| Popis |
EXTENDED ABSTRACT Stem cell derived cardiomyocytes (SCs-CMs).can be prepared by differentiation in vitro from human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). Routinely is checked the expression of cardiac markers by qRT-PCR, immunocytochemistry and western blotting in beating EBs and enzymatically-dissociated CMs. Electrophysiological methods serve well fro conduction and excitation studies, but cannot reflex the mechanics. The contractions of cell clusters resembles functionality, but remains to be studied in detail. In this study, we present quantification method of the spontaneous contraction of SCs-CMs. Moreover, traditional suspension method for 3D clusters - embryoid bodies (EBs) formation results in heterogeneous EB population and heterogenouse cardiac clusters after differentiation, mainly different in size. But formation of EBs through forced aggregation of the cells allows highly uniform population of clusters. This enables involvement of Atomic force microscopy (AFM) when cantilever is directly placed on the contracting cluster. It enables to reflex the laser beam according to the movement and record it on photodiode in real time, thus create equivalent of mechanocardiogram (MCG). Methods: Using a defined number of undifferentiated single cells in AggreWell plate, we formed homogeneous EBs (2000 cells per cluster). The differentiation process is achieved using defined growth factors at different stages to enhance mesodermal differentiation and production of cardiac progenitors. Molecular and functional characterization is then achieved to confirm the cardiac identity of the resulting cells (MYH6, MYH7 and RYR2 genes as well as sarcomeric pattern for cardiac troponin T and alpha-actinin). Atomic force microscopy was performed in standard (Tyrod) media and after inhibition as well as activation to of beta-adrenergic receptors. Temperature sensitivity was tested as continuous controlled cooling of cultivation media, stepwise at 0,5 degree of Celsius in range of 37oC – 27oC. Results: Contrary to suspension method, the forced aggregation using AggreWell plates allows production of highly uniform-sized EBs with up to 80% of contracting clusters. Produced hESC- CMs and hiPSC-CMs expressed cardiac specific markers. Using atomic force microscopy-based technique, we measured the beating properties of hESC-EBs and hiPSC-EBs. A slower beat rate in hESC-CMs was obtained when compared to hiPSC-CMs (51 ± 5 vs 74 ± 7 bpm), while contracting force remained similar (31 ± 7 vs 39 ± 9 nN). Both cell types respond comparably upon stimulation or inhibition of beta-adrenergic pathway and caffeine. Conclusion: Our results indicate that the mechano-biological properties of homogenous beating EBs can be investigated by atomic force microscopy and thus stem cell derived cardiomyocytes can serve as well described disease and drug testing model. The method allowed us to optimize the cultivation and differentiation method reaching highly uniform CMs with high efficiency. REFERENCE: PEŠL, Martin, Ivana AĆIMOVIĆ, Jan PŘIBYL, Renata HÉŽOVÁ, Aleksandra VILOTIĆ, Jeremy FAUCONNIER, Jan VRBSKÝ, Peter KRUZLIAK, Petr SKLÁDAL, Tomáš KÁRA, Vladimír ROTREKL, Alain LACAMPAGNE, Petr DVOŘÁK a Albano MELI. Forced aggregation and defined factors allow highly uniform-sized embryoid bodies and functional cardiomyocytes from human embryonic and induced pluripotent stem cells. Heart Vessels, Japan: Springer Japan, 2013. ISSN 0910-8327. doi:10.1007/s00380-013-0436-9.
|
