iPSCs were generated from embryonic fibroblasts of C57/B6 mice and were provided as a gift by Dr. Kazutoshi Takahashi (Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan). The iPSCs were reprogrammed by the transduction of retroviral vectors encoding four transcription factors, Oct-4, Sox2, c-Myc and Klf4, and cultured in iPSC medium to maintain an undifferentiated state, as previously described (12). Human dermal fibroblasts (HDFs) were isolated from normal human foreskin. All primary human fibroblasts were obtained from each sample prior to tissue fixation in 10% formalin (Nanchang Yulu Co., Jianxi, China) for routine histological examination. The tissue sections were cut into 1–3-mm cubes and incubated with 200 U/ml type I collagenase (Worthington Biochemical Corporation, Lakewood, NJ, USA) for 4 h at 37°C. The fibroblast cell cultures were maintained in Dulbecco’s modified Eagle’s medium (DMEM; 11965-092) supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin (all from Gibco Life Technologies, Grand Island, NY, USA). THP-1 cells (American Type Culture Collection, Manassas, VA, USA) were maintained in RPMI 1640 medium (Gibco Life Technologies) supplemented with 10% FBS, 100 U/ml penicillin, 100 mg/ml streptomycin and 0.5 mM/l β-mercaptoethanol (Gibco Life Technologies). All cell lines were incubated at 37°C in a humidified incubator with 5% CO₂ and cells from passages 6–8 were used. Conditioned medium from iPSCs (2×10⁵ cells/cm²) was diluted to 50, 30 and 0% by HDF-conditioned medium (HDFs-CM).

A total of 15 foreskin samples were collected from the Shanghai Jiaotong University Affiliated Sixth People’s Hospital (Shanghai, China) following approval by the ethics committee for human studies. The patients provided informed consent, and none had a systemic disease or had been previously treated for scars.

Following treatment with 0, 30, 50 or 100% iPSC-CM for 24 h, 4×10⁵ cells HDFs were harvested. The total protein was determined by the microplate bicinchoninic acid method using the BCA Protein Assay kit (Pierce Biotechnology, Inc., Rockford, IL, USA) in accordance with the manufacturer’s instructions.

Cell adhesion assays were perfomed as described previously (13). HDFs were seeded at a density of 3×10⁵ cells/well into 24-well plates until confluence was reached, then were incubated with DMEM supplemented with 10% FBS, 2 mM glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin. THP-1 cells were maintained in RPMI 1640 medium, supplemented with 10% FBS, 100 U/ml penicillin, 100 mg/ml streptomycin and 0.5 mM/L β-mercaptoethanol for 24 h and then were labeled fluorescently using 2.5 mM Cellstain-calcein-AM-solution (Dojindo Molecular Technologies, Inc., Kumamoto, Japan). THP-1 and HDF cells were co-cultured with the THP-1 suspension at a concentration of 5×10⁵ cells/ml and 0, 30, 50 or 100% iPSC-CM was added into each well for 3 h. The plates were centrifuged at 134 × g for 3 min and subsequently incubated at 37°C for 60 min. The medium and the nonadherent THP-1 cells were removed and each well was washed with phosphate-buffered saline three times. Adherent THP-1 cells were then microscopically quantified at a magnification of ×100 in four random visual fields for each well, and were subsequently imaged using an Axiovert 200 inverted fluorescence microscope (Zeiss, Oberkochen, Germany).

HDFs were seeded into 32-mm bacteriological plates (density, 6×10⁴ cells/ml; 2 ml/dish) in DMEM supplemented with 10% FBS, 100 U/ml penicillin and 100 mg/ml streptomycin, sodium ascorbate (50 mg/ml; Gibco Life Technologies) and 0.3 mg/ml acid-extracted collagen I from newborn calf skin (IBFB Pharma GmbH, Leipzig, Germany), as previously described (14), with 0, 30, 50 or 100% iPSC-CM. Furthermore, the contraction efficiency of the iPSC-CM was compared between quiescent and activated HDFs treated with TGF-β1 (Sigma-Aldrich, St. Louis, MO, USA). The cells were cultured at 37°C for 60 min to allow collagen polymerization to occur. The gels were then released from the plates by tilting them slightly. Gradual gel contraction was assessed by measuring the gel area at four time points, including 6, 12, 18 and 24 h. The data are presented as the mean ± standard error of three independent experiments, each conducted in triplicate.

Total RNA was isolated from cultured HDFs using TRIzol reagent (Invitrogen Life Technologies), and the integrity of the RNA was determined by 2% UltraPure agarose gel (Gibco Life Technologies) electrophoresis (15). For RT-qPCR, 2 μg total RNA was reverse transcribed at 37°C for 1 h in a 25-μl reaction medium containing 250 mM Tris-hydrochloric acid (HCl), 375 mM potassium chloride (KCl), 15 mM magnesium chloride (MgCl₂), 50 mM dithiothreitol, 10 mM deoxynucleotide triphosphates (dNTPs), 0.5 μg oligo (dT) 20 primer, 100 U reverse transcriptase (M-MLV) and 25 U ribonuclease inhibitor (all from Takara Bio, Inc., Otsu, Japan) and were subjected to PCR amplification with the primers described in Table I. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was amplified as the internal control. The RT products (0.5-l.0 μg) were amplified with 1 U Taq DNA polymerase (Takara Bio, Inc.) and 1 mM of each primer in a 50 μl reaction mix containing 50 mM KCl, 10 mM Tris-HCl, 1.5 mM MgCl₂ and 0.02 mM each of four dNTPs as follows: Initial denaturation for 3 min at 94°C, 30 cycles of amplification, 1 min of denaturation at 94°C, annealing temperatures of 57°C and 53°C for the collagen Iα1 and GAPDH primers, respectively, 1 min of extension at 72°C and a final 5-min elongation period at 72°C. Parallel PCR assays without reverse transcriptase were performed for each sample to confirm that the PCR products resulted from cDNA rather than from genomic DNA. The PCR products (10 μl) were analyzed by 2% agarose gel electrophoresis (15). The relative abundance of mRNA was calculated by densitometric analysis using Digital Science 1D Image Analysis software, version 3.0 (Kodak, Rochester, NY, USA).

Cells were lysed with radio-immunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Jiangsu, China) supplemented with 1 mM phenylmethylsulfonyl fluoride (Adamas-Beta, Ltd., Shanghai, China). The cell lysates were subject to western blot analysis, which was conducted as described in previous studies (15). The primary antibodies used were as follows: Polyclonal rabbit anti-human α-SMA IgG (1:500; ab15263; Abcam, Cambridge, UK) and monoclonal mouse anti-human collagen I IgG1 (1:200; sc-59772; Santa Cruz Biotechnology, Inc., Dallas, TX, USA).

Statistical analysis was performed using SPSS, version 13.0 (SPSS, Inc., Chicago, IL, USA) and a paired samples t-test was used to identify any differences between the groups. P<0.05 was considered to indicate a statistically significant difference.

To evaluate the cytotoxicity of iPSC-CM on fibroblasts, the total protein products following treatment of HDFs with 0, 30, 50 or 100% iPSC-CM for 24 h were measured. No significant differences were observed in the total protein among the four groups, which suggests that total fibroblast activity is not markedly affected by iPSC-CM (Fig. 1).

HS is formed by the abnormal accumulation of ECM. This predominantly consists of collagen, particularly type I collagen, which is produced by fibroblasts and is vital to HS formation. The proliferative stage of HS is marked by proliferation and activation of fibroblasts, thus, the level of type I collagen may be a marker of fibroblast activation. Myofibroblasts (activated fibroblasts) are identified by α-SMA expression and stress fiber formation (16), and their recruitment, retention and differentiation are commonly triggered by local stimuli of the microenvironment. Those stimuli include TGF-β1, mechanical force and matrix stiffness (17).

To determine whether in vitro TGF-β1-induced fibroblast activation may be suppressed by iPSC-CM in the present study, the level of α-SMA expression was assayed using western blot analysis. The results indicated that iPSC-CM significantly suppressed TGF-β1-induced α-SMA expression in a dose-dependent manner in cultured HDFs (P<0.01; Fig. 2A). Additionally, the alterations in collagen type Iα1 expression and type I collagen protein levels were confirmed by RT-qPCR and western blotting. The data demonstrated that the expression levels of collagen type Iα1 mRNA and collagen I protein were reduced, compared with control in 100% iPSC-CM (P<0.05; Fig. 2B and C). These observations suggest that fibroblast activation may be effectively suppressed by iPSC-CM.

Monocytes and fibroblasts work together during tissue repair, and fibroblasts regulate the responses of monocytes to ECM-derived matrices (18). Monocytes and lymphocytes are the main cells that induce chronic inflammatory reactions in HS. In addition, THP-1 monocytes are routinely used in monocyte assays (19). Therefore, the adhesion levels of THP-1 and cultured HDFs were examined to identify the inhibition of chronic inflammation by iPSC-CM.

In vitro adhesion assays of THP-1 and cultured HDFs demonstrated that iPSC-CM significantly reduced the level of adhesion in a dose-dependent manner (Fig. 3). These results suggest that iPSC-CM may suppress fibroblast activation by inducing the recruitment of inflammatory cells and blocking the direct interaction of inflammatory cells and fibroblasts. However, to confirm the attenuated effect of iPSC-CM on the inflammatory response in HS, in vivo studies are required.

Tissue contraction is dynamic and is characterized by intracellular and extracellular events (20). The contraction of HS is not determined by fibroblast properties alone, but is also dependent on the rate and extent of matrix contraction. To evaluate the effect of iPSC-CM on the contractile ability of HDFs, a 3D collagen gel fibroblast contraction assay was performed. Subsequent to treatment with 0, 30, 50 or 100% iPSC-CM for 6, 12, 18 or 24 h, the contraction of the collagen gels was monitored by measuring the gel area. Significant dose-dependent inhibitory effects of iPSC-CM on the contractile ability of HDFs were observed, and as time progressed, the differences between 100% iPSC-CM and the other three groups remained statistically significant (Fig. 4A). Significant time- and dose-dependent inhibitory effects of iPSC-CM on the contractile ability of HDFs were observed (Fig. 4A). Additionally, the contraction efficiency of iPSC-CM was significantly lower in the quiescent HDFs compared with the activated HDFs, at 18 and 24 h (P<0.05; Fig. 4B). This suggests that iPSC-CM may more efficiently prevent alterations in the contractile ability of activated HDFs treated with TGF-β, compared with quiescent HDFs. These observations further support the hypothesis that iPSC-CM is able to suppress fibroblast activation in vitro.