Effect of human Wharton's jelly mesenchymal stem cell paracrine signaling on keloid fibroblasts

Abstract

Keloid scars are abnormal benign fibroproliferative tumors with high recurrence rates and no current efficacious treatment. Accumulating evidence suggests that human umbilical cord Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) have antifibrotic properties. Paracrine signaling is considered one of the main underlying mechanisms behind the therapeutic effects of mesenchymal stem cells. However, the paracrine signaling effects of WJ-MSCs on keloids have not yet been reported. The aim of this study is to investigate paracrine signaling effects of human WJ-MSCs on keloid fibroblasts in vitro. Human umbilical cords and keloid skin samples were obtained, and WJ-MSCs and keloid fibroblasts were isolated and cultured. One-way and two-way paracrine culture systems between both cell types were investigated. Plasminogen activator inhibitor-I and transforming growth factor-β2 (TGF-β2) transcripts were upregulated in keloid fibroblasts cultured with WJ-MSC-conditioned medium (WJ-MSC-CM) and cocultured with inserts, while showing lower TGF-β3 gene expression. Interleukin (IL)-6, IL-8, TGF-β1, and TGF-β2 protein expression was also enhanced. The WJ-MSC-CM-treated keloid fibroblasts showed higher proliferation rates than their control keloid fibroblasts with no significant change in apoptosis rate or migration ability. In our culture conditions, the indirect application of WJ-MSCs on keloid fibroblasts may enhance their profibrotic phenotype.

Keywords: Fibrosis; Keloid; Scar; Skin; Stem cells; Wharton’s jelly.

Figure 1.

Wharton’s jelly-derived mesenchymal stem cell (WJ-MSC) paracrine signaling effect on the expression of profibrotic genes in human keloid skin fibroblast. (A): One-way paracrine signaling or WJ-MSC-conditioned medium-treated keloid skin fibroblasts versus nonconditioned media-treated keloid skin fibroblasts or control group. (B): Two-way paracrine signaling or Transwell insert system coculture. Results are from six different keloid skin samples. ∗, p ≤ .05; ∗∗, p ≤ .01. Abbreviations: PAI-1, plasminogen activator inhibitor-I; TGF, transforming growth factor.

Figure 2.

Wharton’s jelly-derived mesenchymal stem cell (WJ-MSC) paracrine signaling effect on the expression of antifibrotic genes in human keloid skin fibroblasts. Figure shows downregulation of antifibrotic genes in keloid fibroblasts through one-way paracrine signaling WJ-MSCs (A) and two-way paracrine signaling (B). Messenger RNA levels were studied in six keloid skin samples, but four in the decorin insert system. ∗, p ≤ .05; ∗∗, p ≤ .01. Abbreviation: TGF, transforming growth factor.

Figure 3.

Wharton’s jelly-derived mesenchymal stem cell (WJ-MSC) paracrine effects on the expression of profibrotic proteins in keloid fibroblasts. (A): Upregulation of profibrotic proteins in keloid fibroblasts through one-way paracrine signaling WJ-MSCs, or WJ-MSC-conditioned medium. (B): Upregulation of profibrotic proteins in human keloid skin fibroblasts cocultured with WJ-MSCs via a Transwell insert system. Two different keloid samples were investigated for protein studies. ∗, p ≤ .05. Abbreviations: IL, interleukin; TGF, transforming growth factor.

Figure 4.

Kinetics growth of keloid skin fibroblasts cultured with WJ-MSC-CM. The BrdU proliferation assay (A–C) and CyQUANT assay (D) were performed in three and one keloid skin samples, respectively, after being treated or not treated with WJ-MSC-CM. ∗∗, p ≤ .01; ∗∗∗, p ≤ .001. Abbreviations: BrdU, bromodeoxyuridine; CTRL, control; DAPI, 4′,6-diamidino-2-phenylindole; HPF, high-power field; WJ-MSC-CM, Wharton’s jelly-derived mesenchymal stem cell-conditioned medium.

Figure 5.

Wharton’s jelly-derived mesenchymal stem cell (WJ-MSC) one-way paracrine signaling effects on human keloid skin fibroblast apoptosis and migration. A TUNEL kit was used to measure apoptosis (A, B), and a scratch wound assay (C, D) was performed to examine keloid migration, after treatment with WJ-MSC-CM or nonconditioned medium, in three different keloid samples. Abbreviations: CTRL, control; DAPI, 4′,6-diamidino-2-phenylindole; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling; WJ-MSC-CM, Wharton’s jelly-derived mesenchymal stem cell-conditioned medium.