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Differentiation of cardiomyocytes and generation of human engineered heart tissue.

Nature protocols (2017-05-12)
Kaja Breckwoldt, David Letuffe-Brenière, Ingra Mannhardt, Thomas Schulze, Bärbel Ulmer, Tessa Werner, Anika Benzin, Birgit Klampe, Marina C Reinsch, Sandra Laufer, Aya Shibamiya, Maksymilian Prondzynski, Giulia Mearini, Dennis Schade, Sigrid Fuchs, Christiane Neuber, Elisabeth Krämer, Umber Saleem, Mirja L Schulze, Marita L Rodriguez, Thomas Eschenhagen, Arne Hansen
ZUSAMMENFASSUNG

Since the advent of the generation of human induced pluripotent stem cells (hiPSCs), numerous protocols have been developed to differentiate hiPSCs into cardiomyocytes and then subsequently assess their ability to recapitulate the properties of adult human cardiomyocytes. However, hiPSC-derived cardiomyocytes (hiPSC-CMs) are often assessed in single-cell assays. A shortcoming of these assays is the limited ability to characterize the physiological parameters of cardiomyocytes, such as contractile force, due to random orientations. This protocol describes the differentiation of cardiomyocytes from hiPSCs, which occurs within 14 d. After casting, cardiomyocytes undergo 3D assembly. This produces fibrin-based engineered heart tissues (EHTs)-in a strip format-that generate force under auxotonic stretch conditions. 10-15 d after casting, the EHTs can be used for contractility measurements. This protocol describes parallel expansion of hiPSCs; standardized generation of defined embryoid bodies, growth factor and small-molecule-based cardiac differentiation; and standardized generation of EHTs. To carry out the protocol, experience in advanced cell culture techniques is required.

MATERIALIEN
Produktnummer
Marke
Produktbeschreibung

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Insulin -Lösung human, sterile-filtered, BioXtra, suitable for cell culture
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Fibrinogen aus Rinderplasma, Type I-S, 65-85% protein (≥75% of protein is clottable)
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Transferrin human, powder, BioReagent, suitable for cell culture
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Aprotinin aus Rinderlunge, lyophilized powder, 3-8 TIU/mg solid
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Gelatine aus Schweinehaut, powder, gel strength ~300 g Bloom, Type A, BioReagent, suitable for electrophoresis, suitable for cell culture
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Monoklonales Anti-α-Aktinin (Sarkomer) in Maus hergestellte Antikörper, clone EA-53, ascites fluid
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Natriumselenit, BioReagent, suitable for cell culture, ≥98%
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IWR-1, ≥98% (HPLC)
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Desoxyribonuklease II aus Rindermilz, Type V, essentially salt-free, lyophilized powder, ≥1,000 units/mg protein