Fetal bovine serum (FBS), Dulbecco's modified Eagle's medium (DMEM)/F12, 0.25% trypsin and phosphate-buffered saline (PBS) were purchased from GE Healthcare (Logan, UT, USA). Fluorescein isothiocyanate-conjugated (FITC) mice monoclonal antibodies, including anti-CD29-FITC (cat no. 6604105; 1:50), anti-CD34-FITC (cat. no. IM1870; 1:50), anti-CD44-FITC (cat. no. IM1219U; 1:50), anti-CD45-FITC (cat. no. IM0782U; 1:50), anti-CD90-FITC (cat. no. IM1839U; 1:50), anti-CD105-PE (cat. no. B76299; 1:50), as well as intra-1 (cat. no. A07803; 1:50) and intra-2 (cat. no. A07803; 1:50) were obtained from Beckman Coulter, Inc. (Brea, CA, USA). Monoclonal phycoerythrin (PE)-conjugated antibodies, anti-Bcl-2-PE (cat. no. A15796; 1:50; Invitrogen, Thermo Fisher Scientific, Inc., Waltham, MA, USA) and anti-Survivin-PE (cat. no. 129176; 1:50; eBio-science, Inc., San Diego, CA, USA). Anti-α fetoprotein (AFP) primary antibody and FITC-conjugated IgG (H+L) antibody were provided by LsBio (Seattle, WA, USA) and Invitrogen, Thermo Fisher Scientific Inc., respectively. An RNA extraction kit was purchased from TianGen BioTech Co., Ltd. (Beijing, China). An RNA reverse transcription reaction kit and RNA quantification kit were purchased from Bio-Rad (Hercules, CA, USA). Primers were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China).

Third passage human UC-MSCs were obtained from Beike Biotechnology (Shenzhen, China). These cells were obtained from informed, healthy donors after normal spontaneous vaginal deliveries. The procedure for cell collection and mononuclear cell extraction, cultivation and harvest, has been reported in a previous publication (16). Human HCC HepG2 cells were kindly provided by Professor Lin Wang (17) from the Department of Hepatobiliary Surgery, The 2nd Affiliated Hospital of Kunming Medical University (Kunming, China). UC-MSCs and HepG2 cells were maintained in DMEM/F12 containing 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin (GE Healthcare Life Sciences, Logan, UT, USA). Cells were incubated in a 5% CO₂-humidified incubator at 37°C. The culture medium was changed every two or three days. When cells reached 80–90% confluency, cell passage was conducted using 0.25% trypsin for digestion. Passage seven to eight UC-MSCs were used for the following experiments.

For co-culture of UC-MSCs with HepG2 cells, a Transwell co-culture system (pore size, 0.4 µm; Corning, Corning, NY, USA) was utilized. UC-MSCs were collected by trypsin digestion, resuspended in fresh culture medium, adjusted to the appropriate cell density (2.5×10⁵–4×10⁶ cells/well) and seeded onto a six-well plate (2 ml cell suspension for each well). After the initial seeding of UC-MSCs (24 h), 1×10⁶ HepG2 cells suspended in 2 ml culture medium were seeded into the upper compartment of the Transwell. Cell morphology was observed under a phase contrast microscopy (BX53, Olympus, Tokyo, Japan).

Passage three and passage seven UC-MSCs were used for cell identification. Cells were washed with PBS and digested with 0.25% trypsin. After centrifuging at 300 x g for 5 min, the supernatant was removed and cells were washed twice with PBS. Cells were then incubated with FITC-conjugated primary antibodies against CD29, CD34, CD44, CD45, CD90 or CD105 for 30 min at room temperature in the dark. The expression of cell surface biomarkers was determined using flow cytometric analysis. Fluorescence acquisition was performed in a Coulter-EPICS XL flow cytometer (Beckman-Coulter, Inc.) with a 488 nm argon-ion laser.

After 24, 48 or 72 h of co-culture, HepG2 cells were collected and seeded onto a 96-well plate. Six wells were used for each treatment. Cells were maintained with the culture medium collected from the co-culture system. After 24 h, the co-culture medium was removed and replaced with 180 µl fresh culture medium. Then, 20 µl 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; 5 mg/ml; Beckman-Coulter) was added to each well. After an additional 4 h of incubation at 37°C, the medium was removed and 150 µl dimethyl sulfoxide was added to each well to resuspend the MTT metabolic product. The absorbance of the dissolved formazan was measured at 490 nm (A₄₉₀) using a scanning microplate spectrophotometer (Bio-Rad). The proliferation inhibition rate was calculated using the following formula: Proliferation inhibition rate = (A_{490, Control}-A_{490, Sample}) / A_{490, Control} × 100.

Cell apoptosis was measured by flow cytometry and confirmed by terminal deoxynucleotidyl transferase (TdT)-mediated nick end labeling (TUNEL). Using the Transwell system, UC-MSCs were co-cultured with HepG2 cells. A range of initial seeding densities for UC-MSCs was tested (0.25×10⁶, 0.5×10⁶, 1×10⁶, 2×10⁶ or 4×10⁶ cells/well). After co-culture, HepG2 cells were collected and cell density was adjusted to 1×10⁶ cells/ml. After washing with PBS, cells were fixed with 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA) for 30 min at 4°C. Then, cells were washed with 0.2% bovine serum albumin (BSA; GE Healthcare Life Sciences) in PBS and incubated with 70% ethanol at 20°C for an additional 30 min. After washing twice with 0.2% BSA in PBS, cells were treated with the TdT solution (1.5 µl TdT, 27 µl TdT buffer and 1.5 µl FITC-dUTP; GE Healthcare Life Sciences) or a negative control solution (28.5 µl TdT buffer and 1.5 µl FITC-dUTP) for 1 h at 37°C. After washing, samples were incubated with 20 µl of 10 mg/ml RNase (Sigma-Aldrich) and 20 µl of 0.1% Triton X-100 (Invitrogen; Thermo Fisher Scientific, Inc.) for 15 min at 37°C followed by 100 µg/ml propidium iodide (PI; Sigma-Aldrich) staining for 15–30 min. Fluorescence acquisition and analysis were performed using a flow cytometer (Beckman-Coulter, Inc.).

UC-MSCs were seeded and co-cultured with HepG2 cells for 72 h with a range of initial seeding densities (0.5×10⁶, 0.75×10⁶, 1×10⁶, 1.25×10⁶ or 1.5×10⁶ cells/well). Total RNA was extracted from HepG2 cells using an RNA extraction kit according to the manufacturer's instructions. RNA concentration and purity were assayed by gel electrophoresis. Total RNA (1 µg) was reverse transcribed using a reverse transcription kit according to the manufacturer's instructions. PCR amplification was conducted with specific primers designed by Primer 5.0 software (PREMIER Biosoft, Palo Alto, CA, USA) as follows: Forward: 5′-TGC GTT TCT CGT TGC TTACA-3′ and reverse: 5′-GCT GCC ATT TTT CTG GTGAT-3′ for AFP; forward: 5′-GTG GAT GAC TGA GTA CCT GAA CC-3′ and reverse: 5′-AGA CAG CCA GGA GAA ATC AAAC-3′ for BCL2 gene; forward: 5′-GAC CAC CGC ATC TCT ACA TTC-3′ and reverse: 5′-AAG TCT GGC TCG TTC TCA GTG-3′ for baculoviral IAP repeat containing fifth (BIRC5) gene encoding survivin; and RPS13 was used as a housekeeping gene forward: 5′-GTT GCT GTT CGA AAG CAT CTTG-3′ and reverse: 5′-AAT ATC GAG CCA AAC GGT GAA-3′. The PCR reactions were heated to 94°C for 5 min, and subjected to 30 cycles of 94°C for 30 sec, 60°C for 30 sec and 72°C for 45 sec. Each experimental condition was repeated in triplicate. The amplified product lengths were 81, 124 and 116 bp for AFP, Bcl-2 and Survivin, respectively. The relative expression from amplified RNA samples was calculated using the 2^−ΔΔCq method (18) using Applied Biosystems 7500 thermo-cycler 2.3 (Thermo Fisher Scientific, Inc.).

UC-MSCs were seeded and co-cultured with HepG2 cells for 72 h with a range of initial seeding densities (0.25×10⁶, 0.5×10⁶, 1×10⁶, 2×10⁶ or 4×10⁶ cells/well). HepG2 cells were collected and incubated with 0.5% Tween-20, intra-1 and intra-2 for membrane permeability. Cells were treated with 20 µl AFP-FITC, Bcl-2-PE or Survivin-PE antibodies. The primary antibody (20 µl) was added to tube one, washed with PBS after 30 min, then FITC (H+L) IgG was added. Concurrently, 20 µl IgG-FITC was added into tube two as negative control. After washing three times with a 0.5% Tween-20 solution, fluorescence intensities were analyzed with a flow cytometer (Beckman-Coulter).

Data were analyzed using SPSS 16.0 software (SPSS Inc., Chicago, IL, USA). Statistical significance was determined using repeated measures analysis of variance, a Bonferroni correction was then performed as a post-hoc test. Data are presented as the mean ± standard deviation. P<0.05 was considered to indicate a statistically significant difference.

In vitro cultured UC-MSCs exhibited a spindle-like structure and were arranged in parallel or in swirls (Fig. 1A). These morphological characteristics could be maintained until passage 20 (data not shown), which is consistent with the results of previous studies (19,20). Using flow cytometric analysis, the cell surface expression of stem cell biomarkers, CD29, CD44, CD90, CD105, CD34 and CD45 were determined. As shown in (Fig. 1B and C), UC-MSCs expressed CD29, CD44, CD90 and CD105 but lacked CD34 and CD45, confirming the typical phenotype of MSCs derived from the umbilical cord. No significant difference was observed in the expression of these biomarkers between passage three and passage seven UC-MSCs. These data identified the MSCs derived from the umbilical cord and passage seven to eight UC-MSCs were used for the remaining experiments.

In order to investigate the effects of UC-MSCs on cultured HepG2 HCC cells, the two cell types were co-cultured using a Transwell system. Under normal conditions, cultured HepG2 cells were multi-polar and had a cobblestone-like appearance when they reached confluence (Fig. 2A). However, upon co-culture, the number of the HepG2 cells was reduced and cells displayed an irregular morphology. Additionally, HepG2 cell loss was accelerated when the initial seeding number of UC-MSCs was increased (Fig. 2A). The growth inhibition capability of UC-MSCs on HepG2 cells was further confirmed using an MTT assay. It was demonstrated that UC-MSCs time-dependently inhibited the proliferation of HepG2 cells (P<0.01 for different incubation times; Fig. 2B). Enhanced proliferation inhibition was observed by increasing the initial seeding number of UC-MSCs into the Transwell compartment (P<0.01 compared with different seeding densities). These results suggest that UC-MSCs can inhibit HepG2 cell proliferation.

The UC-MSC-induced apoptosis of HepG2 cells was investigated using a TUNEL assay. As shown in Fig. 3, co-culture of UC-MSCs induced the apoptosis of HepG2 cells in a time-dependent manner (P<0.01 between different time points). Moreover, the initial seeding density of UC-MSCs also influenced the percentage of apoptotic HepG2 cells, as elevated apoptosis was detected with an increased number of UC-MSCs (P<0.01 compared with different initial seeding cell number). It is important to note that over 50% of the HepG2 cells underwent apoptosis following a 72 h incubation with UC-MSCs when 4×10⁶ UC-MSCs were seeded into the co-culture system. These data imply that UC-MSCs promote apoptosis in HepG2 cells.

In order to understand the underlying molecular mechanism of UC-MSC-induced proliferation inhibition and apoptosis induction of HepG2 cells, mRNA and protein expression of AFP, Bcl-2 and Survivin in HepG2 cells were examined by RT-qPCR and flow cytometry, respectively. As shown in Fig. 4, AFP, Bcl-2 and Survivin mRNA expression were observed in control HepG2 cells. After 72 h of co-culture with UC-MSCs, the expression of these three genes in HepG2 cells was gradually reduced and the level of reduction was dependent on initial UC-MSC seeding density. At a low density of UC-MSCs (0.5×10⁶ or 0.75×10⁶ cells/well), mRNA levels of AFP, Bcl-2 and Survivin were reduced when compared with the control (P<0.01), whereas a high initial seeding density of UC-MSCs (1×10⁶, 1.25×10⁶ or 1.5×10⁶ cells/well) essentially eliminated detection of AFP, Bcl-2 and Survivin expression.

To confirm the effects of UC-MSCs on AFP, Bcl-2 and Survivin expression in HepG2 cells, protein levels of these genes were analyzed by flow cytometric analysis after the indicated time period of co-culture. As shown in Fig. 5, co-culture of UC-MSCs significantly downregulated the protein levels of AFP, Bcl-2 and Survivin in HepG2 cells (P<0.01 compared with control). Moreover, downregulation of protein levels appeared to be dependent on the initial seeding density of UC-MSCs, as a higher initial seeding density of UC-MSCs more efficiently decreased HepG2 AFP, Bcl-2 and Survivin protein expression (P<0.01). In addition, co-culture with UC-MSCs time-dependently reduced the expression of these proteins in HepG2 cells within 72 h of co-culture (24 h vs. 48 h, 24 h vs. 72 h, 48 h vs. 72 h; P<0.01). Therefore, it is possible that UC-MSCs may inhibit cell proliferation and promote apoptosis of HepG2 cells via regulating the expression of these proteins.