p53 is an important regulator of normal cell response to stress and frequently mutated in human tumours. Here, we studied the effects of activation of p53 and its target gene p21 in human embryonic stem cells. We show that activation of p53 with small-molecule activator nutlin leads to rapid differentiation of stem cells evidenced by changes in cell morphology and adhesion, expression of cell-specific markers for primitive endoderm and trophectoderm lineages and loss of pluripotency markers. p21 is quickly and dose-dependently activated by nutlin. It can also be activated independently from p53 by sodium butyrate, which leads to the differentiation events very similar to the ones induced by p53. During differentiation, the activating phosphorylation site of CDK2 Thr-160 becomes dephosphorylated and cyclins A and E become degraded. The target for CDK2 kinase in p53 molecule, Ser-315, also becomes dephosphorylated. We conclude that the main mechanism responsible for differentiation of human stem cells by p53 is abolition of S-phase entry and subsequent stop of cell cycle in G0/G1 phase accompanied by p21 activation.
There is accumulating evidence showing that tumours from different tissues contain a small sub-population of cells that possess stem cell characteristics, which can be resistant to treatment and maintain the growth of the tumour. This tumour stem cell concept has important implications for our understanding of tumourigenesis and design of tumour therapy (see Wicha et al., 2006 for a recent review). Although in technical terms the tumour cells, stem cells and tumour stem cells are well defined, the functional relationship between them and their transition mechanisms remain largely obscure. One possibility to clarify these relationships is to study the activity of important components of signal-transduction pathways—oncogenes and tumour suppressor genes—in each of these cell types. Several oncogenes and tumour suppressors, such as Bmi1 (Molofsky et al., 2003), Gfi1 (Hock et al., 2004), Pten (Groszer et al., 2001), Wnt/beta-catenin (Dravid et al., 2005) and Notch (Dontu et al., 2004), have been shown to control the self-renewal of normal untransformed tissue stem cells.
p53 gene is mutated in more than half of human tumours (Hainaut and Hollstein, 2000), which indicates a central role for the p53-dependent pathways in tumour cells. p53 is a tumour suppressor involved in cell cycle regulation and cellular response to different stress stimuli, including DNA damage and oncogenic stress. Owing to its ability to integrate many different signals controlling cell life and death, it has been named the ‘guardian of the genome’ (Lane, 1992). Under normal conditions, p53 is functionally inactive and rapidly degraded by ubiquitin ligase hdm2 (or mdm2 in mouse cells). However, after different types of cell stress hdm2-driven degradation is halted and this results in p53 accumulation and acquisition of transcriptional activity (Vousden and Lu, 2002).
One of the target genes for p53-dependent transcription is p21. p21 is a well-known mediator of p53-dependent cell cycle arrest inhibiting the cyclin-Cdk2 or Cdk4 complexes (Gartel and Radhakrishnan, 2005 for review). It has been shown earlier that p21 can negatively regulate self-renewal of hematopoetic stem cells (Cheng et al., 2000) and adult neural stem cells (Kippin et al., 2005), showing a link between cell cycle regulation and differentiation of adult stem cells.
In this study, we investigated the mechanism of human embryonic stem cell (ESC) differentiation through activation of the p53/p21 pathway by nutlin, a small-molecule inhibitor of p53-hdm2 interaction (Vassilev et al., 2004) that causes rapid elevation of p53, resulting in transcriptional activation. Activation of p53 results in loss of pluripotency and differentiation towards the primitive endoderm and trophectoderm lineages, as indicated by changes in cell morphology and adhesion, expression of cell-specific markers for these lineages and loss of pluripotency markers. Ser-315, the target for CDK2 kinase in the p53 molecule, becomes dephosphorylated. This is accompanied by dephosphorylation of CDK2 Thr-160 and degradation of cyclins A and E. A widely used differentiation inductor, sodium butyrate, was also able to induce p21 and similar cell differentiation, independently of p53, suggesting that activation of p21 is the linchpin underlying the rapid differentiation of human ESC observed after nutlin treatment. We show that the main mechanism responsible for ESC differentiation after p53/p21 activation is the block of cell cycle progression in G1 caused by inhibition of S-phase CDK–cyclin complexes. The downregulation of pluripotency genes NANOG and OCT4 is a later event well separated from early morphological changes, decrease of pluripotency marker GDF3 and induction of differentiation marker CDX2.
Treatment of cells with nutlin leads to changes in ESC morphology
We treated human ESC with two different concentrations of nutlin—10 and 30 μM. These concentrations were optimized by FRET analysis of p53–hdm2 interaction in vivo (data not shown). Different human ESC lines—H9 (Figures 1b, d and f), H1 and hES-NCL1 showed similar rapid change in cell morphology. After 24 h of nutlin treatment, the cells become considerably larger (Figures 1d and f), their cytoplasm to nucleus ratio increases and they progressively lose contacts with neighbouring cells (Figure 1d). As a result, the borders of the ESC colonies become ‘hazy’ when viewed under low magnification (Figure 1b). This rapid morphological effect was observed on cells grown both on feeder cells (mitomycin-inactivated mouse embryo fibroblasts) and on feeder-free growth on plates coated with Matrigel. Sensitivity of cells to nutlin was slightly higher on Matrigel plates due to active intake of nutlin by feeder cells. Progressive cell death was observed during nutlin treatment, as dead cells were removed daily when changing the culture media. The effect of nutlin was dose-dependent: at higher concentrations there were more differentiated cells. Removal of nutlin after 48 h and incubation with normal human ESC medium led to reversal of normal human ESC morphology after 6–7 days (not shown).
Nutlin treatment causes rapid accumulation of transcriptionally active p53 protein
Nutlin treatment causes a significant increase of steady-sate levels of p53 protein (about 3 times) both at 10 and 30 μM concentrations (Figures 2a and b). The accumulating protein is located in cell nucleus (Figure 2c) and is transcriptionally active as it induces transcription of its target genes p21 and HDM2 (Figure 3a) and rapid accumulation of p21 protein (Figures 2a and b). The level of p21 mRNA and protein increases 30 times, whereas the level of HDM2 mRNA increases about 20 times as compared to the untreated cells. The decrease of HDM2 protein (which contrast the increase in HDM2 transcript levels) indicates that a separate cellular mechanism is involved in HDM2 downregulation. The mRNA level of p53 also decreases after nutlin treatment (about 3 times, Figure 3a), which can explain the decrease of p53 protein level after 4 days of treatment of cells with high dose of nutlin (Figure 2a).
Nutlin treatment upregulates the expression of differentiation markers and downregulates cell pluripotency markers in a dose-dependent manner
Nutlin treatment and p53 activation resulted in upregulation of of primitive endoderm markers GATA4 (3 times compared to untreated cells) and GATA6 (5 times) as well as trophectoderm marker CDX2 (6 times) (Figure 3). Most importantly, treatment with nutlin led to downregulation of pluripotency markers, GDF3 (2 times; Figure 3b), NANOG and OCT4 (the levels of both are undetectable after 4 days of treatment; Figures 2a, b and 3a). It is, however, important to mention that 1 day treatment of cells with 30 μM nutlin is not sufficient to downregulate OCT4 and NANOG (Figure 3a), but it is sufficient to induce p53 protein activity, which results in transactivation of p21 (Figure 2a). Moreover, treatment of cells with a lower concentration of nutlin (10 μM) does not cause a decrease of OCT4 protein level within 2 days (Figure 2b). Inhibition of p53 by small interfering RNA (siRNA) caused inhibition of p21 and GATA6 induction (Figure 3c). We also attempted with several p21-specific siRNAs to suppress the increase of p21 mRNA level after nutlin treatment, but without success.
Sodium butyrate treatment changes ESC morphology in a manner similar to nutlin
Sodium butyrate, retinoic acid and vitamin D3 are well known agents able to differentiate a wide variety of cells. We treated H9 cells with 2 mM sodium butyrate, 10 and 30 μM retinoic acid and 1 μM vitamin D3. After 2 days, no changes in cell morphology or gene expression were detected with vitamin D3 (Figures 4 and 5). Retinoic acid caused considerable change in morphology, which was clearly different from the effects of nutlin: the borders of the colonies stay clear and distinct and cell layers on the outer side of the colonies become thicker. Also, the surface of the colonies becomes ‘crinkled’ because of the presence of thicker and thinner cell layers.
The change of morphology caused by sodium butyrate, however, is very similar to changes caused by nutlin (compare Figures 4 and 1). This includes similarity in cell shapes as well as the tendency to lose contacts with neighbouring cells. Moreover, sodium butyrate causes upregulation of p21, GATA4 and GATA6 mRNA levels, whereas the level of p53 mRNA does not change and the level of protein decreases in contrast to nutlin treatment (Figure 5, inset). One day treatment of cells with 2 mM sodium butyrate is enough to induce p21 protein, exactly similarly to nutlin (not shown). It is also evident that the levels of NANOG and OCT4 mRNA do not change as a result of sodium butyrate treatment (Figure 5).
Retinoic acid, which causes changes in morphology different from nutlin/sodium butyrate, also causes different patterns of gene activation in ESCs. It downregulates mRNAs for p21 and pluripotency genes OCT4 and NANOG, whereas no change in HDM2 or differentiation markers GATA4 and GATA6 could be detected (Figure 6).
Nutlin causes dephosphorylation of CDK2 substrate Ser-315 in p53 protein
After treatment of cells with nutlin, the Ser-315 of p53, which is a phosphorylation target of CDK2, becomes dephosphorylated (Figure 1). This is accompanied by dephosphorylation of Thr-160, which is an ‘activating’ phosphorylation of CDK2 kinase and degradation of CDK2 cyclin partners A and E (Figure 1). These cyclins are needed for cell cycle progression from S to G2 phase and from G1 to S phase, respectively. The steady-state levels of CDK2 protein itself do not change.
Cells treated with nutlin accumulate in G0/G1 phase
The cell cycle profile after nutlin treatment was analysed by flow cytometry (Figure 7). Nutlin clearly caused a block of cell entry into S phase. As a result, most of the cells accumulated in G1/G0 phase of the cell cycle. In untreated human ESCs the amount of S-phase cells is very high (48.7%, average of two experiments), but after 4 days of treatment it is significantly downregulated to 13.8%. By then, however, most of the cells (70.9%) have accumulated in G0/G1 phase. Treatment of cells under the same condition with 0.3% dimethyl sulphoxide only (dimethyl sulphoxide is solvent for nutlin) did not cause any changes in cell cyle (not shown).
Cells differentiating due to activation of p53/p21 pathway do not survive in ESC ‘niche’ growth conditions
As seen from Figure 7, an extensive sub-G1 peak of the cell cycle is visible after treatment of cells with nutlin. This indicates the presence of cell death, which is possibly caused by the inability of differentiated cells to survive in an ESC ‘niche’ represented by the media conditions optimized for ESC. We investigated this issue by transient transfection of H9 cells with DNA constructs for overexpression of p53, its transactivation-deficient point mutant His273 and p21 proteins under the control of cytomegalovirus promoter (Figure 8a). Both p53 and p21 caused extensive cell death, whereas the survival of cells transfected with mutant p53 was much better. Analysis of p21 and p53 mRNA expression patterns (Figure 8b) showed strong counter-selection against the cells expressing wild-type p53 or p21.
In this article, we studied the mechanism of human ESC differentiation by activation of the p53/p21 pathway with small chemical compounds. Nutlin has recently been shown to selectively activate the p53 pathway in vitro and in vivo destroying its complex with hdm2, which is itself an E3 ubiquitin ligase and leads to the degradation of p53 (Vassilev et al., 2004). This property has made nutlin a potential drug for cancer treatment aiming at efficiently eliminating the tumour xenografts from nude mice (Tovar et al., 2006).
We show here that nutlin treatment rapidly induces the differentiation of human ESCs (H1, H9 and hES-NCL1). This process is obvious as soon as 24 h after treatment: cells start to change their morphology and adhesion properties, express both primitive endoderm and trophectoderm-specific marker genes and downregulate pluripotency genes. This rapid switch of cell fate was somewhat unexpected, as most of the spontaneous stem cell differentiation events take several days to weeks (see Hyslop et al., 2005b for review).
Recently, Qin et al. (2007) suggested that p53 could directly downregulate the expression of OCT4 transcription, which is important for retaining human stem cell pluripotency. Similarly, it has been shown that in mouse ESCs, p53 may bind to Nanog promoter and therefore inhibit its transcription (Lin et al., 2005). Indeed, bioinformatic analysis revealed the presence of a DNA sequence in the OCT4 gene (starting from position +751 of OCT4 gene IndexTermAGACAAATTC(N)14IndexTermGACCAAGTCC) that is very similar to the defined consenus p53-binding site (Wei et al., 2006). However, our analysis by chromatin immunoprecipitation using p53-specific antibodies did not show any change in the p53 binding at this OCT4 site after nutlin treatment (data not shown).
Our model clearly and principally differs from that of Qin et al. (2007) and this may be a reason for this discrepancy. Qin et al. induced p53 with UV, which caused within 6 h a significant increase of transcriptionally inactive p53 (and therefore there was no upregulation of p21 or hdm2) and downregulation of both OCT4 and NANOG at the transcriptional level. In our experiments, nutlin rapidly induces transcriptionally active p53 (within 1 day) accompanied by increase of p21 and HDM2, whereas the levels of OCT4 and NANOG start to decline later (after 2–3 days) and at higher concentrations of nutlin. In fact, after treatment of cells with 10 μM nutlin for 2 days there was no decrease in the levels of OCT4 protein, although this treatment was sufficient to upregulate levels of transcriptionally active p53. Even after 4 days of low-dose nutlin treatment, the level of OCT4 did not change (data not shown). In addition, after treatment at 30 μM nutlin concentration the OCT4 mRNA levels start to decline significantly only from day 3, which is much later than the changes in differentiation markers used (significant upregulation for GATA4 and GATA6 is already observed from day 1). It is also important to notice that both NANOG and OCT4 transcription can be downregulated with retinoic acid, although no activation of p53 or its transcriptional targets can be seen. These results suggest that the process of differentiation observed after p53 activation is not initiated by downregulation of OCT4 or NANOG. It is more likely that changes in these pluripotency markers demonstrate the loss of pluripotency caused by p53 activation.
It has been shown that p53 can regulate the self-renewal of adult stem cells (Cheng et al., 2000; Kippin et al., 2005; Meletis et al., 2006). These cells express neither OCT4 nor NANOG, and it is therefore clear that mechanisms other than direct downregulation of these genes by p53 must be involved. To reveal the possible mechanism of stem cell differentiation after p53 activation by nutlin, we therefore concentrated our studies on other components of the p53 signalling pathway, first of all to gene p21 (also called WAF). Induction of both p21 mRNA and protein by nutlin is very rapid, occurs at low concentration of nutlin and yields remarkably high levels of p21 (about 25 times increase at mRNA level), whereas the protein is virtually absent in control human ESCs.
p21 is a cell cycle inhibitor binding to both G1-to-S and G2-to-mitosis CDK–cyclin complexes and inhibiting their kinase activity (for a review see Gartel and Radhakrishnan, 2005). p21 has been shown to negatively regulate self-renewal of hematopoietic stem cells (Cheng et al., 2000). Recently, it has been shown that the loss of p21 compromises the quiescence of forebrain stem cell proliferation eventually leading to exhaustion of their proliferative capacity (Kippin et al., 2005).
Several small chemical compounds have been shown to transcriptionally activate p21, both through p53-dependent pathway as well as independently of it. Induced differentiation of myelomonocytic cell line U937 by vitamin D3 is facilitated by transcriptional induction of the p21 gene by vitamin D3 receptor (Liu et al., 1996a). The p21 gene is also a target for retinoic acid, which contains a functional retinoic acid (RA) response element in its promoter and thereby mediates the differentiation of U937 cells by this compound (Liu et al., 1996b). Sodium butyrate is another activator of p21 promoter through Sp1 sites, which occurs independently of p53 (Nakano et al., 1997).
We used vitamin D3, retinoic acid and sodium butyrate and found that human ESCs react differently to these compounds. Vitamin D3 did not have any morphological effect or change of gene expression pattern in the range of concentrations and time intervals used. Retinoic acid caused changes in cell morphology and gene expression patterns very different from those observed after nutlin treatment. Most importantly, the level of p21 decreased in parallel with decrease of cell pluripotency markers, OCT4 and NANOG, in contrast to nutlin treatment, which results in p21 activation and OCT4 and NANOG downregulation. It is therefore clear that the effects of nutlin and retinoic acid to human ESCs are caused by different molecular mechanisms and result in different cell morphology.
Sodium butyrate, however, caused morphological changes in human ESCs that were very similar to nutlin treatment: the cells became larger and changed their shape, their nucleus to cytoplasm ratio decreased and they became progressively nonadhesive. The real-time quantitative PCR analysis showed that sodium butyrate causes the activation of p21 transcription, similarly to nutlin. The levels of p53 mRNA and its other target HDM2, however, did not change and, in contrast to nutlin treatment, the levels of p53 protein decreased. This shows that the effect of sodium butyrate on p21 expression is independent of p53. As the cells were still differentiating along both primitive endoderm and trophectoderm lineages, we conclude that the activity of p53 may be mediated by p21 and differentiation events occurring after nutlin and sodium butyrate treatment are a consequence of p21 activation. This leads to a very interesting question: what are the most immediate changes that take place in human ESCs after p21 activation? GATA4 and GATA6 upregulation is only observed after high concentrations of nutlin unlike p21, which is activated immediately after 24 h of low and high nutlin treatment. This suggests that these differentiation markers are unlikely to be immediate transcriptional targets of p53 and therefore not the initiators of human ESC differentiation observed in this study. A more likely explanation is provided by the changes in cell cycle, which start to occur as soon as 24 h after nutlin treatment and p21 activation. Nutlin treatment and p21 activation abolishes the entry of cells into S phase, which is accompanied by dephosphorylation of Thr-160 of CDK2 and degradation of its partner cyclins E and A. The functional inactivation of CDK2–cyclin complex is also indicated by dephosphorylation of Ser-315 of p53, which is a substrate for CDK2 kinase.
Stem cells and their differentiated progenitors are generally able to survive only in certain biochemical conditions in the cell ‘niche’. The growth media and conditions used in this study were optimized for human ESC growth; therefore, it is not unexpected that differentiation of stem cells is accompanied by significant cell death as shown by cell cycle analysis. Moreover, after incubation of H9 cells for 2 days with 30 μM nutlin and further incubation without the chemical, the cell culture reversed to normal ESC morphology after 6 days (not shown). A possible explanation here is that all the differentiated cells eventually die in the ‘wrong’ cell niche and only ESC survive and proliferate. Transfection of cells with constructs expressing either wild-type p53 or p21 under cytomegalovirus promoter also confirmed this explanation. After the transfection, human ESCs undergo considerable death, whereas the ones expressing p53 or p21 are eliminated. In contrast, the survival of cells expressing a mutant form of p53 unable to induce p21 transcription is much higher.
In summary, our experiments show that p53 is able to cause differentiation of human ESCs due to activation of p21 and subsequent block of S-phase entry (Figure 9). Sodium butyrate causes similar effects independently of p53 due to direct activation of p21. Is the inhibition of cell cycle sufficient to induce differentiation of human ESCs? It seems very likely that for ESCs, differentiation is a ‘default’ pathway and therefore inhibition of S-phase entry is indeed a starting point. This hypothesis is well supported by current ongoing work in our group that has underlined an important role for NANOG in enhancing G1 to S transition in human ESCs, thus linking for the first time the maintenance of pluripotent phenotype to cell cycle regulation and in particular G1 to S transition.
The ability of p53 to differentiate stem cells can explain two important in vivo properties of this protein. First, it has been shown that high activity of p53 causes premature aging of mice (Tyner et al., 2002), which can be explained by loss of normal stem cells. Second, the potential of activated p53 to efficiently eliminate at least some types of tumours (for example, prostate and osteosarcoma xenografts in nude mice Tovar et al., 2006) could be explained by its ability to target the stem cells of these particular tumours. At the same time, the loss of normal stem cells would create a major problem in treating the tumours with this type of p53 activators.
Materials and methods
Cell lines, their maintenance and treatment with low-molecular weight compounds
Human ESCs were grown on mitotically inactivated mouse embryonic fibroblasts and passaged essentially as described in Stojkovic et al. (2004). Few passages before experiments, human ESCs were transferred to Matrigel-coated plates with feeder-conditioned media as previously described in Stojkovic et al. (2004); Hyslop et al. (2005a). Cells were treated with 10 and 30 μM nutlin-3, 2 mM sodium butyrate, 10 and 30 μM retinoic acid (all from Sigma, Dorset, UK) or 1 μM 1α,25-dihydroxyvitamin D3 (Biomol International, Exeter, UK). H9, H1 (both from WiCell Inc., Madison, WI, USA) and hES-NCL1 (Stojkovic et al., 2004) were used in this study.
Cells in 6-well plates were washed with phosphate-buffered saline, fixed with 4% paraformaldehyde for 10 min and permeabilized using 0.2% Triton X-100, 5% fetal calf serum (FCS) in phosphate-buffered saline. After washing three times with 5% FCS in phosphate-buffered saline, the cells were incubated with p53-specific antibody pAb421 (1:200 dilution) for 1 h at room temperature. After washing three times with 5% FCS anti-mouse IGG-FITC conjugate was added (1:100 dilution), cells were kept for 1 h in dark and washed three times with 5% FCS. Microscopy was performed with a Zeiss Axiovert 200 microscope and AxioVision software.
Transient transfection of human ESCs with full p53, mutant p53 and p21 constructs
p53 (human), mutant His273 and p21 cDNAs were cloned into pCG vector (Tanaka and Herr, 1990) as described earlier (Joers et al., 1998). Human ESCs were cultured under feeder-free conditions with feeder-conditioned media free of antibiotics for at least 5 days before transfections and dissociated using Accutase (Chemicon, Temecula, CA, USA, diluted 1:1 in Dulbecco's modified Eagle's medium). Accutase treatment was stopped with 10% FCS in Dulbecco's modified Eagle's medium, the cells were centrifuged 800 r.p.m. 3 min and resuspended in nucleofection solution (Mouse ESC Nucleofection Kit, Amaxa International, Cologne, Germany). Five microgram of each construct was nucleofected into approximately 106 human ESCs using the program A-23. The transfected human ESCs were plated under feeder-free conditions and incubated for 2 days.
siRNAs and transfection
p53 siRNA was obtained from Invitrogen (Carlsbad, CA, USA) (Validated Stealth RNAi DuoPak for p53, final concentration in transfection 40 nM). The cells were collected by Accutase treatment. Transfection of siRNA into human ESCs was carried out using the Mouse ESCs Nucleofection Kit from Amaxa. Our preliminary studies with FITC-labelled control siRNA (Invitrogen) indicated 75% transfection efficiency in human ESCs. After transfection, the cells were cultured for 48 h in mouse embryonic fibroblast-conditioned medium and then treated with nutlin for an additional 48 h in the same medium.
Cell lysis and Western blotting
Cells in 6-well plates were washed with cold phosphate-buffered saline and lysed in RIPA buffer (50 mM Tris–HCl pH 8.0, 150 mM NaCl, 1% IGEPAL CA 630, 0.5% Na-DOC and 0.1% SDS). Before the treatment of cells, 1 mM PMSF and Roche protease inhibitors (1 tablet per 10 ml) were added. After 30 min on ice, the lysates were homogenized and centrifuged 13 000 r.p.m. 15 min. The total protein concentration was determined using a Bradford Kit (Bio-Rad, Hercules, CA, USA) using bovine serum albumin as the concentration standard. Absorption at 595 nm was detected using a Thermo Labsystems Multiskan Ascent. Lysates (20–25 μg total protein) were electrophoresed on a 10% SDS–polyacrylamide gel electrophoresis and electrophoretically transferred to a nylon membrane (Hybond-P, Amersham Biosciences, Piscataway, NJ, USA). Membranes were blocked in Tris-buffered saline with 5% milk and 0.1% Tween. The blots were probed overnight at 4 °C with the following antibodies: for p53 pAb421 (for p53, 2 μg/ml, Eugenics, Brussels, Belgium), pAb1801 (2 μg/ml, Santa Cruz sc-98) or DO-1 (1 μg/ml, Santa Cruz, Santa Cruz, CA, USA). Antibodies for phospho-serine p53 (sc-1706-R, final dilution 1:200), p21 (sc-6246, 1 μg/ml), oct-4 (C-10, 1:200), cdk2 (D-12, 1:200), cyclin A (H-432, 1:200) and β-actin (sc-47778, 1:1000) were also obtained from Santa Cruz. The antibody for hdm2 (2A10) was obtained from Abcam (Cambridge, UK) (2 μg/ml), for NANOG from Aviva Systems Biology (San Diego, CA, USA) (0.5 μg/ml) and for GAPDH from (Abcam, 1:2000). Antibodies for cyclin E were from Upstate (Dorset, UK) (05-363, 1:1000) and for phospho-cdk2 from Cell Signaling (Boston, MA, USA) (1:1000). The next morning, the blots were incubated for 2 h with horseradish peroxidase-conjugated secondary antibodies: donkey anti-goat, goat anti-mouse or goat anti-rabbit (all from Santa Cruz, 1:2000). Antibody/antigen complexes were detected using ECL reagent (Amersham Biosciences) and GeneSnap software (version 4.00.00, SYNGENE) with GeneGnome (SYNGENE). Bands were analysed using the GeneTools software (version 3.00.22, SYNGENE).
Isolation of RNA and Quantitative Real-Time PCR analysis
RNA was isolated using TRIzol Reagent (Invitrogen) according to the manufacturer's protocol. For each well of a 6-well plate 1 ml of TRIzol was added and the lysate was transfered to Eppendorf tubes. After 5 min, 0.2 ml chloroform was added, vortexed, kept for 2 min and then centrifuged 12 000 g 15 min. The upper phase was taken into a fresh Eppendorf tube, RNA was precipitated with 0.5 ml isopropanol, centrifuged and the pellet was washed with 75% ethanol. RNA was dissolved in RNase-free water. 1 μg of RNA was treated with DNaseI, cDNA was synthesized with MMLV Reverse Transcriptase (Promega, Madison, WI, USA) using random hexamers as primers and purified on QIAquick Spin columns (Qiagen, Crawley, UK). Real-time PCR analysis was carried out using DyNAmo SYBR Green qPCR Kit (Finnzymes, Espoo, Finland) and 7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Primers for CDX2, GAPDH, GATA4, GATA6, NANOG and OCT4 have been described earlier (Hyslop et al., 2005a). Primers for p53 were IndexTermAGCCAAGTCTGTGACTTGCA and IndexTermAACCTCCGTCATGTGCTGT, for p21 IndexTermCGAAGTCAGTTCCTTGTGGA and IndexTermCATGGGTTCTGACGGACAT; for hdm2 IndexTermTGCCATTGAACCTTGTGTGATT and IndexTermTGGTTGTCTACATACTGGGCA; for UBC IndexTermATTTGGGTCGCGGTTCTTG and IndexTermTGCCTTGACATTCTCGATGGT; for GDF-3 IndexTermGTCCGCGGGAATGTACTTCG and IndexTermCACCTTGTGGCCATGGGACT. As a standard, human genomic DNA or random-primed cDNA (Clontech, Mountain View, CA, USA) was used.
Cell cycle analysis
Cell cycle analysis was performed using the CyStain DNA 2 step protocol (Partec Gmbh, Germany), which uses DAPI (4,6-diamidino-2-phenylindole) staining. Human ESCs were harvested permeabilized and stained in accordance with manufacturers' instructions and the sample was analysed by flow cytometry (Becton Dickinson, Franklin Lakes, NJ, USA; FACS ARIA). The data were analysed using ModFit (Verity Software House) to generate percentages of cells in G1, S and G2/M phases.
Alkaline phosphatase staining
Alkaline phosphatase staining was carried out using the Alkaline Phosphatase Detection Kit following manufacturer's instructions (Chemicon). Briefly, cells were fixed in 90% methanol and 10% formamide for 2 min and then washed with rinse buffer (20 mM Tris–HCl, pH 7.4, 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 Zeiss microscope and AxioVision software (Carl Zeiss, Jena, Germany).
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We thank Ian Dimmick for help in flow cytometry and Stuart Atkinson for assistance with Quantitative real-time-PCR. This work has been supported by grants from Estonian Science Foundation (ETF6459) and Citrina Foundation (TM) and One North East Regional Developmental Agency (ML).
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