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

Emerging evidence suggests that Ca²⁺ 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 Ca²⁺-handling machinery in hESCs. In this study we used RT-PCR and Western blotting to analyze the expression profiles of genes encoding Ca²⁺-handling proteins; we also used confocal Ca²⁺ imaging and pharmacological approaches to determine the contribution of the Ca²⁺-handling machinery to the regulation of Ca²⁺ signaling in hESCs. We revealed that hESCs expressed pluripotent markers and various Ca²⁺-handling-related genes. ATP-induced Ca²⁺ transients in almost all hESCs were inhibited by the inositol-1,4,5-triphosphate receptor (IP₃R) blocker 2-APB or xestospongin C. In addition, Ca²⁺ 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 Ca²⁺ entry (SOCE) but not voltage-operated Ca²⁺ channel-mediated Ca²⁺ entry was observed. Inhibition of sarco/endoplasmic reticulum (ER) Ca²⁺-ATPase (SERCA) by thapsigargin induced a significant increase in the cytosolic free Ca²⁺ concentration ([Ca²⁺]_i). For the Ca²⁺ extrusion pathway, inhibition of plasma membrane Ca²⁺ pumps (PMCAs) by carboxyeosin induced a slow increase in [Ca²⁺]_i, whereas the Na⁺/Ca²⁺ exchanger (NCX) inhibitor KBR7943 induced a rapid increase in [Ca²⁺]_i. Taken together, increased [Ca²⁺]_i is mainly mediated by Ca²⁺ release from intracellular stores via IP3Rs. In addition, RyRs function in a portion of hESCs, thus indicating heterogeneity of the Ca²⁺-signaling machinery in hESCs; maintenance of low [Ca²⁺]_i is mediated by uptake of cytosolic Ca²⁺ into the ER via SERCA and extrusion of Ca²⁺ out of cells via NCX and PMCA in hESCs.