1 (COL3A1), tenascin-C (TNC), and decorin (DCN), the matrix metalloproteinases MMP1, MMP3, and MMP14, and their tissue inhibitors TIMP1 and TIMP2 in MSC after culture on the different MSC-derived ECM substrates (ECM, Matrigel-ECM, TGFβ1-ECM) or in the respective control conditions without MSC-derived ECM (tissue culture plastic (TCP), Matrigel). Gene expression differed significantly between groups designated with the same letter (p ≤ 0.007 in the post hoc tests). Data were obtained in n = 4 independent experiments.

Figure 4

Matrix metalloproteinases (MMP) in cell culture supernatants. (A): Total matrix metalloproteinase (MMP) activity, measured in the supernatants of MSC after culture on the different MSC-derived ECM substrates (ECM, Matrigel-ECM, TGFβ1-ECM) or in the respective control conditions without MSC-derived ECM (tissue culture plastic (TCP), Matrigel). MMP activity differed significantly between groups designated with the same letter (p ≤ 0.007 in the post hoc tests). (B): Representative image of gelatin zymography of the supernatants; bands within the same kDa range were detected in all samples, but with different intensity (arrows); ladder: Precision Plus Protein Dual Color Standards (Bio-Rad, Hercules, CA, USA); standard 1: 0.5 µg collagenase V; standard 2: 1 µg collagenase V.