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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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 α and β, 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 β-adrenergic pathway.
KeywordsHuman pluripotent stem cell Embryoid body Differentiation Cardiomyocyte Calcium
We would like to thank Dr Livia Eiselleova, Stanislava Koskova, Professor Ales Hampl, Dana Stritecka, and Eva Peslova for their assistance, as well as Professor Majlinda Lako for kindly providing the clone 4 hiPSC. This work was supported by grants from the Ministry of Education, Youth, and Sports of the Czech Republic (CZ.1.07/2.3.00/20.0011 and MSM0021622430), project FNUSA-ICRC (no. CZ.1.05/1.1.00/02.0123) from the European Regional Development Fund, SoMoPro—Marie Curie Actions—South Moravian Region, and by the European Society of Cardiology (ESC) to Albano C. Meli. The research leading to these results obtained a financial contribution from the European Community within the Seventh Framework Program (FP/2007-2013) under Grant Agreement No. 229603. This work was supported by CEITEC—Central European Institute of Technology (CZ.1.05/1.1.00/02.0068) from the European Regional Development Fund. Albano C. Meli was supported by a French Muscular Dystrophy Association Research Grant (AFM). Ivana Acimovic was supported by a PLURICELL grant (CZ.1.07/2.3.00/20.0011).
Conflict of interest
There is no conflict of interest.
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