Human adipose-derived stem cells inhibit bioactivity of keloid fibroblasts

Abstract

Background: A keloid is a fibroproliferative disorder occurring in wounds characterized by an exaggerated response to injury. To date, no effective cure has been identified. As multipotent stem cells, human adipose-derived stem cells (ADSCs) may show the possibility for curing diseases such as fibrosis. This study sought to explore the potential role of human ADSCs in curing keloids.

Methods: After culture in conditioned medium, gene and protein expression of keloid fibroblasts was examined using real-time polymerase chain reaction (RT-PCR) and Western blotting, while analysis of the cell cycle was used to measure the proliferative properties of the cells. Furthermore, ex vivo explant cultures were used to test the effects of ADSC-conditioned medium (ADSC-CM) on CD31+ and CD34+ expression in keloid tissue.

Results: Our experimental results show that ADSC-CM was able to attenuate extracellular matrix-related gene expression as well as decrease protein expression. Cell proliferation was significantly suppressed in our study. CD31+ and CD34+ vessels in ex vivo explants were reduced by 55% and 57% in treatment groups compared with control groups.

Conclusions: Human ADSC-CM significantly inhibited keloid fibroblast-related bioactivities.

Keywords: Adipose-derived stem cells; Conditioned medium; Keloid fibroblasts.

Conflict of interest statement

Ethics approval and consent to participate

The certificate number 2017-311-T231 was issued by the Ethics Review Committee of Shanghai 9th People’s Hospital. We also received informed consent from the patients.

Consent for publication

All authors declare their support for the publication and its contents.

Competing interests

The authors declare that they have no competing interests.

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Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1

Characterization of human adipose-derived stem cells (ADSCs). a Flow cytometric characterization of ADSCs. ADSCs strongly expressed CD29, CD44, CD90, and CD105, and did not express CD31, CD34, or CD45. b ADSCs exhibited a fibroblast-like morphology. c Cells were induced to differentiate into adipocytes (left panel) and osteoblasts (right panel); scale bars = 100 μm

Fig. 2

ADSC-CM inhibits the gene expression of plasminogen activator inhibitor-1 (PAI-1), collagen I (COL I), and tissue inhibitor of metalloproteinase 1 (TIMP1) in keloid fibroblasts. qPCR was employed to analyze the expression of various genes in the cells that were cultured with or without ADSC-CM for 5 days. The gene expression levels are presented relative to the control (CT) level (*P < 0.05)

Fig. 3

ADSC-CM inhibits keloid fibroblast proliferation. The effect of ADSC-CM on the cell cycle profiles was evaluated by flow cytometric analysis. *P < 0.05, versus control

Fig. 4

ADSC-CM depresses the invasive abilities of keloid fibroblasts. a, b As determined by transwell® assay, the migratory keloid fibroblasts were visualized by imaging the nuclei labeled with DAPI. c The number of migrated cells was counted in three randomly selected fields. *P < 0.05, versus control (CT)

Fig. 5

Adipose-derived stem cell-conditioned medium (ADSC-CM) reduces angiogenesis in keloid explant culture. After culturing with (b, d) or without (a, c) ADSC-CM for 8 days, the keloid explants were sectioned and subjected to immunohistochemical analysis using antibodies against CD31 (a, b) and CD34 (c, d). Brown coloration indicates positive staining of related markers. The numbers of CD31-positive endothelial cells and CD34-positive microvascular endothelial cells were counted in three randomly selected fields for each sample under a microscope. Scale bars = 100 μm. *P < 0.05, versus control

Fig. 6

After culturing with (b) or without (a) adipose-derived stem cell-conditioned medium (ADSC-CM), sections were also subjected to H&E staining for histological analysis. Scale bars = 100 μm

Fig. 7

ADSC-CM interferes with intracellular signaling in vitro. To explore the underlying mechanism of the antifibrotic effect of ADSC-CM, the protein level was measured by Western blotting in primary keloid fibroblasts (left panel). Gene expression of transforming growth factor (TGF)-β in the experimental and control (CT) groups (right histogram). *P < 0.05, versus control