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

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.

Immobilization of insulin-like growth factor-1 onto thermosensitive hydrogels to enhance cardiac progenitor cell survival and differentiation under ischemic conditions

Stem cell therapy is a promising approach to treat myocardial infarction. However, direct delivery of stem cells into hearts experiences poor cell engraftment and differentiation, due to ischemic conditions （low nutrient and oxygen） in the infarct hearts. Development of suitable cell carriers capable of supporting cell survival and differentiation under these harsh conditions is critical for improving the efficacy of current stem cell therapy. In this work, we created a family of novel cell carriers based on thermosensitive hydrogels and insulin-like growth factor 1 （IGF-1）, and investigated if these cell carriers can improve cell sur- vival and differentiation under ischemic conditions. The thermosensitive hydrogels were synthesized from N-isopropylacryla- mide, acrylic acid, acrylic acid N-bydroxysuccinicimide ester, and 2-hydroxyethyl methacrylate-oligo（hydroxybutyrate）. The hydrogel solutions can be readily injected through 26G needles, and can quickly solidify at 37 ~C to form highly flexible hy- drogels. IGF-I was immobilized into the hydrogels in order to support long-term cell survival and differentiation. Different amount of IGF- 1 was immobilized by using hydrogels with different content of N-hydroxysuccinicimide ester groups. Cardio- sphere derived cells were encapsulated in the hydrogels and cultured under ischemic conditions. The results demonstrated that a significant improvement of cell survival and differentiation was achieved after IGF-1 immobilization. These IGF-1 immobi- lized hydrogels have the potential to improve cell survival and differentiation in infarct hearts.