Abstract
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.
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Acknowledgements
We thank A. Moretti and K.-L. Laugwitz (University of Munich, Germany) for provision of the C25-hiPSC clone. This study was supported by the Deutsche Forschungsgemeinschaft (grants DFG Es 88/12-1 and DFG HA 3423/5-1), the British National Centre for the Replacement, Refinement, and Reduction of Animals in Research (NC3Rs CRACK-IT grant 35911-259146), the European Research Council (ERC-AG IndivuHeart), the EU (FP7 Biodesign), the German Centre for Cardiovascular Research (DZHK), the German Ministry of Education and Research (BMBF), British Heart Foundation grant RM/13/30157, the German Heart Foundation, the Freie und Hansestadt Hamburg and Era-Net E-RARE (grant 01GM1305).
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K.B. designed and performed the experiments, analyzed the data and wrote the paper. T.S., I.M., B.U., M.C.R., T.W., A.B., D.L.-B., B.K., M.P., G.M., S.L., A.S., D.S., S.F., C.N., E.K., U.S., M.L.S. and M.L.R. contributed to the development or validation of this protocol. A.H. supervised the project. A.H. and T.E. wrote and approved the final paper.
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I.M., T.E. and A.H. are cofounders of EHT Technologies.
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Supplementary Figure 1 Gating strategy used for FACS analysis of hiPSC-cardiomyocytes.
Given are details for forward and side scatter gates of the starting cell population and the gating to isolate cardiac troponin T-FITC-positive cells with the help of unstained cells (A) and cells stained with REA Control (I)-FITC isotype control (B). Absolute numbers of the cells analyzed and percentages of the relevant cell populations are provided. Cells were sorted with a BD FACSCanto II Flow Cytometer and analyzed with the BD FACSDiva Software.
Supplementary Figure 2 Gating strategy used for FACS analysis of hiPSCs.
Given are details for forward and side scatter gates of the starting cell population, the gating to discriminate doublets and aggregates and the gating to isolate TRA-1-60/SSEA-positive cells with the help of cells stained with isotype controls. Absolute numbers of the cells analyzed and percentages of the relevant cell populations are provided. Cells were sorted with a BD FACSCanto II Flow Cytometer and analyzed with the FlowJo Software.
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Breckwoldt, K., Letuffe-Brenière, D., Mannhardt, I. et al. Differentiation of cardiomyocytes and generation of human engineered heart tissue. Nat Protoc 12, 1177–1197 (2017). https://doi.org/10.1038/nprot.2017.033
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DOI: https://doi.org/10.1038/nprot.2017.033
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