Culture and differentiation of hESCs
hESCs were grown on mitotically inactivated mouse embryonic fibroblasts and passaged essentially as previously described (Stojkovic et al., 2004
). EB differentiation was induced by harvesting hESCs with collagenase and culturing them in suspension in knockout DME (Invitrogen) containing 20% FCS (Hyclone), 1 mM l
-glutamine (Invitrogen), 100 mM of nonessential amino acids (Invitrogen), 100 μM β-mercaptoethanol (Sigma-Aldrich), and 1% penicillin-streptomycin (Sigma-Aldrich). One to two passages before experiments, hESCs were transferred to Matrigel (BD)-coated plates with feeder-conditioned media as previously described (Stojkovic et al., 2004
; Hyslop et al., 2005
Karyotype analysis of hESCs
The karyotype of hESCs was determined by standard G-banding procedure.
Stable transfection of hESCs with the full-length cDNA of human NANOG, CDK6, and CDC25A
The full-length cDNA of human NANOG, CDK6, and CDC25A was isolated from cDNA of hESCs using the following oligonucleotides: NANOG forward, 5′-CATGAGTGTGGATCCAGCTTGT-3′; NANOG reverse, 5′-ATCTTCACACGTCTTCAGGTTG-3′; CDK6 forward, 5′-ACTGAATTCACCATGGAGAAGGACGGCCTGTG-3′; CDK6 reverse, 5′-ACTGAATTCTCAGGCTGTATTCAGCTCCGAG-3′; CDC25A forward, 5′-ACTGAATTCACCATGGAACTGGGCCCGGAG-3′; and CDC25A reverse, 5′-ACTGAATTCTCAGAGCTTCTTCAGACGACTG-3′. The cDNAs were cloned into the pTP6 vector. hESCs (H1, H9, and hES-NCL1) were plated on Matrigel-coated plates and cultured in the presence of feeder-conditioned media 4 d before transfection. The transfection of DNA was achieved using the Cell Line Nucleofector kit L (Amaxa) according to the manufacturer's instructions (program A-023). 2 d after the transfection, stable clones were selected using puromycin selection (0.8–1.2 μg/ml) for 10 d. Between 15 and 20 surviving colonies were pooled in each case, and the resulting subline from each cell line was expanded and named, for example, H1 NANOG, H9 NANOG, and hES-NCL1 NANOG. A similar procedure was performed after transfection of the empty vector. Each of the control sublines was named H1 control, H9 control, and hES-NCL1 control. All sublines were maintained with 0.6 μg/ml puromycin to ensure the maintenance of the transgene. Every 8–10 passages, quantitative RT-PCR and Western blot analysis were performed to confirm gene overexpression over time. For simplicity, in most figures, data from one or two overexpressing sublines or controls are shown.
Transient transfection of hESCs with CDK6 and CDC25A luciferase reporter constructs
hESCs were cultured under feeder-free conditions with feeder-conditioned media free of antibiotics for at least 4 d before transfections. hESCs were nucleofected simultaneously with firefly luciferase reporter constructs (6 μg in the case of CDK6 and CDC25A), a transfection control (0.6 μg in the case of the construct containing Renilla luciferase gene driven by the herpes simplex virus thymidine kinase promoter), and 6 μg NANOG cDNA (gift from J.-H. Kim, ChaBiotech Co. Ltd., Seoul, Republic of Korea) using the Cell Line Nucleofector kit L according to the manufacturer's instructions (program A-023). Site-directed mutagenesis for CDK6 and CDC25A luciferase constructs was performed using the QuikChange Site-Directed Mutagenesis kit (Agilent Technologies) according to the manufacturer's instructions. A similar procedure was followed for the transfection of site-directed mutagenesis constructs. After 24 h, cells were lysed using the lysis buffer provided in the Dual Luciferase Detection kit (Promega) according to the manufacturer's instructions. The firefly and Renilla luciferase activities were measured in turn using the LARII and Stop Glow solutions (Promega), and the ratio between the two was calculated.
siRNAs and transfection
siRNAs were obtained from Santa Cruz Biotechnology, Inc. and Invitrogen. The siRNA sequences are shown in Table S1. Transfection with scrambled control siRNAs with similar guanine-cytosine content to gene-specific siRNA sequences provided by the same company were used as a negative control. The transfection of siRNA into hESCs was performed using the high efficiency Cell Line Nucleofector kit L and 80 pmol siRNA (in 2 ml of medium) as outlined in the manufacturer's instructions (program A-023). 24 h after transfection, hESCs were synchronized in G2 to M by incubation in 200 ng/ml of a nocodazole-containing medium for 18 h. The cells were washed three times with normal medium and collected by Accutase (Millipore) treatment at various time points as indicated in the results section.
Flow cytometry analysis of hESCs
For the flow cytometry analysis, the hESCs were collected, processed, and analyzed as previously described (Armstrong et al., 2006
Lysates were electrophoresed on a 10% SDS-PAGE gel and electrophoretically transferred to a polyvinylidene difluoride membrane (Hybond-P; GE Healthcare). Membranes were blocked in Tris-buffered saline with 5% milk and 0.1% Tween. The blots were probed with NANOG (1:1,000; R&D Systems), CDK4 (1:100; Santa Cruz Biotechnology, Inc.), CDK6 (1:100; Santa Cruz Biotechnology, Inc.), cyclin D1, D2, and D3 (1:100; all from Santa Cruz Biotechnology, Inc.), cyclin E, CDK2, CDC25A, c-ABL, retinoblastoma (phosphorylated or not), and c-Myc (1:100; all from Cell Signaling Technology), p15, p16, p18, p19, and Suv39H1 (1:100; all from Cell Signaling Technology), or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody (1:2,000; Abcam) overnight and revealed with horseradish peroxidase–conjugated secondary antibodies, anti–goat (1:2,000; Dako), or anti–rabbit (1:20,000; GE Healthcare). Antibody–antigen complexes were detected using ECL reagent (GE Healthcare). Western blot images were acquired using a luminescent image analyzer (Fujifilm) and LAS-3000 software (Fujifilm). Protein molecular weights are indicated next to the image shown from the Western blotting.
Cell signaling assays
Panorama antibody microarray for cell signaling containing 224 different antibodies spotted in duplicate on nitrocellulose-coated glass was purchased from Sigma-Aldrich. 1 mg of NANOG-overexpressing or control subline cell extracts was collected, labeled with Cy3 and Cy5, respectively, and hybridized to the slides according to the manufacturer's instructions. Cy3 and Cy5 signals were read on the Gene Pix Pro 4.0 (MDS Analytical Technologies). The results from the NANOG sublines were analyzed together using the GeneSpring software (Agilent Technologies). Sample processing was performed using three normalization steps, which involved dye swap where necessary, the division of each spot by the control channel, and the normalization of each spot to the 50th percentile of the entire chip. Filter-on-confidence criteria was used to select the most significantly changed candidates (P < 0.05). A ratio of >1.0 indicates higher expression in both NANOG sublines compared with both respective control hESC sublines, and a ratio <1.0 indicates higher expression in control hESC control sublines compared with NANOG sublines.
LightCycler real-time PCR analysis
Quantitative RT-PCR analysis was performed using QuantiTect SYBR Green PCR Master Mix (QIAGEN) essentially as previously described (Boyer et al., 2005
; Becker et al., 2006
). The LightCycler experimental run protocol used was: PCR activation step (95°C for 15 min), amplification with data acquisition repeated 50 times (94°C for 15 s, annealing temperature for primers for 30 s, and 72°C for 20 s with a single fluorescence data collection), melting curve (60–95°C with a temperature transition rate of 0.1°C/s and continuous fluorescence data collection), and finally cooling to 40°C. The crossing point for each transcript was determined using the second derivative maximum method in the LightCycler software version 3.5.3 (Roche). The GAPDH
crossing point for each sample was used as the internal control of these real-time analyses. The data were analyzed using the comparative threshold cycle method as described in the user bulletin for the LightCycler relative quantification software version 1.01 (Roche). For each gene, the control was set to one, and all other values were calculated with respect to this. PCR reactions were performed using the primers (final concentration of 0.5 μM) described in Table S2 (available at http://www.jcb.org/cgi/content/full/jcb.200801009/DC1
Cells undergoing apoptosis can be enumerated using the annexin V–FITC apoptosis detection kit (BD). The protocol was performed in accordance with the manufacturer's instructions and, in brief, comprises the following steps. Cells were harvested using Accutase, washed twice with ice-cold phosphate-buffered saline, and counted. 105 cells were suspended in 100 μl of 1× binding buffer (supplied), and 5 μl annexin V–FITC and 5 μl propidium iodide solution were added. The mixture was vortexed gently and incubated for 15 min at room temperature in the dark. 400 μl of 1× binding buffer was added, and the cells were analyzed by flow cytometry (FACSCalibur; BD).
Measurement of cell proliferation using BrdU incorporation method
hESC proliferation was measured by incorporation of BrdU (Roche) into the genomic DNA during the S phase (DNA replication) of the cell cycle. hESCs were grown in a 4-well plate to day 2 and incubated in medium containing BrdU for 45 min at 37°C in a humidified atmosphere (5% CO2). Cells were fixed with ethanol and 50 mM glycine, pH 2.0, for 45 min at room temperature and denaturated in 4 M HCl for 10–20 min. Subsequent detection of BrdU was accomplished with antibodies for BrdU (1:5) according to the manufacturer's instructions and visualization at 488 nm using immunofluorescence microscopy. For flow cytometry assay, hESCs were incubated and processed with a BrdU Flow kit (BD) according to the manufacturer's protocol. Cells were stained with FITC or allophycocyanin anti-BrdU and 7-amino-actinomycin. Cells from the same population that were not BrdU labeled were used as a negative control. Flow cytometry analysis was performed using a FACSCalibur and CellQuest software (BD).
Cell cycle analysis
Cell cycle analysis was performed using the CycleTest Plus DNA reagent kit (BD). hESCs were harvested by Accutase treatment and counted with a hemocytometer. 500,000 cells were fixed, permeabilized, and stained in accordance with the manufacturers' instructions, and the sample was analyzed by flow cytometry using a FACSCalibur measuring FL2 area versus total counts. The data were analyzed using ModFit (Tree Star, Inc.) and FlowJo (Tree Star, Inc.) softwares to generate the percentages of cells in G1, S, and G2 to M phases of the cell cycle.
The AP staining was performed using the Alkaline Phosphatase Detection kit (Millipore) according to the manufacturer's instructions. Cells were fixed in 90% methanol and 10% formamide for 2 min and washed with rinse buffer (20 mM Tris-HCl, pH 7.4, and 0.05% Tween 20) once. Staining solution (Naphthol/Fast Red Violet) was added to the wells, and plates were incubated in the dark for 15 min. The bright field images were obtained using a microscope (Axiovert; Carl Zeiss, Inc.) and AxioVision software (Carl Zeiss, Inc.).
ChIP assays were performed mainly as previously described (Atkinson et al., 2005
). In brief, cells were harvested at 70–80% confluence, and ChIP was performed according to the manufacturer's instructions (Millipore). Sonication was optimized to produce chromatin fragments of 500–1,000 bp in length, and DNA from each immunoprecipitation was purified using the Qiaquick DNA Purification kit (QIAGEN). Also included in the experiment was a no antibody control immunoprecipitate to detect any background, and, if it was present, it was subtracted from each immunoprecipitate within that experiment. Pilot experiments performed with no antibody controls and irrelevant antibodies such as IgG revealed no significant differences; thus, no antibody controls were used in all ChIP experiments. Standard errors were generated for quantitative PCR reactions by reading each sample in triplicate. The sequences of the primers used for this purpose are given in Table S3.
hESCs were washed with ice-cold PBS and lysed on ice for 30 min in radio immunoprecipitation assay buffer. Lysates were centrifuged at 14,000 g for 5 min. The supernatant from cell lysates was collected, and the protein concentration was measured using Bradford Reagent (Bio-Rad Laboratories). Protein G–agarose (PGA) beads were washed three times with PBS and incubated for 2 h in a rotor at 4°C in radio immunoprecipitation assay buffer with PMSF and protease inhibitor cocktail (Roche). 400 μg of protein recovered from cell supernatants was precleared with 20 μl PGA slurry for at least 2 h on a rotor at 4°C. PGA beads were removed by centrifugation at 14,000 g for 5 min at 4°C. Immunoprecipitation was performed by overnight incubation/rotation with 2 μg of mouse monoclonal anti-CDK2 antibody (D-12; Santa Cruz Biotechnology, Inc.), rabbit polyclonal anti-CDK4 (C-22; Santa Cruz Biotechnology, Inc.), or rabbit polyclonal anti-CDK6 antibody (C-21; Santa Cruz Biotechnology, Inc.). A no antibody control was also included for each sample. After incubation, 20 μl of PGA beads was added to immunoprecipitated samples and returned to 4°C for 2 h with rotation. PGA beads with bound protein complexes were recovered by centrifugation at 14,000 g for 5 min, and beads were washed once with PBS and 0.2% Triton X-100 and twice with PBS. The sample was divided into two aliquots: one to be used for kinase assays and the second one for Western blotting. For the latter procedure, 40–60 μl of SDS sample buffer was added to the sample before boiling for 5 min. The samples were separated using denaturing acrylamide gels, and Western blotting was performed as indicated above.
Kinase activity assays
Kinase activity assays were performed using the PKLight Assay kit (LT07-500; Cambrex Bio Science Rockland, Inc.) according to the manufacturer's instructions. The PKLight Assay exploits the kinases' intrinsic ATPase activity, resulting in the cleavage of the γ-phosphate moiety of ATP and its subsequent insertion into the target substrate. This results in the phosphorylation of the substrate and the conversion of ATP to ADP. The PKLight Assay measures the consumption of ATP and is based on the bioluminescent measurement of the remaining ATP present in the wells after the kinase reaction. The bioluminescent signal of PKLight Assay is inversely proportional to kinase activity. Phosphorylation of Retinoblastoma or H1 was measured by incubating for 10 min at room temperature 20 μl of immunoprecipitation product for the kinase of interest (see previous section) with 1 mM ATP, kinase buffer (50 mM Tris, pH 7.5, and 5 mM MgCl2), and 5 mg/ml Retinoblastoma or H1 as substrate. 10 μl of kinase stop solution was added to each sample at room temperature for 10 min. Finally, 20 μl of ATP detection reagent was added to each sample at room temperature for 10 min, and the readings were taken using a luminometer. The difference in luminometer reading between the no antibody control and immunoprecipitation product containing the antibody was calculated. This figure, which is indicative of remaining ATP in the solution, was inversely correlated to the kinase activity. The kinase activity for the control sublines was set at 100%, and the respective values for the NANOG sublines were calculated with respect to that.
These were performed using the SensoLyte fluorescein diphosphate (FDP) protein phosphatase assay kit (AnaSpec) according to the manufacturer's instructions. This kit provides a fluorogenic assay for measuring the activity of protein phosphatases such as tyrosine phosphatases and serine/threonine phosphatases that convert the FDP into fluorescein, which has a high extinction coefficient and emission quantum yield, therefore providing high assay sensitivity. Immunoprecipitations were performed using CDC25A antibody (F6; Santa Cruz Biotechnology, Inc.). The immunoprecipitation product was resuspended in 50 μl of phosphatase buffer (20 mM Tris-HCL, pH 8.3, 150 mM NaCL, 2 mM EDTA, 0.01% Triton X-100, 5 mM DTT, and 1 mg/ml BSA). 50 μl of a protein phosphatase–containing sample was mixed with 50 μl of FDP reaction solution. The reaction was incubated at 30°C for 30 min, and 50 μl of stop solution was added to stop the reaction. Fluorescence signal was measured using excitation/emission = 485 nm/538 nm. As a negative control, samples without phosphatase activity (distilled water) were used. The difference in fluorescence readings between the immunoprecipitation product and no antibody control was calculated to deduct background phosphatase activity. The phosphatase activity for the control sublines was set at 100%, and the respective values for the experiment sublines were calculated with respect to that.
Two-tailed pairwise Student's t test was used to analyze results obtained from two samples with one time point. Analysis of variance (single factor or two factors with replication) was used to compare multiple samples (at one time or several time points). The results were considered significant if P < 0.05.
Tumor formation in SCID mice
All procedures involving mice were performed in accordance with institutional guidelines and permission. Approximately 106 hESCs were injected into the testis of adult male SCID mice. After 70–90 d, mice were killed, and tissues were dissected, fixed in Bouins overnight, processed, and sectioned according to standard procedures and stained with either hematoxylin and eosin or Weiger's stain. Material for immunohistochemical analysis was fixed in 4% PFA (Sigma-Aldrich) in PBS (Cambrex Bio Science Rockland, Inc.) overnight, processed, and sectioned to 6 μm according to standard procedures. Sections were cleared using Histoclear (RA Lamb) and rehydrated, and endogenous hydrogen peroxide activity was blocked. Antigen retrieval was performed by microwaving (800 W) tissues in 10 mM of citrate buffer, pH 6 (citric acid [Sigma-Aldrich] and distilled H2O). Endogenous avidin/biotin activity was blocked using a blocking kit (Vector Laboratories). Sections were permeabilized (1% Triton X-100 [Thermo Fisher Scientific] and PBS solution) and blocked (5% normal goat serum [Invitrogen], 0.1% Triton X-100, and PBS), and sections were incubated with the following primary antibodies: AFP (1:100; Sigma-Aldrich), nestin (1:200; Millipore), and SMA (1:200; Sigma-Aldrich). Negative controls were performed with the omission of the primary antibody. A universal ABC detection kit (Vector Laboratories) with a purple-colored Vector VIP substrate (Vector Laboratories) was used to detect the primary antibodies. Sections were briefly counterstained using Mayer's hemalum and briefly blued using 4% alkaline alcohol (4% ammonia [Thermo Fisher Scientific] in 70% alcohol). Sections were dehydrated through a series of alcohols, cleared using Histoclear, and mounted using distyrene/plasticizer/xylene (RA Lamb).
Teratoma sections were visualized using a microscope (Diaphot 300; Nikon) with the following objectives: 4× NA 0.13, 10× NA 0.25, 20× NA 0.40, and 40× NA 1.3. Digital images were recorded using a digital camera (DXM1200; Nikon).
Online supplemental material
Fig. S1 shows karyotype analysis of H1 NANOG and hES-NCL1 NANOG clones after 20 passages in culture. Fig. S2 shows cell proliferation assessed by cell counting over three time points. Fig. S3 shows the maintenance of pluripotency and differentiation capability of NANOG-overexpressing hESC clones. Fig. S4 shows that the C-terminal domain of NANOG is responsible for transactivation of CDK6
. Table S1 shows the sequences of siRNAs used for the down-regulation of CDK6
, and NANOG. Table S2 shows the sequences of oligonucleotides used for the quantitative RT-PCR analysis. Table S3 shows the sequences of oligonucleotides used for the quantitative PCR after ChIP experiments. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200801009/DC1