MEF cells were isolated from 13-day-old C57BL/6 mouse embryos. Cells were mitotically inactivated using mitomycin C (Sigma-Aldrich, St. Louis, MO, USA), as described previously (18). The mitotic inactivation of MEF cells was conducted by treatment with 10 μg/ml mitomycin C for 2 h at 37°C. The cells were washed three times with phosphate-buffered saline (PBS), digested with 0.25% trypsin-EDTA solution (cat no. 25300-054; Invitrogen Life Technologies, Carlsbad, CA, USA) and plated at a density of 1×10⁵/ml, with 2.5 ml in each well of a gelatin-coated six-well dish. Human placentas were obtained with written and informed consent from pregnant females who were negative for human immunodeficiency virus (HIV)-I, hepatitis B and hepatitis C. The study received approval for the appropriate use of human amnion by the institutional Ethics Committee of Shanghai Geriatric Institute of Chinese Medicine (Shanghai, China). Amniotic membranes were mechanically separated from the chorions of placentas, which were obtained from females who had undergone an uncomplicated Cesarean section. HuAECs were harvested from the epithelial layers (with the basement membrane attached) of the obtained amniotic membranes, as described in a previous study, with certain modifications (18). In brief, the membrane was placed in a 250-ml flask containing Dulbecco’s modified Eagle’s medium (DMEM) and cut with a razor to produce 0.5–1.0 cm² segments. The segments were subsequently digested with 0.25% trypsin-EDTA at 37°C for 45 min and the resulting cell suspensions were seeded in a six-well plate in DMEM supplemented with 10% fetal calf serum (FCS; PAA Laboratories GmbH, Pasching, Austria), penicillin (100 U/ml) and glutamine (0.3 mg/ml). Following this, the cells were incubated in a humidified tissue culture incubator containing 5% CO₂ at 37°C. The HuAECs were grown to a density of ~100% and were subsequently used as feeder layers for human iPS cell culture following mitomycin C (Sigma-Aldrich) treatment.

The human iPS cells were generated by our laboratory and derived from CD34⁺ human amniotic fluid cells (HuAFCs) via transduction with lentiviral constructs encoding only Oct4, as previously described (37). iPS cultures were separated from the feeder cells by treatment with 0.125% trypsin-EDTA solution and plated onto and co-cultured with HuAECs or MEFs. The cells were cultured in DMEM:F12 (1:1) medium supplemented with 15% KnockOut™ Serum Replacement (Invitrogen Life Technologies), 1 mM sodium pyruvate, 2 mM L-glutamine, 0.1 mM nonessential amino acids, 0.1 mM β-mercaptoethanol, penicillin (25 U/ml)-streptomycin (925 mg/ml), and mixed, without LIF. The cells were incubated in a humidified tissue culture incubator containing 5% CO₂ at 37°C. All cells were cultured on the same feeder until the 10th passage, prior to being used for subsequent experiments.

The AP activity of human iPS cells, which were cultured on HuAECs or MEFs, was determined using an alkaline phosphatase detection kit (Sigma-Aldrich), in accordance with the manufacturer’s instructions (38).

Total-RNA from each cell was isolated using TRIzol reagent^® (Invitrogen Life Technologies), in accordance with the manufacturer’s instructions. The RNA samples were treated with DNase I (Sigma-Aldrich), quantified and reverse-transcribed into complementary DNA (cDNA) with the ReverTra Ace-α First Strand cDNA Synthesis kit [Toyobo (Shanghai) Biotech Co., Ltd., Shanghai, China]. qPCR was conducted with a RealPlex4 real-time PCR detection system from Eppendorf (Hamburg, Germany), with SYBR^® Green Real-Time PCR Master Mix [Toyobo (Shanghai) Biotech Co., Ltd.] as the detection dye. The qPCR amplification was performed over 40 cycles with denaturation at 95°C for 15 sec and annealing at 58°C for 45 sec. The target cDNA was quantified using the relative quantification method. A comparative threshold cycle (Ct) was used to determine gene expression relative to a control (calibrator); steady-state mRNA levels are expressed as an n-fold difference relative to the calibrator. For each sample, the maker gene Ct values were normalized with the formula: ΔCt=Ct_genes - Ct_18S RNA. To evaluate the relative expression levels, the following formula was used: ΔΔCt=ΔCt_all_groups - ΔCt_blank_control_group. The values used to the plot relative expression of the markers were calculated using the expression 2^−ΔΔCt method. The mRNA levels were calibrated on the basis of levels of 18S ribosomal RNA (rRNA). The cDNA of each gene was amplified with primers as previously described (21,37,39,40).

The small interfering RNA (siRNA)-DNMT1 plasmid was manufactured by Shanghai GenePharma, Ltd. (Shanghai, China) and the methods used for plasmid transfection were in accordance with the company’s instructions. In brief, iPS cells were cotransfected with 0.3 μg siRNA-DNMT1 expression plasmid or siRNA-Mock plasmid, respectively, using Lipofectamine 2000 reagent (Invitrogen, Life Technologies Corporation, Grand Island, NY, USA), in accordance with the manufacturer’s instructions. The cells were seeded in a six-well plate and cultured in DMEM:F12 (1:1) medium supplemented with 15% KnockOut™ Serum Replacement, 1 mM sodium pyruvate, 2 mM L-glutamine, 0.1 mM nonessential amino acids, 0.1 mM β-mercaptoethanol, penicillin (25 U/ml)-streptomycin (925 mg/ml) and mixed, without LIF. The cells were incubated in a humidified tissue culture incubator containing 5% CO₂ at 37°C until 80% confluence was achieved.

Each group of cells was seeded at a density of 3×10⁵ cells per well in six-well plates and cultured until 85% confluent. Following this, each group of cells was washed three times with PBS, prior to being subjected to centrifugation (Allegra X-22^®; Beckman Coulter, Miami, FL, USA) at 1,000 × g for 5 min. The cell pellets were then resuspended in 1 ml PBS, fixed in 70% ice-cold ethanol and stored in a freezer for >48 h. Prior to FCM analysis, the fixed cells were centrifuged, washed twice with PBS and resuspended in PI staining solution (Sigma-Aldrich) containing 50 μl/ml PI and 250 μg/ml RNase A (Sigma-Aldrich). The cell suspensions, which were hidden from light, were incubated for 30 min at 4°C and analyzed using a fluorescence-activated cell sorter (FACS; FCM-500; Beckman Coulter). A total of 20,000 events were acquired for analysis using CellQuest software (BD Biosciences, Franklin Lakes, NJ, USA).

The cells were lysed in DNA lysis buffer [0.5% sodium dodecyl sulfate (SDS), 0.1 M EDTA, 10 mM Tris-HCl (pH 8.0) and 100 ng/ml proteinase K; all from Sigma-Aldrich] and incubated at 55°C for 2 h. The treatment of genomic DNA and the MS-PCR assay were performed as previously described (2,5,9,18,37). In addition, the specific primers for Nanog and Oct-4 were designed as previously described (2,5,9,18,37). The PCR products were separated using 12 g/l ethidium bromide containing agarose gel electrophoresis with 1X Tris acetate EDTA (TAE) buffer, and visualized under UV illumination.

ChIP experiments were conducted using anti-acetylated histone H3 antibody (Upstate Biotechnology, Inc., Lake Placid, NY, USA), anti-trimethylated H3K27 antibody (Abcam, Cambridge, UK) and normal rabbit immunoglobulin G (IgG; Upstate Biotechnology, Inc.) as a negative control. All steps were performed as previously described (2,5,9,18,37). The cells were fixed using 1% formaldehyde for 30 min at 37°C and then quenched using 125 mM glycine for 10 min at room temperature to form DNA-protein cross-links. Following this, the samples were placed on ice and sonicated until chromatin fragments became 200–1,000 bp in size. The samples were then incubated with antibodies at 4°C overnight. The PCR amplification was performed under the following conditions: 33 cycles of denaturation at 95°C for 30 sec, annealing at 55°C for 30 sec and extension at 72°C for 30 sec.

The cultured cells were washed three times with FCS and fixed with 4% paraformaldehyde (Sigma-Aldrich) for 30 min. The cells were then washed using Tris-buffered saline containing 0.1% Triton X-100 [TBST-100 buffer; 25 mM Tris-HCl (pH 8.0), 125 mM NaCl and 0.1% Triton X-100] three times, prior to blocking. Following blocking, the cells were incubated with rabbit anti-human Oct3/4 polyclonal antibody (1:200; Chemicon, Temecula, CA, USA) and rabbit anti-human Nanog polyclonal antibody (1:200; Chemicon) overnight at 4°C, and then with Cy3-conjugated goat anti-rabbit IgG antibody (1:200; Abcam) and 5 μg/ml 4’,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich) at room temperature for 30 min. Following this, the cells were thoroughly washed with TBST-100 and viewed under a fluorescence microscope (DMI3000; Leica Camera Inc., Allendale, NJ, USA).

Cells were lysed using a 2X loading lysis buffer [50 mM Tris-HCl (pH 6.8), 2% sodium dodecyl sulfate, 10% β-mercaptoethanol, 10% glycerol and 0.002% bromphenol blue]. The total quantity of proteins from the cultured cells was subjected to 12% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred onto Hybrid-polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA). Following blocking with 5% (w/v) non-fat dried milk in Tris-buffered saline containing Tween-20 [TBST-20; 25 mM Tris-HCl (pH 8.0), 125 mM NaCl and 0.05% Tween-20], the PVDF membranes were washed four times (15 min each) with TBST-20 at room temperature and incubated with primary antibody. Following extensive washing, the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG secondary antibody (1:1,000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) for 1 h. The membranes were then washed four times (15 min each) with TBST-20 at room temperature, prior to the immunoreactivity being visualized by enhanced chemiluminescence (ECL) using an ECL Chemiluminescent Substrate Reagent kit from Perkin-Elmer Life Science (Norwalk, CT, USA).

All animal procedures were conducted at Shanghai University of Traditional Chinese Medicine (Shanghai, China) with approval from the Institutional Animal Care and Use Committee and in accordance with the institutional guidelines. Human iPS cells (1×10⁶) were inoculated into the hind legs of severe combined immunodeficient (SCID) mice. Teratomas were embedded in paraffin and histologically examined following hematoxylin and eosin staining. The procedure for the teratoma formation experiment was performed as described previously (18).

Each experiment was performed as least three times and the data are presented as the mean ± standard error (SE). The differences were evaluated using Student’s t-tests. P<0.05 was considered to indicate a statistically significant difference.

We have previously described the successful generation of iPS cells from CD34⁺ HuAFCs by transduction with lentiviral constructs encoding only Oct4 (37). In this study, the iPS cells were cultured on HuAEC feeder layers until the 10th passage, prior to use in experiments. After testing the effects of different feeder layers, the pluripotency of iPS cells was assayed. Under the microscope, a number of ESC-like colonies were observed among the feeder cells; these Oct4-green fluorescent protein (GFP)-positive colonies appeared isolated and rounded, consistent with more undifferentiated cells (Fig. 1A). Furthermore, the AP activity of these human iPS cells was high, as represented by the deep blue staining on the surface of the colonies (Fig. 1B). In addition, the IF staining revealed that the expression levels of the pluripotent stem cell markers, Nanog, Oct4 and Sox2, were increased in the iPS colonies (Fig. 1D). Consistent with the IF results, qPCR analysis showed that the expression levels of these stem cell markers were ~60–100-fold higher in human iPS cells than in HuAFCs, which served as an internal control (Fig. 1C). Moreover, the high level of telomerase activity in human iPS cells suggested that their replicative life-span was likely to exceed that of somatic cells. Meanwhile, in the in vivo xenograft experiments, teratomas formed on the legs of SCID mice injected with the iPS cells (Fig. 1E). The iPS-derived teratomas contained cellular representatives of all three germ layers (Fig. 1E). Based on these results, it was concluded that the human iPS cells derived from HuAFCs possessed strong pluripotency.

iPS cells were successively subcultured either on HuAECs or MEFs in order to evaluate the effect of the different feeder layers on pluripotency. The two groups of cells were cultured in uniform conditions and were subcultured in succession until passage 50. Since AP levels decrease as stem cells lose their pluripotency and differentiate, the AP activity of human iPS cells cultured on different feeder layers was analyzed at every 10th passage. The results showed that the AP activity of the iPS cells cultured on MEFs reduced rapidly and there was a significant negative correlation between the AP activity level and the number of cell passages (R²=0.934, P<0.05). However, when the iPS cells were cultured on HuAECs, their AP activity level was not significantly altered (R²=0.793, P>0.05; Fig. 2B). In addition, at the 40th and 50th passage, the AP activity levels of human iPS cells cultured on MEFs (0.417±0.041 and 0.280±0.045, respectively) were significantly lower than those on HuAECs (0.837±0.021 and 0.760±0.022, respectively). To evaluate whether the pluripotency of iPS cells changed, stem cells markers were assayed using qPCR. The results of the qPCR analysis showed that the Nanog and Oct4 expression levels in iPS cells cultured on MEFs reduced rapidly, while those in iPS cells cultured on HuAECs did not markedly change over time (Fig. 2A).

In the early passages (comparing passages 10, 30 and 50), the iPS cells grown on HuAECs and MEFs exhibited similar cell cycle distributions (Fig. 3). Furthermore, the cell cycles of the iPS cells cultured on HuAECs were not markedly different between the 10th, 30th and 50th passages, indicating that long-term culture on HuAECs feeder layers did not affect the process of cell division in the iPS cells. Similarly, when the iPS cells were cultured on MEFs, the FCM analysis showed no significant differences in the cell cycle distribution at each passage. The iPS cells cultured on MEFs were always in the resting stage and early stage of DNA synthesis (G0/G1 stage; Fig. 3). No significant differences were observed between the cell cycles of the iPS cells cultured on HuAECs or on MEFs at the 10th, 30th or 50th passages.

The DNA methylation status of the CpG islands in the Nanog and Oct4 promoters was investigated using sodium bisulfite treatment of the iPS cells cultured on HuAECs or MEFs at every 10th passage. All the CpG islands in the Nanog and Oct4 promoter regions were demethylated with no significant differences from the 10th to the 50th passage in human iPS cells cultured on HuAECs (Fig. 4A). However, in iPS cells cultured on MEFs, these regions of the Nanog and Oct4 promoters were moderately demethylated at passages 10, 20 and 30 (Fig. 4A), prior to becoming hypermethylated at passages 40 and 50. These differences suggested that HuAECs positively regulated the Nanog and Oct4 genes in human iPS cells by maintaining the hypomethylation of promoter CpG islands. In addition, ChIP assays were performed to evaluate the histone H3 acetylation levels of the Nanog and Oct4 promoters in human iPS cells cultured on HuAECs or MEFs. In iPS cells cultured on HuAECs, the acetylation and K27 trimethylation of histone H3 in the Nanog and Oct4 promoters and the 5’untranslated regions (5’UTRs) were similar to those in human iPS cells cultured on MEFs within 20 passages (Fig. 4B). However, from passage 30, histone H3 appeared relatively hyperacetylated and histone K27 appeared hypomethylated in the Nanog- and Oct4-specific sites in human iPS cells cultured on HuAECs, compared with the same regions in cells cultured on MEFs (Fig. 4B). These results suggested that HuAECs were capable of maintaining the Nanog and Oct4 loci in an active transcriptional state through covalent histone modifications over long-term culture. Western blotting was also used to assess the relative protein expression of DNMT1 in iPS cells cultured on different feeder layers. Over multiple passages, DNMT1 protein was more highly expressed in iPS cells cultured on MEFs than in iPS cells cultured on HuAECs (Fig. 4C). At passages 30 and 50, the DNMT1 protein levels in iPS cells cultured on MEFs were 0.676±0.040 and 1.220±0.021 relative to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression levels, respectively. These values were significantly higher than those in iPS cells cultured on HuAEC feeders (0.003±0.001 and 0.021±0.010 relative to GAPDH levels, respectively).

In order to evaluate whether endogenous DNMT1 expression promoted DNA methylation of Nanog and Oct4 and the differentiation of cultured iPS cells, siRNA-DNMT1 and siRNA-Mock were transfected into iPS cells. The efficiency of the transfected siRNA on mRNA and protein expression was assessed using qPCR and western blotting, respectively, and these experiments were performed on all cell groups subcultured up to passage 50. As shown in Fig. 5B, western blotting indicated that the DNMT1 protein expression in iPS cells transfected with siRNA-Mock (1.002±0.089) was higher than that in iPS cells transfected with siRNA-DNMT1 (0.037±0.020, P<0.01, n=3). These results demonstrated that siRNA-DNMT1 specifically interfered with DNMT1 expression in iPS cells. Having confirmed that endogenous DNMT1 expression was suppressed by siRNA, the pluripotent stem cell biomarkers Oct4 and Nanog in iPS cells cultured on MEFs were tested using western blotting. The results revealed that the Oct4 and Nanog protein levels in the siRNA-DNMT1-transfected iPS cells cultured on MEFs were 3.277±0.475 and 3.108±0.719 of GAPDH expression levels, respectively (Fig. 5B). These values were significantly higher than those in the siRNA-Mock-transfected iPS cells (1.002±0.128 and 1.005±0.228 of GAPDH levels, respectively). In addition, the promoter regions of Nanog and Oct4 were observed to be moderately hypomethylated or demethylated in siRNA-DNMT1-transfected iPS cells, while they were moderately hypermethylated in siRNA-Mock-transfected iPS cells (Fig. 5C). These differences suggested that DNMT1 positively regulated the Nanog and Oct4 genes in human iPS cells through the de novo or maintained hypermethylation of the CpG islands, and that high DNMT1 expression in the iPS cells was able to promote their differentiation.