Pluripotent ES (embryonic stem) cells can be expanded in culture and induced to differentiate into a wide range of cell types. Self-renewal of ES cells involves proliferation with concomitant suppression of differentiation. Some critical and conserved pathways regulating self-renewal in both human and mouse ES cells have been identified, but there is also evidence suggesting significant species differences. Cytoplasmic and receptor tyrosine kinases play important roles in proliferation, survival, self-renewal and differentiation in stem, progenitor and adult cells. The present review focuses on the role of tyrosine kinase signalling for maintenance of the undifferentiated state, proliferation, survival and early differentiation of ES cells.

ES (EMBRYONIC STEM) CELLS

ES cells are pluripotent cell lines derived from the ICM (inner cell mass) of the blastocyst [1,2]. They are maintained in culture by addition of factors that promote proliferation in the absence of differentiation, also known as self-renewal (reviewed in [3,4]). mES (mouse ES) cells can retain the functional properties of the pluripotent cells of the early embryo when reintroduced into the blastocyst [5,6], and this capacity for multilineage differentiation can be reproduced in vitro, where they can be induced to differentiate into a wide range of cell types (reviewed in [7]). This unique feature is the basis of various applications of mES cell technology such as in vitro models of mammalian development and germline transgenesis to make knockout mice (reviewed in [7,8]). The more recent isolation of analogous hES (human ES) cells has raised hope that these cells will provide a resource for cell-replacement therapies and drug discovery [9].

The processes involved in self-renewal of ES cells and differentiation are regulated by multiple extracellular and intracellular factors (reviewed in [3,4]). The present review focuses mainly on the role of PTK (protein tyrosine kinase) signalling in mES and hES cells, although some related signalling pathways are described as well.

CELL SIGNALLING BY PTKs

PTKs are found in all multicellular eukaryotic organisms and generally transduce signals from the extracellular environment across the plasma membrane into the interior of the cell (reviewed in [10]). PTKs can be categorized as belonging to either the receptor or non-receptor class, by virtue of whether they possess or lack the receptor-like features of extracellular-ligand-binding and transmembrane domains. Both receptor and non-receptor PTKs can be classified further into families based on their amino acid sequence within the catalytic domain and the presence of common structural domains. The signal transduction pathways that are stimulated by ligand binding to receptor PTKs can ultimately induce diverse cellular responses such as growth, differentiation, migration or survival. The important components of these processes include binding of a growth factor to the extracellular domain of the receptor, receptor dimerization and a subsequent increase in the receptor kinase activity. These events lead to tyrosine phosphorylation of the receptor itself and a variety of intracellular substrates. Tyrosine phosphorylation creates docking sites for SH2 (Src homology 2) or PTB (phosphotyrosine-binding) domains of a variety of signalling proteins, and the specificity of the interaction depends on both the amino acid sequence surrounding the phosphotyrosine and the amino acid sequence of the SH2 or PTB domain. A large family of SH2-domain-containing proteins possesses intrinsic enzymatic activities, such as kinase activity, phosphatase activity or phospholipase activity. The transcription factor family also includes members containing SH2 domains, as do many adaptor and docking proteins. The latter only contain protein-binding domains and utilize these to mediate interactions that link different proteins involved in signal transduction.

LIF (LEUKAEMIA-INHIBITORY FACTOR) PATHWAYS FOR MAINTAINING SELF-RENEWAL OF mES CELLS

The most critical pathways regulating self-renewal in mES cells are those mediated by the cytokine LIF or related cytokines that activate signal transduction from cell-surface cytokine receptors (reviewed in [1113]). LIF is usually provided by a feeder layer of MEFs (mouse embryonic fibroblasts) and/or as a recombinant protein. LIF engages a heterodimeric receptor complex consisting of two related cytokine receptors: the LIF-specific receptor subunit LIFRβ and the gp130 subunit. The binding of the ligand results in dimerization, conformational changes in the intracellular part of the receptor, and recruitment and activation of at least four different downstream pathways: the JAK (Janus tyrosine kinase)/STAT (signal transducer and activator of transcription), Ras/ERK1/2 (extracellular-signal-related kinases 1/2), PI3K (phosphoinositide 3-kinase) and SFK (Src family kinase) pathways (Figure 1).

LIF maintains self-renewal in mES cells

Figure 1
LIF maintains self-renewal in mES cells

The binding of the LIF to the LIFR/gp130 receptor leads to recruitment and activation of at least four different downstream signalling pathways: the JAK1/STAT3 pathway, the Ras/ERK1/2, pathway, the PI3K/Akt pathway and the SFK pathway. BMP4 and Wnt signalling pathways act in combination with the LIF pathway to maintain the self-renewal of mES cells. See text for more details. PIAS, protein inhibitor of activated STAT.

Figure 1
LIF maintains self-renewal in mES cells

The binding of the LIF to the LIFR/gp130 receptor leads to recruitment and activation of at least four different downstream signalling pathways: the JAK1/STAT3 pathway, the Ras/ERK1/2, pathway, the PI3K/Akt pathway and the SFK pathway. BMP4 and Wnt signalling pathways act in combination with the LIF pathway to maintain the self-renewal of mES cells. See text for more details. PIAS, protein inhibitor of activated STAT.

LIF is insufficient to maintain self-renewal in serum-free cultures. There are several components present in serum that help LIF to sustain self-renewal and pluripotency. One such factor is BMP4 (bone morphogenetic protein 4), which has been shown to act in combination with LIF to sustain self-renewal and preserve multilineage differentiation in serum-free mES cell cultures [14]. The critical contribution of BMP4 is to induce the expression of Id genes via the Smad pathway. Forced expression of Id genes liberates ES cells from BMP4 or serum-dependence and allows self-renewal in LIF alone [15].

hES cells express gp130 receptors and respond to LIF by phosphorylation of, for example, ERK1/2, Akt [also known as PKB (protein kinase B)] and STAT3. However, these events do not maintain the expression of pluripotency markers, such as Nanog and Oct3/4 (octamer-motif-binding transcription factor 3/4), or the undifferentiated state of these cells. Thus, in contrast with mES cells, hES cells do not require LIF for self-renewal, although they need to be cultured on MEFs to maintain pluripotency [1618].

JAK/STAT pathway

The JAK family of cytoplasmic tyrosine kinases includes JAK1, JAK2, JAK3 and TYK2. JAK1, JAK2 and TYK2 are expressed in a wide variety of tissues, whereas JAK3 expression is restricted to cells of the haemopoietic system. JAKs are membrane-localized by binding to the cytokine receptor via heterodimeric or homodimeric receptor complexes containing gp130 [11]. The molecular mechanism of activation of JAKs upon cytokine stimulation is not understood and it is still under debate which functional domains exist in the JAKs [19]. Activated JAKs phosphorylate seven tyrosine residues on the intracellular portion of the gp130 receptor chain, which then act as docking sites for SH2-containing proteins that might themselves be phosphorylated by JAKs (reviewed in [1113]). In mES cells, the SH2-containing transcription factors STAT1 and STAT3 are recruited to phosphorylated LIFRs and also become phosphorylated by JAKs. Phosphorylated STATs dimerize and translocate to the nucleus, where they bind to STAT-binding elements in the promoter/enhancer regions of genes regulating self-renewal [1113]. In mES cells, JAK1 is mainly responsible for activating STAT3, which has been shown to be both necessary and sufficient for maintaining mES cells in an undifferentiated state [2023].

Although it is well-documented that LIF and STAT3 are essential for self-renewal of mES cells, the downstream targets remain elusive. Cartwright and co-workers [24] have shown that STAT3 targets the transcription factor c-Myc, which has been found to be important for self-renewal of ES cells. The expression of c-Myc is rapidly down-regulated following LIF withdrawal and overexpression of c-Myc renders the cells undifferentiated, whereas expression of a dominant-negative form of c-Myc antagonizes self-renewal and promotes differentiation [24]. The transcription factor Klf4 (Krüpel-type zinc-finger 4) is another downstream target of STAT3 in undifferentiated mES cells. Overexpression of Klf4 leads to increased Oct3/4 expression and self-renewal of mES cells [25]. Interestingly, c-Myc and Klf4 were part of four factors that were recently found to be able to reprogramme mouse and human somatic cells to become germline-competent iPS (induced pluripotent stem) cells [26,27]. It was shown subsequently, however, that, although c-Myc increases the efficiency of making iPS cells, it was not required. The other two factors were Oct3/4 and Sox2 (SRY-type high-mobility-group box 2) [28]. Oct3/4 is specifically expressed in all pluripotent cells during mouse embryogenesis and in undifferentiated ES cells, and the expression is down-regulated in response to differentiation. Precise levels of Oct3/4 are required to maintain the self-renewal of ES cells and both overexpression and down-regulation alters ES cell fate (reviewed in [13]). LIF does not appear to regulate Oct3/4, but many genes that are regulated by STAT3 also contain Oct3/4-binding sites, suggesting that these two pathways co-operate to regulate ES-cell-specific genes (reviewed in [4]). Nanog, a unique homeobox transcription factor, is another key effector that plays an essential role in maintaining self-renewal of mES and hES cells [2932]. Oct3/4 and Sox2 bind the Nanog promoter and regulate its transcription [33,34]. Analysis of the mouse Nanog promoter region reveals a STAT3-binding site, and it has also been shown that STAT3 can bind this region in vivo [35]. These results suggest that Nanog is a direct target of the LIF/STAT3 pathway and may be responsible for the LIF-induced self-renewal of ES cells, but more studies are needed to confirm this.

JAK2 and TYK2 are not involved in LIF-induced STAT3 activity and self-renewal of mES cells, but instead play a role in the early lineage decision of mES cells to various differentiated cell types [36]. A previous study indicates that the JAK2/STAT3 pathway is essential for the initial stages of cardiomyogenesis, as inhibition of JAK2 or STAT3 resulted in a complete loss of beating areas in EBs (embryoid bodies) [37].

ERK1/2 pathway

The ERK1/2 [also named p42/p44 MAPK (mitogen-activated protein kinase)] pathway regulates many different cellular responses in somatic cells. mES cells have high ERK1/2 activity when they undergo differentiation, and ERK1/2 activation promotes differentiation of ES cells, whereas suppression of the ERK1/2 pathway enhances self-renewal [38]. Inhibition of ERK1/2, however, does not replace the requirement for STAT3 activation, but rather enhances the actions of STAT3. It is not clear whether this effect is direct or indirect, but a recent study has demonstrated that activation of the ERK1/2 pathway can repress Nanog [39].

In mES cells, activation of ERK1/2 via gp130 is dependent on the phosphorylation of the SH2-domain-containing cytoplasmic tyrosine phosphatase SHP2. Phosphorylation of Tyr757 in gp130 recruits SHP2, leading to its tyrosine phosphorylation in a JAK1-dependent manner [40,41]. SHP2 then acts as an adaptor protein, associating with GRB2 (growth-factor-receptor-bound protein 2) and, thereby, activates the Ras/MEK (MAPK/ERK kinase)/ERK1/2 pathway (reviewed in [12]). SHP2 may also associate with the scaffold protein GAB1 (GRB2-associated-binder protein 1), as has been demonstrated in other cell types, which may recruit PI3K, although this interaction remains to be shown in ES cells [42]. Eliminating the SHP2-binding site from a chimaeric gp130 receptor in ES cells blocks the Ras/ERK1/2 pathway and increases self-renewal [43].

PI3K pathway

LIF also activates the PI3K/Akt pathway, which is important for proliferation, survival and self-renewal of mES cells [4447]. It has been hypothesized that GAB1, together with SHP2, recruits PI3K to the gp130 receptor, as discussed above [12]. Inhibition of PI3K signalling in mES cells is associated with a decrease in basal LIF-stimulated phosphorylation of Akt, GSK3α/β (glycogen synthase kinase 3α/β) and S6 proteins, an increase in ERK1/2 phosphorylation and a decline in the ability of the cells to maintain self-renewal [47]. However, PI3K inhibition has no effect on LIF-induced phosphorylation of STAT3 [47]. Interestingly, inhibition of MEK can reverse the effects of PI3K inhibition, suggesting that the PI3K pathway maintains self-renewal through inhibition of the ERK1/2 pathway. In support of the above findings, Watanabe et al. [46] found that a myristoylated active form of Akt can maintain the undifferentiated phenotypes in mES cells without the addition of LIF [46], and Storm et al. [48] have suggested that the PI3K pathway maintains the expression of Nanog. They hypothesize that phosphorylation and inhibition of GSK3 by Akt plays a key role in the regulation of Nanog expression, as blockade of GSK3 activity effectively reverses the effects of PI3K inhibition on Nanog expression and self-renewal [48]. GSK3 is a major inhibitor of the Wnt/β-catenin pathway (Figure 1), which has been shown to be involved in the maintenance of pluripotency in both mES and hES cells [4951]. Moreover, GSK3 phosphorylates and activates p53, which promotes differentiation of mES cells by suppression of Nanog [3,52]. It should be noted, however, that there are several different pools of GSK3 in the cell and it is currently not clear whether the GSK3 pool that is involved in regulating Wnt/β-catenin signalling is the same pool that is inactivated by Akt.

Src family pathway

In addition to the STAT3, ERK1/2 and PI3K pathways, it has been demonstrated that members of the non-receptor SFKs are activated by LIF/gp130 and serum and play roles in self-renewal of ES cells [23,5356]. SFKs consists of ten members and have been shown to regulate diverse cellular processes such as division, motility, adhesion, differentiation and survival [57]. Constitutively activated variants of SFKs, including the viral oncoproteins v-Src and v-Yes, are capable of inducing malignant transformation of a variety of cell types, and SFKs are frequently overexpressed and/or aberrantly activated in epithelial and non-epithelial cancers. At least seven SFK members are expressed in ES cells and many of these undergo dynamic changes in transcriptional and post-transcriptional regulation during EB differentiation in the absence of LIF. Most dramatic are the changes in Hck transcriptional levels, which decrease approx. 30-fold [56]. Moreover, rapid inhibition of tyrosine kinase activity of Hck, Lck and Yes as well as transcriptional silencing of Lyn and Lck have been observed [53,56]. In contrast, differentiation of ES cells neither affects protein expression nor kinase activity of Src or Fyn. LIF activates Hck and Yes in serum-starved mES cells, resulting in the association of Hck and Yes with gp130 through their SH2 domains ([23,53,54], and C. Annerén, unpublished work). C-terminal truncation mutants of gp130 and LIFR fail to induce Hck kinase activity [55]. Serum is also able to induce Yes kinase activity in the absence of LIF in mES cells, indicating that not only LIF regulates SFK activity [53]. Several components present in serum, including FGFs (fibroblast growth factors), Wnts and members of the TGFβ (transforming growth factor β) superfamily have been implicated in regulating self-renewal of mES and hES cells, and many of these may theoretically be responsible for activating Yes and other SFKs, although this remains to be shown. Several studies have shown that a subset of SFKs, such as Hck and Yes, promote self-renewal of ES cells. Induced expression of a kinase-active mutant of Hck maintains ES cells in an undifferentiated state when LIF concentrations are decreased but not absent [23]. Moreover, treating mES and hES cells with the SFK inhibitor SU6656, which is particularly active against Yes [58], decreases ES cell proliferation and survival and the expression of pluripotency genes such as AP (alkaline phosphatase), Oct3/4, FGF4 and Nanog [53,56]. SU6656 also increases the expression of the orphan nuclear receptor gcnf, which is induced by the pro-differentiation factor RA (retinoic acid) and represses oct3/4 gene expression [53,59]. Transfecting mES cells with vectors expressing yes siRNA (small interfering RNA) and a puromycin-resistance gene inhibits the ability to make stable clones in the presence of puromycin, as does transfection with nanog siRNA, suggesting a specific role of Yes in the self-renewal, proliferation or survival of mES cells [53]. Furthermore, transient transfections with yes siRNA induces a decrease in the mRNA levels of nanog, whereas it increases the levels of gcnf [53,59]. A synergistic positive effect on differentiation is also observed when mES cells are cultured in low levels of SU6656 and RA [53]. Inhibition of SFKs with SU6656 in mES cells does not interfere with LIF-induced JAK/STAT3 or ERK1/2 phosphorylation [53,56], suggesting that activation of SFKs by LIF may represent an independent pathway maintaining self-renewal in mES cells.

Meyn et al. [56] have proposed that disparate SFK members may have opposing effects on self-renewal and differentiation of ES cells. Their model (Figure 2) is based on the fact that certain SFK members, such as Src and Fyn, remain highly expressed and active in differentiated EBs, whereas others, such as Yes and Hck, are down-regulated, and on studies with pharmacological inhibitors that have different selectivity profiles for individual SFK members. They showed that complete non-selective inhibition of SFK activity with either the PP2 or A-419259 ATP competitive inhibitors, or high levels of the 4-anilinoquinazoline SKI-1 inhibitor, decrease cell growth and prevent ES cell differentiation. In contrast, the more selective SFK inhibitor SU6656 or low concentrations of SKI-1 caused ES cells to undergo differentiation in the presence of LIF [53,56]. These contradictory results were explained by the fact that SU6656 has a higher selectivity for SFKs linked to self-renewal, e.g. Hck and Yes, but is less potent against, for example, Fyn and Src, which remain active as ES cells differentiate [56,58]. Thus the hypothesis presented by Meyn et al. [56] is that SFKs regulate two independent pathways in ES cells. One pathway, including Src and Fyn, promotes growth and differentiation and another pathway, including Hck and Yes, promotes self-renewal (Figure 2). Although this model is intriguing, it should be noted that high levels of inhibitors can give unwanted inhibition of other PTKs and it cannot therefore be excluded that this may be causing the observed results. A study by Hakuno et al. [60] has shown that the SFK inhibitor PP2 dramatically increases cardiomyocyte differentiation, whereas SU6656 does not have this effect. It was then found that the effect on cardiomyocyte differentiation by PP2 was not due to inhibition of SFKs, as PP2 and SU6656 inhibited Src auto-phosphorylation to a similar degree. Instead, the pro-cardiogenic effect of PP2 was due to inhibition of adhesion- and PDGFβ (platelet-derived growth factor β)-induced FAK (focal adhesion kinase) activation. In contrast, SU6656, even at very high levels, did not inhibit FAK or induce cardiomyocyte differentiation in these experiments. Indeed, it has been shown in several studies that PP2 can induce non-specific inhibition of many PTKs such as the PDGFR (PDGF receptor), EGFR [EGF (epidermal growth factor) receptor], Bcr-Abl, Abl and cKit [58,61,62]. Thus it is possible that other SFK inhibitors also exhibit such non-specific inhibitory effects when given at high levels.

Model for the role of SFK members in self-renewal and differentiation of ES cells, as proposed by Meyn and co-workers [56]

Figure 2
Model for the role of SFK members in self-renewal and differentiation of ES cells, as proposed by Meyn and co-workers [56]

SFKs regulate at least two independent signalling pathways in ES cells. The first one is represented by Src and Fyn and promotes growth and differentiation. The second pathway is represented by Hck and Yes and promotes self-renewal in the presence of LIF (left-hand panel). When both pathways are active in ES cells, the self-renewal pathway dominates, suppressing differentiation and allowing cell expansion. Removal of LIF leads to rapid loss of Hck and Yes activity, allowing differentiation to occur (right-hand panel). Inhibition of Hck and Yes with SU6656 or low concentrations of SKI-1 induces differentiation in the presence of LIF. Inhibition of all SFKs in ES cells inhibits proliferation, but the cells retain the attributes of pluripotent cells, even in the absence of LIF.

Figure 2
Model for the role of SFK members in self-renewal and differentiation of ES cells, as proposed by Meyn and co-workers [56]

SFKs regulate at least two independent signalling pathways in ES cells. The first one is represented by Src and Fyn and promotes growth and differentiation. The second pathway is represented by Hck and Yes and promotes self-renewal in the presence of LIF (left-hand panel). When both pathways are active in ES cells, the self-renewal pathway dominates, suppressing differentiation and allowing cell expansion. Removal of LIF leads to rapid loss of Hck and Yes activity, allowing differentiation to occur (right-hand panel). Inhibition of Hck and Yes with SU6656 or low concentrations of SKI-1 induces differentiation in the presence of LIF. Inhibition of all SFKs in ES cells inhibits proliferation, but the cells retain the attributes of pluripotent cells, even in the absence of LIF.

It has been suggested that Src, Fyn and, possibly, Lck kinases are important mediators in terminal differentiation of glutaminergic neurons derived from ES cells in vitro [63]; however, the functional assays to prove this were all performed with or without the presence of high levels (10 μmol/l) of PP2, which also inhibits other tyrosine kinases as described above.

Suppression of the LIF/STAT3 pathway

Inhibition or termination of LIF signalling is achieved by several different mechanisms. Phosphotyrosines are de-phosphorylated by SHP2, and PIAS (protein inhibitor of activated STAT) can inhibit the transcriptional activity of STAT3 (reviewed in [64]). Moreover, an increasing number of studies have shown that SOCS (suppressors of cytokine signalling) can regulate signalling mediated by gp130. The expression of SOCSs is up-regulated by activated STATs [65], and the SH2 domain in SOCS3 can interact with phosphorylated tyrosines in JAK, STAT or cytokine receptors (reviewed in [66]). The binding of SOCS3 to JAK and STAT may negatively regulate their activity, leading to their degradation via the ubiquitin–proteasome system [67]. SOCS3-null mES cell lines have increased LIF-induced activation of both STAT3 and ERK1/2. This leads to impaired self-renewal and greater differentiation into primitive endoderm. Attenuation of ERK1/2 signalling by the addition of MEK inhibitors to SOCS3-null cell cultures rescues the differentiation phenotype [68]. In conclusion, the overall balance of conflicting activation of STAT3 and ERKs might determine the efficiency of the self-renewal of ES cells. As long as the balance is in favour of JAK1/STAT3 activation, self-renewal is sustained and differentiation is inhibited [12].

FGF AND IGF (INSULIN-LIKE GROWTH FACTOR) PATHWAYS FOR MAINTAINING SELF-RENEWAL OF hES CELLS

As described above, LIF does not induce self-renewal of hES cells. Instead, hES cell lines require supplementation with FGF2, also named bFGF (basic FGF), which binds to members of the FGFR (FGF receptor) tyrosine kinase family. Unlike LIF, which is used to sustain undifferentiated proliferation of mES cells [15], the molecular basis for how FGF2 supports self-renewal is still unclear. Studies using cDNA microarray analysis have shown elevated levels of components of the FGF/FGFR signalling pathway (FGFR1–FGFR4, and FGF2, 11 and 13) in undifferentiated hES cells compared with their differentiated progeny, human tissue and mES cells [6971]. A previous study showed, using real-time PCR, that FGFR1–FGFR4 are indeed all expressed in undifferentiated hES cell cultures, with FGFR1 being the most abundant [72]. However, the same group also showed, in contrast with the gene array data, that initiation of differentiation is accompanied by transcriptional up-regulation of all four FGFRs, whereas the expression of FGF2 was unchanged [72]. Inhibition of FGFR1 by addition of SU5402 to hES cell cultures suppresses activation of downstream protein kinases, down-regulates Oct3/4 expression, up-regulates p27 and causes rapid cell differentiation [72,73]. Undifferentiated hES cells, in contrast with mES cells, contain unusually high basal activity of ERK1/2. When hES cells are exposed to exogenous FGF2, ERK1/2 activity is elevated further and levels of the downstream target c-fos are increased. However, this ERK1/2 activity is not accompanied by changes in differentiation or proliferation [72,74].

FGFR signalling can also induce differentiation of hES cells. FGF2 induces development of ectodermal and mesodermal cells from hES cells and can also support hES cell differentiation into neural lineages [7580]. However, in all of these experiments, FGF2 is added to pre-differentiated hES cells in EBs, suggesting that hES cells use exogenous FGF2 in different ways depending on their state of differentiation.

In mES cells, FGFR signalling appears to be crucial for early epithelial differentiation, as mES cells expressing a dominant-negative truncated mutant of FGFR2 fail to differentiate into the two epithelial layers of the EB. A concomitant decrease in Akt and PLCγ-1 (phospholipase Cγ-1) activation by the truncated FGFR2 mutant suggests that FGF signalling and epithelial differentiation is connected with the PI3K pathway [81]. Another study indicated that FGFR1 plays a role in endoderm formation, as a targeted disruption of FGFR1 in mES cells blocked the formation of the visceral endoderm, organization of the columnar epithelium and cavitation in EBs [82]. Several studies also suggest a role of FGFR1 in haemopoietic and cardiomyocyte, but not vascular, development. FGFR1-null mES cells have a severely impaired ability to generate pulsating cardiomyocytes in 9-day-old EBs, and the expression of early and late cardiac genes is decreased in these cells compared with wild-type EBs. Moreover, treating ES cells with inhibitors of FGFR tyrosine kinase signalling, such as the FGFR1 inhibitor SU5402, also prevents cardiomyocyte differentiation, without affecting the expression of the haemopoietic/endothelial cell marker flk1 [83]. Moreover, haemopoietic development is disturbed in FGFR1-null EBs compared with wild-type stem cells, whereas vascularization is enhanced in these cells [84]. FGF4 is also required for appropriate differentiation of mES cells, and inactivation of the fgf4 gene in mES cells alters endoderm differentiation [85]. Interestingly the fgf4 gene is a direct target of Oct3/4 and Sox2 [86]. Recently, it was demonstrated that FGF4, and its associated activation of the ERK1/2 signalling cascade, induces mES cells to exit the self-renewal programme [87]. Interfering with FGF or ERK1/2 activity did not impair mES cell self-renewal, but restricted their ability to commit to differentiation [87].

A proteomic screen, to identify other candidate hES-cell-supportive factors, revealed that the most prevalent family of growth factors, present in MEF-conditioned medium, are IGFs, of which IGF2 was the most abundant. The receptor for IGF2, the tyrosine kinase IGF1R (IGF1 receptor), has also been identified in a genomic analysis of hES cells [71]. Recently, Bendall and co-workers [73] presented a model showing how FGF2 may maintain the self-renewal of hES cells by activating the IGF2 pathway. They distinguished two morphologically distinct regions in culture plates of hES cells grown in feeder-free cultures, which they claim could provide some insight into paracrine signalling events regulating the self-renewal of hES cells (Figure 3). One region consists of the undifferentiated Oct3/4-expressing hES cells in central tightly packed colonies. The other region, surrounding the colonies, consists of hdF cells (human differentiated fibroblast-like cells) which do not express Oct3/4. Interestingly, immunocytochemical analysis of hES cell cultures showed that FGFR1 is only expressed in the hdF cells surrounding the Oct3/4-expressing cell colonies, suggesting that, although FGFRs are important in hES cell culture systems, they are not expressed by the self-renewing pluripotent hES cells [73]. They then showed that IGF1R, on the other hand, is expressed exclusively in the undifferentiated hES colony cells and correlates with pluripotent stem cell markers such as Oct3/4, SSEA4 (stage-specific embryonic antigen 4), Tra-1-81 (tumour recognition antigen-1-81) and Tra-1-60 (tumour recognition antigen-1-60) [73]. Although FGFR1 inhibition with SU5402 increased cell differentiation of hES cells but had little effect on proliferation, blocking IGFR impeded cell expansion and decreased SSEA3 expression and Akt phosphorylation [73]. They then demonstrated that IGF2 is expressed by hdF cells in response to FGF2 and that addition of IGF2 alone in defined conditions was sufficient to sustain expansion self-renewal and pluripotency of hES cells. Moreover, TGFβ1 was also found to be expressed by hdF cells in response to FGF2 in hES cell cultures [73]. TGFβ appears to be important for self-renewal of hES cells, similar to BMP4 in mES cells [15,88]. On the basis of their results Bendall and co-workers proposed the model in which hES cells, cultured without MEFs and in the presence of FGF2, spontaneously differentiate into hdF cells. In response to FGF2, hdF cells express hES-cell-supportive factors, including IGF2 and TGFβ, which act in concert to sustain self-renewal of undifferentiated hES cells in the central colonies.

Model for the role of FGF2 in maintaining the self-renewal of hES cells, as proposed by Bendall and co-workers [73]

Figure 3
Model for the role of FGF2 in maintaining the self-renewal of hES cells, as proposed by Bendall and co-workers [73]

Two morphologically distinct regions can be distinguished in plates of hES cells grown in feeder-free cultures in the presence of FGF2: Oct3/4-expressing central colonies and surrounding hdF cells. IGF1R is exclusively expressed in undifferentiated cells in central colonies, whereas FGFR1 is predominantly expressed in the surrounding hdF cells that have already started to differentiate and have lost Oct3/4 expression. hES cells spontaneously differentiate into hdF cells that, upon stimulation, with FGF2 start to express endogenous hES-cell-supportive factors, including IGF2 and TGFβ. IGF2 binds IGF1R and is alone sufficient in maintaining expansion and pluripotency of hES cell cultures. TGFβ is also important for the self-renewal of hES cells.

Figure 3
Model for the role of FGF2 in maintaining the self-renewal of hES cells, as proposed by Bendall and co-workers [73]

Two morphologically distinct regions can be distinguished in plates of hES cells grown in feeder-free cultures in the presence of FGF2: Oct3/4-expressing central colonies and surrounding hdF cells. IGF1R is exclusively expressed in undifferentiated cells in central colonies, whereas FGFR1 is predominantly expressed in the surrounding hdF cells that have already started to differentiate and have lost Oct3/4 expression. hES cells spontaneously differentiate into hdF cells that, upon stimulation, with FGF2 start to express endogenous hES-cell-supportive factors, including IGF2 and TGFβ. IGF2 binds IGF1R and is alone sufficient in maintaining expansion and pluripotency of hES cell cultures. TGFβ is also important for the self-renewal of hES cells.

As early as 1995, Takahashi and co-workers [89] demonstrated that the IGF2 pathway could co-operate with LIF to sustain the self-renewal of rat ES cells. They showed that the combination of mouse LIF and rat IGF2 promoted the growth of the ICM of the blastocyst and was effective for establishment of pluripotent cell lines derived from the ICM. IGF signalling has also been suggested to be important for the self-renewal of mES cells. IRS1 (insulin receptor substrate 1) is a major downstream signalling protein for insulin and IGF1Rs and conveys signals to the PI3K/Akt and ERK1/2 pathways. It has been found to be expressed and phosphorylated during self-renewal of mES cells, and differentiation of mES cells is associated with a decrease in IRS1 expression [90]. Targeting IRS1 with irs1 siRNA results in a decrease in Oct3/4 protein expression and AP activity, and a concomitant decrease in phosphorylation of the PI3K signalling proteins GSK3 and Akt. Moreover, a marked down-regulation of Id1 and Id2 protein expression, important components of the BMP4 signalling pathway, is observed [90]. Conversely, overexpression of IRS1 inhibits mES cell differentiation [90], and treating mES cells with IGF1 or insulin induces phosphorylation of IGF1R and ERK1/2 [91]. In contrast, Prelle et al. [92] have shown that overexpressing IGF2 in mES cells promotes myogenic differentiation.

EGF PATHWAY FOR MAINTAINING mES CELL GROWTH

EGF is a powerful mitogen that elicits DNA synthesis and proliferation in a variety of cell types. The effects of EGF are related to the activity of the EGFR tyrosine kinase (ErbB1). EGF induces DNA synthesis in mES cells via a PLC/PKC (protein kinase C), Ca2+ influx and ERK1/2 signalling pathway [93]. Moreover, EGF increases the levels of GLUT1 (glucose transporter 1) and, thus, the 2-deoxyglucose uptake through PKC, p38 MAPK and ERK1/2 pathways. GLUT1 is the major glucose transporter in the ICM of the blastocyst, which contains the ES cells [94], and EGF may thus be responsible for helping the ES cells meet the high metabolic demands of the actively dividing cells [95]. Inhibition of EGFR signalling in ES cells induces selective apoptosis and decreased growth rates of certain differentiated cell types; thus increasing the proportion of cell types that do not appear to require EGFR kinase activity for their production or survival. Skeletal and cardiac muscle are found to be the main differentiated tissue produced from EGFR- mutant clones, whereas other cell types are sparse or absent [96].

POSSIBLE ROLE OF THE SCF (STEM CELL FACTOR)/Kit PATHWAY FOR SELF-RENEWAL OF mES CELLS

The SCF receptor cKit is a receptor tyrosine kinase that plays a role in proliferation, differentiation and survival of a range of stem and progenitor cells (reviewed in [97]). A correlation between cKit expression and pluripotency of mES cells has been reported [98,99]. cKit-null mES cells and mES cells neutralized with a cKit antibody die when induced to differentiate in the absence of LIF in monolayer cultures, and this effect could be reversed in mutant cells by activation of a hybrid cKit receptor [99,100]. Sorting mES cells on the basis of cKit expression showed that cKit-negative cells express lower levels of the pluripotency genes bmp4 and nanog and elevated levels of genes known to be up-regulated during ES cell differentiation, such as pim3 and pim2, compared with unfractionated cells or cells expressing high levels of cKit [99]. However, SCF/cKit signalling is not sufficient to maintain the self-renewal of mES cells in the absence of LIF [99].

TIE2, FLK1 (FOETAL LIVER KINASE 1) AND PDGFR PATHWAYS FOR LINEAGE COMMITMENT OF ES CELLS

Tie2, FLK1 and PDGFRs are all involved in lineage commitment of pre-differentiated ES cells and do not appear to play a role in the self-renewal of ES cells. Tie2 and FLK1 mark the haemangioblast and, although Tie2 is important for ES-cell-derived endothelial, but not haemopoietic, cell survival [101], FLK1 is crucial for establishment of endothelial and haemopoietic lineages and perhaps for their common progenitor [102,103]. PDGF is an angiogenetic factor and stimulates blood vessel formation in EBs [102,104], but has also been shown to promote cardiogenesis in mES cells [105].

NTs (NEUROTROPHINS) AND TRK RECEPTORS MEDIATE ES CELL SURVIVAL

A limitation to hES cell studies has been the difficulty in trypsinizing and clonally deriving these cells. hES cells sustain self-renewal when cultured in tightly packed colonies on MEFs, but the survival of single hES cells is very low. In fact, the clonal survival of single hES cells is only approx. 0.4% [106], and single cell cultures often lead to high rates of karytopic abnormalities, differentiation and increased cell death [107]. The knowledge of ES-cell-survival factors is still limited, but recently Pyle et al. [106] have demonstrated a role for NTs in hES cell survival. They showed that hES cells express TrkB and TrkC [but not TrkA and p75NGFR, where NGFR is NGF (nerve growth factor) receptor], which are tyrosine kinase receptors for the NGFR family of NTs [70,106,108,109]. Pyle et al. [106] showed that the addition of BDNF (brain-derived neurotrophic factor), NT3 and NT4, which are ligands for TrkB and TrkC, to trypsinized single hES cells activated the PI3K/Akt survival pathway and significantly increased the cloning ability of these cells (36-fold increase). The surviving clones exhibited maintained pluripotency and developmental potential. In addition, treating hES cells with pharmacological inhibitors against Trk receptors induced apoptosis. Interestingly, it was found that MEFs express several NTs, including BDNF, NT3, NT4 and NGF, and that neutralizing antibodies to BDNF, NT3 and NT4 could decrease the clonal survival of hES cells cultured on MEFs in a dose-dependent manner. Antibodies to NGF, however, had no significant effect on the number of AP-positive colonies, which was explained by the low expression levels of the NGF receptor TrkA and p75NGFR in hES cells [106].

The ability of NTs to support clonal survival of hES cells may facilitate many uses of hES cells, such as genetic selection or high-throughput screening [106]. It is not yet known what role NTs play in mES cells, but one study has suggested that they may play a role in mouse oocyte maturation leading to improved embryonic development [110].

BCR-ABL TYROSINE KINASE IN THE SELF-RENEWAL OF ES CELLS

Chimaeric tyrosine kinase fusion protein Bcr-Abl, responsible for the development of CML (chronic myelogenous leukaemia), has the capacity to induce a haemopoietic proliferative potential when expressed in differentiating ES cells [111,112]. Induced expression of Bcr-Abl in EBs increases the number of multipotent and myeloid-lineage-committed progenitors in a dose-dependent manner while suppressing the development of committed erythroid progenitors. When Bcr-Abl is expressed in undifferentiated ES cells, however, it phosphorylates and activates STAT3 and promotes self-renewal of ES cells in the absence of LIF [113115]. Bcr-Abl also increases MEKK1 (MEK kinase 1) levels in ES cells, and studies in MEKK1-knockout ES cells suggest that MEKK1 is essential for Bcr-Abl-induced STAT3 activation and self-renewal [113].

CONCLUSIONS

The present review summarizes some of the current knowledge on the role of PTK signalling in mES and hES cells (Table 1). Although some PTKs appear to play similar roles in hES and mES cells, many of them exhibit disparate functions and expression patterns. Maintaining self-renewal of ES cells, in particular, is differently regulated in mES and hES cells. mES cells are maintained by activation of the LIF pathways with support from BMP4, whereas maintaining self-renewal and survival of hES cell is mediated by NTs and FGF2. Some downstream cytoplasmic signalling pathways are also differently activated and play disparate roles in mES and hES cells, such as the ERK1/2 and the JAK/STAT3 pathways. Although there is evidence suggesting significant species differences, some critical and conserved pathways regulating self-renewal of both mES and hES cells have also been identified. For instance, Wnt signalling through GSK3β can, independently of the LIF pathway, maintain both mouse and human stem cell populations [4951]. Moreover, cell survival is mediated by the activation of the PI3K/Akt pathway in both mES and hES cells, although this pathway is activated by LIF in mES cells and by NTs and IGF2 in hES cells. The SFK Yes is expressed and active in undifferentiated mES and hES cells, and the activity decreases when the cells differentiate. In mES cells, Yes activity is induced by LIF or serum [53]. The factor(s) responsible for activating SFKs in hES cells remains to be shown, but possible candidates are FGF2, IGF2 or other components present in serum. Yes inhibition induces differentiation of both mouse and human stem cells [53]. Although different receptor activations and cell signalling pathways are active in hES and mES cells, they appear to merge to the same conserved transcriptional pathways. This is supported by the recent studies demonstrating that overexpression of the same transcription factors in mES and hES cells, Oct3/4 and Sox2, Klf4 and c-Myc, could reprogramme mouse and human somatic cells to become iPS cells [26,28]. c-Myc and Klf4 are both downstream targets of the LIF/STAT3 pathway in mES cells, but it is not known how these are targeted in hES cells. However, degradation of c-Myc has been shown to be mediated by the Wnt target GSK3β [24,116] and, therefore, Wnt signalling in mES and hES cells may contribute to the total level of c-Myc. Reprogramming of human somatic cells to become iPS cells has also been accomplished by overexpressing Nanog and Lin28 (instead of c-Myc and Klf4), together with Oct3/4 and Sox2 [32]. Thus, as it appears that some factors can substitute for each other, it is likely that they either share the same targets or activate one another. Indeed, it is known that the combined action of Sox2 and Oct3/4 activate Nanog [34]. Moreover, Klf4 can co-operate with Oct3/4 and Sox2 to activate gene expression [117] and may also be indirectly involved in up-regulating Nanog protein expression by repressing p53 [118]. Although it is not completely understood how these transcription factors are activated in hES and mES cells, they are probably targeted by several upstream signalling pathways that act in different constellations in mES and hES cells. Many of these pathways will most definitely include PTKs.

Table 1
Summary of tyrosine kinases found in ES cells and their function
Tyrosine kinaseFunction in ES cellsReferences
Receptor tyrosine kinase   
 FGFR1 Expressed in Oct3/4-negative hdF cells in hES cell cultures, which express IGF2 and TGFβ in response to FGF2. Important for differentiation of mES cells into ectodermal and mesodermal cells when activated in differentiated EBs. [72,73,7580
 IGF1R Expressed in undifferentiated hES cells and correlates with pluripotency. Activation of IGF1R with IGF2 is sufficient to maintain the self-renewal of hES cells. [71,73,89
 EGFR Induces DNA synthesis and 2-deoxyglucose uptake in mES cells. [9395
 PDGF Angiogenetic factor stimulating blood vessel formation in EBs. Promotes cardiogenesis in mES cells. [102,104,105
 cKit Prevents apoptosis and enhances cell-cycle progression. Expression correlates with pluripotency but activation is not sufficient for maintaining the self-renewal of ES cells in the absence of LIF. [98100
 Tie2 Marks the haemangioblast. Important for ES-cell-derived endothelial, but not haemopoietic, cell survival. [101
 Flk-1 Marks the haemangioblast. Crucial for establishment of endothelial and haemopoietic lineages and perhaps for their common progenitor. [102,103
 TrkB and TrkC Maintain survival and improve cloning of hES cells. Several NTs that bind and activate TrkB and TrkC are expressed by MEFs. [106
SFK   
 Src and Fyn Maintain ES cell growth. May induce differentiation, as inhibition of these sustain the self-renewal of ES cells. Important mediators in terminal differentiation of glutaminergic neurons derived from mES cells [56,63
 Hck and Yes Maintain the self-renewal and cell growth of mES cells. [23,5356
JAK family   
 JAK1 Activates STAT3 and maintains self-renewal of mES cells reviewed in [1113
 JAK2 and TYK1 Involved in early lineage decisions. JAK2 is important for cardiomyogenesis. [36,37
Fusion protein   
 Bcr-Abl Phosphorylates and activates STAT3, and promotes the self-renewal of mES cells in the absence of LIF. [113115
Tyrosine kinaseFunction in ES cellsReferences
Receptor tyrosine kinase   
 FGFR1 Expressed in Oct3/4-negative hdF cells in hES cell cultures, which express IGF2 and TGFβ in response to FGF2. Important for differentiation of mES cells into ectodermal and mesodermal cells when activated in differentiated EBs. [72,73,7580
 IGF1R Expressed in undifferentiated hES cells and correlates with pluripotency. Activation of IGF1R with IGF2 is sufficient to maintain the self-renewal of hES cells. [71,73,89
 EGFR Induces DNA synthesis and 2-deoxyglucose uptake in mES cells. [9395
 PDGF Angiogenetic factor stimulating blood vessel formation in EBs. Promotes cardiogenesis in mES cells. [102,104,105
 cKit Prevents apoptosis and enhances cell-cycle progression. Expression correlates with pluripotency but activation is not sufficient for maintaining the self-renewal of ES cells in the absence of LIF. [98100
 Tie2 Marks the haemangioblast. Important for ES-cell-derived endothelial, but not haemopoietic, cell survival. [101
 Flk-1 Marks the haemangioblast. Crucial for establishment of endothelial and haemopoietic lineages and perhaps for their common progenitor. [102,103
 TrkB and TrkC Maintain survival and improve cloning of hES cells. Several NTs that bind and activate TrkB and TrkC are expressed by MEFs. [106
SFK   
 Src and Fyn Maintain ES cell growth. May induce differentiation, as inhibition of these sustain the self-renewal of ES cells. Important mediators in terminal differentiation of glutaminergic neurons derived from mES cells [56,63
 Hck and Yes Maintain the self-renewal and cell growth of mES cells. [23,5356
JAK family   
 JAK1 Activates STAT3 and maintains self-renewal of mES cells reviewed in [1113
 JAK2 and TYK1 Involved in early lineage decisions. JAK2 is important for cardiomyogenesis. [36,37
Fusion protein   
 Bcr-Abl Phosphorylates and activates STAT3, and promotes the self-renewal of mES cells in the absence of LIF. [113115

Finally, many tyrosine kinases play crucial roles in self-renewal or cell fate decisions in mES and hES cells. However, the tyrosine kinase family is very big and diverse, with members demonstrating vastly different cell functions. The role of many of them in ES cells and exactly how they exert their action remains to be investigated.

Abbreviations

     
  • AP

    alkaline phosphatase

  •  
  • BDNF

    brain-derived neurotrophic factor

  •  
  • BMP4

    bone morphogenetic protein 4

  •  
  • EB

    embryoid body

  •  
  • EGF

    epidermal growth factor

  •  
  • EGFR

    EGF receptor

  •  
  • ERK

    extracellular-signal-related kinase

  •  
  • ES cell

    embryonic stem cell

  •  
  • FAK

    focal adhesion kinase

  •  
  • FGF

    fibroblast growth factor

  •  
  • FGFR

    FGF receptor

  •  
  • FLK1

    foetal liver kinase 1

  •  
  • GRB2

    growth-factor-receptor-bound protein 2

  •  
  • GAB1

    GRB2-associated-binder protein 1

  •  
  • GSK3

    glycogen synthase kinase 3

  •  
  • hdF cell

    human differentiated fibroblast-like cell

  •  
  • hES cell

    human ES cell

  •  
  • ICM

    inner cell mass

  •  
  • IGF

    insulin-like growth factor

  •  
  • IGF1R

    IGF1 receptor

  •  
  • iPS cell

    induced pluripotent stem cell

  •  
  • IRS1

    insulin receptor substrate 1

  •  
  • JAK

    Janus tyrosine kinase

  •  
  • Klf4

    Krüpel-type zinc-finger 4

  •  
  • LIF

    leukaemia-inhibitory factor

  •  
  • LIFR

    LIF receptor

  •  
  • MAPK

    mitogen-activated protein kinase

  •  
  • MEF

    mouse embryonic fibroblast

  •  
  • MEK

    MAPK/ERK kinase

  •  
  • MEKK

    MEK kinase

  •  
  • mES

    cell, mouse ES cell

  •  
  • NGF

    nerve growth factor

  •  
  • NGFR

    NGF receptor

  •  
  • NT

    neurotrophin

  •  
  • Oct

    octamer-motif-binding transcription factor

  •  
  • PDGF

    platelet-derived growth factor

  •  
  • PDGFR

    PDGF receptor

  •  
  • PI3K

    phosphoinositide 3-kinase

  •  
  • PKC

    protein kinase C

  •  
  • PLC

    phospholipase C

  •  
  • PTB

    phosphotyrosine-binding

  •  
  • PTK

    protein tyrosine kinase

  •  
  • RA

    retinoic acid

  •  
  • SCF

    stem cell factor

  •  
  • SFK

    Src family kinase

  •  
  • SH2

    Src homology 2

  •  
  • SHP

    SH2-domain-containing cytoplasmatic tyrosine phosphatase

  •  
  • siRNA

    small interfering RNA

  •  
  • SOCS

    suppressors of cytokine signalling

  •  
  • Sox

    SRY-type high-mobility-group box

  •  
  • SSEA

    stage-specific embryonic antigen

  •  
  • STAT

    signal transducer and activator of transcription

  •  
  • TGF

    transforming growth factor

I thank Dr Erik Forsberg and Dr Christoffer Tamm for comments on the manuscript. The research in my laboratory is supported by the Swedish Research Council, the Juvenile Diabetes Research Foundation, the Swedish Diabetes Association and the Jaensson's Foundation.

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