Placentas at term (38–40 weeks gestation; n=10) were obtained from healthy donor mothers following the provision of informed consent and according to the procedures of the institutional review board. The present study was approved by the Ethics Committee of The First Affiliated Hospital of Jinzhou Medical University (Jinzhou, China). Briefly, the cells were isolated using a tissue culture method (15) and cultured in 5% CO₂ at 37°C, following which the placenta-derived cells at the fourth passage were treated with 0.25% trypsin-EDTA, harvested, and washed twice with phosphate-buffered solution (PBS). The cells were incubated on ice for 20 min with pre-diluted PE-labeled mouse anti-human antibodies CD13 (cat. no. 560998), CD73 (cat. no. 561014), CD90 (cat. no. 561970), CD166 (cat. no. 559263), HLA-DR (cat. no. 555561) and FITC-labeled mouse anti-human antibodies CD14 (cat. no. 555397) (all from BD Biosciences, San Jose, CA, USA), CD29 (cat. no. 11-0299-41; eBiosiences, San Diego, CA, USA), CD31 (cat. no. 560984), CD44 (cat. no. 560977), CD45 (cat. no. 561865), CD105 (cat. no. 561443) and HLA-ABC (cat. no. 557348). Control groups were incubated with FITC-(cat. no. 555786) and PE-conjugated mouse anti-human IgG (cat. no. 555787) (all from BD Biosciences). The labeled cells were analyzed using flow cytometry (BD FACSCanto II; Becton-Dickinson Co., Franklin Lakes, NJ, USA).

Briefly, the proliferation of hPDMSCs was measured using an MTS assay following treatment with SM and its most commonly examined components, including danshensu (DSS), salvianolic acid B (SA B), protocatechuic aldehyde (PCAD), tanshinone I (TS I), tanshinone IIA (TS IIA) and cryptotanshinone (CTS), at different concentrations for 4 days. As concentrations of SM <4 mg/l, DSS <10 mg/l, SA B <10 mg/l, PCAD <0.5 mg/l, TS I <1 mg/l, TS IIA <0.1 mg/l and CTS <0.1 mg/l had minimal effect on cell proliferation (Fig. 2A), 4 mg/l SM, 10 mg/l DSS, 10 mg/l SA B, 0.5 mg/l PCAD, 1 mg/l TS I, 0.1 mg/l TS IIA and 0.1 mg/l CTS concentrations were preferred to enable superior assessment of the effective components of SM in promoting cardiomyogenic differentiation in experiments. The hPDMSCs were inoculated in 96-well plates, 24-well plates and 10-cm culture dishes at a density of 1×10³ cells/cm² and cultured in 5% CO₂ at 37°C. The following day, the cells were respectively treated with 4 mg/l SM, 10 mg/l DSS, 10 mg/l SA B, 0.5 mg/l PCAD, 1 mg/l TS I, 0.1 mg/l TS IIA and 0.1 mg/l T CTS in complete medium containing Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Gibco, Gaithersburg, MD, USA), 1 mmol/l L-glutamine, 0.1 mmol/l, β-mercaptoethanol and 1% non-essential amino acids for the induction of cardiomyogenic differentiation. The aforementioned medium was replaced every 4 days. The morphologic characteristics of the cells were analyzed under a microscope (Leica DMI1; Leica Microsystems Inc; Mannheim, Germany) every day. As the absorbance values in an MTS assay can indirectly reflect the number of living cells, with the absorbance values being positively associated with the number of living cells, cell proliferation was measured using the MTS assay every 4 days. The experiment was terminated at day 20.

The cells were fixed in PBS containing 4% paraformaldehyde (PFA) for 20 min, permeabilized in PBS containing 0.2% Triton X-100 for 10 min, and blocked in a serum-free blocking solution for 5 min at room temperature. The cells were then incubated with primary antibody against α-SCA (1:100 dilution; cat. no. ab9465; Abcam, Cambridge, UK) overnight at 4°C. Following extensive washing with PBS, the cells were incubated with HRP-conjugated goat anti-rabbit IgG (1:100 dilution; cat. no. ab6721; Abcam). Finally, the nuclei were stained by incubation with hematoxylin. The results were analyzed under a microscope (Leica DMI1; Leica Microsystems Inc.).

The cells were washed three times with PBS and fixed in PBS containing 4% PFA for 30 min. The fixed cells were washed three times with PBS and permeabilized in PBS containing 0.2% Triton X-100 for 10 min. The cells were then washed three times with PBS and blocked in a serum-free blocking solution for 5 min at room temperature. The blocked cells were incubated with cardiac troponin-I (cTnI) primary antibody (1:50 dilution; cat. no. ab47003; Abcam) overnight at 4°C. Following extensive washing with PBS, the cells were incubated with FITC-conjugated goat anti-rabbit IgG (1:50 dilution; cat. no. ab6717; Abcam) for 1 h at room temperature. Following extensive washing with PBS, the nuclei were stained by incubation with propidium iodide (PI). The results were analyzed using a fluorescence microscope. The differentiation ratio of cardiomyocytes was calculated as the fraction of cTnI-positive cells in total cells, which were in a counterpart visual field. The rate was calculated as the average of >10 separate fields.

The cells were washed twice with PBS and then collected. Total proteins were extracted with cell lysis buffer (cat. no. P0013b; Beyotime) according to the manufacturer's protocol. The protein quantity was determined by BCA assay (BioTek, Winooski, VT, USA). Equal quantities of protein (30 µg) were respectively separated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 8 or 10% gel and then transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 5% non-fat milk for 1 h and then incubated overnight at 4°C with the following primary antibodies: GAPDH (1:5,000 dilution; cat. no. ab9485), GATA-binding protein 4 (GATA4; 1:1,000 dilution; cat. no. ab84593), atrial natriuretic factor (ANF; 1:1,000 dilution; cat. no. ab14348), cTnI (1:1,000 dilution; cat. no. ab47003), glycogen synthase kinase-3β (GSK-3β; 1:10,000 dilution; cat. no. ab32391), phosphorylated (p-)GSK-3β (1:10,000 dilution; cat. no. ab75814) or β-catenin (1:10,000 dilution; cat. no. ab10000), all from Abcam. This was followed by incubation with HRP-conjugated goat anti-rabbit IgG (1:5,000 dilution; cat. no. ab6721; Abcam) for 2 h at room temperature. Antibody detection was performed using a chemiluminescence detection kit (Omega Lum G; Aplegen Inc., Pleasanton CA, USA).

Each experiment was repeated at least three times. All data are presented as the mean ± standard deviation. A one-way analysis of variance was used for comparisons among the groups. Statistical analysis was performed using SPSS 13.0 software. P<0.05 was considered to indicate a statistically significant difference.

The placenta-derived cells grew to form colonies and exhibited an adherent fibroblastoid appearance. The results of the flow cytometric analysis demonstrated that the placenta-derived cells were positive for CD13, CD29, CD44, CD73, CD90, CD105, CD166 and HLA-ABC, and negative for CD14, CD31, CD45 and HLA-DR (Fig. 1). The surface markers of these placenta-derived cells were exactly the same as those of previously reported bone marrow- and cord blood-derived mesodermal cells (16).

To better assess whether SM and its active components inhibited hPDMSC proliferation and stimulated cell differentiation, cell proliferation was evaluated using an MTS assay and cell morphological characteristics were analyzed using microscopy. The cells in the negative control group maintained an increased growth rate and maintained fibroblast-like morphology. However, the cells treated with SM and its active components exhibited a slower growth rate, with certain cells beginning to exhibit altered morphology. These cells gradually increased in size to form a stick-like appearance. On day 20, the cells became enlarged and exhibited a number of branches connecting with adjoining cells (Fig. 2A). Among the SM components, TS IIA had the most marked effect on the cells (Figs. 2B and 3).

In order to corroborate the cardiomyogenic differentiation of hPDMSCs, immunohistochemistry was performed to detect α-SCA, and western blot analysis was performed to detect the protein expression levels of GATA4, ANF and cTnI. The immunohistochemistry revealed that some of the treated cells stained positive for α-SCA, and there were more α-SCA-positive cells in the TS IIA treatment group (Fig. 4A). Compared with the negative control group, the protein expression levels of GATA4, ANF and cTnI were increased in the treatment groups, however, the protein expression levels of GATA4, ANF and cTnI were marginally higher in the cells treated with TS IIA (Fig. 4B and C).

In order to determine whether Wnt/β-catenin signaling was important in SM and its active components in promoting the cardiomyo-genic differentiation of hPDMSCs, the protein expression levels of GSK-3β, p-GSK-3β and β-catenin were examined. The results showed that the expression levels of GSK-3β and p-GSK-3β were increased to different degrees, whereas the expression of β-catenin was decreased to different degrees in the treatment groups. In addition, the ratios of p-GSK-3β to GSK-3β were increased to different degrees, particularly in the TS IIA treatment group (Fig. 4B and C).

For further analysis of the effective components of SM in promoting cardiomyogenic differentiation, immunofluorescence was performed to detect cardiomyogenic differentiation rate. cTnI was not expressed in the control group. The expression levels of cTnI in the treatment groups were different, with the cardiomyogenic induction rate of the TS IIA being marginally higher (Fig. 5).