title: Characterization of calcium signals in human embryonic stem cells and in their differentiated offspring by a stably integrated calcium indicator protein.
abstract: Intracellular calcium signaling pathways play a major role in cellular responses such as proliferation, differentiation and apoptosis. Human embryonic stem cells (hESC) provide new possibilities to explore the development and differentiation of various cell types of the human body. Intracellular calcium responses to various ligands and the calcium signaling pathways, however, have not been thoroughly studied in embryonic stem cells and in their differentiated progenies. In our previous work we demonstrated that the use of the fluorescent calcium indicator Fluo-4 with confocal microscopy allows sensitive and reliable measurements of calcium modulation in human embryonic stem cells and stem-cell derived cardiomyocytes. Here we developed a human embryonic stem cell line stably expressing a genetically encoded Ca(2+) indicator (GCaMP2) using a transposon-based gene delivery system. We found that the differentiation properties were fully preserved in the GCaMP2-expressing hESC lines and Ca imaging could be performed without the need of toxic dye-loading of the cells. In undifferentiated hES cells the calcium signals induced by various ligands, ATP, LPA, trypsin or angiotensin II were comparable to those in Fluo-4 loaded cells. In accordance with previous findings, no calcium signal was evoked by thrombin, histamine or GABA. Cardiomyocyte colonies differentiated from hES-GCaMP2 cells could be recognized by spontaneous contractions and Ca(2+) oscillations. GCaMP2-expressing neural cells were identified based on their morphological and immuno-staining properties and Ca signals were characterized on those cells. Characteristics of both the spontaneous and ligand-induced Ca(2+) signals, as well as their pharmacological modification could be successfully examined in these model cells by fluorescence imaging.
authors.affiliation: Institute of Molecular Pharmacology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary. apati@biomembrane.hu
authors.forename: Ágota
authors.initials: Á
authors.lastname: Apáti
authors.forename: Katalin
authors.initials: K
authors.lastname: Pászty
authors.forename: Luca
authors.initials: L
authors.lastname: Hegedűs
authors.forename: Orsolya
authors.initials: O
authors.lastname: Kolacsek
authors.forename: Tamás I
authors.initials: TI
authors.lastname: Orbán
authors.forename: Zsuzsa
authors.initials: Z
authors.lastname: Erdei
authors.forename: Kornélia
authors.initials: K
authors.lastname: Szebényi
authors.forename: Adrienn
authors.initials: A
authors.lastname: Péntek
authors.forename: Ágnes
authors.initials: Á
authors.lastname: Enyedi
authors.forename: Balázs
authors.initials: B
authors.lastname: Sarkadi
mesh_terms: D000814:Aniline Compounds / Q000737:chemistry; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects; D002454:Cell Differentiation; D002460:Cell Line; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism*; D006632:Histamine / Q000494:pharmacology; D006801:Humans; D018613:Microscopy, Confocal; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013917:Thrombin / Q000494:pharmacology; D014966:Xanthenes / Q000737:chemistry; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology
chemical_list: D000814:Aniline Compounds; C409648:Fluo 4; D011993:Recombinant Fusion Proteins; D014966:Xanthenes; D005680:gamma-Aminobutyric Acid; D006632:Histamine; D013917:Thrombin; D002118:Calcium