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

Abstract

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.

Differential compartmentalization of BMP4/NOGGIN requires NOGGIN trans-epithelial transport

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PGC-1α activator ZLN005 promotes maturation of cardiomyocytes derived from human embryonic stem cells

“…Briefly, hESCs (H7, WiCell) were cultured in mTeSR1 media (Stem Cell Technologies, Vancouver, Canada) on Matrigel coated dishes (hESC qualified; Corning, New York, NY, USA) based method as previously described [66][67][68]. Cardiomyocyte differentiation was followed AGING the protocol reported previously [69], hESCs with density of 80% confluence were digested into single cells by Accutase (Cat No.…”

Dysregulation of Neuronal Calcium Signaling via Store-Operated Channels in Huntington's Disease

“…The knockdown of both STIM isoforms reduced SOCE and inhibited the entry of mouse embryonic stem cells into a neural lineage (Hao et al, 2014 ). In human embryonic stem cells (hESCs), SOCE but not VGCC-mediated Ca 2+ entry was observed, thus confirming that SOCE could play an essential role in Ca 2+ signaling that is important for the self-renewal and differentiation of hESCs (Huang et al, 2017 ) and supporting the role of molecular components of SOCE in brain development.…”