FGF-signalling in the differentiation of mouse ES cells towards ...

The pluripotent stateThe pluripotency of ES cells along with their capability to self-renew indefinitely are the mostimportant characteristics of ES cells. The pluripotent state is characterised morphologically bytightly associated cells growing in rounded clusters. Molecularly, they are characterised by theexpression of markers, some of which are found also in the ICM of the developing embryo. Themost commonly used are Oct4, Nanog, Stage specific embryonic antigen-1 (SSEA-1), Sox2 andAlkaline phosphatase (Solter and Knowles 1978; Pease et al. 1990; Nichols et al. 1998; Avilion etal. 2003; Chambers et al. 2003; Mitsui et al. 2003). To test if ES cells have maintained theirpluripotency over time when in culture, they are evaluated in several ways, each providing a morestringent test but at the same time taking more resources. They can be evaluated by i)morphology; ii) a positive stain with antibodies for the pluripotent markers; iii) subcutaneousinjection in mice and formation of teratomes with cells of all three germ layers; iv) injection intothe ICM of a developing embryo where they give rise to chimaeras with contributions to tissues ofall three germ layers and the germ line (Ohtsuka and Dalton 2008). The last test is considered the‘golden standard’ but is time-consuming and is therefore not routinely carried out except in theestablishment and analysis of newly generated transgenic cell lines or by investigating whether acertain (manipulated) ES cell line is entirely pluripotent.Maintaining the pluripotent state in an ES cell culture is done through prevention ofdifferentiation (or induction of self-renewal properties) and promotion of proliferation. LIF isadded to the culture medium of pluripotent ES cells and is the main factor involved in keepingcells pluripotent (Figure 1-4A). It activates the JAK/STAT3-signalling pathway, resulting intranscription of genes involved in self-renewal, one of these being C-myc (Niwa et al. 1998;Cartwright et al. 2005). LIF also activates the important Phosphoinositol 3 kinase (PI3K), leadingto activation of Ras/ mitogen-activated protein kinase (MAPK) and AKT pathways. The latter isinvolved in relief of C-myc repression by glycogen synthase kinase-3 (GSK-3), which seems to bevery important in maintaining self-renewal capability (Umehara et al. 2007). BMP is a secondimportant factor for maintaining self-renewal, its effect possibly depending on the cultureconditions in which it acts (Ohtsuka and Dalton 2008). BMP activates SMAD1/5/8-signallingleading to expression of Inhibitor of differentiation (Id)-genes which block at least neuraldifferentiation (Figure 1-4A; (Ying et al. 2003a)). BMPs also act by suppressing the p38 MAPK,which would otherwise promote differentiation (Qi et al. 2004; Kunath et al. 2007). FGF4 isexpressed in pluripotent ES cells in an autocrine fashion and the activation of the ERK1/2-signalling cascade by FGF must be suppressed in order to maintain cells in the plupipotent state(Ma et al. 1992; Kunath et al. 2007; Nichols et al. 2009). The LIF-gp130 receptor complex sees tothis.12