br Materials and methods br Results br

Materials and methods
Results
Discussion Our study evaluated the significance of G protein-coupled signalling pathways on undifferentiated hESC maintenance under self-renewing conditions using a spectrum of pharmacological drugs and small PS341 (the effects of which are summarised in Fig. 5). In a primary screen using established pharmacological drugs and alkaline phosphatase staining as a surrogate marker of stemness we implicated downstream elements of the Gα-q/11 subunit pathway, specifically PLCβ, intracellular free calcium, [Ca++]i, and CAMKII kinase, as well as the Gα-i/o subunit as of particular significance for maintenance of this phenotype. By contrast, perturbation of the Gαs-AC–PKA pathway had more general affects on total cell growth and/or viability. The significance of free intracellular calcium signalling was confirmed in independent experiments demonstrating i) correlation with other stemness markers (SSEA4), ii) the potential to compensate for an absence of exogenously supplied bFGF following agonist-mediated activation of a transgenic hESC line expressing an exogenous Gα-q/11 subunit-coupled GPCR, iii) the ability of small molecule drugs known to elevate intracellular free calcium to substitute for bFGF in a defined medium, and iv) the ability of at least one of these drugs to elevate phosphorylation of anticipated (Calmodulin) and stem cell renewal relevant (Stat-3) substrates . Our study thus substantiates a previously unappreciated role for calcium-mediated signal transduction in undifferentiated hESC maintenance and further studies to manipulate these pathways to support long term hESC maintenance and pluripotency. Our primary screen involving 6days of sustained treatment with a spectrum of pharmacological agents served to indicate the general significance of G protein-linked signal transduction pathways to hESCs. Thus, cholera toxin, an activator of Gαs, causing activation of AC had no effect on cell growth/viability generally or undifferentiated hESC maintenance specifically, at the concentrations evaluated (0.1–5μgml). However, both treatment with forskolin, which activates AC, and pertussis toxin, which inhibits the AC inhibitor Gαi/o interfered with cell growth/viability. These findings suggest that in hESCs grown under self-renewal conditions, Gαs is not particularly important for self-renewal whereas sustained elevation of cAMP can interfere with cell growth/viability generally. This was corroborated by the observation that interference with PKA, whose activation would be stimulated by elevated cAMP, promoted cell growth. Our results contrast with the available evidence highlighting a role for the Gαs-AC–PKA pathway in mouse ESC renewal. In mouse ESCs, cAMP and PKA have been implicated in self-renewal in the absence of LIF signalling (Faherty et al., 2007). Stimulation of Gαs protein by cholera toxin during differentiation in embryoid bodies increased cell proliferation and prevented the time-related decline in pluripotency transcription factors (Layden et al., 2010). Forskolin has also been reported as a key factor in stabilising a LIF-dependent mESC-like phenotype in hESCs following transduction with OCT4, Sox2 and Klf4. This effect was executed at least partly through induction of Klf2 and Klf4 expression (Hanna et al., 2010). The inhibitory effect of pertussis toxin on cell viability was apparent at low and intermediate concentrations (100 and 200ngml) whereas at high concentration (500ngml), it negatively affected undifferentiated hESC maintenance only. Pertussis toxin catalyses the ADP-ribosylation of Gαi/o, thereby precluding GTP exchange and dissociation of the Gα subunit from the Gβ and Gγ subunits which normally follows GPCR-mediated activation. Pertussis toxin has previously been shown to antagonise Sphingosine-1-phosphate (S1P) and PDGF stimulation of hESC self-renewal in serum-free culture, the former being a ligand of Gαi/o-coupled receptors (Pebay et al., 2005). In self-renewal culture conditions equivalent to those used in our study but involving only treatments over 2days (not 6 as in our study), it has been reported that treatment of hESCs and induced pluripotent stem cells (iPSCs) with pertussis toxin altered colony morphology and organisation but did not affect cell proliferation, apoptosis, expression of pluripotency markers or ability to differentiate into germinal lineages in vitro (Nakamura et al., 2009). In the same study, effects on colony morphology were not apparent following treatment with the Gαs activator, cholera toxin, which was also consistent with our findings. In mouse, S1P stimulates mESC renewal via activation of extracellular signal-regulated ERK1 and ERK2. This can be inhibited by both pertussis toxin, affecting Gαi/o, as well as by inhibition of PKC, downstream of the Gα-q/11 pathway (Rodgers et al., 2009).