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Emerging evidence suggests that Ca 2+ signals are important for the self-renewal and differentiation of human embryonic stem cells (hESCs). However, little is known about the physiological and pharmacological properties of the Ca 2+ -handling machinery in hESCs. In this study we used RT-PCR and Western blotting to analyze the expression profiles of genes encoding Ca 2+ -handling proteins; we also used confocal Ca 2+ imaging and pharmacological approaches to determine the contribution of the Ca 2+ -handling machinery to the regulation of Ca 2+ signaling in hESCs. We revealed that hESCs expressed pluripotent markers and various Ca 2+ -handling-related genes. ATP-induced Ca 2+ transients in almost all hESCs were inhibited by the inositol-1,4,5-triphosphate receptor (IP 3 R) blocker 2-APB or xestospongin C. In addition, Ca 2+ transients were induced by a ryanodine receptor (RyR) activator, caffeine, in 10%-15% of hESCs and were blocked by ryanodine, whereas caffeine and ATP did not have additive effects. Moreover, store-operated Ca 2+ entry (SOCE) but not voltage-operated Ca 2+ channel-mediated Ca 2+ entry was observed. Inhibition of sarco/endoplasmic reticulum (ER) Ca 2+ -ATPase (SERCA) by thapsigargin induced a significant increase in the cytosolic free Ca 2+ concentration ([Ca 2+ ] i ). For the Ca 2+ extrusion pathway, inhibition of plasma membrane Ca 2+ pumps (PMCAs) by carboxyeosin induced a slow increase in [Ca 2+ ] i , whereas the Na + /Ca 2+ exchanger (NCX) inhibitor KBR7943 induced a rapid increase in [Ca 2+ ] i . Taken together, increased [Ca 2+ ] i is mainly mediated by Ca 2+ release from intracellular stores via IP3Rs. In addition, RyRs function in a portion of hESCs, thus indicating heterogeneity of the Ca 2+ -signaling machinery in hESCs; maintenance of low [Ca 2+ ] i is mediated by uptake of cytosolic Ca 2+ into the ER via SERCA and extrusion of Ca 2+ out of cells via NCX and PMCA in hESCs.

Functional expression of the Ca2+ signaling machinery in human embryonic stem cells