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Expression and reconstitution of the bioluminescent Ca2+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

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Science China(Life Sciences) 2016年第8期59卷 811-824页

作者： Harvey Y.S.Chan Man Chun Cheung Yi Gao Andrew L.Miller Sarah E.Webb Division of Life Science&State Key Laboratory of Molecular Neuroscience HKUSTClear Water BayHong KongChina stem Cell&Regenerative Medicine Consortium Li Ka Shing Faculty of MedicineThe University of Hong KongPokfulamHong KongChina Marine Biological Laboratory Woods Hole MA 02543USA

In order to develop a novel method of visualizing possible Ca2＋ signaling during the early differentiation of hesCs into cardi- omyocytes and avoid some of the inherent problems associated with using fluorescent reporters, we expressed the biolumines- cent Ca2＋ reporter, apo-aequorin, in hes2 cells and then reconstituted active holo-aequorin by incubation withf-coelenterazine. The temporal nature of the Ca2＋ 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 Ca2＋ transients （generated by re- lease from intracellular stores） were detected in 1-12-day-old cardiospheres but transients were generated in cardiospheres following stimulation with KC1 or CaC12, indicating that holo-f-aequorin was functional in these cells. Furthermore, following the addition of exogenous ATP, an inositol trisphosphate receptor （IP3R） agonist, small Ca2＋transients were generated from day 1 onward. That ATP was inducing Ca2＋ 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 minima/Ca2＋ 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.

关键词： Ca2+ signaling apo-aequorin expression bioluminescence hes2 human embryonic stem cells hesC-derived cardiospheres IP3 and ryanodine receptors

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