Suppression of Erk signalling promotes ground state pluripotency in the mouse embryo

Abstract

Embryonic stem (ES) cells can be derived and propagated from multiple strains of mouse and rat through application of smallmolecule inhibitors of the fibroblast growth factor (FGF)/Erk pathway and of glycogen synthase kinase 3. These conditions shield pluripotent cells from differentiation-inducing stimuli. We investigate the effect of these inhibitors on the development of pluripotent epiblast in intact pre-implantation embryos. We find that blockade of Erk signalling from the 8-cell stage does not impede blastocyst formation but suppresses development of the hypoblast. The size of the inner cell mass (ICM) compartment is not reduced, however. Throughout the ICM, the epiblast-specific marker Nanog is expressed, and in XX embryos epigenetic silencing of the paternal X chromosome is erased. Epiblast identity and pluripotency were confirmed by contribution to chimaeras with germline transmission. These observations indicate that segregation of hypoblast from the bipotent ICM is dependent on FGF/Erk signalling and that in the absence of this signal, the entire ICM can acquire pluripotency. Furthermore, the epiblast does not require paracrine support from the hypoblast. Thus, naïve epiblast and ES cells are in a similar ground state, with an autonomous capacity for survival and replication, and high vulnerability to Erk signalling. We probed directly the relationship between naïve epiblast and ES cells. Dissociated ICM cells from freshly harvested late blastocysts gave rise to up to 12 ES cell clones per embryo when plated in the presence of inhibitors. We propose that ES cells are not a tissue culture creation, but are essentially identical to preimplantation epiblast cells.

Wnt Pathway Regulation of Embryonic Stem Cell Self-Renewal

“…They posited that mESCs could exist in a ground state of selfrenewal if extracellular signals were blocked. This model was supported by the observations that activation of ERK signaling promoted differentiation of mESCs and that mESC differentiation was constrained by adding an Fgf receptor (Fgfr) inhibitor and an ERK inhibitor (PD0325901) to a chemically defined media (Ying et al 2008;Nichols et al 2009). Using only Fgfr and ERK inhibitors did not sustain mESC cultures, because cells died without undergoing self-renewal divisions.…”

“…Adding a GSK3 inhibitor alone, such as BIO or CHIR99021, sustained a short burst of self-renewal for only one or two passages (Ying et al 2008). Remarkably, the combination of the three inhibitors, which was subsequently reduced to only two (PD0325901 þ CHIR99021), was quite effective; these so-called two-inhibitor (2i) conditions supported the derivation of new mESC lines from blastocyst outgrowths and expansion into large cultures from a single cell of an already established mESC line (Ying et al 2008;Nichols et al 2009). Mouse ESCs grown in 2i conditions were cultured for several weeks with no apparent defects, and they maintained the ability to contribute to chimeric mice when injected into blastocysts (Ying et al 2008).…”

“…Austin Smith and colleagues noted that signals present in commonly used mESC cultures (serum-containing media) actually included significant differentiation-inducing signals (Ying et al 2003(Ying et al , 2008Kunath et al 2007;Nichols et al 2009). They posited that mESCs could exist in a ground state of selfrenewal if extracellular signals were blocked.…”

Calcium signaling in human pluripotent stem cells

“…3). The balanced, moderate level of activated ERK sustains the pluripotent state [51,57], while a strong reduction of PI3K activity causes elevated ERK activity followed by differentiation of PSCs (as shown in mouse studies [58,59]). …”

Switching on pluripotency: a perspective on the biological requirement of Nanog

“…E4.5 Nanog2/2 ICMs also showed apoptosis and failed to form a hypoblast, but a few cells retained a capacity to specify into trophoblast when cultured as outgrowths. Nanog2/2 embryos failed to respond to selective inhibitors of mitogen-activated protein (MAP) kinase and GSK3 (2i) with LIF, a culture medium that induces the expansion of the naive pluripotent epiblast and that is optimal for the propagation of Nanog2/2 ES cells [12,18]. These findings demonstrate that, in the absence of Nanog, pluripotency is not specified and as a result the ICM undergoes apoptosis.…”