PI3K/Akt, Raf/Mek/Erk, Activin/Smad and Wnt/β-catenin signaling pathways have all been implicated in regulating human stem cell pluripotency. However, in a broader context, how these pathways cooperate to maintain the balance between self-renewal and differentiation has been unclear. Using a simple, defined culture system which forms the basis of StemPro® hESC SFM (Wang et al., 2007
), we describe a novel cross-talk mechanism where PI3K/Akt suppresses the activation of pro-differentiation pathways centering around Raf/Mek/Erk and canonical Wnt signaling. The signaling cross-talk described here is widely applicable to all hESC media types, including MEF-CM, StemPro® and mTesr1®. Only two extrinsically-activated signaling pathways are required to maintain pluripotency. First, activation of Smad2,3 and its downstream targets, such as Nanog, by Activin A/Nodal (Xu et al., 2008
; Vallier et al., 2009
) and second, the activation of PI3K/Akt signaling by factors such as Igf-1, heregulin and Fgf2. These two signaling requirements can be identified in all hESC media formulations described to date. For example, serum contains high levels of Igfs and knockout serum replacement (KSR) contains high levels of insulin. Sources of Activin A include MEF feeder layers, MEF-CM in addition to its inclusion in defined media formulations (Ludwig et al., 2006
; Wang et al., 2007
; Yao et al., 2006
). hESCs also produce Nodal, a TGFβ member that can signal through the Activin A/Alk4 receptor to activate Smad2,3. Wnt ligands produced by human pluripotent cells appear to be sufficient for activation of downstream targets such as β-catenin and Snail in the absence of PI3K/Akt signaling, although addition of exogenous Wnt3a has been reported to enhance rates and efficiencies of mesendoderm differentiation (D’Amour et al., 2005
Together, our studies show that PI3K/Akt regulates the ability of Activin A/Smad2,3 to control the balance between self-renewal and differentiation (see ). Under self-renewing conditions, PI3K/Akt suppresses Erk and Wnt signaling, allowing Smad2,3 to activate a specific subset of target genes required for self-renewal. In the absence of PI3K/Akt signaling however, Erk and Wnt pathways are activated and effectors such as β-catenin and Snail can permit Smad2,3 to activate genes that direct early differentiation and EMT. Besides the activation of Wnt signaling, loss of PI3K increases the threshold of phosphorylated Smad2,3, enabling it to target a subset of genes that are not active under self-renewing conditions. Precisely how Akt regulates Smad2,3 thresholds is unclear, but may involve the direct interaction and sequestration of Smad3 out of the nucleus (Conery et al., 2004
; Remy et al., 2004
). How pluripotency genes become inactivated under these conditions is also unclear but could involve a negative feedback loop of some kind once early differentiation genes are activated. Interestingly, pluripotency factors such as Nanog and Oct4 have been recently implicated in the initiation of differentiation (Teo, et al., 2011
; Yu et al., 2011
; Thomson et al., 2011
), which may account for their continued expression during the initial stages of differentiation.
Despite its well-established role in antagonizing pluripotency in mESCs, the role of Erk in human pluripotent cells has been more open to question (Armstrong et al., 2006
; Li et al., 2007
; Ding et al., 2010
; Na et al., 2010
). While we find that elevated Erk activity promotes mesendoderm differentiation, we cannot rule out the possibility that low levels of Erk signaling are important for hESC maintenance. In this scenario Erk signaling would be maintained below a threshold level to permit self-renewal but, once this level is surpassed, mesendoderm differentiation would initiate. Recent work has suggested that Erk signaling may promote the expression of Nanog during the initial stages of mesendoderm differentiation (Yu et al., 2011
). Our work is consistent with this finding.
For some time the exact roles of Fgf2 and Wnt signaling in maintenance of human pluripotent cells have remained unclear. We believe a confounding problem in dissecting the roles of many signaling pathways has been the complexity and inconsistencies of media formulations such as MEF-CM. In our assays, Fgf2 has no consistent effect on Akt, Erk or Gsk3β at low concentrations (F10
). However, at high concentrations (F100
) it regulates Akt, Erk and Gsk3β in a manner comparable to that of Igf-1 and heregulin. Several reports have described the need for high levels of Fgf2 in feeder-free media formulations, consistent with our findings (Levenstein et al., 2006
; Yao et al., 2006
). In our studies we routinely used heregulin in place of Fgf2 because it functions at low concentrations and therefore provided cost benefits but, cells can be maintained long-term (>10 passages) in AIF100
media (data not shown). Signaling in AIF100
and HAI media was indistinguishable. The reason for requiring two sources of PI3K activators for long-term self-renewal is not understood. One possibility is that sustained PI3K signaling requires multiple activators of PI3K. This is suggested by studies indicating that different receptor tyrosine kinases activate PI3K with different kinetics (see Zhang et al., 2002
). Sustaining PI3K signaling over time may therefore require two activators with different activation kinetics.
We have addressed the role of Wnt/Gsk3β signaling in hESCs in numerous ways using, (1) multiple chemical inhibitors (BIO, GSKi-XV, CHIR99021), (2) the Wnt antagonist, Dkk1 and (3) a genetic approach using a DN-GSK3. Additionally, these analyses were performed using multiple cell lines and multiple media conditions (HAI, StemPro®, MEF-CM, mTesr1®). Collectively we find that Wnt signaling is antagonistic to self-renewal and promotes differentiation of hESCs, but only if Erk and Activin/Smad signaling pathways are active.
Our experiments show that different concentrations of Gsk3 inhibitors have different biological effects and reconcile conflicting reports in the literature (Sato et al., 2004
; James et al., 2005
; Xiao et al., 2006
; Villa Diaz et al., 2009
; Ding et al., 2010
; Dravid et al., 2005
; Sumi et al., 2008
; Hay et al., 2008
; Bone et al., 2011
). These findings are in agreement with a recent report showing that Gsk3 inhibitors have dose-dependent effects; at low concentrations they stabilize pluripotent cells and at high concentrations they promote differentiation (Tsutsui et al., 2011
; Li et al., 2011
). At the biochemical level, these findings can be explained by the presence of different Gsk3 complexes that perform separate biochemical functions (Voskas et al., 2011). In this scheme, the biological effects of separate Gsk3 complexes would be subject to different signaling thresholds. In pluripotent cells, Myc is stabilized at low concentrations of inhibitor, while β-catenin target gene activation requires higher concentrations. Here, Gsk3 complexes controlling Myc are part of the canonical PI3K/Akt pathway and serve to antagonize self-renewal pathways (Singh and Dalton, 2009
; Cartwright et al., 2005
; Smith et al., 2010
; Takahashi and Yamanaka, 2006
). Increased Myc stability therefore provides an explanation for how hESC self-renewal is promoted at low inhibitor concentrations. High concentrations of Gsk3 inhibitors activate β-catenin resulting in loss of pluripotency markers and increased levels of mesendoderm markers, consistent with the well-established role of Wnt in differentiation and development. The pool of Gsk3 required for this is part of the canonical Wnt pathway and quite separate from that which regulates substrates such as Myc. Together, these findings explain many of the discrepancies in the literature as to the function of Wnt/β-catenin signaling and Gsk3 inhibitors in hESCs.
Recent studies suggest that β-catenin is not required to maintain the pluripotency of mESCs (Lyashenko et al., 2001; Wray et al., 2011
). However, low levels of β-catenin may promote pluripotency of ‘naïve’ ESCs by blocking Tcf3-based repression (Yi et al., 2011
), and not through β-catenin activation of target genes. Additional work has also found that inhibition of Wnt promotes the conversion of ‘naïve’ mESCs to ‘primed’ EpiSCs (ten Berge et al., 2011
). Importantly, hESCs are considered to be in a ‘primed’ pluripotent state, similar to mouse EpiSCs (Tesar et al., 2007
). Our data showing that inhibition of Wnt signaling by Dkk1 promotes hESC pluripotency is therefore in agreement with these recent findings.
A prediction of our model is that cross-talk signaling between the Erk and Wnt pathways also controls mesoderm induction during early vertebrate development. While the Raf/Mek/Erk and Wnt pathways have long been known to control early mesoderm induction in vertebrate embryogenesis through transcriptional regulators such as Eomesodermin, Xbra and Mix1 (Heasman, 2006
), the relationship between these pathways has not been clearly established. Although PI3K/Akt is unlikely to be involved in this scenario, our studies in human pluripotent cells in vitro have allowed us to uncover what we propose to be a mechanism that broadly applies to mesendoderm induction in vivo. As an extension of our model, we propose that Erk activation crosstalks with Gsk3β to activate canonical Wnt signaling during mesoderm and endoderm induction. Furthermore, studies in Xenopus have shown that the ability of Activin to induce mesoderm in animal cap assays is dependent upon Erk activity (LaBonne and Whitman, 1994
). While the molecular mechanism for this is unclear, we predict that Erk, in conjunction with Wnt, promotes β-catenin activation such that it may cooperate with Smad2,3 complexes to activate mesodermal gene expression during embryonic development.
In summary, we have defined a framework to explain how cell-signaling pathways coordinate the balance between self-renewal and differentiation. Central to this framework is the activity of PI3K/Akt which allows Activin A/Smad2,3 signaling to promote self-renewal. In the absence of PI3K signaling Smad2,3 collaborates with Wnt pathway effectors to promote differentiation. This model has far-reaching implications for cell fate commitment not only in pluripotent cells in vitro but also for cell fate determination in early embryonic development.