ES (embryonic stem) cell lines are derived from the epiblast of pre-implantation embryos and like the inner cell mass cells from which they are derived exhibit the remarkable property of pluripotency, namely the ability to differentiate into all cell lineages comprising the adult organism. ES cells and their differentiated progeny offer tremendous potential to regenerative medicine, particularly as cellular therapies for the treatment of a wide variety of chronic disorders, such as Type 1 diabetes, Parkinson's disease and retinal degeneration. In order for this potential to be realized, a detailed understanding of the molecular mechanisms regulating the fundamental properties of ES cells, i.e. pluripotency, proliferation and differentiation, is required. In the present paper, we review the evidence that PI3K (phosphoinositide 3-kinase)-dependent signalling plays a role in regulation of both ES cell pluripotency and proliferation.
- bone morphogenetic protein (BMP)
- embryonic stem cell (ES cell)
- phosphoinositide 3-kinase (PI3K)
ES (embryonic stem) cells
ES cell lines exhibit the unique property of pluripotency, i.e. the ability to differentiate into all cell lineages comprising the adult organism [1,2]. Pluripotency is underpinned by ES cell self-renewal, the term given to the symmetrical division of ES cells to generate two identical, undifferentiated daughter cells. In essence, self-renewal is proliferation that is accompanied by the suppression of differentiation. Regulation of ES cell self-renewal is of great interest because the ability to maintain and expand pluripotent ES cells is essential if the therapeutic potential offered by ES cell-derived progeny to regenerative medicine is to be realized.
Regulation of murine ES cell pluripotency
Pluripotency of mES (murine ES) cells is regulated by a complex network of extrinsic factors, signalling pathways and transcription factors. LIF (leukaemia inhibitory factor) was the first cytokine shown to play a role in maintenance of pluripotency , via activation of STAT3 (signal transducer and activator of transcription 3) and induction of c-Myc [3–5]. However, like many cytokines, LIF also activates the ERKs (extracellular-signal-regulated kinases) ERK1 and ERK2, which appear to promote differentiation , leading to the suggestion that the balance between STAT3 and ERK signals is important in determining ES cell fate . More recently it has been demonstrated that BMPs (bone morphogenetic proteins) BMP2 and BMP4 collaborate with LIF to facilitate mES cell self-renewal, via BMP2/BMP4 activation of Smad4, inducing expression of the Id transcriptional repressors, which actively suppress neuronal differentiation . Wnt signalling has also been implicated, largely as a result of the use of the small molecule BIO [6-bromo-indirubin 3′-oxime; an inhibitor of GSK-3 (glycogen synthase kinase 3)] that facilitates activation of the canonical Wnt signalling pathway . The Src tyrosine kinase also appears to play a role in maintenance of self-renewal of mES cells .
Among the transcriptional regulators, Oct-4 (octamer-binding protein-4), Sox-2 and Nanog have all been implicated in the maintenance of mES cell pluripotency . Oct-4 is expressed by undifferentiated mES and hES (human ES) cells, its expression being lost upon differentiation . Nanog (from the Norse Tir nan Og meaning ‘ever young’) was identified by expression cloning  and in silico analyses  as a homeodomain protein that can maintain pluripotency of mES cells independently of LIF. Recent large-scale screening studies have mapped promoters co-bound by Nanog, Oct-4 and Sox-2 in hES cells  and Nanog/Oct-4 co-bound promoters in murine ES cells , demonstrating that Oct-4 and Nanog target some of the same genes in ES cells. Ivanova et al.  have identified a further five genes, Tcl1, Tbx3, Esrrb, Dppa4 and Unigene Mm.343880, whose down-regulation leads to loss of pluripotency of mES cells. Further evidence suggests that epigenetic mechanisms also play a part in determining ES cell fate . Thus it is increasingly clear that a network of transcription factors and other regulators influence and control the fate of both mES and hES cells .
Regulation of ES cell proliferation
Studies of murine and Rhesus monkey ES cells have demonstrated that the cell cycle of ES cells is not subject to the same regulatory checkpoints as the cell cycle of somatic cells [18–20]. mES cells transit through the cell cycle very rapidly (∼8 h), spending as little as 1–2 h in G1-phase and as a consequence approx. 50% of ES cells in a growing population are in S-phase [18,19]. The unorthodox cell cycle of mES cells, which shows no periodicity in cyclin E expression and no retinoblastoma restriction point [18,19], has made it a challenge to define the molecular mechanisms that regulate ES cell proliferation and the relationship between the signals regulating proliferation and pluripotency is still relatively poorly understood. Recent data have demonstrated that an ES-cell-specific Ras-family member, termed ERas (ES cell-expressed Ras), plays a role in controlling proliferation of mES cells  as does mTOR (mammalian target of rapamycin) [22,23]. Both of these pathways involve PI3K (phosphoinositide 3-kinase)-dependent signalling.
The PI3Ks are a family of lipid kinases, comprising three subclasses and it is typically the class IA subgroup that is activated in response to growth factor and cytokine signalling . Class IA PI3Ks are heterodimers, consisting of a regulatory subunit (for which there are three genes, generating five isoforms, p85α, p55α, p50α, p85β and p55γ) and a catalytic subunit, of which there are three isoforms, p110α, p110β and p110δ, each encoded by a separate gene. The primary product of class IA PI3K action is the phosphoinositide PI(3,4,5)P3 (phosphatidylinositol 3,4,5-trisphosphate), which recruits PH domain (pleckstrin homology domain)-containing proteins to the plasma membrane, facilitating activation of a range of downstream signalling cascades . Functionally, PI3Ks have been implicated in a wide array of physiological processes including proliferation, development, growth and migration . Accumulating evidence from mice that have the activity of a selected class IA catalytic or regulatory isoform ablated have demonstrated that specific isoforms play selective roles both during development and in the function of somatic cells. For example, p110δ plays a key role in T-, B- and mast-cell biology [24,26], p110γ in inflammatory cells  and p110α in insulin signalling [27,28]. Interestingly, ablation of the p110β isoform leads to lethality at the pre-implantation stage and although the molecular basis of this lethality is unknown, this observation suggests that PI3K signalling plays a critical role during early embryogenesis . One caveat to the gene-targeted mice that completely lack a specific class IA PI3K isoform is the observation that expression of other subunits can also be affected, complicating interpretation of the data in some studies. For a more detailed discussion see .
PI3K signalling and ES cell proliferation
A role for PI3Ks in control of mES cell proliferation was first suggested when it was discovered that ES cells deficient in PTEN [phosphatase and tensin homologue deleted on chromosome 10; a lipid phosphatase that dephosphorylates the 3′ position of the inositol ring, thereby reducing PI(3,4,5)P3 levels and acting as a negative regulator of PI3K signalling] displayed a 5–10% decrease in the time taken to complete a cell division cycle . Interestingly, PTEN-null ES cells also show decreased dependency on serum for division, raising the possibility that serum-containing factors play an important role in activation of PI3Ks in ES cells. Ablation of the ES cell-specific form of Ras, ERas, also results in decreased ES cell proliferation, while expression of an active form of p110α in ERas-deficient ES cells restores proliferation to levels similar to those of wild-type ES cells . However, ERas cannot be the only activator of class IA PI3Ks in ES cells, since PKB (protein kinase B) activity (PKB is a critical downstream mediator of PI3K signals) is still observed in ERas-null ES cells , although significantly decreased. Hence, the evidence from these essentially gain-of-function approaches suggest that an elevation in PI(3,4,5)P3 levels can enhance proliferation of ES cells. Further data support a role for PI3Ks in regulation of ES cell proliferation since inhibition of PI3Ks with 25 μM of the broad specificity inhibitor LY294002 decreases ES cell proliferation, cells accumulating in G1-phase of the cell cycle . mTOR signalling plays a key role in regulation of mES cell proliferation, since inducible deletion of mTOR in mES cells or treatment with rapamycin leads to dramatic decreases in ES cell proliferation [22,23]. In contrast however, knockout of the pik3r1 gene (which produces the p85α, p55α and p50α regulatory subunits) leads to only a modest decrease in ES cell proliferation, whereas ES cells deficient in PDK1 (phosphoinositide-dependent kinase 1), one of the key downstream targets of class IA PI3K signalling, have no reported proliferative phenotype . One caveat to these latter two studies is that compensatory changes could have arisen during selection of the ES cell clones derived and used for these studies such that alternative pathways controlling proliferation are up-regulated in these clones. While this important possibility should be taken into consideration, current evidence collectively suggests that PI3K-dependent signalling does play a role in controlling ES cell proliferation , similar to the role of PI3Ks in somatic cells .
PI3K signalling and ES cell self-renewal
In contrast with the reports implicating PI3K signalling in regulation of ES cell proliferation, our group has reported that PI3K-dependent signals are required for optimal maintenance of self-renewal, since inhibition of PI3K signalling (with either 5 μM LY294002 or upon expression of a dominant-negative class IA PI3K mutant) in the presence of LIF led to loss of self-renewal , with cells under going multi-lineage differentiation (E. Kingham and M. J. Welham, unpublished work). Our findings have recently been supported by the work of other groups. For example, expression of a myristoylated form of PKB maintains pluripotency of murine ES cells in the absence of LIF and also maintains self-renewal of monkey ES cells . In addition, PKB was identified in an RNA interference-based screen for positive regulators of ES cell self-renewal . Importantly, a role for PI3K signalling in maintenance of hES cell pluripotency and survival has been reported [35,36]. To date there has been little commonality demonstrated between the signals regulating murine versus hES cell pluripotency, so the demonstration that PI3K signalling plays a role in maintenance of pluripotency in both cell types is significant.
Mechanistically, our group has identified a potential role for activation of ERK signalling upon inhibition of PI3Ks in promoting the loss of self-renewal observed . In further studies, we have found that PI3K inhibition leads to down-regulation of the intrinsic regulator, Nanog, at both the RNA and protein levels. We have also revealed a role for GSK-3 in PI3K-dependent regulation of self-renewal and Nanog expression. Significantly, activation of an inducible form of Nanog prevents the loss of self-renewal observed in the presence of PI3K inhibitors, consistent with the relationship between PI3K signalling and maintenance of Nanog expression being a functional one .
We have taken a number of approaches to further investigate the molecular mechanisms whereby PI3Ks regulate ES cell fate. Using Affymetrix microarray expression analyses we have defined the PI3K-dependent transcriptome in ES cells generating a dataset of genes whose expression is altered upon inhibition of PI3K signalling in ES cells. We have also investigated the ability of other growth factors and cytokines to activate PI3K signalling in ES cells. Our previous work has shown that LIF can stimulate PI3K signalling in ES cells, while LIF or serum treatment facilitates co-precipitation of the p85 PI3K regulatory subunits with phosphotyrosine proteins, indicative of recruitment/activation of class IA PI3Ks . To investigate which extracellular factors may further contribute to activation of PI3Ks in ES cells we have used serum-free conditions to cultivate ES cells, as described in . Using this system, we have found that LIF, BMP4 and insulin can all activate PI3K signalling, judged in part by their ability to induce phosphorylation of S6 ribosomal protein at serine residues Ser235/Ser236. In each case, S6 phosphorylation can be inhibited by wortmannin (see Figure 1). These results confirm that multiple upstream factors can activate PI3Ks in ES cells and, in view of the observation that PTEN-null ES cells exhibit decreased dependence on serum for their growth, suggest that PI3Ks are normally activated by multiple signals in ES cells.
Interestingly, under the serum-free conditions used, ES cells needed to be maintained at densities of more than ∼1.5×104 cells/cm2 in order to retain viability, leading us to consider a contribution from either cell–cell contact or autocrine factors in maintenance of ES cells. To study this, conditioned medium was collected from ES cells for 24 h (either in the presence or absence of LIF) and then used to treat naïve ES cells. Expression of Nanog was monitored by quantitative PCR, as a measure of the ability to maintain expression of pluripotency genes, and the effects were compared with those of fresh medium±LIF. Our results (Figure 2) suggest that ES cells can produce autocrine factors that co-operate with LIF to ensure maximal expression of Nanog. We have also studied the effects of PDK1 (phosphoinositide-dependent kinase 1) deficiency on ES cell pluripotency. In contrast with the initial report , we find that pdk-1-null ES cells exhibit enhanced proliferation compared with parental ES cells and while self-renewal of pdk1-null ES cells is decreased at low doses of LIF, this is not apparent at optimal doses of LIF (H. K. Bone and M. J. Welham, unpublished work). Most surprisingly, we discovered that pdk-1 ES cells exhibit elevated levels of Nanog mRNA and protein (H. K. Bone and M. J. Welham, unpublished work). These observations suggest the pdk1-null ES cells may have undergone compensatory alterations during either their initial derivation and/or subsequent culture and these findings highlight to us the difficulty in comparing ES lines with targeted homozygous disruption of specific genes.
How can the evidence supporting a role for PI3Ks in regulation of both mES cell proliferation and regulation of pluripotency be rationalized? We have observed that doses of LY294002 greater than 10 μM decrease ES cell proliferation , while others have shown similar effects with 25 μM LY294002 . At lower doses, LY294002 perturbs self-renewal . mTOR is a key regulator of ES cell proliferation [22,23] and can also be inhibited by LY294002, but in general this requires higher doses of LY294002 than the dose that inhibits class IA PI3K isoforms β and δ . Hence, it is possible that the effects of higher doses of LY294002 on proliferation result from additional inhibition of mTOR. Taking these findings into consideration we propose two models that may explain the involvement of PI3Ks in the control of both proliferation and self-renewal of ES cells. In model 1, distinct PI3K isoforms could couple selectively with either ERas or mTOR, to regulate ES cell proliferation, while other isoforms couple specifically with GSK-3/Nanog pathways, thereby contributing to regulation of self-renewal. In an alternative model, the PI3K-dependent pathways controlling pluripotency and proliferation could be regulated by different threshold levels of PI(3,4,5)P3. A modest reduction in PI(3,4,5)P3 levels could perturb PI3K-dependent signals involved in regulation of self-renewal, while proliferation is not affected. A more significant reduction in PI(3,4,5)P3 levels could lead to a decline in the PI3K-dependent signals regulating proliferation, resulting in decreased ES cell proliferation. Future investigations will help delineate the mechanisms by which PI3Ks contribute to regulation of ES cell fate.
We thank all the members of the Welham laboratory for discussions and encouragement. This work was supported by funding from BBSRC (Biotechnology and Biological Sciences Research Council), EU FP6, MRC and Wellcome Trust.
3rd Focused Meeting on PI3K Signalling and Disease: Biochemical Society Focused Meeting held at Bath Assembly Rooms, U.K., 6–8 November 2006. Organized and Edited by B. Hemmings (Friedrich Miescher Institute for Biomedical Research, Switzerland), B. Vanhaesebroeck (Ludwig Institute for Cancer Research, U.K.), S. Ward (Bath, U.K.) and M. Welham (Bath, U.K.).
Abbreviations: BMP, bone morphogenetic protein; ES, embryonic stem; ERas, ES cell-expressed Ras; ERK, extracellular-signal-regulated kinase; GSK, glycogen synthase kinase; hES, human ES; LIF, leukaemia inhibitory factor; mES, murine ES; mTOR, mammalian target of rapamycin; Oct-4, octamer-binding protein-4; PI3K, phosphoinositide 3-kinase; PI(3,4,5)P3, phosphatidylinositol 3,4,5-trisphosphate; PKB, protein kinase B; PTEN, phosphatase and tensin homologue deleted on chromosome 10; SHP-2, Src homology 2 domain-containing protein tyrosine phosphatase 2; STAT3, signal transducer and activator of transcription 3
- © 2007 The Biochemical Society