A total of 12 umbilical cords from full-term deliveries were obtained from the Affiliated Hospital of Guizhou Medical University (Guiyang, China) from January-December 2012. The gender ratio of the collected umbilical cords was 1:1. Written informed consent was obtained from parents and the experiments were performed and approved by the Ethical Committee of Guizhou Medical University (Guiyang, China). The isolation and culture of hUCMSCs was performed as previously described (24). The umbilical cords were washed twice with pre-cooled PBS at 4°C, the umbilical vessels were removed and the Wharton's jelly was minced into ~2.5 mm³ sections. The small sections were plated in 100-mm dishes (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany) in 5-mm wide gaps and cultured in Dulbecco's modified Eagle's medium/Nutrient mixture F12 (DMEM/F12; GE Healthcare Life Sciences, Chalfont, UK) supplemented with 20% fetal bovine serum (FBS; GE Healthcare Life Sciences), 100 U/ml penicillin (Beyotime Institute of Biotechnology, Beijing, China) and 100 mg/ml streptomycin (Beyotime Institute of Biotechnology) at 37°C with 5% CO₂. Cells at 85% confluence were digested with 0.25% trypsin (GE Healthcare Life Sciences) and transferred into 75 cm² cell culture flasks (Sigma-Aldrich; Merck Millipore) and cultured with DMEM/F12 medium with 10% FBS at 37°C with 5% CO₂.

The passage 4 of hUCMSCs was plated into 6-well (5×10⁴ cells/well) or 24-well (1×10⁴ cells/well) plates and randomly divided into two groups: i) Control group; and ii) experimental group. The cells were treated with 5 µg/ml αIR-3 (cat. no. MABS192; Merck Millipore) in the experimental group and without αIR-3 in the control group for 24 h. Cell viability, apoptosis, cell cycle and levels of Akt/glycogen synthase kinase (GSK)-3β activation were subsequently quantified.

hUCMSCs (1×10⁴) cultured on coverslips were fixed in 4% paraformaldehyde for 10 min and permeabilized with 0.1% Triton X-100 for 10 min at room temperature. Following washing with PBS, the cells were incubated with 3% hydrogen peroxide for 10 min at room temperature to block the endogenous peroxidase activity. Following washing twice with PBS, the cells were blocked with 10% goat serum (Wuhan Boster Biological Technology, Ltd., Wuhan, China) for 20 min and then incubated with rabbit anti-human polyclonal IGF-1 antibody (cat. no. ab9572; 1:200; Abcam, Cambridge, MA, USA;) and rabbit anti-human polyclonal IGF-1 receptor β antibody (cat. no. 3027; 1:200; Cell Signaling Technology, Inc., Danvers, MA, USA) overnight at 4°C. The cells were washed with PBS three times and then incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (cat. no. A0208; 1:5,000; Beyotime Institute of Biotechnology) for 30 min at room temperature. The cells were washed four times with PBS and incubated with streptavidin biotin complex (SABC) solution (Beyotime Institute of Biotechnology) for 20 min at room temperature and subsequently washed with PBS four times. The cells were incubated with 100 µl 3,3′-diaminobenzidine substrate solution (Beyotime Institute of Biotechnology) for 5 min and observed under an inverted microscope (NIKON TS100; Nikon Corporation, Tokyo, Japan), followed by dehydration with 95% ethanol, clearing with xylene and sealing with neutral gum (Beyotime Institute of Biotechnology).

Following treatment with αIR3, hUCMSCs were digested with 0.25% trypsin, collected, washed with pre-cold PBS and fixed with cold 70% ethanol. The cells were washed with pre-cold PBS three times, resuspended in 500 µl PBS and treated with 50 µl of RNase-A (100 µg/ml; Sigma-Aldrich; Meck Milipore) and 200 µl of propidium iodide (50 µg/ml; Beyotime Institute of Biotechnology) solution with 0.1% Triton-X 100 in the dark. Cell cycle was analyzed using a flow cytometer at 488 nm (BD Biosciences, San Jose, CA, USA).

Apoptosis of hUCMSCs was quantified using a flow cytometry assay using a Dead Cell Apoptosis kit with fluorescein isothiocyanate-Annexin V/(Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) following the manufacturer's protocol. Analysis of apoptosis (Annexin V positivity) was conducted using a flow cytometer at 488 and 535 nm.

Proteins were extracted from the treated hUCMSCs using RIPA lysis buffer (Sigma-Aldrich; Merck Millipore) supplemented with protease inhibitor (Sigma-Aldrich; Merck Millipore), phosphatase inhibitor (Sigma-Aldrich; Merck Millipore) and phenylmethylsulfonyl fluoride (PMSF; Sigma-Aldrich; Merck Millipore). The protein concentration was quantified using a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology). Proteins (30 µg/lane) were diluted in 2X SDS-PAGE protein loading buffer (Beyotime Institute of Biotechnology), heated at 95°C for 5 min, loaded into 10% SDS-PAGE gel (Beyotime Biotechnology) and separated by electrophoresis. Following electrophoresis, the proteins were transferred onto polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked with 5% non-fat milk in Tris-buffered saline with Tween-20 (TBST) for 4 h at room temperature and subsequently incubated with rabbit anti-human phosphorylated (p)-Akt polyclonal antibody (cat. no. ab18206; 1:1,000; Abcam), rabbit anti-human p-glycogen synthase kinase 3β (GSK-3β) polyclonal antibody (cat. no. 9336; 1:1,000; Cell Signaling Technology, Inc.), rabbit anti-human p21 polyclonal antibody (cat. no. ab7960; 1:1,000; Abcam), rabbit anti-human cyclin D1 polyclonal antibody (cat. no. 2922; 1:1,000; Cell Signaling Technology, Inc.), rabbit anti-human p-p70 S6 kinase (P70S6K) polyclonal antibody (cat. no. 9025; 1:1,000; Cell Signaling Technology, Inc.), rabbit anti-human p-ERK1/2 monoclonal antibody (cat. no. 4370; 1:1,000; Cell Signaling Technology, Inc.) in blocking solution at 4°C overnight. The blots were washed 3 times with TBST and incubated with HRP-conjugated goat anti-rabbit secondary antibody (cat. no. BA1054; 1:5,000, Wuhan Boster Biological Technology) in a blocking solution for 1 h at room temperature. The immunoreactive bands were washed with TBST, visualized with western blotting enhanced chemiluminescence reagent (EMD Millipore) and subsequently exposed to X-ray film (Thermo Fisher Scientific, Inc.). The blots were normalized to the expression of β-actin, which was detected by the same blots through washing with a stripping buffer (Beyotime Institute of Biotechnology) and subsequent incubation with rabbit anti-mouse β-actin polyclonal antibody (cat. no. ab8227; 1:2,000; Abcam). All protein bands were quantified by densitometry using ImageJ software (version 1.48h3; National Institutes of Health, Bethesda, MD, USA).

Statistical analysis was performed using SPSS version 15.0 software (SPSS, Inc., Chicago, IL, USA). Data are presented as the mean ± standard deviation from 4–6 independent experiments. Univariate comparisons of means were evaluated using the Student t-test. P<0.05 was considered to indicate a statistically significant difference.

The morphology of primary and passaged hUCMSCs was determined at 2 and 7 days after the Wharton's jelly sections were plated. As presented in Fig. 1A, round cells dissociated from the Wharton's jelly sections and attached to the bottom of the culture dishes 2 days after the small sections were plated. By day 7, cells assumed triangular and spindle shape and a number of cells had long dendrites (Fig. 1B). Cells at passage 4 exhibited long spindle shape and upon reaching confluence, formed a pattern similar to a whirlpool (Fig. 1C). Immunocytochemistry staining revealed that IGF-1 was expressed in hUCMSCs, particularly in the nuclei (Fig. 1D). IGF-1R was also positively expressed in hUCMSCs, primarily located on the membrane and absent in the nuclei (Fig. 1E).

The present study quantified cell viability using an MTT assay. As presented in Table I, treatment with the IGF-1R-specific blocker αIR-3 for 24 h significantly reduced the hUCMSCs cell viability when compared with the control group (P<0.05). Additionally, cell cycle analysis revealed that the number of cells in the G2/M phase was reduced in the experimental group compared with the control group, which also indicated a reduced proliferation of hUCMSCs following treatment with αIR-3 (Fig. 2).

Cell apoptosis was quantified by analysis of Annexin V activity using flow cytometry. The increase of Annexin V activity indicated an early stage of apoptosis (25). As presented in Fig. 3, treatment with the IGF-1R-specific blocker, αIR-3, markedly increased Annexin V positivity (from 0.25% in the control group to 10.40% in the experimental group).

Previous studies have reported that the binding of IGF-1 to its receptor may mediate the activation of the PI3K/Akt and MAPK/ERK signaling pathways (15,16). The findings of the present study revealed that treatment with the IGF-1R-specific blocker αIR-3 significantly reduced the expression levels of p-Akt, p-Gsk-3β and p-P70S6K (P<0.05; Fig. 4; Table II), However, no significant difference was identified in p-ERK1/2 expression when the experimental group was compared with the control group (P>0.05; Fig. 4; Table II). These findings indicated that the effect of autocrine IGF-1 on hUCMSCs cell viability may be due to the activation of the Akt/Gsk-3β/P70S6K signaling pathway; however, not the activation of MAPK/ERK signaling pathway. In addition, treatment with αIR-3 also significantly reduced cyclin D1 expression levels and increased p21 expression levels (P<0.05; Fig. 4; Table II). These two proteins are important factors determining cell viability and apoptosis (26).