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Sci China Life Sci. 2016 Aug;59(8):811-24. doi: 10.1007/s11427-016-5094-6. Epub 2016 Jul 19.

Expression and reconstitution of the bioluminescent Ca(2+) reporter aequorin in human embryonic stem cells, and exploration of the presence of functional IP3 and ryanodine receptors during the early stages of their differentiation into cardiomyocytes.

Author information

1
Division of Life Science & State Key Laboratory of Molecular Neuroscience, HKUST, Clear Water Bay, Hong Kong, China.
2
Stem Cell & Regenerative Medicine Consortium, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong, China.
3
Marine Biological Laboratory, Woods Hole, MA, 02543, USA.
4
Division of Life Science & State Key Laboratory of Molecular Neuroscience, HKUST, Clear Water Bay, Hong Kong, China. barnie@ust.hk.

Abstract

In order to develop a novel method of visualizing possible Ca(2+) signaling during the early differentiation of hESCs into cardiomyocytes and avoid some of the inherent problems associated with using fluorescent reporters, we expressed the bioluminescent Ca(2+) reporter, apo-aequorin, in HES2 cells and then reconstituted active holo-aequorin by incubation with f-coelenterazine. The temporal nature of the Ca(2+) signals generated by the holo-f-aequorin-expressing HES2 cells during the earliest stages of differentiation into cardiomyocytes was then investigated. Our data show that no endogenous Ca(2+) transients (generated by release from intracellular stores) were detected in 1-12-day-old cardiospheres but transients were generated in cardiospheres following stimulation with KCl or CaCl2, indicating that holo-f-aequorin was functional in these cells. Furthermore, following the addition of exogenous ATP, an inositol trisphosphate receptor (IP3R) agonist, small Ca(2+) transients were generated from day 1 onward. That ATP was inducing Ca(2+) release from functional IP3Rs was demonstrated by treatment with 2-APB, a known IP3R antagonist. In contrast, following treatment with caffeine, a ryanodine receptor (RyR) agonist, a minimal Ca(2+) response was observed at day 8 of differentiation only. Thus, our data indicate that unlike RyRs, IP3Rs are present and continually functional at these early stages of cardiomyocyte differentiation.

KEYWORDS:

Ca2+ signaling; HES2 human embryonic stem cells; IP3 and ryanodine receptors; apo-aequorin expression; bioluminescence; hESC-derived cardiospheres

PMID:
27430888
DOI:
10.1007/s11427-016-5094-6
[Indexed for MEDLINE]

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