"Regulatory  Networks  in  Embryo-Derived  Pluripotent  Stem  Cells".

Michele Boiani & Hans R. Schöler

Max-Planck Institute for Molecular Biomedicine, Department of Cell and Developmental Biology, Mendelstrasse 7/Von-Esmarch Strasse 56, 48149 Münster, Germany.
correspondence to: Hans R. Schöler:    schoeler@mpi-muenster.mpg.de 

Mammalian development requires the specification of over 200 cell types from a single totipotent cell. Investigation of the regulatory networks that are responsible for pluripotency in embryo-derived stem cells is fundamental to understanding mammalian development and realizing therapeutic potential. Extracellular signals and second messengers modulate cell-autonomous regulators such as OCT4, SOX2 and Nanog in a combinatorial complexity. Knowledge of this circuitry might reveal how to achieve phenotypic changes without the genetic manipulation of Oct4, Nanog and other toti/pluripotency -associated genes.

Multicellular organisms require the existence and precise control of STEM CELLS that maintain tissue HOMEOSTASIS by replacing terminally differentiated, aged or injured cells. In addition to naturally occurring somatic stem-cell systems, such as the haematopoietic system in the fetus and the adult, stem cells can be obtained from peri- and early post-implantation-stage EMBRYOS. From an ontogenic perspective, embryo-derived PLURIPOTENT stem cells might be considered to represent an archetypal genomic state from which other patterns of genome activity ensue later in development. These embryo-derived stem cells have been categorized using different names (EMBRYONIC STEM CELLS (ESCs), embryonal carcinoma cells (ECCs), and embryonic germ cells (EGCs); Fig. 1).

Figure 1: Origin of Stem Cells in the mammalian embryo.

Figure 1: Origin of Stem Cells in the mammalian embryo.

In this figure, the pluripotent cells of the embryo are tracked in green. From left to right, the morula-stage mouse embryo (embryonic day 2.5; E2.5) holds a core of pre-ICM (inner cell mass) cells that turn into ICM cells at cavitation/blastulation (E3–E4). At this stage, embryonic stem cell (ESC) and TROPHOBLAST STEM CELL (TSC) cell lines can be derived in vitro, and implantation occurs in vivo. The two blastocyst-derived stem-cell populations of the mouse are purported to lack the ability to interconvert. In contrast to mouse ESCs, human ESCs can be efficiently directed to differentiate into trophoblast cells, although culture conditions for establishing human TSCs have not been found so far [129]. As the blastocyst fully expands (and undergoes implantation in vivo), the ICM delaminates giving rise to a primitive ectoderm and a primitive endoderm layer. At this stage, pluripotent cell lines that are known as embryonal carcinoma cells (ECCs) can be derived from the primitive ectoderm — whether they are distinct from ESCs has not been resolved. At E6 and subsequent stages, the experimental ability to derive ESCs, TSCs and ECCs from the mouse embryo is progressively lost, and the in vivo embryo will start gastrulating. This process involves the formation of a mesoderm layer between ectoderm and endoderm, and the formation of the primordial germ cells (PGCs). Interestingly, ESCs have been suggested to be the earliest germ cells emerging in vitro [130]. So, the ability of ESCs to 'form' advanced germ-cell stages in vitro [131-133] might not be surprising after all, and argues that ESCs (or their in vivo equivalent) give rise to specialized cell lineages by changes that direct them forwards or backwards along an existing pathway. In line with this idea it is noteworthy that pluripotent cell lines can be derived from later germ-cell stages, namely embryonic germ cells (EGCs) from PGCs, and germline stem cells (GSCs) from neonatal testis [57]. This figure was modified with permission from Ref. 134 © (2002) Elsevier.

http://stemcells.nih.gov/info/scireport/appendixE.asp

This is exemplified by the elements of the most well known signal-transduction pathway in mouse ESCs — the glycoprotein-130 (gp130) receptor-mediated pathway, which is also found in many unrelated tissues. There is considerable interest in finding ways to perpetuate the pluripotency of embryo-derived cell lines, and such knowledge is crucial for many potential biomedical applications related to tissue regeneration ('regenerative medicine'). However, this knowledge is also required to appreciate how, in vivo, stem cells orchestrate the generation, maintenance and regeneration of tissue, while preventing or suppressing aberrant growth and avoiding depletion. These tasks are likely to entail combinatorial complexity and nonlinear, or previously unidentified, interactions between known extracellular signals, second messengers and downstream effectors (Box 1).

We often assume that understanding cellular decisions requires the identification of novel molecules and phenomena. Although unique proteins are necessary for pluripotency in embryonic stem cells (ESCs) (for example, octamer-binding transcription factor-4 (OCT4) and Nanog) these proteins seem to be the downstream effectors of upstream signalling events. At the level of microenvironmental sensing and signal processing, unique fate-specific exogenous factors and intracellular signalling proteins seem to be rare. Several lines of evidence indicate that molecular signals might exert their developmental control through the magnitude and/or duration of their stimulation of membrane-spanning proteins. That is, through non-qualitative and nonlinear interactions whereby known, rather than new, molecules exert effects that are dependent on the dose and mode of their exposure to stimulating signals [107-109].

Embryo-derived stem cells would not be of practical interest if they could not re-enter development. Although the ESC era began officially in 1981, the journey to find whether mammalian embryos could be altered by exogenous cells that could re-enter development had started earlier (Box 2). 

Investigators had tried to manipulate mouse-embryo development using native cells [110, 111] or embryonal carcinoma cells (ECCs) of explanted TERATOMA [112-116], which could integrate into the embryo after injection into the blastocyst cavity. After being placed in such a microenvironment, ECCs can lose their malignant phenotype and enter normal development [113, 117] (although tumours might recur in the adults [116]). Some ECC cell lines have been maintained for eight years as ascites that were transplanted every two to three weeks into a new host [114], and which retained the ability to participate in normal mouse development after blastocyst injection. But other ECC cell lines lost the ability to differentiate [118], or differentiated only under specialized conditions [119], and accumulated chromosomal aberrations [116]. More recently, F9 and P19 ECCs were described that had subtle genetic changes compared to mouse euploid totipotent teratocarcinoma (METT)-1 cells [120], which were not previously detected due to the resolution of the assays [121]. Results would be easier to obtain with a robust, pluripotent cell line that could be stably maintained in vitro for a long time. This was accomplished independently by three groups in 1981 (Refs 2, 3) and in 1984 (Ref. 122) with the 'creation' of a new type of stem cell, embryonic stem cells (ESCs), in the culture dish.

Pluripotent cells are first recognized in the ICM of the mouse blastocyst at embryonic day 3.5 (E3.5), and are present as late as in the pre-gastrulating embryo (Fig. 1). Until approx E5.5, at least some primitive ectoderm cells remain pluripotent. In the rapidly changing embryo, these are not necessarily obvious as stem-cell populations, nor do they exist for very long. However, their stem-cell capacity can be revealed by experimental intervention that essentially 'captures' the cells in a stem-cell state. Martin used normal mouse blastocysts cultured in ECC-conditioned medium [2], whereas Evans and Kaufman used a co-culture system in which cells derived from the ICM of delayed mouse blastocysts were grown on a layer of mitotically inactivated mouse embryonic fibroblast (MEF) FEEDER CELLS in the presence of blood serum [3]. In the absence of conditioned medium, the self-renewing and pluripotent ESC phenotype cannot be preserved, which leads to preferential neuronal differentiation.

Pluripotency of ESCs can be judged by their ability to integrate, after transplantation, into the ICM of E3.5 blastocysts and, similar to the ICM, to express alkaline phosphatase, E-cadherin, stage-specific embryonic antigen-1 (SSEA1) and octamer-binding transcription factor-4 (OCT4)4 (Table 1). Pluripotency has only been shown conclusively in the mouse — ESCs are completely integrated into a developing embryo after intra-blastocyst injection and produce a high rate of CHIMAERISM in tissues of the developing fetus. Depending on the definition of 'extra-embryonic', ESCs also have a limited contribution in extra-embryonic tissues [5, 6]. In fact, through the ICM that they colonize, ESCs can contribute to extra-embryonic MESODERM and therefore to composite annexes such as the amnion, the allantois and the yolk sac. Mouse ESCs do not give rise to the TROPHECTODERM, which is consistent with the first cell-lineage decision that separates ICM and trophectoderm in the blastocyst.

In a similar way to mice, cell lines resembling ESCs have also been established from certain primates [7], including humans [8]. However, cell lines from embryos of other mammalian species (such as rabbits, pigs, cattle and sheep) have been notoriously more difficult to establish or propagate without losing pluripotency [9] or to assay conclusively for developmental potency (using intrablastocyst injection), and therefore should be defined as 'ESC-like cells'. There is not even homogeneity among mice, and ESCs from certain mouse strains can be isolated and maintained in culture with relative ease, whereas others require more elaborate culture conditions [10]. Even from a single mouse blastocyst, diverse ESC cell lines can arise [11]. The ability to derive ESCs after somatic-cell nuclear transfer (SCNT) — achieved first in mice [12, 13] and now in humans [14] — might provide a more uniform source of ESCs.

Signalling that controls nuclear decisions

In addition to general pathways of signal transduction that operate in most cell types, such as extracellular matrix (ECM) signalling that is based on cell-membrane receptors of the integrin family (for example, integrin beta1; Ref. 15), several exogenous factors that are involved in nucleus-directed signalling pathways are known to modulate stem-cell pluripotency both in vivo and in vitro. Remarkably, the genes that express proteins that are involved in signal transduction and regulation make up the largest proportion of the TRANSCRIPTOME of ESCs, and comprise 17% of the genes that are enriched in pluripotent versus differentiated human ESCs. These genes encode several ligand–receptor pairs and secreted inhibitors of the fibroblast growth factor (FGF), transforming growth factor (TGF)-beta/bone morphogenetic protein (BMP), Nodal and Wingless type (WNT) signalling pathways [16, 17].

The LIF signal cascade. In vitro, leukaemia inhibitory factor (LIF; also known as differentiation inhibitory activity, Dia [18]) is essential for maintaining the undifferentiated state of mouse ESCs. Interestingly, LIF is only able to sustain ESCs in the presence of serum, suggesting that additional factors are required. Provided either by a feeder layer of MEFs or a cell line (for example, the STO-SNL cell line) or as a human recombinant protein, LIF exerts its effect by binding to the LIF receptor (LIFR)–gp130 heterodimer receptor on the cell membrane and activating the signal transducer and activator of transcription-3 (STAT3; Fig. 2).

Figure 2:  Combinatorial signalling pathways involved in maintaining mouse ESC pluripotency.

Figure 2:  Combinatorial signalling pathways involved in maintaining mouse ESC pluripotency.

Cell-surface receptors initiate signals that are conveyed (thin black lines) to the nucleus and affect key pluripotency transcription factors such as octamer-binding transcription factor-4 (OCT4) and Nanog, and self-renewal transcription factors such as signal transducer and activator of transcription-3 (STAT3). So far, only the leukaemia inhibitory factor (LIF) receptor (LIFR)–STAT3 pathway has been defined in detail. The cytokine LIF functions by binding to LIFR at the cell surface, which causes it to heterodimerize with another transmembrane protein, glycoprotein-130 (gp130). This is followed by the activation of kinases that amplify and drive the signal to the nucleus, or that recruit other factors for docking to the activated LIFR–gp130 receptor. On binding LIF, the intracellular domains of the LIFR–gp130 heterodimer can recruit the non-receptor Janus tyrosine kinase (JAK) and the antiphosphotyrosine immunoreactive kinase (TIK) and become phosphorylated. The phosphorylated intracellular domains of the LIFR–gp130 heterodimer function as docking sites for proteins that contain Src-homology-2 (SH2) domains, which include the transcription factor STAT3. At the nuclear level, STAT3, OCT4 and Nanog cause changes in gene expression that result in (pointed arrow) or counteract (truncated line) phenotypic traits of embryonic stem cells (ESCs). OCT4 functions in connection with SRY-related high-mobility group (HMG)-box protein-2 (SOX2) and other cofactors to determine whether target genes are activated or repressed. Aspects of this figure are speculative as, in some cases (bone morphogenic protein-4; BMP4), the surface receptors are known but the transducers are not, and in others both have yet to be identified (OCT4, Nanog). The current lack of information is highlighted by a question mark. GAB1, GRB2-associated binding protein-1; GSK3, glycogen synthase kinase-3; Id, inhibitor of differentiation, JAK, janus kinase; MEK, mitogen-activated protein kinase (MAPK) and extracellular signal regulated kinase (ERK) protein kinase; SMAD, similar to mothers against decapentaplegic homologue; SHP2, SH2-domain-containing protein tyrosine phosphate-2; WNT, wingless type protein.

Six key STATs (STAT1–6) have been identified and — with the exception of STAT4, which is restricted to myeloid cells and testis — STATs are expressed ubiquitously [19]. In vivo studies depict a different scenario, in which the LIF cascade is not required for pre-gastrulation mouse development (Fig. 3).

Figure 3:  The phenotypic effects of gene-targeting experiments in mice.

Figure 3:  The phenotypic effects of gene-targeting experiments in mice.

This figure depicts the phenotypic effects of gene-targeting experiments in mice that provided insights into the pluripotency of embryos and embryonic stem cells. Oct4-/- (octamer-binding transcription factor-4), Nanog-/-, Stat3-/- (signal transducer and activator of transcription-3), gp130-/- (glycoprotein-130), Lif-/- (leukaemia inhibitory factor) and Lifr-/- (LIF receptor) embryos can develop to various stages in vivo— as highlighted by the outer ring in the figure, but they all fail (truncated line from the ring to the centre) to yield ESCs (embryonic stem cells) in vitro (see centre of the figure). So, ESCs need factors that neither the ICM (inner cell mass) nor the primitive ectoderm require. It is important to make clear that ICM and ESCs might be equivalent but are not equal. ESCs might only represent some aspects of the natural embryo, and studying cells and phenomena in vivo is indispensable. E1.5, E3.5, E8.5 and E18.5 represent the number of days of embryonic (E) development, although developmental stages following E10.5 should properly be referred to as fetal.

Although LIF is required in vitro to derive and preserve pluripotent ESCs, in vivo mouse embryos that lack LIF can develop to a stage subsequent to ESC derivation. These contrasting findings suggest that alternative pathways might be involved in maintaining pluripotency in vivo and in vitro. Alternatively, the pluripotent-cell population of the blastocyst is transient under normal circumstances, so disruption of a cytokine pathway in vivo might not exert its detrimental effect. In support of this are experiments that challenge the blastocyst with unusual circumstances known as 'DIAPAUSE' (in which development becomes slower, implantation is delayed and the embryo must preserve its pluripotent founder-cell population to be able to implant and later form a fetus). After the end of diapause, Lif-/- blastocysts are unable to resume development [20].

In the presence of LIF in the culture medium, STAT3 binds to phosphotyrosine residues on activated LIFR–gp130 heterodimer receptors [21] and undergoes phosphorylation and dimerization itself. Phosphorylated STAT3 dimers then translocate to the nucleus, where they function as transcription factors. A conditionally active form of STAT3 that is induced by tamoxifen (a STAT3–oestrogen-receptor (ER) fusion protein) [22] can be used to maintain the pluripotent phenotype when LIF is removed from the culture media. However, ESC cell lines that express STAT3–ER at a lower level are not fully maintained in the undifferentiated state by tamoxifen. So, a minimal dose of STAT3 seems to be required for ESC propagation and pluripotency.

In addition to the pathway leading to STAT3 nuclear translocation, the intracellular domains of the LIFR–gp130 heterodimer can, on binding LIF, recruit the nonreceptor tyrosine kinase Janus (JAK) and the antiphosphotyrosine immunoreactive kinase (TIK) and activate other pathways (Fig. 2). The treatment of ESCs with LIF also induces the phosphorylation of extracellular signal-regulated protein kinases, ERK1 and ERK2 (Ref. 23), and increases mitogen-activated protein kinase (MAPK) activity [24]. This occurs through activation of a bridging factor between the cytokine and the MAPKs — the widely expressed tyrosine phosphatase SRC-HOMOLOGY-2 (SH2)-DOMAIN-containing protein tyrosine phosphatase-2 (SHP2; Ref. 25).

Despite the ability of LIF–STAT3 signalling to support the self-renewal of mouse ESCs, it does not prevent the differentiation of human ESCs [26] (Table 1). In particular, even with human-derived LIF in the culture medium, OCT4 gene activity is reduced and human ESCs differentiate [27]. It is unclear whether LIF has different effects in human ESCs. For example, it might not be sufficiently effective at supporting undifferentiated growth or it might be actively inhibited [28]. Although the LIF–(LIFR–gp130)–STAT3 signalling pathway is not universal (the human requirement for this pathway is not equivalent to the mouse requirement) and it is not obligatory for mouse ESC pluripotency (forced expression of Pem can compensate for the absence of LIF; see below), it provides a striking example of how activation of a cytokine pathway can be used to alter cell fate and potency. Ultimately, this indicates that it is possible to achieve phenotypic changes without the genetic manipulation of important pluripotency-associated genes.

BMP4. Our current knowledge of bone morphogenetic protein-4 (BMP4) and related factors has numerous gaps, and there is no definitive view of how this factor fits into a general molecular scheme of pluripotency. Similar to LIF, BMP4 is considered to be a key anti-neurogenesis factor in the embryo, and its affect on cultured ESCs is reminiscent of LIF, in that ESCs differentiate into neurons in its absence [29]. Furthermore, mouse embryos without BMP4 develop past the stage at which ESCs can be derived [30]. In the presence of LIF, BMP4 contributes to the LIF cascade, enhancing the self-renewal and pluripotency of ESCs is by activating the gene encoding the transcription factor SMAD4 (similar to mothers against decapentaplegic homologue-4), which, in turn, activates members of the Id (inhibitor of differentiation) gene family ]29] (Fig. 2). This interaction is facilitated in the presence of serum. By contrast, in the absence of LIF, BMP4 counteracts the LIF cascade, interacting with different SMAD transcription factors (for example, SMAD1, 5 and 8) that have an inhibitory effect on the Id genes. Results also indicate that BMP4 can regulate cell fate in relation to cell density [31] by activating distinct cytoplasmic signals. In conclusion, BMP4 seems to be the serum-derived factor in ESC cultures that conatin serum + LIF [29]. And the balance between LIF and BMP4, and how this balance of signals is sensed at the cell surface, are jointly responsible for maintaining the undifferentiated state of mouse ESCs. Notably, BMP proteins are suggested to be targets of another ligand cascade that is initiated on binding of WNT to its receptor (see below) [32].

WNT proteins. WNT proteins are secreted glycoproteins that have widespread roles in tissue differentiation and organogenesis [33]. In vivo, Wnt5a and Wnt11 are expressed in mouse embryos that undergo the morula-to-blastocyst transition, but they are only weakly expressed or are not expressed at all in embryos that do so in vitro. This is consistent with the morbidity of embryos that are cultured in vitro. The upregulation of Wnt11 is temporally coordinated with the surge of maternal oestradiol at E4 — the time of implantation [34]. The canonical WNT pathway is activated on binding of the WNT protein to the Frizzled receptor on the cell membrane (Fig. 2). Activation of the pathway leads to inhibition of glycogen-synthase kinase-3 (GSK3), subsequent nuclear accumulation of beta-catenin and the expression of target genes. Sato and colleagues used a new and specific reversible inhibitor of GSK3, 6-bromoindirubin-3'-oxime (BIO), to demonstrate that activation of the canonical WNT pathway maintains the undifferentiated phenotype in both mouse and human ESCs, and sustains expression of the pluripotent-state-specific transcription factors OCT4, REX1 (also known as zinc-finger protein-42; ZFP42) and Nanog [27] in the absence of supplemented LIF. The genetic manipulation of WNT signalling in ESCs — by either inactivating the adenomatous polyposis coli (APC) complex (a multiprotein complex tumour suppressor that mediates signal transduction from the WNT receptor on the cell surface to beta-catenin in the nucleus) or by introducing a dominant-active form of beta-catenin — results in the inhibition of neural differentiation in vitro [32]. This also occurs after activation of the downstream targets of WNT signalling, such as cyclin-D1 (Ref. 35), MYC [36] and BMP proteins [32]. Neural differentiation can be partially restored by addition of the BMP4 antagonist Noggin [32].

Emerging transcriptional regulators of ESCs. Most recent studies have pointed to one signalling pathway through which nutrients and environmental cues — that is, amino acids [37, 38] and inositols 39] — regulate survival of early mouse embryos and ESCs. The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that was originally identified as a target of rapamycin in Saccharomyces cerevisiae and was then found to be highly conserved among eukaryotes [40, 41]. This signalling pathway ultimately regulates the interaction between mTOR and two downstream substrates, eukaryotic translation-initiation factor 4E (eIF4E)-binding protein-1 and ribosomal-protein-S6 kinase [42]. Inositols also participate in another pathway that is centered on the phosphatase and tensin homologue deleted on chromosome 10 (Pten) gene.

Inactivation of PTEN increases the proliferation of mouse primordial germ cells and enhances the derivation of EGCs [43]. In ESCs, it leads to increased entry into S phase and increased cell survival [44]. PTEN is a phosphatase that has a wide range of functions [45] and its lipid-phosphatase activity is associated with tumour suppression. The chief substrate of PTEN is phosphatidylinositol-3,4,5-triphosphate (PtdIns(3,4,5)P3). PTEN therefore terminates phosphatidylinositol 3-kinase (PI3K) signalling by dephosphorylation of the PtdIns(3,4,5)P3 second messenger [43]. The serine/threonine kinase AKT is a downstream effector of PI3K that phosphorylates mTOR in vitro, indicating a direct linkage between mTOR and the PI3K–AKT pathway, which is negatively regulated by PTEN [46]. Through a double-gene approach, in which both Pten and Akt1 were mutated, it was demonstrated that AKT1 upregulation is essential for the tumorigenic phenotype observed for Pten-null ESCs [44].

Also emerging is the function of microRNAs (miRNAs) in the transcriptional regulatory circuitry of ESCs. Recent studies indicate that miRNAs have important roles in gene regulation as more than a third of mammalian protein-encoding genes are conserved miRNA targets [47, 48]. Moreover, ESCs that lack the machinery that processes miRNA transcripts are unable to differentiate [49]. However, this new topic should be discussed in a more specific report than this review.

Nuclear (transcription factor) pathways

Signalling pathways eventually lead to the nucleus and, in the case of ESCs, result in the transcriptional induction or repression of genes that are responsible for implementing stem-cell pluripotency. Recurring questions relate to the existence of other, as-yet-unidentified, 'stemness' genes (that is, genes that confer stem-cell properties on a cell) and how to find them [50, 51]. Oct4 was recognized as one such gene long before most of the current methods of gene-expression analysis were available (Box 3). 

The protein encoded by Oct4, also known as Oct3 and Pou5f1 was first described independently by Schöler, Rosner and Okamoto [53-55]. The mouse gene — located at the t-locus on chromosome 17 — is specifically expressed in the germline and encodes a homeodomain-containing protein that was called 'OCT' because of its ability to bind to and activate transcription through the 'octamer' DNA sequence 5'-ATGCAAAT-3'. Members of this transcription-factor family share a conserved DNA-binding domain, the POU domain, which was originally identified in the transcription factors PIT1, OCT1, OCT2 and UNC86. The POU domain consists of two structurally independent subdomains — the POU-specific domain (POUS) and the homeodomain (POUH), which are connected by a flexible linker. Interestingly, OCT4 binding of octamer-containing DNA sequences can either repress or activate transcription depending on the flanking sequences [123], which is consistent with its critical role in maintaining pluripotency and controlling differentiation.

Methods of SUPPRESSION-SUBTRACTIVE HYBRIDIZATION have been used to identify several putative downstream genes of Oct4 (Refs 61,62). These include the gene encoding the extracellular matrix (ECM) protein osteopontin (Spp1) [124], which is expressed in the primitive endoderm; heart and neural crest derivatives expressed-1 (Hand1), which is expressed in early trophectoderm [60, 64]; fibrobast-growth factor-4 (Fgf4) which is expressed in the ICM [125[; F-box protein-15 (Fbx15), which is expressed in embryonic stem cells and later in testis [88]; and Rex1, which is also known as zinc-finger protein-42 (Zfp42; Ref. 79). It is becoming more and more clear that this crucial transcription factor is not sufficient on its own, but that the presence of interacting cofactors determines whether the target genes are activated or repressed by OCT4. For instance, a possible viral partner that is required for remote transcriptional activation by OCT4 is the adenoviral protein E1Am [126, 127]. SRY-related high-mobility group (HMG)-box protein-2 (SOX2) stimulates undifferentiated-embryonic-cell transcription factor-1 (Utf1) and Fgf4 gene expression [64, 128] and represses OCT4-mediated activation of Spp1 (Ref. 124) by acting cooperatively with OCT4.

After the initial report of Wang and colleagues [68] the role of ENK (early embryo-specific NK, where NK represents NK-2, which is a synonym of the Drosophila melanogaster gene ventral nervous system defective) remained unclear. Chambers and colleagues [70] took advantage of leukaemia inhibitory factor (LIF) receptor (LIFR)-deficient embryonic stem cells (ESCs). Rare ESCs that could survive and form colonies on mouse embryonic fibroblast (MEF) feeder cells without binding LIF were expanded in culture, and cDNA libraries were constructed from the ESC–MEF co-cultures. The inserts from the libraries were subcloned into episomal-plasmid vectors that were based on the replicative function of the polyoma virus. The vectors were used to transfect ESCs that were expressing the polyoma large T antigen, and these cells were then selected for growth in the absence of LIF. This led to the isolation of surviving, LIF-independent cells, which were found to express Nanog that was not integrated in the nucleus. Using a different strategy, Mitsui and colleagues [71] searched for genes that were enriched in undifferentiated ESCs by performing digital (in silico) differential display of expressed sequence tag (EST) libraries in undifferentiated ESCs versus somatic tissues, as well as in undifferentiated versus differentiated ESCs. In the Nanog overexpression experiments performed by Chambers and colleagues, a Nanog transgene was used and loxP sites flanked this transgene. This elegant trick allowed the Nanog transgene to be removed by Cre/loxP RECOMBINATION. As the removal of the Nanog transgene restored a dependence on LIF for pluripotency, it also showed that a secondary mutation that might have been caused accidentally by previous genetic manipulations (Cre/loxP) was not responsible for the effect caused by the Nanog overexpression. The overexpression of Nanog also renders ESCs independent of bone morphogenic protein-4 (BMP4) (which is usually provided together with LIF in the absence of serum). In contrast to wild-type ESCs, which markedly downregulate the inhibitors of differentiation (Id) genes in serum-free, BMP4-free and LIF-free culture conditions, ESCs that overexpress Nanog contain reduced but appreciable (Id1) or even increased (Id2 and Id3) mRNA levels of Id genes [29].

The transcription factor OCT4. OCT4 is a POU-DOMAIN transcription factor that is expressed by all pluripotent cells during mouse embryogenesis, and is also abundantly expressed by undifferentiated mouse ESCs and ECC cell lines [52-55], as well as in EGC cell lines [56]. So far, however, experiments show that Oct4 expression is generally weaker in germline stem cells (GSCs) [57]. OCT4 has also been established as a marker for human pluripotent ESCs — microarray and quantitative reverse-transcription (Q-RT)-PCR analyses indicate that it is downregulated in the differentiated versus the undifferentiated state of stem cells [16]. This observation should be treated with caution, as the observation that neuronal differentiation occurs with sustained expression of Oct4 (Ref. 58) disputes the widespread belief that downregulation of Oct4 is required for the differentiation of somatic lineages. OCT4-deficient mouse embryos only develop to a stage that looks like a blastocyst, and although cells are allocated to the interior, these blastocysts are actually only composed of trophectoderm cells (Fig. 3). As these structures lack a genuine ICM, they cannot be used to produce ESC cell lines [59]. OCT4 has therefore been viewed as being involved in preventing trophectoderm and perhaps somatic-cell differentiation from the ICM, as well as being crucial for maintaining the pluripotent state during embryonic development. In mouse ESCs, the manipulation of Oct4 expression through inducible or repressible Oct4 transgenes indicates that the relative amount of OCT4 protein ultimately determines cell fate [60]. However, the target genes that are actually responsible for implementing OCT4 decisions are only partly known [61, 62]. Similarly, the potential interactions of OCT4 with other (co)factors — except for SOX2 (SRY-related high-mobility group (HMG)-box protein-2; Fig. 2) — remain unclear.

The transcription factor SOX2. SOX2 is a member of the HMG-domain DNA-binding-protein family that is implicated in the regulation of transcription and chromatin architecture [63]. SOX2 forms a ternary complex with either OCT4 or the ubiquitous OCT1 protein on the enhancer DNA sequences of Fgf4 (Ref. 64). This allows SOX2 to participate in the regulation of the ICM and its progeny or derivative cells. Consistent with this role, Sox2 is expressed in ESCs, but it is also expressed in neural stem cells. When gene targeting was used to inactivate Sox2, the primitive ectoderm was defective, but it could be rescued (albeit only to survive longer) by the injection of wild-type ESCs into the Sox2-/- blastocysts [65]. By examining the phenotypic consequences in Sox2-mutant embryos and in chimaeras, a cell-autonomous requirement for the gene was found in both primitive and extra-embryonic ectoderm (the MULTIPOTENT precursors of all embryonic and of the TROPHOBLAST cell types, respectively). Similar roles were also found for another gene that is expressed in the founder-cell population of the embryo — Foxd3 (a transcription factor gene that is also known as Genesis and belongs to the forkhead family of transcription factors with a winged-helix DNA-binding structure). Marked loss of primitive ectoderm cells occurred in the Foxd3-/- blastocysts, and Foxd3-/- ESCs could not be derived, although expression of Oct4, Sox2 and Fgf4 appeared to be normal in the Foxd3-/- ICM [66]. Data suggest a direct interaction between OCT4 and FOXD3 as is seen to occur between OCT4 and SOX2 (Ref. 67), but this requires further substantiation. These phenotypes might not represent the primary functions of these genes, as an earlier role of SOX2 and FOXD3 within the ICM might be masked by the persistence of maternal protein. Altogether, the data indicate that maternal components could be involved in establishing early cell-fate decisions, and that a combinatorial code, requiring SOX2 and OCT4 (Fig. 2), specifies the first three cell lineages that arise soon after implantation.

The transcription factor Nanog. Investigations into the maintenance of pluripotency led to the identification of a novel gene encoding a HOMEODOMAIN-containing transcription factor that confers a LIF-independent ability for cell renewal and pluripotency on mouse ESCs. This gene was first described by Wang and colleagues as ENK [68] (early embryo-specific NK, where NK represents NK-2, which is a synonym of the Drosophila melanogaster gene ventral nervous system defective) due to its homology with members of the NK gene family [69]. The presence of a homeodomain had indicated that ENK functions as a transcriptional regulator. The original name was changed after independent cloning by two laboratories and subsequent functional analysis. It was named Tir nan Og or Tir Na Nog, after the mythological Celtic land of the 'ever young' [70, 71] (Box 4).

Nanog mRNA is present in primordial germ and EGCs, is also revealed in situ by riboprobe hybridization of genital ridges [70], and it can be detected, by using RT-PCR, in adult tissues [72]. Nanog protein was not found in Stella-positive mouse primordial germ cells [73], despite Stella itself (which is also known as PGC7 or DPPA3) being considered a marker of pluripotency[74]. Nanog mRNA was not detected at later stages — neither in mature ovaries and testes [71] nor in GSC cell lines derived from neonatal testis [57]. This suggests that the function of Nanog in germ cells is progressively extinguished as they mature, and that the very few molecules that are detected by highly sensitive methods at later stages have an insignificant, if any, biological role. Undifferentiated, wild-type ESCs normally express Nanog. However, the physiological levels of Nanog in ESCs do not prevent their differentiation after LIF withdrawal. So, under physiological conditions, Nanog seems to be one of several factors that are expressed in pluripotent cells and are downregulated at the onset of differentiation. Although expressed sequence tags (ESTS) of NANOG have been found to occur more frequently in undifferentiated than in differentiated human ESCs, NANOG was not in the group of 532 genes that were identified as being specific to human ESCs [16]. Nanog-/- mouse ESCs differentiate slowly into extra-embryonic endoderm lineages, which is consistent with the absence of a primitive ectoderm in Nanog-/- embryos that were analysed at E5.5 in vivo [71] (Fig. 3). So Nanog expression is responsible for the maintenance of a primitive ectoderm in the embryo. Unlike wild-type ESCs and those forced to express Oct4, mouse ESCs that are overexpressing Nanog are resistant, but not completely refractory, to the spontaneous differentiation that occurs after LIF withdrawal or by chemical induction (for example, after treatment with 3-methoxybenzamide or all-trans retinoic acid). The persistence of Nanog therefore seems to delay, rather than block, the differentiation of ESCs; that is, the threshold of differentiation is increased rather than abolished. In contrast to Nanog overexpression, the reduced expression seen in Nanog+/- ESCs results in labile pluripotency whereby spontaneous differentiation is more likely to occur after longer times spent in culture ('passages') [73]. So, the amount of Nanog per cell is crucial for stably maintaining an undifferentiated state even in the presence of LIF. Similar to OCT4, it has been proposed that Nanog might regulate differentiation through the transcriptional repression of genes that promote differentiation (for example, endodermal GATA-binding protein-6, Gata6) a proposal that was based on the presence of Nanog-binding sites in the control regions of these genes as well as that of Rex1/Zfp42 (Ref. 71). Little is known about regulation of the Nanog gene, except that the transcriptional activator and tumour suppressor p53 binds to the promoter of Nanog, thereby enabling p53-dependent suppression of Nanog expression [75]. Loss of p53 results in a 100-fold increase in susceptibility to testicular teratoma [76]. So teratoma formation might be due to derepression of genes such as Nanog. In agreement with this, GSCs in vitro gave rise to multipotent GSCs when isolated from p53-deficient, but not from wild-type mice [57]. Cues or agents that induce DNA damage also downregulate Nanog and this has been shown to maintain genetic stability in ESCs by inducing the differentiation of ESCs into other cell types. Interestingly, a p53-binding element was also found directly upstream of PTEN [77] (see above).

STAT3, OCT4, SOX2 and Nanog are not alone. In the absence of LIF, the forced expression of Pem [78] — another gene besides Oct4 and Nanog that produces a homeodomain-containing protein — blocks the differentiation of ESCs both in vitro and in vivo (Nanog overexpression also blocks the differentiation in the absence of LIF). The block of ESC differentiation resulting from the forced expression of Pem was caused by either domain region of PEM — the homeodomain as well as the non-homeodomain — although the latter appears to be the more responsible. ESCs that constitutively express Pem are unable to form tumours after subcutaneous injection, which indicates a role for PEM in regulating the transition between undifferentiated and differentiated cells of the early mouse embryo. Due to the fleeting existence of either the ICM or the primitive ectoderm cell population, the early role of PEM in vivo is less apparent than its later role in the post-implantation stages. After implantation, PEM expression is found in the ectoplacental cone, PRIMITIVE ENDODERM (also known as the hypoblast) and both parietal and visceral endoderm (but not in primitive ectoderm), thereby indicating that PEM has extra-embryonic functions.

Combinatorial signals for pluripotency

In mouse ESCs, the activation of the LIFR–gp130 pathway by cytokines in the ECM, and the maintenance of Oct4 and Nanog expression are the key elements of a genetic programme that confers 'stemness'. As we have discussed above, other pathways (such as those involving WNT, BMP4 and inositol) and factors (such as SOX2 and mTOR) are superimposed onto the key elements (Fig. 2), thereby adding combinatorial complexity. Having reviewed the elements of the programme separately, we now describe a combinatorial model of their function.

Combinatorial signalling of Nanog and OCT4. Increased expression of Oct4 causes mouse ESCs to differentiate into extra-embryonic endoderm and mesoderm, whereas increased expression of Nanog enhances self-renewal and maintenance of the undifferentiated state. On the other hand, lowered expression of Oct4 causes mouse ESCs to differentiate into trophectoderm, as defined by cell morphology and expression of marker genes (for example, caudal-type homeobox transcription factor-2, Cdx2). However, Nanog-deficient embryos do not form a primitive ectoderm and Nanog-/- cells differentiate into visceral and parietal endoderm (GATA6- and GATA4-positive cells, respectively). Taken together, these observations indicate that the two genes operate independently of one another but that their functions are coordinated. The relationship between OCT4 and Nanog was therefore examined experimentally.

The overexpression of Nanog in cells that lacked both of the endogenous Oct4 alleles and that were propagated by a doxycycline-responsive Oct4 transgene [70] could not reverse the differentiation that occurred in the presence of doxycycline — that is, in the absence of OCT4. Therefore, the expression of Oct4 is required for Nanog-mediated self-renewal in ESCs. However, Nanog expression was maintained in the blastocyst ICM cells from Oct4-deficient embryos, although whether they carried any residual pluripotency is doubtful. This indicates that other pluripotency-mediating cell-specific factors might contribute to alternative transcriptional regulatory mechanisms for Nanog. It has been shown that OCT1 and OCT6 are expressed in pluripotent embryonic cells [79, 80] and have the capacity to bind to octamer elements in the Fgf4, Sox2, undifferentiated-embryonic-cell transcription factor-1 (Utf1), and Rex1promoter regions [81, 82].

In summary, the primary function of OCT4 and Nanog might be the repression of embryonic-cell differentiation and a combinatorial signal from both proteins leads to renewal and pluripotency of the primitive ectoderm. Assuming that OCT4 and Nanog sequentially suppress the differentiation of the ICM into extra-embryonic lineages — first to trophectoderm (OCT4) then to primitive endoderm (Nanog) — as their levels drop in the primitive ectoderm, further developmental control genes might mediate the subsequent differentiation of the primitive ectoderm, such as Sox2 (Ref. 65) and Foxd3 (Ref. 66).

Nanog and STAT3 function in parallel. In cells overexpressing Nanog, the phosphorylation levels, kinetics and distribution of STAT3 do not differ in comparison to wild-type ESCs. Similarly, Nanog levels are not affected in cells with altered STAT3 signalling [70] — for example, if the gp130 co-receptor is mutated and lacks the ability to recruit STAT3, or if gp130 is hyperactive after the induction of the target gene suppressor of cytokine signalling-3 (Socs3). Similarly, inhibiting JAK, an upstream activator of STAT3, did not affect the ability of Nanog to promote ESC renewal. In summary, Nanog does not activate STAT3, and STAT3 does not induce Nanog expression. However, the activation of STAT3 through LIFR enhances the proliferation of Nanog-overexpressing cells and alters their morphology — they change from being evenly distributed in a monolayer to being tightly clustered and adhesive. So these signals might be adding to each other, but are not identical. Similarly, inhibition of ERK enhances the ability of LIF to promote the self-renewal of ESCs. However, inhibiting ERK does not change the effects that altering Nanog expression has on ESCs. Taken together, evidence from overexpression and inhibition experiments that involved Nanog, gp130, JAK and other molecules indicates that Nanog and STAT3 probably function in parallel, but not in the same pathway, although they might have common targets.

No direct interaction between OCT4 and STAT3. Expression of a dominant-negative form of Stat3 induces the differentiation of ESCs [21]. Furthermore, in the absence of LIF, expression of the conditionally active STAT3 fusion protein STAT3–ER prevents differentiation and promotes ESC self-renewal after tamoxifen treatment [22]. The constitutive expression of other genes, such as Nanog and Pem, also prevents the LIF-withdrawal-induced differentiation of ESCs. By contrast, the forced maintenance of Oct4 expression cannot prevent the differentiation of ESCs that is induced by either the withdrawal of LIF or the expression of dominant-negative STAT3 (Ref. 60). These findings purport a lack of interaction between OCT4 and STAT3 and the occurrence of an interaction between Nanog and STAT3, the nature of which remains to be defined.

WNT and OCT4. OCT4 has a crucial function and several known effects in embryo development. For example, Oct4 expression is known to increase in germ-cell nuclear factor (Gcn f)-deficient mouse embryos (which is consistent with Oct4 gene derepression) [83]. Despite this, however, OCT4 has been a genetic 'orphan' because of the lack of an association with any signalling pathway or any other gene that might function upstream of it. However, the WNT signalling pathway might provide some clues to how extracellular and cell-surface events have an effect on Oct4. Wnt3-deficient embryos express high levels of OCT4 (Ref. 84). The entire Wnt3-deficient embryo remains bilayered at gastrulation and continues to express Oct4 throughout, which indicates that the loss of signalling from the WNT receptor at the cell surface results in perpetuation of the undifferentiated state. APC, which forms part of a multiprotein tumour suppressor that is associated with colorectal cancer, mediates signal transduction from the WNT receptor on the cell surface to beta-catenin in the nucleus [85]. Alleles that represent increasingly severe mutations in Apc — as judged by elevated levels of active beta-catenin — increasingly affect the developmental potential of ESCs, and restrict the differentiation potential of ESCs in vivo and in vitro [86]. A threshold model has been put forward whereby Apc alleles with various strengths (due to different mutations) would result in different levels of beta-catenin in the nucleus, which then cause phenotypic changes [32]. This threshold model resembles that of OCT4 for ESC differentiation. Taken together, the effect of a Wnt3 mutant on OCT4 levels in mouse ESCs, and the involvement of both WNT and OCT4 in threshold-mediated responses, indicates the intriguing possibility that the WNT signalling cascade and the role of OCT4 as a cell-fate determinant are directly linked.

Oct4 and Sox2. The proteins encoded by these genes act cooperatively at promoters. As discussed above, the octamer and sox elements have recently been shown to be required for the upregulation of mouse and human Nanog transcription [87]. Adjacent octamer and sox elements have also been identified as cis-regulatory elements in the Fgf4 (Ref. 64), Sox2 (Ref. 82), Utf1 (Ref. 81), and Fbx15 (Ref. 88) genes, which are expressed in ECC and ESCs and during embryogenesis.

Pluripotency pathways in other systems

On the basis of the knowledge reviewed here, it is sensible to ask, and it will be interesting to determine, whether pluripotency can be established and propagated in vitro by methods other than deriving ESCs from zygotic embryos. The current lack of definitive control over embryo-derived stem-cell fate finds an interesting parallel in the lack of definitive control over the nuclei of differentiated cells that regain pluripotency after transfer into oocytes (cloning). The recent advances in the derivation of human pluripotent stem-cell lines [14, 89] and the successes in mammalian embryo cloning by NUCLEAR TRANSFER, provide an attractive possibility for restoring tissue function by cellular transplantation using SCNT — often referred to as therapeutic cloning. CLONAL blastocysts might be used for the establishment of almost perfectly matched ESC cell lines that can be induced to differentiate in vitro and provide the patient with cells for an autologous graft. Therapeutic cloning is not an unreasonable suggestion as it has been previously demonstrated to be feasible in mice [90, 91] and recently in humans [89]. Before this procedure can be considered for clinical application, it should be determined how complete the reprogramming process actually is. The control of Oct4 and Nanog expression is instrumental for successful cloning, in which a UNIPOTENT cell nucleus becomes pluripotent after transfer into an ooplasm, provided that a set of developmental pluripotency-associated genes are expressed [92, 93]. The requirement for Oct4 and Nanog for the maintainence of pluripotency indicates that the somatic-cell-derived alleles must become reactivated through nuclear reprogramming after SCNT into oocytes. It has been shown that reactivation of the silent somatic alleles of Oct4 and Nanog occurs only partially in mouse clones [73, 92]. Interestingly, the frequency of Nanog reactivation surpasses that of Oct4 (Ref. 73). By contrast, X-chromosome inactivation appeared to occur as it would during normal development [94]. However, even though X inactivation was shown to occur properly in somatic-cell-derived clonal embryos of the mouse, subtle abnormalities in expression and METHYLATION of several imprinted genes were shown to occur in mouse clones that were derived from somatic cells [95] and ESCs [96]. However, viable clonal 'offspring' can survive to adulthood despite the widespread dysregulation in expression of these genes.

Within a clonal embryo, reprogramming in adjacent blastomeres seems to occur independently of each other, and at the blastocyst stage OCT4 is expressed by some cells and not by others in the ICM — a phenomenon known as 'mosaicism'. Altogether, the partial reprogramming of one key gene being associated with the partial reprogramming of another one, and so on, indicates that there is a subset of clones in which there are at least some cells that are reprogrammed for all genes. ESC-derivation, differentiation and transplantation therapy after SCNT does not require full-term embryonic development. It requires the formation of blastocysts, the derivation of ESCs from them, and then differentiation of the ESCs into specific cell types. So the partial conversion of the unipotent programme into a pluripotent one might be sufficient for therapeutic cloning.

We were asking if pluripotency can be established and propagated in vitro by methods other than deriving ESCs from zygotic embryos — and the answer is yes, by SCNT. Similarly, it is also sensible to ask, and it will be interesting to determine, whether methods other than cloning by SCNT into oocytes can achieve this result. When pluripotent stem cells are used instead of oocytes as recipients for nuclear transfer that is mediated by cell–cell fusion, the hybrid cells show a loss of stage- and tissue-specific markers of the somatic-cell parent (gene extinction) and a de novo activation of traits and genes which are characteristic of pluripotent cells. X inactivation, for example, is a process that is tightly linked with differentiation and occurs in the soma of females. By demonstrating the re-activation of the X chromosome after cell fusion, and its subsequent random inactivation during differentiation, it was possible to prove that pluripotent stem cells can reset the inactivation mark correctly [97, 98]. Furthermore, when induced to differentiate, either in vitro or in vivo, a broad spectrum of differentiated cells could be obtained despite their tetraploid condition. The hybrid cells can also be examined for their developmental potential by injecting them into host blastocysts. By generating viable chimaeras, it was possible to produce cell hybrids in different tissues, which indicates the ability of these injected cells to contribute to all cell lineages. There is an alluring possibility that cell fusion might be naturally occurring in vivo, such that the adult stem cells of one tissue can generate differentiated cells that are specific to another tissue by fusing with local cells [99-102] and then segregating. However, cell fusion might not explain all such occurrences of apparent transdifferentiation [103].

Ultimately, it will be interesting to determine whether pluripotency can be established and propagated in vitro by methods other than nuclear transfer — whether into oocytes or other cells (fusion) — using cellular and regulatory systems other than those of ESCs, and perhaps without the presence of extracellular factors. For example, the multipotent adult progenitor cells, MAP-C cells, which are derived from adult bone marrow stroma, seem to regain pluripotency in vitro in the absence of other companion cells that might secrete pluripotency factors. However, this is a long-term process and the level of MAP-C Oct4 expression is 1,000-fold less than that seen in ESCs [104].

In conclusion, it appears that pluripotency is achieved through the combination of properly sequenced processes that control chromatin accessibility, chromatin modifications, and activation and repression of specific genes. This is complicated by the requirement for the finely tuned regulation of the relative levels of expression. Nanog has been celebrated as being as important as OCT4, which for many years was thought to be the sole transcription factor that was strongly and unquestionably associated with pluripotency. However, OCT4-binding sites have recently been found in the Nanog upstream control region, which exemplifies the central theme of this review. We suggest that it is improbable that screening a 'pluripotent' cDNA library would lead to the discovery of what actually implements pluripotency. Several questions regarding the mechanisms of pluripotency remain open. Given that the intrinsic regulators OCT4 and Nanog are essential but not sufficient for specifying a pluripotent cellular state, we must discover which genes lie upstream and are in control of Oct4 and Nanog expression. Are there other genes that mediate pluripotency, particularly, are there any 'master' genes? Manipulation of such a 'master' gene could serve as a tool to comply with ethical concerns raised by human cloning as such a gene would be a target for ANT (altered nuclear transfer) [105]. This approach may constitute the panacea of modern human regenerative medicine although there is no consensus on the validity of this proposal [106].

Note added in proof:

During the preparation of this review, Boyer et al. [135] mapped the transcription factors OCT4, SOX2 and Nanog to the promoters of a large population of genes that define, either positively or negatively, human ESCs. The authors showed that promoter regions are co-occupied by OCT4, SOX2 and Nanog in both transcriptionally active genes that have a role in defining ESC identity (undifferentiated phenotype) and inactive genes that promote their development once de-repressed (differentiation). The co-presence of OCT4, SOX2 and Nanog at gene promoters is interpreted by the cell in a combinatorial manner through autoregulatory and feed-forward loops. This study gives us a more comprehensive map of the core regulatory circuitry of pluripotency.

BLASTOCYST A pre-implantation mouse embryo of about 40–80 cells at day 3–4 after fertilization (in the case of human blastocysts the pace of development would be slower). The blastocyst consists of a hollow sphere made up of an outer layer of cells (the trophectoderm), a fluid-filled cavity (the blastocyst cavity or 'blastocoel'), and a cluster of cells at one pole on the interior (the inner cell mass).

CHIMAERISM The contribution to embryonic and fetal tissues of exogenous sources of cells with a different genotype, including cells that come from a different organism or species. For example, the injection of cells from one embryo into the blastocyst cavity of another embryo.

CLONAL (CLONED) Describes the genetically identical progeny of a single mitotic cell. Also applied to an embryo to indicate its derivation from the splitting of another embryo or by nuclear transfer into a recipient oocyte.

CRE/LOXP RECOMBINATION SYSTEM A site-specific recombination system derived from Escherichia coli bacteriophage P1. Two short DNA sequences (loxP sites) are engineered to flank the target DNA. Activation of the Cre-recombinase enzyme catalyses recombination between the loxP sites, leading to excision of the intervening sequence.

DIAPAUSE In general, a period of physiologically enforced dormancy between periods of activity. In the pre-implantation-stage embryo, it is a transient/reversible state of reduced vitality during which the blastocyst floats in the uterus without implanting.

EMBRYO In the mouse, the embryo is the developing organism from the time of fertilization until E10.5, when it becomes known as a fetus. The stages are: pre-implantation embryo, until E3.5; post-implantation embryo, from E3.5–E10.5; fetus, E10.5 onwards.

EMBRYONIC STEM CELLS (ESCs). Derivative cells of the ICM of the pre-implantation embryo that have the potential to become all specialized cell types of the adult organism. As established cell lines, ESCs have been cultured under in vitro conditions that allow proliferation without differentiation for months or years.

EST (EXPRESSED SEQUENCE TAG) A unique stretch of DNA within a coding region of a gene that is useful for identifying full-length genes and serves as a landmark for gene mapping.

FEEDER CELLS Cells used in co-culture to maintain pluripotent stem cells. Feeder cells usually consist of 'mouse embryonic fibroblasts' (MEFs), which are actually fetal mesenchymal cells obtained from E13.5 fetuses. These cells can proliferate for only a few passages in vitro (primary MEFs) or be immortalized (STO (SIM mouse thioguanine- and ouabain-resistant)-SNL (STO, NEO, LIF) cells).

HOMEODOMAIN A domain in a protein that is encoded for by a homeobox—it consists of about 60 amino-acid residues and recognizes and binds to specific AT-rich DNA sequences.

HOMEOSTASIS A property of cells or organisms to regulate the internal environment in response to external cues to maintain a stable condition inside.

INNER CELL MASS (ICM). The prominent cluster of cells inside the blastocyst. In vivo, these cells give rise to the fetus. In vitro, the ICM cells are the source for ESCs.

MESODERM Germ layer; the middle layer of a group of cells derived from the primitive ectoderm of the fully expanded blastocyst; in general, it gives rise to bone, muscle, connective tissue and the middle layer of the skin. There is also an extra-embryonic mesoderm.

METHYLATION Covalent modification of the DNA nucleotide cytosine by addition of a methyl (CH3) group.

MULTIPOTENT Ability of a single stem cell to differentiate along multiple lineages and give rise to different tissues. It has broad potential but less than a pluripotent cell.

NUCLEAR TRANSFER The procedure of removing the nucleus of a donor cell and transplanting it into another. Typically this involves a somatic cell (donor) and an oocyte (recipient) and is instrumental to the cloning procedure therefore known as SCNT (somatic cell nuclear transfer).

PLURIPOTENT The ability of a single stem cell to develop into all different cell types of the body but not into the extra-embryonic cell types.

POU DOMAIN A specific region or amino-acid sequence in a protein that is associated with specific sequences such as the octamer motif (5'-ATGCAAAT-3') on DNA. It was originally identified in the transcription factors PIT1, OCT1, OCT2 and UNC86.

PRIMITIVE ECTODERM Also known as the epiblast. The upper layer of a group of cells derived from the inner cell mass of the blastocyst; it gives rise to all cells of the fetus.

PRIMITIVE ENDODERM Also known as the hypoblast. Lower layer of a group of cells derived from the inner cell mass of the blastocyst. It gives rise to visceral (lining the inner cell mass) and parietal extra-embryonic (lining the trophectoderm) endoderm.

SRC-HOMOLOGY-2 (SH2) Src-homology-2 (SH2) domains are modules of approx100 amino acids that bind to specific phosphotyrosine-containing peptide motifs. SH2-domain proteins have numerous roles such as adaptors, scaffolds, kinases, phosphatases, Ras signalling, transcription, ubiquitination, cytoskeletal regulation, signal regulation, phospholipid second-messenger signalling, and yet others.

STEM CELLS Cells that have the ability to divide for indefinite periods in the undifferentiated state but retain the potential to give rise to specialized cells.

SUPPRESSION-SUBTRACTIVE HYBRIDIZATION A method to identify DNA/RNA that is present in one sample but not in the others.

TERATOMA Also known as terratocarcinoma. A tumour composed of tissues from the three embryonic germ layers, which is usually found in the ovary and testis. It can be produced experimentally in animals by the injection of pluripotent stem cells, to determine the abilities of the stem cells to differentiate into various types of tissues.

TRANSCRIPTOME The full complement of messenger RNA (mRNA) and non-coding RNAs, transcripts in the cell, weighted by their expression levels.

TROPHECTODERM The outer cell layer of a blastocyst, made up of a mural and polar component. Gives rise to trophoblast cells upon implantation in the uterine wall.

TROPHOBLAST The extra-embryonic, highly proliferative tissue that is responsible for implantation, contributing to the placenta that controls the exchange of oxygen and metabolites between mother and embryo.

TROPHOBLAST STEM CELLS (TSCs). Cells derived from the trophectoderm of the blastocyst or early postimplantation trophoblast that have the potential to form all the cell types of the trophoblast-derived layer of the placenta.

UNIPOTENT Refers to a cell that can only differentiate in one specific way.

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We regret not being able to cite all the relevant publications due to space constraints. We wish to thank J. Rossant for her valuable comments on the manuscript, and acknowledge the members of our laboratory for helpful discussions and A. Malapetsa for help in the preparation of the manuscript. This work was supported by the Max-Planck Society.

Competing interests statement. The authors declare no competing financial interests.

Michele Boiani is a scientist and project leader at the Max-Planck Institute for Molecular Biomedicine in Münster, Germany. As reflected by his published work, he has always had two main research interests: the use of micromanipulation techniques on oocytes, and the analysis of gene expression during early embryo development. He received his predoctoral education in reproductive biology (1994–1999) under the guidance of Anne Grete Byskov at the Rigshospitalet in Copenhagen, Denmark and Carlo Alberto Redi at the University of Pavia, Italy. As a postdoctoral fellow he joined Hans Schöler's group in 2000 at the University of Pennsylvania, Philadelphia, USA, where he became more interested in developmental pluripotency. His research focus is on 'reprogramming' of nuclear potency in somatic-cell-derived clones of the mouse.

Hans Robert Schöler studied biology at the University of Heidelberg (1982 diploma). He gained a Ph.D. at the Center for Molecular Biology in Heidelberg, Germany (1985). Between 1986–1988 he was Head of Research Group at Boehringer Mannheim (now Roche) in Tutzing. From 1988–1991 he was a staff scientist at the Max-Planck Institut for Biophysical Chemistry in Göttingen, Germany. Between 1991–1999 he was head of a Research Group at European Molecular Biology Laboratory (EMBL). From 1999–2004 he was Professor and 'Marion Dilley and David George Jones Chair' in reproduction medicine, and Director of the Center for Animal Transgenesis and Germ Cell Research at the University of Pennsylvania, School of Veterinary Medicine, Pennsylvania, USA. Since April 2004 he has been the Director of the Max-Planck Institute for Molecular Biomedicine, Department for Cell and Developmental Biology in Münster and Professor of the Medical Faculty of the Westfälische-Wilhelms-Universität Münster, Germany. His research focus is on germline development and pluripotency.

    * Embryo-derived stem cells can be considered archetypal stem cells. Among them, embryonic stem cells (ESCs) are derived at an earlier stage of embryo development than others, such as embryonal carcinoma cells (ECCs) and embryonic germ cells (EGCs). Compared to adult or fetal stem cells, embryo-derived stem cells are easier to identify, to isolate and to use as established cell lines.

    * The golden feature of ESCs is their pluripotency, as defined as the ability to give rise to any derivative of the three primary embryonic germ layers and to germ cells. Pluripotency can be maintained indefinitely if ESCs are cultured under correct conditions, and can be demonstrated in mice when ESCs injected into blastocysts become incorporated in the conceptus and participate in normal development. Notably, disorganized proliferation and tumour formation of ESCs might also occur, regarded here as 'the other side' of pluripotency.

    * ESCs behave normally within a blastocyst but give rise to tumours when transplanted ectopically, for example, under the skin, it therefore implies that exogenous cues have a bearing on cell fate. Whether they do so by priming or by selecting ESC states is not known.

    * Most of the extrinsic and intrinsic regulators that are known to operate in mouse ESCs (for example, LIF and BMP4 extracellular ligands; OCT4, SOX2 and Nanog transcription factors) have a dose-dependent effect on pluripotency. Genes that are required to maintain pluripotency in human ESCs are mostly unknown, with the exception of OCT4. Combinatorial interactions and nonlinear responses, which have been described in mice, compound this situation.

    * The current lack of definitive control over stem-cell fate finds an interesting parallel in the lack of definitive control over nuclei of differentiated cells regaining pluripotency after transfer into oocytes (cloning).

    * An understanding of how pluripotency is secured in ESCs is likely to indicate ways to reinstate pluripotency in somatic cells. The oocyte is the common reactor that is used to reinstate pluripotency on somatic nuclei. However, in the future we might not even need oocytes to reinstate pluripotency, as has been shown for somatic cells fused with ESCs and for bone marrow cells that can become multipotent adult progenitor cells (MAP-C cells) in culture.

* An understanding of the ESC circuitry might reveal how to achieve phenotypic changes without genetic manipulation of Oct4 and Nanog and other pluripotency-associated genes.

This new comprehensive review of mouse cell embryogenesis by Michele Boiani and Hans Schöler reveals the role of multi-gene regulatory networks in maintaining the pluripotent state in embryonic mouse stem cells, and in mediating the progressive cellular changes of mouse embryogenesis. Protein and RNA molecules are the chief agents of repression and activation during mammalian gene regulation (1-5).

1. Song X, Sun Y, and Garen A, "Roles of PSF protein and VL30 RNA in reversible gene regulation".

2. Parada LA, McQueen PG,  and Misteli T, "Tissue-specific spatial organization of genomes", Genome Biology, vol. 5, no. 7, r44 (June 21, 2004).

3. Frenster JH, and Hovsepian JA, "Ultrastructure  of Closed Loops within Euchromatin of Isolated Lymphocyte Nuclei".

4. Hovsepian JA, and Frenster JH, "Sense and Antisense during RNA Initiation of the DNA Transcription Bubble".

5. Czihak G, "Evidence for inductive properties of the micromere RNA in sea urchin embryos", Naturwissenschaften vol. 52, no. 6, pp. 141-142 (1965).
 
 

1. Song X, Sun Y, and Garen A, "Roles of PSF protein and VL30 RNA in reversible gene regulation".

2. Parada LA, McQueen PG,  and Misteli T, "Tissue-specific spatial organization of genomes", Genome Biology, vol. 5, no. 7, r44 (June 21, 2004).

3. Frenster JH, and Hovsepian JA, "Ultrastructure  of Closed Loops within Euchromatin of Isolated Lymphocyte Nuclei".

4. Hovsepian JA, and Frenster JH, "Sense and Antisense during RNA Initiation of the DNA Transcription Bubble".

5. Czihak G, "Evidence for inductive properties of the micromere RNA in sea urchin embryos", Naturwissenschaften vol. 52, no. 6, pp. 141-142 (1965).

Links to Euchromatin Activator RNA Reviews:
Links to Euchromatin Activator RNA Research:
Links to Ultrastructural Probes of DNase I-Sensitive Sites:
Links to RNA as a Therapeutic Agent:
Links to Hodgkin Lymphoma Immuno-Pathology:
Links to Activated T-Lymphocyte Immunotherapy:
Links to Medical Systems Biology:
Links to Selective Gene Transcription:
Links to RNA-Induced Epigenetics:
Links to RNA-Induced Embryogenesis:
Links to RNA and Biological Causality:
Links to Reprogramming and Neoplasia:

A Brief History of Activator RNA:

"Ultrastructural Probes of Active DNA Sites, and the RNA Activators of DNA". (PowerPoint Presentation).