Abstract
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.
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Acknowledgements
This work was supported by grants from the National Natural Science of China (No 81520108004, 81470422 and 31030050 to HTY, No 31401167 to MZ), the National Basic Research Program of China (No 2014CB965100 to Huang-tian YANG), the National Science and Technology Major Project (No 2012ZX09501001 to Huang-tian YANG), the National Key Research and Development Program (2016YFC1301200 to Huang-tian YANG), and the Natural Science Foundation of Shanghai (No 17ZR1435500 to Jin-jun HUANG).
We thank WiCell Research Institute for providing the H7 and H9 hESCs and Dr He-ping CHENG (Peking University, Beijing, China) for providing the Flash Sniper software.
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Huang, Jj., Wang, Yj., Zhang, M. et al. Functional expression of the Ca2+ signaling machinery in human embryonic stem cells. Acta Pharmacol Sin 38, 1663–1672 (2017). https://doi.org/10.1038/aps.2017.29
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DOI: https://doi.org/10.1038/aps.2017.29
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