title: Forced aggregation and defined factors allow highly uniform-sized embryoid bodies and functional cardiomyocytes from human embryonic and induced pluripotent stem cells.
abstract: 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 Ca(2+) imaging and atomic force microscopy, we demonstrate the functionality of RyR2 to release Ca(2+) 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.
authors.affiliation: Department of Biology, Faculty of Medicine, Masaryk University, Brno, Czech Republic.
authors.forename: Martin
authors.initials: M
authors.lastname: Pesl
authors.forename: Ivana
authors.initials: I
authors.lastname: Acimovic
authors.forename: Jan
authors.initials: J
authors.lastname: Pribyl
authors.forename: Renata
authors.initials: R
authors.lastname: Hezova
authors.forename: Aleksandra
authors.initials: A
authors.lastname: Vilotic
authors.forename: Jeremy
authors.initials: J
authors.lastname: Fauconnier
authors.forename: Jan
authors.initials: J
authors.lastname: Vrbsky
authors.forename: Peter
authors.initials: P
authors.lastname: Kruzliak
authors.forename: Petr
authors.initials: P
authors.lastname: Skladal
authors.forename: Tomas
authors.initials: T
authors.lastname: Kara
authors.forename: Vladimir
authors.initials: V
authors.lastname: Rotrekl
authors.forename: Alain
authors.initials: A
authors.lastname: Lacampagne
authors.forename: Petr
authors.initials: P
authors.lastname: Dvorak
authors.forename: Albano C
authors.initials: AC
authors.lastname: Meli
mesh_terms: D002118:Calcium / Q000378:metabolism; D002454:Cell Differentiation; D002460:Cell Line; D048430:Cell Shape; D048429:Cell Size; D058732:Embryoid Bodies / Q000502:physiology*; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000502:physiology*; D032383:Myocytes, Cardiac / Q000166:cytology / Q000502:physiology; D018995:Myosin Heavy Chains / Q000378:metabolism; D015203:Reproducibility of Results; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D020107:Troponin T / Q000378:metabolism
chemical_list: D019837:Ryanodine Receptor Calcium Release Channel; D020107:Troponin T; D018995:Myosin Heavy Chains; D002118:Calcium