Nanog is a homeodomain transcription factor that is expressed specifically in undifferentiated embryonic stem (ES) cells and has been shown to be essential in the maintenance of pluripotency in mouse ES cells. To examine the function of NANOG in primate ES cells, we generated transgenic monkey ES cell lines expressing three- to seven-fold higher levels of NANOG protein compared to wild-type ES cells. These NANOG over-expressing cell lines retained their undifferentiated state in the absence of a feeder layer, as shown by expression of undifferentiated ES cell markers such as alkaline phosphatase (ALP) and OCT-4. We also demonstrated that in vitro differentiation of transgenic cell lines was mostly restricted to the ectodermal lineage, as examined by reverse transcriptase-polymerase chain reaction (RT-PCR). Knockdown experiments using NANOG small interfering (si) RNA resulted in induction of differentiation markers such as AFP, GATA4 and GATA6 for the endoderm and CDX2 for the trophectoderm. These results suggest that NANOG plays a crucial role in maintaining the pluripotent state of primate ES cells.
Embryonic stem (ES) cell lines were first established in 1981 from the inner cell mass (ICM) of preimplantation mouse embryos (Evans & Kaufman 1981). Because ES cells have the ability to proliferate indefinitely and differentiate into almost any cell type both in vitro and in vivo, they have been very valuable tools for studying mechanisms of cell differentiation during embryogenesis. Since human ES cell lines were established and shown to have similar differentiation potential to mouse ES cells (Thomson et al. 1998), they have attracted much attention as possible unlimited cell sources for cell transplantation therapies to treat patients with degenerative diseases (Smith 2001). We subsequently established cynomolgus monkey ES cell lines as an animal model for preclinical research of human ES cell therapies and showed that they are very similar to human ES cells in most respects, including their expression of molecular markers in the undifferentiated state and their differentiation potency (Suemori et al. 2001). Both human and monkey ES cells currently require growth on a feeder layer for long-term maintenance in culture, rendering the large-scale culture of ES cells that would be necessary for human ES cell therapies very labor-intensive. However, the use of feeder cells could result in contamination of ES cells with infectious agents, rendering them unusable for therapeutic applications in humans. Recently, several groups have identified factors that are able to sustain the undifferentiated state of human ES cells in the absence of a feeder layer (Sato et al. 2004; James et al. 2005; Xu et al. 2005). However, little is known about the long-term efficacy of such factors; therefore, growth on a feeder layer currently remains the most dependable way stably to maintain primate ES cells. In order to establish reliable culture methods for primate ES cells that do not require feeder cells, the molecular mechanisms that sustain the self-renewal and pluripotency of primate ES cells must be elucidated.
Nanog, a homeodomain transcription factor, was shown to be an essential factor in sustaining the pluripotency of mouse ES cells (Chambers et al. 2003; Mitsui et al. 2003). Targeted disruption of the nanog gene in mouse ES cells results in differentiation primarily along the primitive endoderm lineage, suggesting that Nanog is a crucial factor that prevents ES cells from differentiating into primitive endoderm (Mitsui et al. 2003). Several groups have reported observing NANOG expression in undifferentiated human ES cells (Richards et al. 2004; Sato et al. 2004; Zaehres et al. 2005). To elucidate the functions of NANOG in pluripotency of primate ES cells, we carried out experiments in over-expression and knockdown of the NANOG gene in monkey ES cells. Results indicated that NANOG is involved in the maintenance of self-renewal of monkey ES cells.
NANOG is expressed in specifically undifferentiated monkey ES cells
In mouse ES cells, Nanog has been shown to be essential for the self-renewal of ES cells, and elevated expression of Nanog is sufficient to maintain self-renewal of ES cells independent of LIF signaling (Chambers et al. 2003; Mitsui et al. 2003). However, the function of NANOG in monkey ES cells is still unclear. We used the CMK6 cynomolgus monkey ES cell line, which is maintained in an undifferentiated state when cultured on a feeder layer. CMK6 cells formed tightly packed colonies with each cell exhibiting a high nuclear/cytoplasmic ratio, and expressed undifferentiated ES cell markers such as ALP and OCT-4 protein. When CMK6 cells were cultured without a feeder layer for 3 days, cells formed colonies with a flattened morphology and almost lost expression of ALP and OCT-4 as compared with undifferentiated ES cells (Fig. 1A). Next, we compared the expression level of NANOG protein in undifferentiated and differentiated CMK6 cells. Whereas high expression of NANOG protein was observed in undifferentiated ES cells, it was undetectable in differentiated cells (Fig. 1B).
Because it has been previously reported that a subpopulation of mouse ES cells expresses Oct-4 but not Nanog proteins in culture (Hatano et al. 2005), we examined co-localization of NANOG and OCT-4 protein by immunostaining in monkey ES cells. Both NANOG and OCT-4 were localized to the nucleus. NANOG expression was observed in all cells with slight variation in the level of expression compared to OCT-4 protein expression (Fig. 1C).
Constitutive expression of NANOG maintains self-renewal of monkey ES cells
To examine the function of NANOG in primate ES cells, we generated transgenic monkey ES cell lines that constitutively over-express NANOG. An expression vector consisting of the human NANOG cDNA driven by a CAG promoter and a PGK-neo selectable marker was transfected into CMK6 cells. G418-resistant transgenic cell lines were examined for their NANOG protein expression level by Western blot analysis. Ten NANOG transgenic cell lines were examined and showed up to seven-fold higher NANOG protein expression than wild-type (wt) or mock (empty vector) transgenic cells. Because expression levels of endogenous and exogenous NANOG could not be separately quantified, three clones with different expression level of NANOG protein from clones showing more than two-fold higher expression of NANOG, were chosen and used for further analysis. The clones (6N1, 6N2, 6N3) displayed approximately five-, three- and seven-fold, respectively, higher expression than wild-type cells (Fig. 2A). The NANOG transgenic and mock transgenic cell lines were morphologically indistinguishable from wild-type cells. NANOG transgenic clones were positive for expression of undifferentiated ES cell markers such as ALP, OCT-4, TRA-1-60, and SSEA-4 (Figs 1A and 2C), suggesting that they have retained their undifferentiated state.
To examine whether over-expression of NANOG is able to sustain the self-renewal of monkey ES cells, NANOG transgenic ES cells were cultured in the absence of a feeder layer, and the level of NANOG protein was determined by Western blot analysis (Fig. 2B). While NANOG expression was reduced in mock transgenic and wild-type CMK6 cells, the level of NANOG protein remained constant in NANOG transgenic cells. NANOG transgenic cells formed OCT-4-positive colonies with a typical undifferentiated ES cell morphology, and expressed other undifferentiated ES cell markers such as ALP, SSEA-4, and TRA-1-60 (Fig. 2C). In contrast, wild-type and mock transgenic cells rapidly differentiated in the absence of a feeder layer, as judged by their flattened morphology and reduction of OCT-4 protein expression. Similar results were obtained using another monkey ES cell line, CMK9. For quantitative analysis, we counted the number of undifferentiated and differentiated colonies formed after plating ES cell lines to feeder-free plates. Although plating efficiency was similar among cell lines, mock transgenic cells formed only a small number of undifferentiated colonies, and NANOG transgenic clones formed 60–95% undifferentiated colonies in both CMK6 and CMK9 cells (Fig. 2D). The 6N1 NANOG transgenic cell line was maintained in an undifferentiated state without a feeder layer for 41 passages, corresponding to 4 months (Fig. 2E). Similar results were obtained in 6N2 and 6N3 cell lines. These results suggest that constitutive over-expression of NANOG is sufficient for maintenance of the self-renewal and stem cell identity.
To examine whether over-expression of NANOG affects to the clonal propagation, we performed colony forming assays. ES cells were dissociated into single cells and plated on to a feeder layer. After 7-day culture, colonies were stained for ALP. Few differentiated colonies were observed and the numbers of ALP positive undifferentiated colonies were counted (Fig. 2F,G). NANOG transgenic cell lines showed about 8% of cloning efficiency, whereas mock transgenic and wild-type cells showed about 5% of cloning efficiency. These results indicated NANOG over-expression appeared to slightly increase the efficiency of clonal propagation of primate ES cells, but the efficiency was still much lower than that achieved by passaging of ES cells as cell clumps.
Constitutive expression of NANOG suppresses differentiation of ES cells
When mouse ES cells are allowed to form aggregates in a suspension culture, they differentiate to form early embryo-like structures called embryoid bodies (EBs). A previous report showed that over-expression of nanog caused mouse ES cells to inhibit diverting to endoderm lineages when differentiated into EBs (Hamazaki et al. 2004). We therefore investigated whether constitutive expression of NANOG affects the differentiation potential of monkey ES cells by generating EBs from NANOG transgenic ES cells and examining the expression of markers for the endoderm, mesoderm, ectoderm and trophectoderm.
EBs were generated from mock transgenic and NANOG transgenic cells to induce cell differentiation. EBs from NANOG transgenic cells displayed obviously different morphology from those from mock transgenic cells (Fig. 3A). EBs from mock transgenic cells had a distinguishable outer layer, which resembled primitive endodermal cells. On the other hand, EBs from NANOG transgenic cells remained as smooth spheres lacking a distinguished outer layer.
RNA was prepared from 10-day-old EBs and analyzed by RT-PCR for expression of differentiated cell markers, including albumin (ALB), α-fetoprotein (AFP), and GATA4 for endoderm tissue, musashi1 (MSI1) and neurofilament 68 kDa (NF) for the ectoderm, α-myosin heavy chain (α-MHC) for the mesoderm, and CDX2 for the trophectoderm (Fig. 3B). EBs produced from mock transgenic cells showed up-regulation of all the marker genes examined, whereas none of the markers except ectoderm differentiation markers were induced in EBs made from NANOG transgenic cells. Induction of ectoderm markers and reduction of OCT4 were observed in EBs from a NANOG transgenic clone, 6N1, but not in the others. This might be attributable to clonal difference among cell lines. Otherwise, similar results were obtained in three NANOG transgenic cell lines. Altogether, over-expression of NANOG appeared to suppress differentiation of ES cells into endoderm, mesoderm and trophectoderm lineages.
Reduction of NANOG expression induces differentiation of ES cells
To determine whether NANOG is required for maintaining the undifferentiated state of ES cells, we transfected NANOG-specific siRNA into wild-type CMK6 ES cells. Because the cells transfected with NANOG siRNA did not display distinct morphological changes, we performed RT-PCR analysis to detect changes in expression of differentiation markers. As shown in Fig. 4A, NANOG expression was specifically suppressed by RNA interference. In ES cells transfected with NANOG siRNA, the endodermal markers AFP, GATA4 and GATA6 and the trophectoderm marker CDX2 were induced compared to expression in control cells transfected with GAPDH siRNA (Fig. 4B). The mesodermal marker α-MHC was not detected in NANOG knockdown cells, and expression of ectoderm markers, such as MSI1 and NF, was not affected by reduction of NANOG. These results suggest that NANOG maintains the undifferentiated state of monkey ES cells by inhibiting differentiation into extraembryonic tissues, such as trophectoderm and primitive endoderm.
While many studies have investigated the molecular mechanisms underlying the maintenance of the undifferentiated state of mouse ES cells, little is known about the genes that regulate the self-renewal and pluripotency of human and non-human primate ES cells. Although Nanog is known to be an essential factor for maintaining pluripotency in mouse ES cells (Chambers et al. 2003; Mitsui et al. 2003), its role in primate ES cells is still unclear.
Here, we showed that NANOG plays a similar role in monkey ES cells to that in mouse ES cells. In monkey ES cells, NANOG is expressed in undifferentiated ES cells and down-regulated upon differentiation as observed in mouse ES cells (Chambers et al. 2003; Mitsui et al. 2003). Monkey ES cell lines constitutively expressing NANOG maintain their undifferentiated state in the absence of a feeder layer for over 4 months, suggesting that NANOG is sufficient for the self-renewal of monkey ES cells. Recently, a report showed that over-expression of NANOG maintains pluripotency of human ES cells (Darr et al. 2006). This report supports our findings in monkey ES cells.
We used clones with different levels of NANOG expression, but significant differences in maintenance of the undifferentiated state among NANOG transgenic cell lines were not found. We also showed that reduction of NANOG by siRNA results in differentiation into trophectoderm and primitive endoderm in monkey ES cells. Recently, similar results have been reported in human ES cells (Hyslop et al. 2005; Zaehres et al. 2005). Altogether, NANOG is supposed to maintain the undifferentiated state of primate ES cells by inhibiting differentiation into extraembryonic lineages, though our findings on NANOG over-expression would be ideally confirmed by using a regulatable gene expression system.
NANOG transgenic cell lines showed slightly higher efficiency in clonal propagation than mock transgenic cells. Because previous reports suggested that Nanog affects proliferation of mouse cells (Zhang et al. 2005; Loh et al. 2006), NANOG might affect proliferation of primate ES cells to some extent. However, the efficiency of propagation by clonal passaging was much lower than that by standard passaging, showing that over-expression of NANOG is not sufficient for clonal propagation.
Mounting evidence points towards the existence of multiple factors that regulate Nanog expression in ES cells. For example, Oct-4 can regulate Nanog expression through octamer binding sites in the nanog promoter (Kuroda et al. 2005; Rodda et al. 2005). The tumor suppressor protein p53 was also shown to suppress expression of the nanog gene by directly binding to its promoter region, resulting in cell differentiation (Lin et al. 2005). In addition to these intrinsic factors, extracellular signaling molecules involved in the maintenance of self-renewal of primate ES cells have been reported. For example, TGF-β superfamily growth factors were reported to inhibit differentiation of ES cells through SMAD2/3 activation (James et al. 2005). The relationship between these intra- and extra-cellular signaling molecules in regulation of NANOG expression has not been fully addressed. Although some of the molecules involved in self-renewal of ES cells are shared between mouse and primate, significant differences between mouse and primate ES cells have also been reported (Chambers & Smith 2004; Sumi et al. 2004). More work is required to elucidate the molecular mechanisms that regulate expression of NANOG and sustain pluripotency in primate ES cells.
Culture of ES cells and formation of embryoid bodies
The monkey ES cell lines CMK6 and CMK9 were established and cultured as previously described (Suemori et al. 2001; Sumi et al. 2004). Briefly, ES cells were cultured on mitomycin C-treated mouse embryonic fibroblast (MEF) feeder cells in ES medium consisting of DMEM/F-12 supplemented with 20% KnockOutTM Serum Replacement (KSR, Invitrogen, Carlsbad, CA), 2 mm l-glutamine, 1% MEM nonessential amino acids and 0.1 mmβ-mercaptoethanol. The cells were passaged every 3–4 days by dissociation into small clumps of 10–50 cells.
To produce embryoid bodies (EBs), ES cells were treated with a dissociation solution CTK, containing 1 mg/mL collagenase type IV, 0.25% trypsin, 1 mm CaCl2, and 20% KSR in PBS and detached from culture plates by gentle pipetting to avoid dissociation of colonies. The cells were cultured in suspension in Petri dishes. EBs were grown in ES medium for 10 days and then collected for preparation of total RNA.
ES cells were dissociated into small clumps (10–50 cells), and 2 × 104 cells were plated to 35 mm culture plates coated with MatrigelR (Becton Dickinson, Bedford, MA, USA). After culture for 3 days in ES medium, the cells were fixed and were stained for undifferentiated markers. For quantitative analysis of undifferentiated state, colonies with uniform OCT-4 staining were counted as undifferentiated colonies. OCT-4 negative colonies with or without small amount of OCT-4 positive cells on the edge of the colonies were counted as differentiated colony.
For long-term culture, cells were cultured on Matrigel-coated plates in ES medium. The cells were passaged every 3–4 days by dissociation into small clumps of 10–50 cells.
For clonal propagation assay, ES cells were dissociated into single cells using 0.05% trypsin/EDTA, and 1 × 103 cells were plated on to a feeder layer in a 35 mm culture plate. The cells were cultured for 7 days and stained for ALP, and the number of ALP positive colonies was counted.
Construction of expression plasmids and transfection into ES cells
To create a pCAG/PGKneo construct, a neomycin-resistance (neo) cassette driven by a PGK-1 promoter derived from pKJ2 was inserted into a pCAGGS vector (Boer et al. 1990; Niwa et al. 1991). To generate a plasmid encoding human NANOG, cDNA prepared from KhES-1 human ES cells (Suemori et al. 2006), was cloned by RT-PCR using the following primers: forward, 5′- GCGAATTCAACATGAGTGTGGATCCAGCTTG-3′; and reverse, 5′-GCGAATTCTCACACGTCTTCAGGTTGCATG-3′. The PCR product was inserted into the pGEMR-T vector (Promega, Madison, WI, USA), then excised using EcoRI and inserted into an EcoRI-digested pCAG/PGKneo vector. The sequences of these expression plasmids were confirmed by sequencing.
To introduce the expression vectors into monkey ES cells, cells were plated on to a feeder layer 24 h before transfection. Linearized pCAGGS-NANOG or control vector plasmid (10 µg) was transfected into monkey ES cells using LipofectamineTM 2000 (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's protocol. G418 (Sigma, St. Louis, MO, USA) selection (100 µg/mL) was begun 48 h after transfection. After selection for 10 days, the surviving colonies were picked individually to a 24-well plate and expanded.
Western blot analysis
To quantitate NANOG protein expression, ES cells were plated on to extracellular matrix (ECM) prepared from MEFs as previously described (Sumi et al. 2004) in order to remove feeder cells. ES cells were maintained in an undifferentiated state on MEF-ECM at least for 1 week. Lysates prepared from ES cells cultured for 3 days on Matrigel or MEF-ECM were separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane. The membrane was blocked with 5% skim milk and first probed with a rabbit polyclonal antibody against anti-human-NANOG antibody (ReproCELL, Tokyo, Japan), a mouse monoclonal anti-Oct-4 antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), a mouse monoclonal anti-β-Actin antibody (Sigma). After reaction with a horseradish-peroxidase (HRP)-conjugated goat anti-rabbit antibody, and goat anti-mouse antibody (DakoCytomation, Glostrup, Denmark), detection was performed using Western Blotting Luminol Reagent (Santa Cruz Biotechnology Inc.).
Immunocytochemical and Immunocytofluorescense analysis
ES cells were fixed with 3.7% formaldehyde in PBS for 20 min. ALP activity was detected using the VectorR Blue substrate kit (Vector Laboratories, Burlingame, CA, USA). For OCT-4 staining, the cells were permeabilized with 0.2% Triton X-100 in PBS for 10 min. The cells were incubated with anti-Oct-4, anti-SSEA-4 (CHEMICON International Inc., Temecula, CA, USA), or anti-TRA-1-60 (CHEMICON International Inc.) antibodies in PBS. After incubation with an HRP-conjugated secondary antibody and visualization with diaminobenzidine substrate, cells were examined by a light microscopy.
For immunofluorescence staining, cells were cultured for 3–4 days with a feeder layer on glass slides and fixed with 4% paraformaldehyde. After permeabilization with 0.2% Triton X-100 for 5 min, cells were incubated with primary antibodies in PBS containing 1% bovine serum albumin, 3% goat serum and 0.1% Triton X-100 overnight at 4 °C, followed by incubation with secondary fluorescence-conjgated antibodies at room temperature. Alexa FluorTM 488-conjugated anti-mouse immunoglobulin G (IgG) and Alexa FluorTM 546-conjugated anti-rabbit IgG antibodies were purchased from Molecular Probes (Eugene, OR, USA). Cells were washed 3 times with PBS, mounted and examined under a fluorescence microscope.
Small interfering RNA and transfection
NANOG siRNA (SMARTpoolR, DHARMACON, Chicago, IL, USA) and GAPDH siRNA (SilencerTM, Ambion, Austin, TX, USA) were prepared at a concentration of 40 µm, according to the manufacturer's instructions. Twenty-four hours before transfection, ES cells were plated (2 × 103 cells/35 mm culture plate) on Matrigel-coated plates and then cultured in medium conditioned by MEFs. Transfection complex prepared with 10 µL of the siRNA duplex and LipofectamineTM 2000 at a ratio 1 : 1 in 250 µL OptiMEMR (Invitrogen, Carlsbad, CA, USA) was transfected into ES cells. After the cells were cultured with transfection complex for 24 h, the medium was replaced with MEF-conditioned medium. Subsequently, the cells that cultured for 24 h or 48 h in MEF-conditioned medium were collected for RT-PCR analysis.
Total RNA was extracted from ES cells or EBs using the RNeasyR kit (Qiagen, Valencia, CA, USA), according to the manufacturer's protocol. cDNA was synthesized from 2 µg of total RNA using OmniscriptR reverse transcriptase (Qiagen). PCR reactions were optimized to allow semiquantitative comparisons within the log phase of amplification. Gene-specific primers were designed based on published sequences as follows: GATA4 (329 bp), 5′-ATGGGACGGGTCACTATCTG-3′ and 5′-AAGGCTCTCACTGCCTGAAG-3′; GATA6 (392 bp), 5′-GCCAACTGTCACACCACAAC-3′ and 5′-GCGAGACTGACGCCTATGTA-3′, CDX2 (564 bp), 5′-GAACCTGTGCGAGTGGATGCG-3′ and 5′-GGTCTATGGCTGTGGGTGGGAG-3′; NANOG (584 bp), 5′-AAGACAAGGTCCCGGTCAAG-3′ and 5′-CCTAGTGGTCTGCTGTATTAC-3′; albumin (229 bp), 5′-GCATCCTGATTACTCTGACATG-3′ and 5′-CTTGGTGTAACGAACTAATTGC-3′; α-fetoprotein (232 bp), 5′-GGGAGCGGCTGACATTATTA-3′ and 5′-CACCCTGAGCTTGACACAGA-3′; α-myosin heavy chain (413 bp), 5′-GTCATTGCTGAAACCGAGAATG-3′ and 5′-GCAAAGTACTGGATGACACGCT-3′; musashi1 (498 bp), 5′-CGAGCTCGACTCCAAAACAATTGACC-3′ and 5′-TCTACACGGAATTCGGGGAACTGGTA-3′; neurofilament 68 kDa (379 bp), 5′-GTTCAAGAGCCGCTTCAC-3′ and 5′-CACGCTGGTGAAACTGAG-3′; OCT4 (219 bp), 5′-GAGAACAATGAGAACCTTCAGGAGA-3′ and 5′-TTCTGGCGCCGGTTACAGAACCA-3′; GAPDH (454 bp), 5′-GGATTTGGCCGTATTGG-3′ and 5′-TCATGGATGACCTTGGC-3′, and HPRT (516 bp), 5′-ATGCTGAGGATTTGGAAAGGGTGTTTATTC-3′ and 5′-TGAAGTATTCATTATAGTCAAGGGCATATC-3′.
This work was supported in part by The National BioResource Project of the Ministry of Education, Culture, Sports, Science and Technology of Japan and by the Japan Society for the Promotion of Science.