In our experiments in mTESR a complex medium

In our experiments in mTESR, a complex medium which is at least more biologically defined than fibroblast conditioned medium supplemented with a serum replacement (i.e. KOSR™) used in our primary screen, SERCA inhibitors and LPA, promoted undifferentiated hESC stemness. Only LPA stimulated a significant increase in the phosphorylation of Calmodulin and STAT3. Phosphorylation of the latter was also evident for TGF-β alone or together with bFGF. LPA is a bio-active lysophospholipid and GPCR ligand like S1P. It can be generated extracellularly and intracellularly by various pathways and its effects are dependent on the differential pgc-1α inhibitor of its receptors and the G proteins they are linked with. Many of its signalling effects are mediated through the activation of extracellular signal-regulated kinases 1/2, PLC, small GTPases, calcium mobilisation and activation or inhibition of adenylate cyclase ((Lee et al., 2007); reviewed by (Pitson and Pebay, 2009)). LPA can also act as an intracellular second messenger via the nuclear transcription factor peroxisome proliferator-activated receptor γ (PPARγ) (McIntyre et al., 2003). In human ESCs there are at least 6 receptors for LPA and 5 for S1P, most of which appear to be expressed. Notable among LPA receptors is LPA4 (also known as GPR23) which according to a meta-analysis of the hESC transcriptome is specifically enriched in hESCs (Assou et al., 2007). In mouse ESCs LPA induces Ca++ mobilisation and c-myc expression via PLC (Todorova et al., 2009). To our knowledge, no biological effects of LPA on hESC have been previously reported (Pebay et al., 2005; reviewed by Pitson and Pebay, 2009). LPA has been shown to activate Stat3 in other cell types (Seo et al., 2010), as has Gαs and Gα-q mediated signal transduction (reviewed by (Callihan et al., 2011)). Our inability to detect significant effects of the SERCA inhibitors on the phosphorylation status of calcium signal transducing and stem cell renewal associated substrates probably related to the masking effects of signal transduction elicited by other growth factor and components in mTESR medium, namely Lithium Chloride, GABA, Pipecolic Acid, and insulin (Ludwig et al., 2006). Differential outcomes in our primary screen following interference with calcineurin vs CAMKII kinase suggest that general (i.e. cell proliferation and viability) and specific roles (stem cell renewal) for Ca++ may be discriminatable at this juncture, although further work is needed to dissect the network of relevant effectors including specific and/or overlapping roles for the different SERCA and IP3 receptors which are expressed. In addition to its SERCA inhibitory activity, chlorpromazine also has some CAMKII inhibitory activity, possibly explaining why this drug only appeared to promote self renewal at low concentrations. CAMKII kinases are multi-functional regulators of transcription factors, glycogen metabolism and neurotransmitter secretion. They are distinct in possessing an autocatalytic activity which serves as a molecular memory device whose effect persists long after its activation (Braun and Schulman, 1995). This raises the interesting possibility that stimulation of hESC renewal could be achieved by periodic activation of CAMKII signalling. Investigating this would also benefit from technology enabling better control of paracrine cell stimulation in vitro.
Conclusion It has only been in the last few years that it has been possible to control pluripotent stem cell renewal and differentiation using chemical modulators of intracellular signalling pathways (reviewed by (Ng and Surani, 2011)). In mESCs independence from LIF can be achieved using inhibitors of the FGF/ERK and glycogen synthetase kinase-3 (GSK3) signalling pathways (Nichols and Smith, 2009). Our study implicates Gα-i/o- and Gα-q/11-subunit-mediated signalling, notably PLCβ, calcium and CAMKII kinase in short term hESC culture. Activation of Gα-q/11-subunit coupled GPCR elevates intracellular free calcium and compensates for reduced bFGF. Small molecule modulators of intracellular free calcium also appear to substitute for bFGF and we provide evidence that LPA phosphorylates Calmodulin and STAT3, as does TGF-β when presented alone or together with bFGF. Further investigation of second messenger pathways affecting calcium homeostasis are needed in minimally defined media supportive of hESC renewal together with strategies for controlling paracrine signalling in vitro. This will contribute to the development of chemically-defined systems supportive of human pluripotent stem cell renewal.