Functional expression of the Ca signaling machinery in human embryonic stem cells.

Emerging evidence suggests that Ca 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-handling machinery in hESCs. In this study we used RT-PCR and Western blotting to analyze the expression profiles of genes encoding Ca-handling proteins; we also used confocal Ca imaging and pharmacological approaches to determine the contribution of the Ca-handling machinery to the regulation of Ca signaling in hESCs. We revealed that hESCs expressed pluripotent markers and various Ca-handling-related genes. ATP-induced Ca transients in almost all hESCs were inhibited by the inositol-1,4,5-triphosphate receptor (IPR) blocker 2-APB or xestospongin C. In addition, Ca 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 entry (SOCE) but not voltage-operated Ca channel-mediated Ca entry was observed. Inhibition of sarco/endoplasmic reticulum (ER) Ca-ATPase (SERCA) by thapsigargin induced a significant increase in the cytosolic free Ca concentration ([Ca]). For the Ca extrusion pathway, inhibition of plasma membrane Ca pumps (PMCAs) by carboxyeosin induced a slow increase in [Ca], whereas the Na/Ca exchanger (NCX) inhibitor KBR7943 induced a rapid increase in [Ca]. Taken together, increased [Ca] is mainly mediated by Ca release from intracellular stores via IP3Rs. In addition, RyRs function in a portion of hESCs, thus indicating heterogeneity of the Ca-signaling machinery in hESCs; maintenance of low [Ca] is mediated by uptake of cytosolic Ca into the ER via SERCA and extrusion of Ca out of cells via NCX and PMCA in hESCs.