Embryonic stem cells (ESCs) originate as undifferentiated inner mass cells within a blastocyst. Pluripotency and indefinite self-renewal are the two distinctive properties of ESCs that have captured the imagination of scientists and non-scientist alike. The pluripotency of these cells empowers them to differentiate into all derivatives of the primary germ layers, giving rise to all cells of the organism, for as long as they are specified to. Such properties foster great potential in aiding the development of therapeutic solutions for confronting degenerative disease, whilst depending our understanding of human cellular biology. Nonetheless, the growth of ESCs, as a pluripotent population necessitates the presence of regulatory mechanisms that ensure balanced transmission of signals for survival, proliferation and self-renewal of stem cells.
Recent investigations into the molecular and cellular mechanism of ESCs have identified numerous factors deemed important for the maintenance of ESC pluripotency and self-renewal; including extracellular signalling factors, transcription factors, cell cycle regulators, microRNAs, epigenetic modifiers and genes linked with chromosomal instability. Here, we will look at the currently understood mechanisms of ESC pluripotency and self-renewal, and aim to explain the means by which different regulators of ESCs may interact.
Signalling Transduction Pathways
Figure 1 | An illustration of the LIF/JAK/STAT3 signalling Pathway. (Adapted from Hirai et al. 2011)
The self-renewal and pluripotency of ESCs is influenced by a variety of extrinsic signalling factors that maintain such characteristics through regulation of genes involved in pluripotency.
A key player in the maintenance of ESC’s self-renewal and pluripotency is a member of the IL-6 cytokine family known as Leukaemia inhibitory factor (LIF) (Williams et al. 1988). The stimulation of the LIF receptor by its ligand, LIF, leads to the activation of three major signal transduction pathways: The JAK (Janus Kinase)/STAT3 (signal transducer and activator of transcription 3), SHP2 (SH2 (Src homology 2) domain-containing tyrosine phosphatase 2)/MAPK (mitogen activated protein kinase) and PI3K (phosphoinositide 3-kinase)/AKT pathways (Hirai et al. 2011).
Following its activation by LIF, LIFR recruits the transmembrane glycoprotein 130 (gp130) to form a heterodimer (Figure 1), this heterodimer activates the gp130 bound JAK by trans-phosphorylation (Murphy et al. 2011). Amongst the JAK family, JAK1 is particularly important for the self-renewal capabilities of mESCs, this is shown in experiments where a higher levels of LIF is required in culture to maintain Jak1-/- mouse ESCs (mESC) in comparison to its wild type counterpart (Ernst et al. 1996). Activation of JAKs leads to the phosphorylation of tyrosine residues on gp130, which creates a binding site for STAT3. Consequently, JAKs phosphorylate the STAT3 recruited to the binding site, resulting in a homodimerized STAT3 that binds to a sequence at the enhancer of its target genes to regulate their expression (X. Chen et al. 2008).
The activation of STAT3 on its own is necessary and sufficient for self-renewal and pluripotency of mESCs; its necessity was demonstrated by Niwa et al (1998), where the use of a dominant negative STAT3 mutant hindered the self-renewing capacity of mESCs. Later, its sufficiency was displayed using experiments inducing STAT3 phosphorylation in absence of LIF leading to self renewal and prevention of differentiation (Matsuda et al. 1999).
Whilst the downstream targets of LIF-STAT3 pathway remain to be fully elucidated, the transcription factor c-Myc appears to be an important downstream target of this pathway. In a study by Cartwright et al (2005) it was shown that LIF withdrawal results in a dramatic down regulation of c-Myc and consequently leads to its phosphorylation and degradation. In addition, the stable expression of c-Myc in absence of LIF was shown to promote mESC self-renewal; whereas, the dominant negative form of c-Myc obstructs self-renewal and promotes ESC differentiation (Cartwright et al. 2005).
However, in humans, STAT3 activation by LIF is not sufficient for maintaining pluripotency of human ESCs (hESCs). In a study by Humphrey et al (2004) it was shown that STAT3 activation does not prevent hESCs differentiation, hinting at the presence of STAT3 independent pathways for maintaining pluripotency in hESCs.
It has also been suggested that hESCs exist in a “primed state” of pluripotency, as compared to the “naïve state” seen in mESCs, this is to say that hESCs are in a slightly less flexible state of pluripotency, and it is proposed that LIF aids the acquisition of thennaïve state of pluripotency by hESCs (Nichols & Smith 2009).
Years after the establishment of LIF-STAT3 as an important factor for ESC pluripotency and self-renewal; it was observed that in serum free cultures, LIF is insufficient to block ESC differentiation, hinting at the presence of other factors in the serum that complement LIF action. BMP4 is a member of the TGFβ superfamily, and a candidate for the factor that co-affects with LIF in the serum. BMP4 acts via the activation of intracellular proteins Smad1/5/8. Once BMP4 is added to the serum free culture it allows LIF to maintain mESC pluripotency (Ying et al. 2003).
Intriguingly, BMP4 has opposite effects in mouse and human ESC; such that it promotes hESC to differentiate into trophoblast orpprimitive endoderm (Xu et al. 2002). Inhibition of BMP4 by its inhibitors such as Noggin, allows hESC to remain pluripotent. In addition, GDF3, a secreted factor of the TGFβ family, is overexpressed in hESC and reduced in mESCs. In other words, a high level of GDF3 maintains pluripotency in hESC, but not in mouse. This may suggest that GDF3 may be an inhibitor of BMP4, and may account for the different function of GDF3 and BMP4 in ESCs (Levine & Brivanlou 2006).
Furthermore, ERK activity is elevated in mESCs that undergo differentiation, and the suppression of this pathway is shown to promote mESC self-renewal (Lodge et al. 2005). On the other hand, hESCs show high basal levels of ERK activity in their undifferentiated state, which is thought to impact the basic fibroblast growth factor (bFGF) signalling that has been shown to promote hESCs self-renewal (Lodge et al. 2005).
Amongst the members of the FGF family, FGFR1 expression is most abundant in undifferentiated hESCs, and it has been shown that high a level of bFGF is sufficient to maintain hESCs pluripotency. It has been suggested that noggin, a BMP antagonist, synergises with bFGF to repress BMP signalling to facilitate hESC’s undifferentiated proliferation (Xu et al. 2005). This again, highlights the differences in mechanisms of ESC pluripotency in mouse and human.
The phosphoinositide 3-kinase (PI3K) pathwayyis imperative for the proliferation, survival and maintenance of pluripotency in ESCs. Inhibition of PI3K/Akt signalling in hESCs leads to their differentiation, even in the presence of LIF and feeder cells (Tanaka et al. 2002). Eras is a stimulant of PI3K which is found in abundance in ESCs; these findings hint at the importance of PI3K/Akt signalling for the maintenance of pluripotency in ESCs. In addition, PI3K/Akt signalling is important for the suppression of apoptosis in mESCs, since the density of ESCs is critical to their self-renewal capacity, this may be an alternative mechanism through which PI3K promotes ESC self-renewal (Gross et al. 2005).
Wnt signalling has been shown to maintain the undifferentiated state of ESCs and sustain the expression of ESC markers. The endogenously activated Wnt in ESCs starts to plummet in levels once the cells start to differentiate (Sato et al. 2004). The action of Wnt/β-catenin signalling on ESC self-renewal is analogous in both human and mouse, this is in contrast to LIF-STAT3 and BMP signalling, however Wnt is able to upregulate STAT3 expression, exhibiting a synergistic action withLLIF-STAT3 (Hao et al. 2006). In addition, c-Myc, a target gene of STAT3, is also upregulated as a consequence of Wnt signalling; this indicates that c-Myc might be a converging point for Wnt and LIF-STAT3 signalling (Cartwright et al. 2005).
TGFβ and its correlates are expressed at high levels in undifferentiated hESCs (Sato et al. 2004). It has been shown that activin A, a member oftthe TGFβ superfamily, is able to maintain hESCs in an undifferentiated state in the absence of STAT3 activity. Activin’s action is mediated through the Smad signalling pathway, particularly via Smad2/3 in undifferentiated cells (Beattie et al. 2005). Smad2/3 is able to suppress BMP4 in hESCs by suppressing the Smad1/5 activity (which is necessary for BMP4 function) (Beattie et al. 2005). However in the mouse, variations in Smad2/3 levels do not affect Oct4 levels, implying the non-necessity of Smad2/3 for mESCs pluripotency.
Transcription Factors
Figure 2 | (A) An illustration of feed-forward transcriptional regulatory circuitry in hESCs. Blue circles represent regulators; orange rectangles represent gene promoters. Solid arrows show regulator-promoter binding. Geneseencoding regulators are connected to their regulatorsbby dashed arrows. (B) The interconnected auto regulatory loop formed by OCT4, SOX2, and NANOG. (Adapted from Boyer et al. 2005)
Experiments in the last decade have uncovered various regulatory circuitries involved in the self-renewal and differentiation of ESCs. Amongst these, transcription factors Oct4, Sox2 andNNanog are thought to be central to the transcriptional regulatory hierarchy that determines ESC differentiation, partly due to their unique and intricate expression during early development. The actions of these factors are regulated by environmental signals, as well as auto regulation. It is thought that the expression of these factors, leads to the activation of self-renewal genes and repression of pro-differentiation genes, hence maintaining the pluripotent state of ESCs.
Oct4 (also known as Pou5fl) is a member of the POU (Pit-Oct-Unc) family of transcription factors. Oct4 expression starts in the early embryo and is later limited to pluripotent cells, including ESCs and germ cells. Both human and mouse ESCs display high expression levels of Oct4, however upon differentiation of ESCs, Oct4 levels diminish and the cells loose pluripotency. To maintain the pluripotent state of ESCs, Oct4 must be expressed at precise levels, such that the loss of Oct4 leads to the inapt differentiation of ESCs into trophectoderm, whilst its overexpression dictates a primitive endoderm and mesoderm fate; thus for pluripotency, Oct4 protein levels must be kept within a narrow window of abundance.
Oct4 regulation appears to be independent of LIF and JAK-STAT signalling; however, the presence of STAT binding sites on a number of Oct4 target genes, hints at a possible cooperation between the two factors. It has been suggested that ESG1 (embryo specific gene1), a downstream target of both Oct4 and STAT3, acts to incorporate the two factors through yet unknown mechanisms (Tanaka et al. 2002).
Oct4 and Sox2 contain a regulatory complex that is essential for controlling gene expressions that influence cell pluripotency, including themselves. This auto regulation may be critical to the maintenance of precise Oct4 levels in primitive cells (Okumura-Nakanishi et al. 2005). Furthermore, Nanog, a homeobox-containing transcription factor is also deemed important for ESC pluripotency and activation of Oct4 expression (Pan et al. 2006).
Nanog is expressed in pluripotent cells, and similar to Oct4, its expression is absent in differentiated cells. Mouse ESCs that are deprived of Nanog differentiate into endoderm lineages; whereas mESCs subject to overexpression of Nanog undergo self-renewal independent of LIF, albeit with reduced capability, thus signifying the importance of Nanog in regulating the pluripotent state (Ying et al. 2003). Amid the lack of adequate understanding regarding Nanog mediated mechanism that drive pluripotency, it is proposed that Nanog may act as a transcription repressor of downstream genes implicated in cell differentiation, including Gata4 and Gata6 (Mitsui et al. 2003).
However, Nanoggcan also be an activator of genes involve in self-renewal, such as Rex1 (Zfp-42), this acidic zing finger gene is also a target of Oct4/Sox2 signalling; this suggest that Rex1 mayb be an intersection of Nanog and Oct4/Sox2 activity (Shi et al. 2006). Recent experiments involving Chip analysis and RNAi-mediated knockdowns have suggested that Oct4/Sox2 signalling may target the Nanog promoter directly, hinting at the presence of interconnected networks between these three factors (Kuroda et al. 2005).
Investigations into the mechanisms of Nanog, Oct4 and Sox3 signalling have revealed an interconnected intrinsic core regulatory circuit that is key for maintaining ESC pluripotency (Figure 2). Oct4 activityyis regulated via interaction with other transcription factors that are expressed in pluripotent cells, such as Sox2; this member of the HMG-domain DNA-bindinggprotein family acts with Oct4 to form a ternary complex targeting the enhancer element of their target genes. In addition to regulating Oct4 and Sox2 expression, the Oct4/Sox2 complex, has been found to target Nanog via the Oct4/Sox2 binding site discovered in the Nanog promoter (Kuroda et al. 2005).
Therefore, Oct4 maintains a steady concentration of Nanog in ESCs by activating and repressing the Nanog promoter at below and above steady levels respectively (Pan et al. 2006). This could explain the link between Oct4 overexpression and ESC differentiation. In addition, FoxD3, a transcriptional repressor is able to augment Nanog activity, by repressing the repressive actions of Oct4 on Nanog promoter at the steady state (Pan et al. 2006). Hence, the interactions and regulations between such transcription factors are essential for adjusting precise levels of gene expression, and thus maintaining ESC pluripotency.
In addition, microRNAs are also implicated in pluripotency of ESCs. A microRNA is a small non-coding RNA molecule, which is involved in transcriptional and post-transcriptional regulation of gene expression (K. Chen & Rajewsky 2007). Studies have shown that certain sets of microRNA are exclusively expressed in pluripotent ESCs, and are absent in differentiated cells; in addition, at the time of differentiation, they downregulate the “pluripotent genes” and activate lineage specific ones (Cheng et al. 2005); such that the loss of mature microRNAs in Dicer1 null mice hinders ESC’s ability to differentiate into the three primary germ layers (Kanellopoulou et al. 2005). Despite our limited understanding of the mechanisms of microRNA action, they appear to be important for both the self-renewing and pluripotent abilities of ESCs through regulation of enzymes that affect chromatin structure (Cheng et al. 2005).
Chromatin and epigenetic Modification
Chromatin remodelling and modification is a key factor in determining gene activity. The differentiation of ESCs to developmentally more restricted states involves changes in patterns of gene expression, such changes result in selective expression of transcription factors via chromatin and epigenetic modifications including, DNA methylation of CpG dinucleotides and covalent histone modification.
The chromatin structure, dynamics and nuclear architecture within the ESCs are different to that of somatic cells. The abundance of acetylated histone modifications in ESCs represent an accessible euchromatin (open conformation) state that is open to nucleases activity (Boyer et al. 2006). During retinoic acid-induced differentiation of ESCs, there is an upregulation of the heterochromatin (closed conformation) marker tri-methylated residue K9 of histone H3 (H3-triMeK9), accompanied with diminishing levels of euchromatin markers acetylated histone H3 and H4 (Meshorer & Misteli 2006).
Considering that the chromatin nuclei structure in pluripotent ESCs is that of an open conformation, it has been shown that lineage specific genes are replicated earlier in pluripotent cells than differentiated cells, this is combined with abundance of methylated H3K4 and acetylated H3K9, as well as H3K27 trimethylation. The latter methylation is deemed critical for the silencing of lineage specific genes in ESCs, to maintain a pluripotent state (Azuara et al. 2006).
DNA methylation can lead to gene silencing. This is usually achieved by methylation of CpG islands at the promoter of genes. DNA methyltransferases (Dnmts) are critical to the methylation process. The loss of Dnmt1, Dnmt3a/b or CpG island-binding protein (CGBP) results in severe hypomethylation in ESCs resulting in loss of differentiation capacity (Carlone et al. 2005). In differentiated cells, the Oct4 promoter/enhancer is heavily methylated; this is in contrast with the hypomethylation of the same region in ESCs, this allows continued Oct4 expression in these cells to maintain their pluripotency, in contrast to the differentiated cells.
Furthermore, polycomb group proteins (PcG) are chromatin modification factors that have been recently implicated in ESCs self-renewal and pluripotency. Polycomb complexes (PRC1 and PRC2-PRC3) have the ability to repress chromatin-remodelling activity and facilitate its oligomerization or condensation. Studies in ESCs have shown that the PcG components PRC1 and PRC2, repress the activity of a large number of differentiation promoting genes including transcription factors and signalling factors. The inhibition of these PcG components results in the elimination of the repressive action of the Polycomb complex. Therefore PcG are important for the prevention of premature expression of differentiation genes; however this is achieved in a dynamic fashion such that at the appropriate time of differentiation, the PcG targets which where once repressed are allowed to be activated (Buszczak & Spradling 2006) (Boyer et al. 2006).
Furthermore, the cell cycle in embryonic stem cells is abbreviated when compared to that of somatic cells. TheeG1 phase is shorter in ESCs; during this phase, ESCs are more susceptible to differentiation by retinoic acid; this spectacle of shortened G1 duration may contribute to the self-renewal ability of ESCs. In addition telomerase activity has also been linked to ESC pluripotency and self-renewal. Telomeres are critical for chromosomal integrity. It has been shown that telomerase activity is elevated in ESCs, whereas it diminishes in these cells as they begin to differentiate, and is normally considerably lower in somatic cells (Bhattacharya et al. 2004). Telomerase activity is believed to be regulated by TGFβ and bFGF, for example bFGF can up-regulate telomerase activity in hESCs (Kurz et al. 2003), although it may not be necessary for its activation.
Conclusion
Experiments in the last decades have uncovered important mechanisms involved in the pluripotency and self-renewal of human and mouse embryonic stem cells. These cells display similar markers of pluripotent stem cell lines such as Oct4, Sox2, and Nanog, accompanied with high levels of telomerase activity.
Whilst the key intrinsic factors that control the pluripotency of hESCs and mESCs seem to be similar, there are notable differences in their extrinsic requirements, particularly the inability of LIF/STAT3 signalling to maintain hESCs pluripotency.
Future studies will undoubtedly pave the way for the expansion of our currently limited knowledge concerning the complex molecular mechanism of ESC pluripotency and self-renewal, where they will prove as a powerful tool for applications in the scientific and clinical disciplines.
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