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, 39 (1), 129-140

The Effects of Adipose Stem Cell-Conditioned Media on Fibrogenesis of Dermal Fibroblasts Stimulated by Transforming Growth Factor-β1

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The Effects of Adipose Stem Cell-Conditioned Media on Fibrogenesis of Dermal Fibroblasts Stimulated by Transforming Growth Factor-β1

Bo Yuan et al. J Burn Care Res.

Abstract

Adipose-derived stem cells (ASCs) have been shown to enhance wound healing by human dermal fibroblasts; however, the interactions between ASCs and fibroblasts during injury remain unclear. Fibroblasts were treated with ASC-conditioned medium (ASC-CM) with and without transforming growth factor-β1 (TGF-β1) stimulation. Fibroblast proliferation, apoptosis, differentiation and expression of extracellular matrix genes and proteins, type I collagen, and type III collagen were measured. Also, wound-healing effect of ASC-CM was verified with in vivo animal study. ASC-CM inhibited proliferation and enhanced apoptosis of fibroblasts under TGF-β1 stimulation. Furthermore, 10% ASC-CM inhibited α-smooth muscle actin expression in fibroblasts, whereas 100% ASC-CM increased collagen, especially type III, expression in fibroblasts. ASC-CM was found to contain more basic fibroblast growth factor than hepatocyte growth factor, and 100% ASC-CM increased hepatocyte growth factor gene expression in fibroblasts. These results suggest ASCs affect fibrogenesis by dermal fibroblasts stimulated with TGF-β1 via paracrine signaling by adipocytokines present in ASC-CM. These results also suggest that higher concentrations of ASC-CM increase collagen production and inhibit fibroblast proliferation to avoid excessive fibrogenesis. We demonstrated that a lower ASC-CM concentration attenuated fibroblast differentiation. Additionally, 100% ASC-CM significantly reduced the wound size in an in vivo wound-healing model. In this study, we provided evidence that ASCs modulate fibrogenesis by fibroblasts via paracrine signaling, suggesting that application of ASCs during wound healing may improve the quality of wound repair.

Figures

Figure 1.
Figure 1.
Characterization of cell surface markers of ASCs by flow cytometry and differentiation of ASCs. Microscopy demonstrated that the ASCs have a fibroblast-like morphology (A). Flow cytometry showed that ASCs were positive for CD49d, CD73, CD90, and CD105 but were negative for CD34. (B). Fourteen days after induction, ASCs rounded and lipids collected in cytoplasm, which was confirmed by positive-staining with Oil Red O (C); in chondrogenesis differentiation medium, ASCs formed chondrogenic pellets, which were stained with Alcian Blue (D). Scale bars: 100 μm. ASC, adipose-derived stem cells.
Figure 2.
Figure 2.
Quantification of collagen production by HFF-1 cells after 72 hours of stimulation with TGF-β1 was performed by Sirius Red staining. Collagen production was increased with 5 and 10 ng/mL of TGF-β1 when compared with the control group (0 ng/mL of TGF-β1). More collagen was produced with 10 ng/mL of TGF-β1 than with 5 ng/mL of TGF-β1. Solid bars represent analysis of variance with Tukey’s posttest among groups. *P value ≤ .001; n = 3. HFF-1, human foreskin fibroblast; TGF-β1, transforming growth factor-β1.
Figure 3.
Figure 3.
Real time of HFF-1 cell proliferation in ASC-CM was monitored using an xCELLigence (ACEA) platform. All of the experiments were performed using E-plates according to the protocol in the ACEA manual and N = 3. (A and B) HFF-1 cells were seeded at 500 cells/well with or without ASC-CM and TGF-β1 (with appropriate controls and blanks). Solid bars represent analysis of variance with Tukey’s posttest among groups. *P value ≤ .001; n = 3. ASC-CM, adipose-derived stem cells–conditioned medium; HFF-1, human foreskin fibroblast; TGF-β1, transforming growth factor-β1.
Figure 4.
Figure 4.
Analysis of HFF-1 cell apoptosis in ASC-CM. (A) Visualization of apoptotic HFF-1 cells with various dilutions of ASC-CM. The red stain (propidium iodide) indicates the nucleus. The green (fluorescein-labeled anti-dUTP antibody) and the yellow (derived by merging green and red) stains indicate TUNEL-positive HFF-1. (B) Quantitative data from the 72-hour endpoint demonstrated that increasing ASC-CM concentrations promoted apoptosis. Solid bars represent analysis of variance with Tukey’s posttest among groups. *P value ≤ .05; †P value ≤ .01; ‡P value ≤ .001; n = 4. Scale bars: 100 μm. ASC-CM, adipose-derived stem cells–conditioned medium; HFF-1, human foreskin fibroblast; TUNEL, transferase dUTP nick end labeling.
Figure 5.
Figure 5.
Expression of α-SMA in fibroblasts in ASC-CM. α-SMA gene (A) or protein (B) expression was measured by real-time PCR or Western blot with TGF-β1 stimulation with varying ASC-CM concentrations and with of ASC-CM alone. α-SMA expression was inhibited with TGF-β1 stimulation and 10% ASC-CM. Solid bars represent analysis of variance with Tukey’s posttest among groups. *P value ≤ .05; †P value ≤ .01; ‡P value ≤ .001; n = 5 (A) and n = 3 (B). α-SMA, α-smooth muscle actin; ASC-CM, adipose-derived stem cells–conditioned medium; PCR, polymerase chain reaction; TGF-β1, transforming growth factor-β1.
Figure 6.
Figure 6.
Expression of collagen types 1 and 3 by fibroblasts in ASC-CM. (A) Col-1 α1, (B) Col-3 α1 gene expression in HFF-1 cells and (C) Col-1, (D) Col-3 protein secretion in supernatants were measured by real-time PCR or Western blot with TGF-β1 stimulation with varying ASC-CM concentrations and with ASC-CM alone. Col-3 gene expression and protein secretion increased with 100% ASC-CM and TGF-β1. Solid bars represent analysis of variance with Tukey’s posttest among groups. *P value ≤ .05; †P value ≤ .01; ‡P value ≤ .001; n = 5 (A, B) and n = 3 (C, D). ASC-CM, adipose-derived stem cells–conditioned medium; Col-1 α1, collagen type 1 α1; Col-3 α1, collagen type 3 α1; HFF-1, human foreskin fibroblast; PCR, polymerase chain reaction; TGF-β1, transforming growth factor-β1.
Figure 7.
Figure 7.
FGF-2 and HGF gene expression in fibroblasts in ASC-CM. (A) FGF-2, (B) HGF were measured by real-time PCR. When comparing groups treated with and without TGF-β1, TGF-β1 appears to be a crucial regulator FGF-2 gene expression. There was a close relationship between concentrations of ASC-CM and HGF gene expression. 100% ASC-CM increases HGF gene expression relative to 0 and 10% ASC-CM. Solid bars represent analysis of variance with Tukey’s posttest. *P value ≤ .05; †P value ≤ .01; ‡P value ≤ .001; n = 4 (A) and n = 5 (B). ASC-CM, adipose-derived stem cells–conditioned medium; FGF-2, basic fibroblast growth factor; HFF-1, human foreskin fibroblast; HGF, hepatocyte growth factor; PCR, polymerase chain reaction; TGF-β1, transforming growth factor-β1.
Figure 8.
Figure 8.
100% ASC-CM promoted wound healing in an animal injury model. (A) Six 6-mm full-thickness wounds were created on the back of Sprague Dawley rats. Four of them were treated daily with 0, 10, 50, and 100% ASC-CM, respectively, since the third day after wounding, and the remaining two were allowed to heal naturally. (B) On the seventh day after wounding, the wound size seemed to be correlated with different treatments. (C) Quantitative data from the 7 days after wounding demonstrated that 100% ASC-CM significantly enhanced wound healing, compared with blank control or 0% ASC-CM group. Solid bars represent analysis of variance with Tukey’s posttest. *P value ≤ .05; n = 7. ASC-CM, adipose-derived stem cells–conditioned medium.

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