Ethics committee approval was obtained for the collection of abscised human hemangiomas from The Second Affiliated Hospital of Anhui Medical University (Hefei, China; no. PJ-bb2017-026). The tissues were immediately used for cell isolation and culture for in vitro experiments.

The proliferating IH tissues removed from the patients were immediately immersed in growth medium consisting of high glucose Dulbecco's modified Eagle's medium (GE Healthcare Life Sciences, Shanghai, China), 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), and 1% penicillin-streptomycin (PS; Beyotime Institute of Biotechnology, Haimen, China) at 4°C and quickly taken to the laboratory. The fat or skin tissues were removed and the samples were rinsed three times in PBS. The tissues were minced and digested with 0.2% collagenase (SERVA Electrophoresis GmbH, Heidelberg, Germany) at 37°C for 2 h until the samples were chylous, and were passed through a 100-µm cell strainer. The cells expressing CD133 were separated from the resulting single-cell suspension using a magnetic bead technique (Miltenyi Biotec, Inc., Auburn, CA, USA) and cultured on fibronectin-coated plates in endothelial growth medium-2 (EGM-2; Lonza Group, Ltd., Basel, Switzerland) supplemented with 20% FBS and 1% PS in a 37°C incubator with 5% CO₂.

HemSCs in the logarithmic growth phase were seeded into 96-well tissue culture plates (Corning, Inc., Corning, NY, USA) at an initial density of 2×10³ cells. After 24 h of serum starvation, IGF-1 (Peprotech, Inc., Rocky Hill, NJ, USA), dissolved in EBM-2 containing 5% FBS, was added at 0, 10, 20, 100 and 200 ng/ml. After 72 h of culture, Cell Counting kit-8 (CCK-8) reagent (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) was added to each well and the absorbance at 490 nm was read using a microplate reader (BioTek ELx 800; BioTek Instruments, Inc., Winooski, VT, USA).

The growth kinetics of IGF-1-treated HemSCs were examined by seeding HemSCs into 96-well plates (Corning, Inc.) at a density of 1.5×10³ cells/well. Following serum starvation, the cells were treated with 100 ng/ml IGF-1 containing 5% FBS. CCK-8 was added after 0, 1, 3, 5 and 7 days of culture and the cells were incubated at 37°C for a further 4 h. The optical density at 490 nm was measured with a microplate reader (BioTek ELx 800).

Cells (1×10⁴) cultured on coverslips were fixed in 4% poly-formaldehyde for 15 min at room temperature. Following air-drying for 5 min, the cells were permeabilized in 0.5% Triton X-100 for 10 min. Endogenous peroxidase activity was inactivated by treatment with 3% hydrogen peroxide at room temperature for 5–10 min. The slides were blocked with 10% FBS for 15–20 min at room temperature, and the blocking solution was aspirated without further washing. The cells were washed with PBS, and the slides were incubated overnight at 4°C with the following primary antibodies: Anti-IGF-1 (1:500, cat. no. bs-0014R), anti-IGF-1R (1:500, cat. no. bs-0227R) and anti-β-actin (1:500, cat. no. bs-0016R; all BIOSS, Beijing, China). Antibodies were replaced with PBS in the negative control group. Horseradish peroxidase-conjugated secondary antibody working solution (cat. no. PV-6000; OriGene Technologies, Inc., Beijing, China) were incubated for 10–15 min at 37°C. The cells were observed by bright-field microscopy (×400) for 3–10 min immediately following addition of diaminobenzidine (DAB) solution (50 µl DAB:1 ml DAB substrate; stored in the dark). The cells were hematoxylin-stained for 0.5–1 min at room temperature, and gum-sealed following drying. The cells were photographed under bright field illumination (×400, Olympus IX71; Olympus Corporation, Tokyo, Japan).

The adipogenic differentiation of IGF-1-treated HemSCs was evaluated by seeding 5.0×10⁴ cells in 6-well plates in EGM-2/10% FBS. When cells were oversaturated, the original medium was switched to an adipogenic differentiation media (Cyagen Biosciences, Inc., Santa Clara, CA, USA) containing either IGF-1 (100 ng/ml), IGF-1 (100 ng/ml) plus OSI-906 (1 µM; Selleck Chemicals, Houston, TX, USA; an IGF-1R inhibitor), IGF-1 (100 ng/ml) plus LY294002 (10 µM; MedChemExpress, Monmouth Junction, NJ, USA), LY294002 (10 µM; MedChemExpress; PI3K inhibitor), or no treatment, and the cells were incubated for 10 days. At room temperature, the cells were washed gently with PBS twice, fixed with 4% fresh formaldehyde in PBS for 15 min and stained with Oil Red O for 30 min. Cells were examined with an inverted microscope (×200), and the numbers determined using Image J software (1.51s; National Institutes of Health, Bethesda, MD, USA). The cells were photographed using an Eclipse E800 microscope (Nikon Corporation, Tokyo, Japan).

HemSCs were grown on 10-cm tissue culture plates (Corning, Inc.) in EGM-2/10% FBS containing IGF-1 (100 ng/ml), IGF-1 (100 ng/ml) plus OSI-906 (1 µM), IGF-1 (100 ng/ml) plus LY294002 (10 µM), or no treatment. Total protein was extracted using radioimmunoprecipitation assay lysis buffer (1 mM; BIOSS), and protein concentration was determined with a Bradford Protein Assay kit (Beyotime Institute of Biotechnology). Cell protein extracts (5 µl/lane) were subjected to SDS-PAGE on 10% polyacrylamide gels. The samples were transferred to polyvinylidene fluoride membranes, which were blocked with 5% skim milk for 2 h, incubated with primary antibodies overnight at 4°C, and subsequently incubated with IRDye 800CW-conjugated goat anti-rabbit IgG secondary antibody (cat. no. bs-40295G-IRDye8; 1:20,000; BIOSS) for 1 h at room temperature. The primary antibodies were: Anti-PPARγ (cat. no. bs-0530P; 1:500; BIOSS), anti-CCAAT/enhancer-binding protein (C/EBPα; cat. no. bs-0016R; 1:1,000), anti-C/EBPβ (cat. no. bs-1396R; 1:1,000), anti-adiponectin (cat. no. bs-0471R; 1:1,000; BIOSS), anti-IGF-1 (cat. no. bs-0014R; 1:1,000; all BIOSS), anti-phosphorylated (p)AKT (cat. no. 9271; 1:500), anti-AKT (cat. no. 9272; 1:500) and anti-β-actin (cat. no. 3700; 1:500; all Cell Signaling Technology, Inc., Danvers, MA, USA). Finally, the signal was detected using enhanced chemiluminescence reagents (Beyotime Institute of Biotechnology), and band density was analyzed with ImageJ software (1.51s; National Institutes of Health, Bethesda, MD, USA).

The data are expressed as the mean ± standard deviation (n=3 for each experiment). SPSS version 23 (IBM Corp., Armonk, NY, USA) was used to perform statistical analysis. Where appropriate, an unpaired Student's t-test was used to assess the differences between two groups, and one-way analysis of variance followed by Fisher's Least Significant Difference test was applied for the analysis of the mean values among multiple groups. P<0.05 was considered to indicate a statistically significant difference.

The CCK-8 assay was performed to determine whether IGF-1 affected in vitro HemSC proliferation. The addition of IGF-1 at 100–200 ng/ml stimulated the proliferation of HemSCs, with the 100 ng/ml concentration exhibiting the best response among all the groups (Fig. 1A). Cell growth curves confirmed that treatment with 100 ng/ml IGF-1 promoted cell proliferation between days 3 and 7 when compared with the non-treatment group (Fig. 1B).

Fig. 2A presents the positive expression of IGF-1 and IGF-1R in HemSCs. IGF-1 was localized primarily to the cytoplasm, while IGF-1R was localized to the cytoplasm and the cell membrane.

The HemSCs exhibited increased cell numbers and greater lipid droplet density when treated with adipogenic differentiation medium containing IGF-1, compared with adipogenic differentiation medium alone or with media containing IGF-1 plus OSI-906, IGF-1 plus LY294002, or LY294002 alone (Fig. 2B). The IGF-1 treatment thus appeared to stimulate adipogenesis and lipid accumulation in the HemSCs, whereas treatment with the OSI-906 IGF-1R inhibitor or LY294002, a specific inhibitor of PI3K, suppressed this IGF-1-induced lipid accumulation.

Western blotting confirmed an increase in C/EBPα, C/EBPβ, PPARγ and adiponectin expression in response to IGF-1 when compared with the control group. This increase in transcription factors and adipocyte-specific proteins indicated that IGF-1 enhanced adipogenesis in HemSCs and this enhancement was suppressed in the presence of OSI-906 or LY294002. In addition, LY294002 alone was able to suppress HemSC adipogenesis and inhibit the expression of C/EBPα, C/EBPβ, PPARγ and adiponectin proteins (Fig. 3A). The IGF-1 treatment increased the phosphorylation of AKT, but this increase was suppressed by co-treatment with LY294002 or OSI-906, whereas the amounts of total AKT protein were unaltered (Fig. 3B). Therefore, IGF-1 appeared to induce adipocyte differentiation in HemSCs by upregulating the phosphorylation of AKT via an IGF-1R-PI3K signaling pathway.