AggreWell plate aggregation and defined factors allow highly uniform-sized and functional cardiac embryoid bodies from human ESCs and iPSCs
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| Year of publication | 2012 |
| Type | Conference abstract |
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| Description | Objectives: In vitro, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) can be differentiated into functional cardiomyocytes (CMs). Differentiation of hESCs and hiPSCs to the cardiac lineage can be done using different methods: co-culture with mouse visceral endoderm-like cells (END-2 cells), through formation of embryoid bodies (EBs) or culturing as a monolayer on matrigel supplemented with defined factors. Efficiency of these methods depends on developmental potential of each cell line and may vary between lines. In this study, we present cardiac differentiation of hESCs and hiPSCs by formation of EBs through forced aggregation of the cells. Material and methods: From hESCs and hiPSCs plated on matrigel, EBs are formed by forced aggregation in AggreWell plates with app. 2000 cells per EB. Formed EBs are plated on low-adhesive dishes the following day. The differentiation process is achieved using defined growth factors (Activin A, BMP4, VEGF, IWR1 and FGF2) at different stages as previously shown to enhance mesodermal differentiation and cardiac progenitors. PCR, western blot and immunocytochemistry are performed to evaluate the gene and protein profile while microelectrode array (MEA), atomic force microscopy (AFM) and confocal microscopy are used to investigate the biophysical cardiac properties. Results: The forced aggregation using AggreWell plates allows highly uniform-sized EBs with up to 50% of contracting clusters from 5 tested lines including 3 hESC and 2 hiPSC. First contracting EB appears between day 14 and 20 of differentiation. Our hESC-CMs and hiPSC-CMs expressed cardiac specific markers such as cardiac troponin T and cardiac ryanodine receptor / calcium release channel (RyR2). Electrophysiological maturity of hESC-CMs is determined by MEA. Thus, our hESC-CMs are able to respond to activation (isoproterenol) as well as to inhibition (metoprolol) of beta-adrenergic receptors. To quantify the contractile and elastic properties of the cardiac EBs, we use the AFM technique. To assess the functionality of the sarcoplasmic reticulum, spontaneous (calcium sparks) and stimulated (calcium waves) calcium events are investigated using confocal microscope. Exposure of enzymatically-dissociated hESC-CMs to caffeine, a RyR2 agonist, induces a quick and large calcium wave followed by a long and total absence of calcium transient. These results suggest that the recorded calcium transients are RyR2-dependent. Conclusions: Our preliminary results indicate that the forced aggregation using AggreWell plates is a relevant method to generate highly uniform-sized EBs from both hESCs and hiPSCs. The intracellular calcium release through RyR2 appears functional although further studies are needed to fully characterize the calcium handling at the sarcoplasmic reticulum level in these cells. Moreover, similarly to previous studies, we observe significant variability between stem cell lines (hESC and iPSC) to differentiate in CMs. |
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