Effects of conditioned medium obtained from human adipose-derived stem cells on skin inflammation

Abstract

Introduction: Cell therapy using adipose-derived mesenchymal stem cells (ASCs) is a promising avenue of regenerative medicine for the treatment of various diseases. It has been considered that ASCs exert their therapeutic effects through the secretion of multiple factors that are critical for tissue remodeling or the suppression of inflammation. Recently, conditioned medium (CM) from ASCs that contains a complex of secreted factors has received attention as a cost-effective alternative to cell therapy.

Methods: We investigated the effects of CM obtained from ASCs (ASCs-CM) using human dermal fibroblasts (hDFs) and human epidermal keratinocytes with or without interleukin (IL)-1β and examined mRNA levels of marker genes. We also examined alterations in cell proliferation and morphology of hDFs following treatment with ASCs-CM. We further investigated the effects of ASCs-CM treatment on prevention of skin inflammation using a mouse model.

Results: In hDFs and human epidermal keratinocytes, the ASCs-CM treatment suppressed pro-inflammatory factors and enhanced regenerative and remodeling factors with or without interleukin (IL)-1β exposure. The ASCs-CM treatment also enhanced cell proliferation of hDFs and prevented morphological changes in response to IL-1β exposure. Furthermore, in a mouse model of skin inflammation, treatment with ASCs-CM reduced the inflammatory reactions, including redness and thickness.

Conclusions: CM from ASCs may represent a potential alternative to ASC therapy for the treatment of inflammatory skin conditions.

Keywords: ASC therapy; Adipose-derived mesenchymal stem cells (ASCs); Conditioned medium (CM); Human dermal fibroblasts (hDFs); Interleukin (IL)-1β; Skin inflammation.

Fig. 1

Effects of conditioned medium (CM) obtained from adipose-derived stem cells (ASCs) on human dermal fibroblasts (hDFs). mRNA levels of marker genes in hDFs treated with ASCs-CM at concentrations of 0, 250, 500, and 1000 μg/ml for 24 (A) and 48 h (B) in the presence of interleukin (IL)-1β (1 ng/mL) or vehicle. All data are expressed as the mean ± SD (n = 3). ∗P < 0.05 vs. 0 μg/ml, determined by ANOVA followed by Dunnett's post hoc test.

Fig. 2

Effects of ASCs-CM on cell proliferation and morphological changes of hDFs. (A) CCK-8 assay of hDFs treated with ASCs-CM at concentrations of 0, 250, 500, and 1000 μg/ml for 48 h. All data are expressed as the mean ± SD (n = 4). ∗P < 0.001 vs. 0 μg/ml determined by the Kruskal–Wallis test and Dunn's post hoc test for unequal variance. (B) Crystal violet staining after the CCK-8 assay. (C) Morphology of hDFs treated with 250 μg/ml ASCs-CM for 7 days with or without 1 ng/ml IL-1β. Scale bars, 100 μm.

Fig. 3

Effects of ASCs-CM on human epidermal keratinocytes. (A) mRNA levels of marker genes in human epidermal keratinocytes treated with ASCs-CM at concentrations of 0, 250, 500, and 1000 μg/ml for 48 h. All data are expressed as the mean ± SD (n = 3). ∗P < 0.05 vs. 0 μg/ml, determined by ANOVA followed by Dunnett's post hoc test. (B) CCK-8 assay of human epidermal keratinocytes treated with ASCs-CM at concentrations of 0, 250, 500, and 1000 μg/ml for 48 h. All data are expressed as the mean ± SD (n = 4). ∗P < 0.001 vs. 0 μg/ml determined by the Kruskal–Wallis test and Dunn's post hoc test for unequal variance. (C) Crystal violet staining after the CCK-8 assay.

Fig. 4

Effects of ASCs-CM on prevention of skin inflammation induced by phorbol myristate acetate (PMA) using a mouse model. (A) Gross appearance of ears at day 0 (before induction), 1, 2, and 7. Right and left ears were treated with 20 μl of PBS (vehicle) and 20 μl of 30 mg/ml CM (600 μg), respectively. Both ears were then exposed to 20 μl of PMA solution. (B) Thickness of the ears at day 0, 1, 2, and 7. ∗P < 0.05 vs. PBS at each time point, determined by two-way ANOVA for unequal variance. (C) Hematoxylin and eosin (H&E) staining of the ears at day 0 and 7. Inset boxes in H&E staining indicate enlarged images below. NTC, no treatment control. Scale bar, 100 μm.