Original Article

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

Authors: HUANG Ji-jun1,2, WANG Yi-jie2, ZHANG Min2, ZHANG Peng2, LIANG He2, BAI Hua-jun2, YU Xiu-jian2, YANG Huang-tian1,3,4
1 Heart Center, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai 200233, China
2 Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences & Shanghai Jiao Tong University School of Medicine, Shanghai 200031, China
3 Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences & Shanghai Jiao Tong University School of Medicine, Shanghai 200031, China
4 Second Affiliated Hospital, Zhejiang University, Hangzhou 310009, China
Corresponding to: YAG Huang-tian: htyang@sibs.ac.cn,
DOI: 10.1038/aps.2017.29
Received: 24 November 2016
Accepted: 13 March 2017
Advance online: 17 July 2017

Abstract

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.
Keywords: Ca2+ signaling; Ca2+ release; Ca2+ extrusion; Ca2+ channels; Ca2+ pumps; human embryonic stem cells