Adipose-derived stromal cells increase the formation of collagens through paracrine and juxtacrine mechanisms in a fibroblast co-culture model utilizing macromolecular crowding

• Published in: Stem cell research & therapy Vol. 13; no. 1; p. 250
• Main Authors: Søndergaard, Rebekka Harary, Højgaard, Lisbeth Drozd, Reese-Petersen, Alexander Lynge, Hoeeg, Cecilie, Mathiasen, Anders Bruun, Haack-Sørensen, Mandana, Follin, Bjarke, Genovese, Federica, Kastrup, Jens, Juhl, Morten, Ekblond, Annette
• Format: Journal Article
• Language: English
• Published: England BioMed Central Ltd 11.06.2022; BioMed Central; BMC
• Subjects: Acids; Actin; Adipose-derived stromal cells; Analysis; Angiogenesis; Antibodies; Ascorbic acid; Cell culture; Cells, Cultured; Coculture Techniques; Collagen; Collagen (type I); Collagen - metabolism; Collagen Type I - metabolism; Enzyme-linked immunosorbent assay; Extracellular matrix; Extracellular Matrix - metabolism; Fibroblasts; Fibronectin; Fibronectins; Fibronectins - metabolism; Flow cytometry; Gelatinase A; Humans; Immunocytochemistry; Immunomodulation; Lysates; Macromolecular crowding; Macromolecules; Matrix metalloproteinase; Matrix Metalloproteinase 2 - genetics; Matrix Metalloproteinase 2 - metabolism; Metalloproteinase; Metalloproteinases; Organic acids; Paracrine and juxtacrine co-cultures; Paracrine signalling; Peptides; Pharmacy; Protein arrays; Proteins; Smooth muscle; Stromal cells; Stromal Cells - metabolism; Transforming Growth Factor beta - metabolism; Transforming growth factor-b; Transforming growth factors; Wound healing; Macromolecular crowding; Fibroblasts; Extracellular matrix; Adipose-derived stromal cells; Metalloproteinases; Paracrine and juxtacrine co-cultures
• ISSN: 1757-6512; 1757-6512
• DOI: 10.1186/s13287-022-02923-y

Adipose-derived stromal cells (ASCs) possess a multitude of regenerative capabilities, which include immunomodulation, angiogenesis, and stimulation of extracellular matrix (ECM) remodeling. However, the underlying mechanisms leading to ECM remodeling remain largely elusive and highlight the need for functional in vitro models for mode of action studies. Therefore, the purpose of this study was to develop an in vitro co-culture model to investigate the capabilities of ASCs to modulate fibroblasts and ECM. An ECM in vitro model with ASCs and normal human dermal fibroblasts (NHDFs) was established utilizing macromolecular crowding, ascorbic acid, and TGF-β stimulation. Paracrine and juxtacrine co-cultures were created using transwell inserts and cell cultures with direct cell-cell contacts. The cultures were screened using RT PCR Profiler Arrays; the protein levels of myofibroblast differentiation marker alpha smooth muscle actin (αSMA) and ECM remodeling enzymes were analyzed using western blot on cell lysates; the formation of collagen type I, III, VI, and fibronectin was investigated using ELISA on culture supernatants; and the deposition of collagens was analyzed using immunocytochemistry. TGF-β stimulation of NHDF monocultures increased the expression of 18 transcripts relevant for ECM formation and remodeling, the protein levels of αSMA and matrix metalloproteinase-2 (MMP-2), the formation of collagen type I, III, VI, and fibronectin, and the deposition of collagen type I and VI and decreased the protein levels of MMP-14. Inclusion of ASCs in the ECM co-culture model increased the formation of collagen type I and III through paracrine mechanisms and the formation of collagen type VI through juxtacrine mechanisms. The co-culture model provides effective stimulation of NHDF monocultures by TGF-β for enhanced formation and deposition of ECM. In the model, ASCs induce changes in ECM by increasing formation of collagen type I, III and VI. The obtained results could guide further investigations of ASCs' capabilities and underlying mechanisms related to ECM formation and remodeling.