NANOG Expression Analyses
The presence of a duplication pseudogene, NANOGP1, and 10 processed pseudogenes that are closely related to NANOG [7, 8] could provide, in principle, competing transcript targets in siRNA knockdown studies. By designing specific primers for each of the 10 processed pseudogenes, we found that none of them was expressed in either human ES or EC cells (data not shown). However, like NANOG, NANOGP1 seems to be expressed in both human ES and EC cells (Fig. 1A). Only one band, at the expected molecular weight for NANOG (34.6 kDa), was detected by Western blot analysis (Fig. 1B); the absence of a band corresponding to a full-length NANOGP1 protein (expected at ∼27 kDa) is consistent with the prediction by Booth and Holland  that NANOGP1 is unable to be translated into a full-length protein. Immunofluorescence analyses with a NANOG antibody revealed that NANOG is localized in the nuclei of EC and ES cells (Fig. 1C), consistent with bioinformatic analyses that predict a nuclear localization signal (PVKKQKT) at amino acids 92–98 of NANOG protein.
Figure Figure 1.. Expression of NANOG and NANOGP1 in human ES and EC cells. (A): Expression of NANOG and NANOGP1 in human ES and EC cells assessed by RT-PCR. PCR oligonucleotides were designed to span a region between exon 3 and exon 4, which is shorter in NANOGP1 compared with NANOG transcript due to use of an alternative splicing site. (B): NANOG protein expression in human ES and EC cells by Western blot analysis. (C): Immunohistochemistry with NANOG antibody. Nuclear localization was observed in NT2-SP12 EC cells and H1 ES cells but not in the NC, which was stained with the Rhodamine-conjugated secondary antibody only. Cell nuclei shown by DAPI staining. Bar = 20 μm. (D): NANOG expression in human oocytes and preimplantation embryos. NANOG was only found to be present in the inner cell mass of expanded blastocysts and not trophectoderm cells. The NC was stained with the secondary antibody only. Cell nuclei shown by DAPI staining. Bar = 50 μm. Abbreviations: DAPI, 4′,6′-diamidino-2-phenylindole; EC, embryonic carcinoma; ES, embryonic stem; NC, negative control; RT-PCR, reverse transcription–polymerase chain reaction.
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Immunostaining of human preimplantation embryos and oocytes shows that the NANOG protein was absent from unfertilized oocytes, 2–16 cell embryos, and early morulae (Fig. 1D); however, very restricted expression was observed in the ICM of blastocysts, as has been described for mouse Nanog [5, 6, 12].
siRNA-Mediated NANOG Knockdown in Human EC and ES Cells
Previous studies in murine ES cells have suggested that stable downregulation of Nanog using the RNA interference (RNAi) technique has resulted in an inability to generate stable clones, which may reflect a role for Nanog in survival and renewal of ES cells . In view of this, we used short double-stranded RNA oligonucleotides (corresponding to a NANOG-specific region outside the homeodomain-encoding and SMAD4 domain–encoding regions; see Materials and Methods) to investigate the role of NANOG in human ES and EC cells. To rule out the possibility that effects of NANOG siRNA observed in this study are due to increased toxicity to the cells or increased contamination of this particular siRNA during chemical synthesis, we used several controls, and these included a mutated version of the NANOG siRNA (Fig. 2B). Previous reports have indicated that RNAi with control genes such as GFP or β2-microglobulin in human EC or ES cells does not result in nonspecific effects, changes in cell-surface antigen, or differentiation [18, 19]; therefore, GFP siRNA in addition to untreated and mock-transfected cells was used as additional negative control in this study (Fig. 2B). The most effective short double-stranded RNA corresponded to a sequence shared by NANOG and the duplication pseudogene, NANOGP1. This would suggest that NANOGP1 transcript, despite not being translated into a protein, would be downregulated as result of the RNAi approach. To ensure maximal silencing, ES and EC cells were transfected twice over a 48-hour interval. Evaluation of transfection efficiency 48 hours after the second transfection and using a Rhodamine-labeled NANOG siRNA (Fig. 2A) revealed a relatively high efficiency (in EC cells, 70% ± 1.53%; in ES cells, 65% ± 2.08%). Similar results were obtained with Rhodamine-labeled GFP and mutated NANOG siRNA (data not shown). This is similar to the transfection efficiency achieved by lentiviral delivery of siRNA in human ES cells . Real-time PCR analysis confirmed statistically significant downregulation of NANOG and NANOGP1 in cells transfected with NANOG siRNA compared with all control groups (Fig. 2B), which do not show significant downregulation of NANOG. There was a 63-, 34-, and 18-fold reduction of NANOG in the hES-NCL1, H1, and NT2-SP12 cells treated with siRNA to NANOG, respectively, compared with cells treated with GFP siRNA; corresponding reductions in NANOGP1 transcript levels were 90-, 46-, and 15-fold in the hES-NCL1, H1, and NT2-SP12 cell lines, respectively. There were no statistically significant changes in NANOG expression between cells treated with mutated NANOG siRNA, GFP siRNA, or mock-transfected cells, which indicates that they could be used as reliable negative controls. In a few instances (3 of 60 pairwise comparisons), we did detect statistically significant changes in NANOG expression between untreated cells and the other control groups. Although this occurred at low frequency, it did indicate that untreated cells might not provide the best negative control for these experiments. It is for this reason that we have used cells treated with GFP siRNA as negative control for most of the experiments, as described below. To further confirm NANOG downregulation, we carried out Western blotting analysis, which showed reduction of NANOG protein in both EC and ES cells (Fig. 2C). Furthermore, NANOG downregulation was maintained up to 6 days after transfection (supplemental online Fig. 1).
Figure Figure 2.. NANOG downregulation in human ES and EC cells by RNAi. (A): Uptake of rhodamine-labeled NANOG siRNA in EC cells (NT2-SP12). Bar = 100 μm. (B):NANOG and NANOGP1 expression assessed by real-time RT-PCR 1 day after transfection in cells treated with either NANOG siRNA, mutated NANOG siRNA, or GFP siRNA or mock-transfected cells. The transfections were performed in triplicates, and the average of normalized ratio of target gene (either NANOG or NANOGP1)/GAPDH was calculated. The data represent the mean ± SEM from three experiments. The value for the cells treated with control GFP siRNA was set to 1 (100%), and all other values were calculated with respect to this. Statistical significance of the results was assessed using Student's t-test (p < .05). *Statistically significant changes in expression between the NANOG siRNA–treated cells and all control groups. (C): Western blot analysis of NANOG protein levels in NT2-SP12 (EC cells) and hES-NCL1 (ES cells) 2 days after transfection with either NANOG (N) or GFP (G) siRNA. GAPDH was used as a loading control. (D): Morphology of hES-NCL1 and NT2-SP12 4 days after transfection with either NANOG or GFP siRNA. (1): NT2-SP12 transfected with GFP siRNA; typical EC morphology was observed. (2): NT2-SP12 transfected with NANOG siRNA; spindle-shaped cells are present, indicative of differentiation. (3): hES-NCL1 transfected with GFP siRNA, showing typical dense ES morphology. (4) Spindle and flattened enlarged differentiated cells are present in the hES-NCL1 transfected with NANOG siRNA. Bar = 100 μm. (E): Changes in SSEA-4 expression as result of NANOG downregulation. Flow cytometry analysis of NANOG siRNA–treated cells (blue) and GFP siRNA–treated cells (green) shows downregulation of SSEA-4 expression in NANOG siRNA–treated cells (mean intensity of fluorescence, 66.78) compared with GFP-treated cells (mean intensity of fluorescence, 135.2). Abbreviations: EC, embryonic carcinoma; ES, embryonic stem; GFP, green fluorescent protein; RNAi, RNA interference; RT-PCR, reverse transcription–polymerase chain reaction; siRNA, small interfering RNA; SSEA, stage-specific embryonic antigen.
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Nanog Knockdown Induces Differentiation in Human ES and EC Cells
The cell lines hES-NCL1, H1, and NT2-SP12 were transfected with either NANOG or GFP siRNA and monitored over 6 days. EC cells transfected with either mutated NANOG or GFP siRNA as well as mock-transfected cells remained undifferentiated for up to 6 days after transfection. In contrast, 2 days after transfection with NANOG siRNA, the cells began to appear spindle-shaped, and by 6 days, the proportion of spindle-shaped cells had increased (Fig. 2D). Human ES cells transfected with GFP siRNA, mutated NANOG siRNA, and mock-transfected cells remained largely undifferentiated (Fig. 2D), although a small proportion of morphologically differentiated cell types began to appear by 4 days after transfection. In contrast, 2 days after transfection with NANOG siRNA, enlarged flattened cells began to appear with an increased proportion of differentiated cells by days 4 and 6 (Fig. 2D). We analyzed the transfected cells by flow cytometry for the expression of cell-surface antigens typical for the human ES and EC cells, such as stage-specific embryonic antigen (SSEA)-4 and tumor-rejection antigen (TRA)-1-60, and as can be seen from Figure 2E, cells treated with NANOG siRNA had downregulated SSEA-4 expression (mean intensity, 66.78) compared with GFP siRNA–treated cells 4 days after transfection (mean intensity, 135.20), suggesting loss of pluripotency in human ES and EC cells as result of NANOG downregulation.
We sampled RNA transcripts at 2-day periods and conducted RT-PCR analysis for markers of pluripotency, such as SOX2, OCT4, and REX1, and of differentiation, such as PAX6 (neuroepithelium), BRACHYURY (mesoderm), fibroblast growth factor (FGF)-5 (primitive ectoderm), GATA6 and GATA4 (primitive endoderm), and CDX2 and GATA2 (trophectoderm) (data not shown). We noticed that in ES and EC cells transfected with NANOG siRNA, GATA4, GATA6, GATA2, and CDX2 were upregulated compared with cells transfected with GFP siRNA from day 4 after transfection. To enable a better analysis of these differentiation events, we used real-time PCR for all of the marker genes mentioned above (Fig. 3A). Real-time PCR analysis for the mesoderm marker BRACHYURY, primitive ectoderm marker FGF5, and neuroectoderm marker PAX6 showed no significant differences between the NANOG and GFP siRNA–transfected cells 4 days after transfection (data not shown), suggesting that NANOG downregulation is unlikely to induce differentiation along these lineages.
Figure Figure 3.. NANOG downregulation results in differentiation to trophectodermal lineages and extraembryonic endoderm. (A): Real-time RT-PCR analysis at day 4 after transfection for pluripotency markers (OCT4, SOX2, REX1, TERT), extraembryonic endoderm markers (GATA6, GATA4, LAMININ B1, AFP), and trophectoderm markers (CDX2, GATA2, hCGα, and hCGβ). The transfections were performed in triplicates, and the average of normalized ratio of target gene/GAPDH was calculated. The data represent the mean ± SEM from three experiments. The value for the cells treated with control GFP siRNA was set to 1 (100%), and all other values were calculated with respect to this. Statistical significance of the results was assessed using Student's t-test (p < .05). *Statistically significant changes in expression between the NANOG siRNA–treated cells and all control groups. (B): Real-time PCR analysis for NANOG, TERT, hCGβ, and LAMININ B1 at three passages after transfection. The transfections were performed in triplicates, and the average of normalized ratio of target gene/GAPDH was calculated. The data represent the mean ± SEM from three experiments. The value for the cells treated with control GFP siRNA was set to 1 (100%), and all other values were calculated with respect to this. Statistical significance of the results was assessed using Student's t-test (p < .05). *Statistically significant changes in expression between the NANOG siRNA and GFP siRNA–treated cells. Abbreviations: GFP, green fluorescent protein; RT-PCR, reverse transcription–polymerase chain reaction; siRNA, small interfering RNA.
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As assessed by real-time RT-PCR, NANOG downregulation induced OCT4 downregulation at day 4 after transfection, indicating loss of pluripotency in human ES cells; however, no downregulation of SOX2 or REX1 (with the single exception of REX1 in EC cells) was observed at this time. Sox2 expression has been found in trophoblast stem cells and shown to play a critical role in the development of the placenta . Rex1 was also shown to be critical for early placenta and preimplantation development in bovine embryos . NANOG downregulation induces the expression of early trophectodermal markers CDX2 and GATA2 as well as later markers hCG-alpha and hCG-beta (Fig. 3A) as early as day 4, consistent with induction of trophectoderm differentiation. The maintenance of SOX2 and REX1 expression could therefore be indicative of this type of differentiation and could not be used as estimate of pluripotency in these cells. In line with this observation, we noticed that SOX2 and REX1 expression was maintained after three passages of NANOG siRNA–transfected cells, whereas the expression of extraembryonic endoderm and trophectoderm markers was significantly upregulated in NANOG siRNA–treated cells compared with GFP siRNA–treated cells (Fig. 3B). Our own work has shown that the RT unit of telomerase, TERT, is downregulated upon differentiation of human ES and EC cells and can therefore serve as a biomarker for human ES/EC cells (Walter and Lako, unpublished observations). We did not observe significant changes in the expression of TERT at 4 days after transfection as a result of NANOG downregulation (Fig. 3A); however, significant downregulation was noticed after three passages (Fig. 3B). These results suggest that investigation of pluripotency and differentiation markers should be carried out at least at two time points after transfections because the kinetics of their regulation during differentiation process might be slightly different.
Expression of extraembryonic endoderm transcription factors GATA4 and GATA6, parietal endoderm marker LAMININ B1, and visceral endoderm marker AFP was significantly upregulated by real-time RT-PCR analysis 4 days after transfection with NANOG siRNA compared with GFP siRNA, consistent with the induction of extraembryonic endoderm differentiation (Fig. 3A). It should be noted that no AFP was detected in either the GFP or NANOG siRNA–transfected NT2-SP12 cells; however, this is perhaps due to monolayer culture conditions as opposed to aggregates, which show AFP upregulation . These findings are consistent with the proposed role of Nanog as suppressor of primitive endoderm differentiation in murine embryos and ES cells [5, 6]. These results obtained by quantitative PCR were confirmed by Western blot analysis (Fig. 4A) or immunocytochemistry on NANOG siRNA–transfected and GFP siRNA–transfected cells (Figs. 4B, 4C). AFP was only detected in the NANOG siRNA–transfected H1 and hES-NCL1 cells and was absent from human ES cells transfected with GFP siRNA (Fig. 4B). Some cells positive for GATA6 (Figs. 4A, 4C), GATA4, and CDX2 (data not shown) were detected at the periphery of the colonies of GFP siRNA–transfected H1, hES-NCL1, and NT2-SP12 cell lines, and this is consistent with a small amount of spontaneous differentiation occurring in these cultures as result of the length of siRNA experiment. However, many more cells positive for GATA6 (Figs. 4A, 4C), GATA4, and CDX2 were observed at human ES and EC cells transfected with NANOG siRNA. To correlate NANOG reduction with differentiation in individual cells, costaining of NANOG siRNA–treated cells with NANOG antibody and GATA6 was carried out (Fig. 4D). This analysis showed that by day 4 after transfection, cells with GATA6 expression had much reduced NANOG expression, providing further evidence of differentiated state in these cells induced by NANOG downregulation.
Figure Figure 4.. NANOG downregulation results in induction of AFP and GATA6. (A): Western blot analysis of GATA6 protein levels in hES-NCL1 (ES cells) 4 days after transfection and NT2-SP12 (EC cells) 6 days after transfection with either NANOG (N) or GFP (G) siRNA. GAPDH was used as a loading control. (B): Immunohistochemistry with AFP antibody. AFP was only detected in human ES cells (H1) transfected with NANOG siRNA 4 days after transfection and not in human ES cells transfected with GFP siRNA. Bar = 100 μm. (C): Immunohistochemistry with GATA6 antibody. A few GATA6-positive cells are present in human ES cells (H1) transfected with GFP siRNA 4 days after transfection, but many more GATA6-positive cells are present at the periphery of human ES cells (H1) transfected with NANOG siRNA. Bar = 100 μm. (D): Immunocytochemistry with GATA6 and NANOG antibody in hES-NCL1 cells treated with NANOG siRNA at day 4 after transfection. Cells that show strong GATA6 expression have much-reduced NANOG expression. Bar = 20 μm. Cell nuclei shown by DAPI staining. Abbreviations: DAPI, 4′,6′-diamidino-2-phenylindole; EC, embryonic carcinoma; ES, embryonic stem; GFP, green fluorescent protein; siRNA, small interfering RNA.
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To investigate whether the effects of NANOG downregulation are irreversible, the transfected cells were maintained for three passages in culture. Real-time RT-PCR analysis at this stage indicated that NANOG expression was downregulated 1.8-fold and 3.2-fold in hES-NCL1 and NT2-SP12 cells, respectively (Fig. 3B), compared with a 63- and 18-fold decrease detected at day 4 after transfection. Despite this, the expression of trophectodermal marker hCG-beta and extraembryonic endoderm LAMININ B1 was upregulated in both ES and EC cells transfected with NANOG siRNA compared with cells treated with GFP siRNA (Fig. 3B). As other reports have suggested, the observed effect is likely to result from overgrowth of ES cells compared with more slowly growing differentiated cells . The presence of differentiated cells that express trophectodermal and endodermal markers after multiple passages suggests that the effects of NANOG downregulation are irreversible; however, rescue-type experiments combined with selection strategies need to be carried out.