title: A role for intracellular calcium downstream of G-protein signaling in undifferentiated human embryonic stem cell culture.
abstract: Multiple signalling pathways maintain human embryonic stem cells (hESC) in an undifferentiated state. Here we sought to define the significance of G protein signal transduction in the preservation of this state distinct from other cellular processes. Continuous treatment with drugs targeting G(αs)-, G(α-i/o)- and G(α-q/11)-subunit signalling mediators were assessed in independent hESC lines after 7days to discern effects on normalised alkaline phosphatase positive colony frequency vs total cell content. This identified PLCβ, intracellular free calcium and CAMKII kinase activity downstream of G(α-q/11) as of particular importance to the former. To confirm the significance of this finding we generated an agonist-responsive hESC line transgenic for a G(α-q/11) subunit-coupled receptor and demonstrated that an undifferentiated state could be promoted in the presence of an agonist without exogenously supplied bFGF and that this correlated with elevated intracellular calcium. Similarly, treatment of unmodified hESCs with a range of intracellular free calcium-modulating drugs in biologically defined mTESR culture system lacking exogenous bFGF promoted an hESC phenotype after 1week of continuous culture as defined by co-expression of OCT4 and NANOG. At least one of these drugs, lysophosphatidic acid significantly elevates phosphorylation of calmodulin and STAT3 in this culture system (p<0.05). These findings substantiate a role for G-protein and calcium signalling in undifferentiated hESC culture.
authors.affiliation: Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, EH16 4SB, UK.
authors.forename: Alexander
authors.initials: A
authors.lastname: Ermakov
authors.forename: Steve
authors.initials: S
authors.lastname: Pells
authors.forename: Paz
authors.initials: P
authors.lastname: Freile
authors.forename: Veronika V
authors.initials: VV
authors.lastname: Ganeva
authors.forename: Jan
authors.initials: J
authors.lastname: Wildenhain
authors.forename: Mark
authors.initials: M
authors.lastname: Bradley
authors.forename: Adam
authors.initials: A
authors.lastname: Pawson
authors.forename: Robert
authors.initials: R
authors.lastname: Millar
authors.forename: Paul A
authors.initials: PA
authors.lastname: De Sousa
mesh_terms: D002118:Calcium / Q000378:metabolism*; D002454:Cell Differentiation; D002478:Cells, Cultured; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; D019204:GTP-Binding Proteins; D002118:Calcium