Inhibition of pathological phenotype of hypertrophic scar fibroblasts via co-culture with adipose derived stem cells

Abstract Hypertrophic scar (HS) is a dermal fibroproliferative disease characterized by fibroblast over-proliferation, overproduction and deposition of the extracellular matrix (ECM). Growing evidence demonstrated that adipose derived stem cells (ASCs) secrete a plethora of trophic and anti-fibrotic factors, which suppress inflammation and ameliorate fibrosis of different tissues. However, few studies investigate their effect on repressing HS activity. This study evaluated the suppressing effect of ASCs on HS fibroblast bioactivity and the possible mechanism via a co-culture model. HS-derived fibroblasts (HSFs) and ASCs were isolated from individual patients. HSFs or HSFs treated with transforming growth factor-β1 (TGF-β1) were co-cultured with ASCs and the change of HSF cellular behaviors, such as cell proliferation, migration, contractility and gene/protein expression of scar-related molecules, were evaluated by cell counting assay, cell cycle analysis, scratch wound assay, fibroblast-populated collagen lattice (FPCL) contractility assay, real-time quantitative polymerase chain reaction (RT-qPCR), ELISA, and western blotting assay. After 5 days of ASC co-culture treatment, the expression levels of collagen I (Col 1), collagen III (Col 3), fibronectin (FN), TGF-β1, interleukin-6 (IL-6), interleukin-8(IL-8), connective tissue growth factor (CTGF) and alpha-smooth muscle actin (α-SMA) in HSFs decreased significantly while the expression levels of decorin (DCN) and MMP1/TIMP1 ration increased significantly. Besides, after 5 days of exogenous TGF-β1 stimulation, the expression levels of collagen 1, fibronectin, TGF-β1, IL-6, CTGF and α-SMA in HSFs increased significantly. Impressively, all these increased gene expression levels were reversed by 5 days of ASCs co-culture treatment. Additionally, the proliferation, migration and contractility of HSFs were all significantly reduced by ASC co-culture treatment. Furthermore, the protein levels of TGF-β1 and intracellular signal pathway related molecules，such as p-smad2, p-smad3, p-Stat3 and p-ERK, were down-regulated significantly in HSFs after 5 days of ASCs co-culture treatment.This study demonstrated that co-culture of HSFs with ASCs not only inhibited proliferation, migration and contractility of HSFs but also decreased the expression levels of HSF-related or TGF-β1-induced molecules. Additionally, the anti-fibrotic effect on HSFs was likely mediated by the inhibition of multiple intracellular signaling. The results of this study suggest the therapeutic potential of ASCs for HS treatment, which is worth of further investigation.