The Ethics Committee of the Second Affiliated Hospital of Anhui Medical University (PJbb2017-004) approved the collection of abscized human hemangiomas. The tissues were used instantly for cell isolation and cultivation in in vitro experiments.

The proliferating IH tissues resected from the patients were immediately immersed in a growth medium at 4°C [Dulbecco's modified Eagle's medium, high glucose, 10% fetal bovine serum (FBS; Biological Industries, Kibbutz Beit Haemek, Israel), and 1% penicillin-streptomycin (PS; Beyotime Institute of Biotechnology, Haimen, China)] and then quickly taken to our laboratory. The fatty and skin tissue were resected, the samples were rinsed three times in phosphate-buffered saline (PBS) and then minced. A 0.2% compound of collagenase (cat. no. 17454; Serva Electrophoresis GmbH, Heidelberg, Germany) was used to digest the samples at 37°C for 2 h until they were chylous. They were then filtered through a 100-micron cell strainer. From this single cell suspension, cells expressing CD133 were selected using a magnetic beads technology (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) and then cultured on fibronectin-coated plates (Corning Incorporated, Corning, NY, USA) in EGM-2 media (cat. no. CC-4176; Lonza Group, Ltd., Basel, Switzerland) that was supplemented by 20% FBS and 1% PS.

Logarithmic growth phase HemSCs were seeded into 96-well tissue culture plates (Corning Incorporated) at an initial density of 2×10³. After serum starvation for 24 h, IGF-2 (cat. no. 100-12-50UG; PeproTech, Inc., Rocky Hill, NJ, USA) at 0, 10, 20, 100, and 200 ng/ml, respectively, was added to EBM-2 containing 5% FBS in the experimental groups. After 72 h in the culture, CCK-8 reagents (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) were added to disparate wells under different treatments. The absorbency was read at 490 nm using a microplate reader (Biotek ELx 800; BioTek Instruments, Inc., Winooski, VT, USA).

To examine the growth kinetics of IGF-2-treated HemSCs, HemSCs were seeded into 96-well plates (Corning Incorporated) at a density of 1.5×10³ cells/well. After serum starvation, the cells were treated with 100 ng/ml IGF-2 containing 5% FBS. CCK-8 was added at 0, 1, 3, 5, and 7 days after culture and incubated at 37°C for 4 h. The results of the OD values at 490 nm were tested by a microplate reader (Biotek Elx 800; BioTek Instruments, Inc.).

When the cells had increased by 70% in 24 well plates, they were washed in PBS followed by fixing in 4% poly-formaldehyde for 15 min, drying in air for 5 min, and permeabilizing in 0.5% Triton X-100 for 10 min. Endogenous peroxidase activity was inactivated using 3% hydrogen peroxide for 5–10 min at room temperature. The slides were incubated in blocking solution (reagent A) for 15–20 min, and then the blocking solution was out-welled without washing. Between each step, the cells were washed in PBS, the slides were incubated overnight at 4°C, and the primary antibodies were diluted by PBS to achieve the required concentration. The primary antibodies were replaced by PBS in the negative control group. The secondary antibodies were incubated for 10–15 min at 37°C. The cells were observed under a bright-field microscope for 3–10 min immediately after the DAB solution (cat. no. K166724B; 50 µl DAB: 1 ml DAB substrate, kept away from light) was added to the slides, which underwent hematoxylin staining for 0.5–1 min and gum seal after drying at room temperature. The cells were photographed using bright-field microscope illumination (Olympus IX71; Olympus Corporation, Tokyo, Japan).

To evaluate the adipogenic differentiation of IGF-2-treated HemSC, 5.0×10⁴ cells were seeded on 6-well plates in EGM-2/10% FBS. When the cells were oversaturated, the original medium was changed to adipogenic differentiation media (cat. no. HUXMF-90031; Cyagen Biosciences, Santa Clara, CA, USA) with IGF-2 (100 ng/ml), IGF-2 (100 ng/ml) plus OSI-906 (cat. no. S1091; 1 µM; Selleck Chemicals, Houston, TX, USA), or no treatment for 10 days. The oil red o-stained cells were observed using an inverted microscope. Photos were taken using the Eclipse E800 microscope (Nikon Corporation, Tokyo, Japan).

HemSCs were grown on 10-cm tissue culture plates (Corning Incorporated). The cells were cultured in EGM-2/FBS-10% containing IGF-2 (100 ng/ml), IGF-2 (100 ng/ml) plus OSI-906 (1 µM), IGF-2 (100 ng/ml) plus LY294002 (cat. no. HY-10108; 10 µM; MedChem Express Co., Ltd., Shanghai, China), or no treatment. Protein extracts were subjected to SDS-PAGE on 8–15% polyacrylamide gels. The samples were then transferred to PVDF membranes. The membranes were blocked by 5% non-fat milk for 2 h and then incubated first with primary antibodies overnight at 4°C and then with secondary antibodies. The following primary antibodies were used: Anti-PPARγ (cat. no. bs-0530P), anti-C/EBPα (cat. no. bs-1630R), anti-C/EBPβ (cat. no. bs-1396R), anti-adiponectin (cat. no. 0471R; all from BIOSS, Beijing, China), and anti-pAKT (cat. no. 9271), anti-AKT (cat. no. 9272), anti-IGF-2 (cat. no. 25690), and anti-β-actin (cat. no. 3700; Cell Signaling Technology, Inc., Danvers, MA, USA).

HemSCs were maintained on fibronectin-coated 10-cm plates in EGM-2/FBS-20%. The medium was changed every two days. HemSCs at passage numbers 5 to 15 were used in all experiments. During the media changes, propranolol hydrochloride (cat. no. P8688; Sigma-Aldrich, St. Louis, MO, USA) was added to the culture media at the indicated concentrations.

Total RNA was isolated using the TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The cDNA was synthesized by the reverse transcription of 2 µg of total RNA using a PrimeScript™ RT reagent (Takara, Dalian, China). A semi-quantitative real-time polymerase chain reaction was conducted using the Applied Biosystems 7500 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) and the SYBR Premix Ex TaqTM (Takara). The results were normalized to β-actin expression levels, and the samples were analyzed in triplicate. The gene expression levels of C/EBPα, C/EBPβ and PPARγ were then determined (Table I).

The results of two independent experiments were expressed as the mean ± SD (n=3 for each experiment). In comparing the two groups, an unpaired Student's t-test was used to assess the differences, and analysis of variance was applied for the analysis of the mean values among multiple groups. A value of P<0.05 was considered to indicate a statistically significant difference.

A CCK-8 assay was performed to determine whether IGF-2 influenced the proliferation of HemSCs in vitro. The results showed that 10–200 ng/ml of IGF-2 promoted the proliferation of HemSCs. The concentration at 100 ng/ml was the most appropriate among all the groups (Fig. 1A). When 100 ng/ml IGF-2 was used for the cell growth curve, the IGF-2-treated cells promoted increased cell proliferation compared to the non-treatment group from day 1 to day 7 (Fig. 1B).

As shown in Fig. 2A, the expression of IGF-2, IGF-2R, IGF-1R, and IR was positive in the HemSCs. In cell localization, the dyeing of IGF-2 was primarily in the cytoplasm. IGF-2R, IGF-1R, and IR were in both the membrane and the cytoplasm.

In order to determine whether the induction of IGF-2 accelerated the differentiation of adipocytes, HemSCs was exposed to IGF-2 (100 ng/ml), IGF-2 (100 ng/ml) plus OSI-906 (1 µM), or no treatment for 10 days in adipogenic differentiation media (Cyagen Biosciences). Phase-contrast microscopy was used after oil red o-staining. The results indicated that IGF-2 treatment stimulated the accumulation of lipogenesis in the HemSCs, which was shown by the increase in cell numbers and the density of lipid droplets in the HemSCs treated with adipogenic differentiation media containing IGF-2 compared to the adipogenic differentiation media alone or the adipogenic differentiation media containing IGF-2 plus OSI-906 (1 µM) group (Fig. 2B). The results further confirmed the adipogenic effects of IGF-2 on HemSCs. The results also showed that the IGF-1R inhibitor (OSI-906, 1 µM) suppressed the IGF-2 inducing lipid accumulation.

We also aimed to determine whether IGF-2 could regulate the adipogenic differentiation of HemSCs into adipocytes via IGF receptors. The results of the western blot analysis showed that the expressions of disparate transcription factors and adipocyte specific proteins were increased in the IGF-2 treated group compared to the control group. C/EBPα, C/EBPβ, PPARγ, and adiponectin were increased, which indicated that IGF-2 enhanced adipogenesis in HemSCs. Interestingly, the IGF-2-treated medium in the presence of OSI-906 or LY294002 or LY294002 alone could repress the IGF-2-induced adipogenesis and inhibit the protein expressions of C/EBPα, C/EBPβ, PPARγ, and adiponectin (Fig. 3A). In addition, the p-AKT level was attenuated compared with that in the IGF-2 treated cells (Fig. 3B). These findings suggested that the IGF-2 mediated the induction of adipocyte differentiation by upregulating the phosphorylation of AKT via the IGF-1R-PI3K signaling pathway in the HemSCs.

HemSCs were grown in EGM-2/20% FBS at vrious nanomolar concentrations of propranolol (0, 50, and 100 µM). During 24 h of adipogenic differentiation, the gene expression levels of C/EBPα, C/EBPβ (CCAAT/enhancer-binding protein) and PPAR (peroxisome proliferator-activated receptor) were observed. In the 50-µM propranolol solution, the C/EBPα RNA level was no changed compared with the control group, whereas it was increased after intervention in 100 µM propranolol solution compared to the control group. The RNA levels of C/EBPβ and PPARγ were significantly elevated compared with the control group after intervention with 50 and 100 µM of propranolol solution (Fig. 4B). The changes in the levels of protein expression of C/EBPα, C/EBPβ, and PPARγ were similar to those in RT-qPCR in the detection of the adipogenic differentiation index in HemSCs. However, no significant change in C/EBPα was found (Fig. 4A). Interestingly, several concentrations of propranolol-treated HemSCs showed marked increases in IGF-2 compared to the control group (Fig. 4C). Furthermore, IGF-2 may have been involved in this effect, which was shown by its increased levels following the treatment by pronanolol.