Molecular and Functional Characterization of Uniform-Sized Beating Embryoid Bodies and Cardiomyocytes from Human Embryonic and Induced Pluripotent Stem Cells
| Autoři | |
|---|---|
| Rok publikování | 2014 |
| Druh | Konferenční abstrakty |
| Fakulta / Pracoviště MU | |
| Citace | |
| Popis | In vitro human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) are able to differentiate into functional cardiomyocytes (CMs). Traditional suspension method for 3D embryoid bodies (EBs) formation results in heterogeneous EB population. Here, we hypothesized that forming homogenous EB in size and shape allows studying the mechano-biological properties of spontaneously beating clusters containing CMs. Using a defined number of single cells in AggreWell plate, we formed homogeneous EBs that can be efficiently differentiated into functional CMs by application of defined growth factors. We checked the expression of cardiac markers by qRT-PCR, immunocytochemistry and western blotting in both beating EBs and enzymatically-dissociated CMs and found increased expression of MYH6, MYH7 and RYR2 genes as well as sarcomeric pattern for cardiac troponin T and alpha-actinin. Using atomic force microscopy-based technique, we measured the beating properties of hESC-EBs and hiPSC-EBs and found a slower beat rate in hESC-CMs when compared to hiPSC-CMs (51 ± 5 vs 74 ± 7 bpm) and similar contraction force (31 ± 7 vs 39 ± 9 nN). Both cell types respond upon stimulation or inhibition of beta-adrenergic pathway and caffeine. Using Ca2+ imaging we also demonstrated the functionality of RyR2 to release Ca2+ from the sarcoplasmic reticulum and evaluated the contribution of IP3R. Thus, upon xestospongin C and histamine (IP3R modulators), spontaneous Ca2+ transients are maintained confirming the role of RyR2. Using the patch-clamp technique, we evaluated the cardiac population constituting the EBs and found that more than 75% of excitable cells exhibit atrial-like action potentials. Our results indicated that the mechano-biological properties of homogenous beating EBs can be investigated. They also indicated that spontaneous beating EBs contain mostly functional and organized atrial CMs. |
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