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Forced aggregation and defined factors allow highly uniform-sized embryoid bodies and functional cardiomyocytes from human embryonic and induced pluripotent stem cells

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PEŠL Martin AĆIMOVIĆ Ivana PŘIBYL Jan HÉŽOVÁ Renata VILOTIĆ Aleksandra FAUCONNIER Jeremy VRBSKÝ Jan KRUZLIAK Peter SKLÁDAL Petr KÁRA Tomáš ROTREKL Vladimír LACAMPAGNE Alain DVOŘÁK Petr MELI Albano

Druh Článek v odborném periodiku
Časopis / Zdroj Heart Vessels
Fakulta / Pracoviště MU

Lékařská fakulta

Citace
WWW http://link.springer.com/article/10.1007/s00380-013-0436-9#
Doi http://dx.doi.org/10.1007/s00380-013-0436-9
Obor Genetika a molekulární biologie
Klíčová slova human pluripotent stem cell embryoid body differentiation cardiomyocyte calcium
Popis In vitro human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) can differentiate into functional cardiomyocytes (CMs). Protocols for cardiac differentiation of hESCs and hiPSCs include formation of the three-dimensional cell aggregates called embryoid bodies (EBs). The traditional suspension method for EB formation from clumps of cells results in an EB population heterogeneous in size and shape. In this study we show that forced aggregation of a defined number of single cells on AggreWell plates gives a high number of homogeneous EBs that can be efficiently differentiated into functional CMs by application of defined growth factors in the media. For cardiac differentiation, we used three hESC lines and one hiPSC line. Our contracting EBs and the resulting CMs express cardiac markers, namely myosin heavy chain alpha and beta, cardiac ryanodine receptor/calcium release channel, and cardiac troponin T, shown by real-time polymerase chain reaction and immunocytochemistry. Using Ca2+ imaging and atomic force microscopy, we demonstrate the functionality of RyR2 to release Ca2+ from the sarcoplasmic reticulum as well as reliability in contractile and beating properties of hESC-EBs and hiPSC-EBs upon the stimulation or inhibition of the beta-adrenergic pathway.
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