Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Differentiation of cardiomyocytes and generation of human engineered heart tissue

Nature Protocols volume 12, pages 1177–1197 (2017)Cite this article

Subjects

An Author Correction to this article was published on 05 August 2019

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Quantitative analysis of cardiomyocytes differentiated from hiPSCs (day 17).
Figure 2: Expression of pluripotency markers by hiPSCs.
Figure 3: Schematic of the protocol for differentiation of cardiomyocytes from hiPSCs and EHT generation.
Figure 4: Graphical illustration of differentiation and EHT generation.
Figure 5: Bright-field images at different time points of cardiac differentiation with five different cell lines, showing the typical morphology.
Figure 6: Estimation of EB volume after EB formation.
Figure 7: Pictures of EHTs from hiPSC-CMs of three different control cell lines taken during a contractility measurement (left) and representative force recording (right).

Similar content being viewed by others

References

  1. Burridge, P.W., Keller, G., Gold, J.D. & Wu, J.C. Production of de novo cardiomyocytes: human pluripotent stem cell differentiation and direct reprogramming. Cell Stem Cell 10, 16–28 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Mercola, M., Colas, A. & Willems, E. Induced pluripotent stem cells in cardiovascular drug discovery. Circ. Res. 112, 534–548 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Knollmann, B.C. Induced pluripotent stem cell-derived cardiomyocytes: boutique science or valuable arrhythmia model? Circ. Res. 112, 969–976 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Hansen, A. et al. Development of a drug screening platform based on engineered heart tissue. Circ. Res. 107, 35–44 (2010).

    Article  CAS  PubMed  Google Scholar 

  5. Schaaf, S. et al. Human engineered heart tissue as a versatile tool in basic research and preclinical toxicology. PLoS One 6, e26397 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Hirt, M.N. et al. Increased afterload induces pathological cardiac hypertrophy: a new in vitro model. Basic Res. Cardiol. 107, 307 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  7. Mannhardt, I. et al. Human engineered heart tissue: analysis of contractile force. Stem Cell Rep. 7, 29–42 (2016).

    Article  CAS  Google Scholar 

  8. Uzun, A.U. et al. Ca(2+)-currents in human induced pluripotent stem cell-derived cardiomyocytes effects of two different culture conditions. Front. Pharmacol. 7, 300 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  9. Weinberger, F. et al. Cardiac repair in guinea pigs with human engineered heart tissue from induced pluripotent stem cells. Sci. Transl. Med. 8, 363ra148 (2016).

    Article  PubMed  CAS  Google Scholar 

  10. Thomson, J.A. et al. Embryonic stem cell lines derived from human blastocysts. Science 282, 1145–1147 (1998).

    CAS  PubMed  Google Scholar 

  11. Ludwig, T.E. et al. Derivation of human embryonic stem cells in defined conditions. Nat. Biotechnol. 24, 185–187 (2006).

    Article  CAS  PubMed  Google Scholar 

  12. Chen, G. et al. Chemically defined conditions for human iPSC derivation and culture. Nat. Methods 8, 424–429 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Xu, C. et al. Feeder-free growth of undifferentiated human embryonic stem cells. Nat. Biotechnol. 19, 971–974 (2001).

    Article  CAS  PubMed  Google Scholar 

  14. Frank, S., Zhang, M., Scholer, H.R. & Greber, B. Small molecule-assisted, line-independent maintenance of human pluripotent stem cells in defined conditions. PLoS One 7, e41958 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Lanner, F. & Rossant, J. The role of FGF/Erk signaling in pluripotent cells. Development 137, 3351–3360 (2010).

    Article  CAS  PubMed  Google Scholar 

  16. Vallier, L., Alexander, M. & Pedersen, R.A. Activin/Nodal and FGF pathways cooperate to maintain pluripotency of human embryonic stem cells. J. Cell Sci. 118, 4495–4509 (2005).

    Article  CAS  PubMed  Google Scholar 

  17. Eiselleova, L. et al. A complex role for FGF-2 in self-renewal, survival, and adhesion of human embryonic stem cells. Stem Cells 27, 1847–1857 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Ding, V.M. et al. FGF-2 modulates Wnt signaling in undifferentiated hESC and iPS cells through activated PI3-K/GSK3beta signaling. J. Cell Physiol. 225, 417–428 (2010).

    Article  CAS  PubMed  Google Scholar 

  19. Zoumaro-Djayoon, A.D. et al. Investigating the role of FGF-2 in stem cell maintenance by global phosphoproteomics profiling. Proteomics 11, 3962–3971 (2011).

    Article  CAS  PubMed  Google Scholar 

  20. Miltenyi Biotec. Human FGF-2 IS. <http://www.miltenyibiotec.com/en/products-and-services/macs-cell-culture-and-stimulation/cytokines-and-growth-factors/premium-and-research-grade/human-fgf-2-is.aspx.

  21. Burridge, P.W. et al. A universal system for highly efficient cardiac differentiation of human induced pluripotent stem cells that eliminates interline variability. PLoS One 6, e18293 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Burridge, P.W. et al. Chemically defined generation of human cardiomyocytes. Nat. Methods 11, 855–860 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Yang, L. et al. Human cardiovascular progenitor cells develop from a KDR+ embryonic-stem-cell-derived population. Nature 453, 524–528 (2008).

    Article  CAS  PubMed  Google Scholar 

  24. Laflamme, M.A. et al. Cardiomyocytes derived from human embryonic stem cells in pro-survival factors enhance function of infarcted rat hearts. Nat. Biotechnol. 25, 1015–1024 (2007).

    Article  CAS  PubMed  Google Scholar 

  25. Mummery, C. et al. Differentiation of human embryonic stem cells to cardiomyocytes: role of coculture with visceral endoderm-like cells. Circulation 107, 2733–2740 (2003).

    Article  CAS  PubMed  Google Scholar 

  26. Kattman, S.J. et al. Stage-specific optimization of activin/nodal and BMP signaling promotes cardiac differentiation of mouse and human pluripotent stem cell lines. Cell Stem Cell 8, 228–240 (2011).

    Article  CAS  PubMed  Google Scholar 

  27. Moretti, A., Laugwitz, K.L., Dorn, T., Sinnecker, D. & Mummery, C. Pluripotent stem cell models of human heart disease. Cold Spring Harb. Perspect. Med. 3 http://dx.doi.org/10.1101/cshperspect.a014027 (2013).

  28. Zweigerdt, R., Olmer, R., Singh, H., Haverich, A. & Martin, U. Scalable expansion of human pluripotent stem cells in suspension culture. Nat. Protoc. 6, 689–700 (2011).

    Article  CAS  PubMed  Google Scholar 

  29. Olmer, R. et al. Suspension culture of human pluripotent stem cells in controlled, stirred bioreactors. Tissue Eng. Part C Methods 18, 772–784 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Lian, X. et al. Directed cardiomyocyte differentiation from human pluripotent stem cells by modulating Wnt/β-catenin signaling under fully defined conditions. Nat. Protoc. 8, 162–175 (2013).

    Article  CAS  PubMed  Google Scholar 

  31. Kempf, H. et al. Controlling expansion and cardiomyogenic differentiation of human pluripotent stem cells in scalable suspension culture. Stem Cell Rep. 3, 1132–1146 (2014).

    Article  CAS  Google Scholar 

  32. Kempf, H., Kropp, C., Olmer, R., Martin, U. & Zweigerdt, R. Cardiac differentiation of human pluripotent stem cells in scalable suspension culture. Nat. Protoc. 10, 1345–1361 (2015).

    Article  CAS  PubMed  Google Scholar 

  33. Chen, V.C. et al. Development of a scalable suspension culture for cardiac differentiation from human pluripotent stem cells. Stem Cell Res. 15, 365–375 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Lanier, M. et al. Wnt inhibition correlates with human embryonic stem cell cardiomyogenesis: a structure-activity relationship study based on inhibitors for the Wnt response. J. Med. Chem. 55, 697–708 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Tulloch, N.L. et al. Growth of engineered human myocardium with mechanical loading and vascular coculture. Circ. Res. 109, 47–59 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Kensah, G. et al. Murine and human pluripotent stem cell-derived cardiac bodies form contractile myocardial tissue in vitro. Eur. Heart J. 34, 1134–1146 (2013).

    Article  CAS  PubMed  Google Scholar 

  37. Nunes, S.S. et al. Biowire: a platform for maturation of human pluripotent stem cell-derived cardiomyocytes. Nat. Methods 10, 781–787 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Thavandiran, N. et al. Design and formulation of functional pluripotent stem cell-derived cardiac microtissues. Proc. Natl. Acad. Sci. USA 110, E4698–4707 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Turnbull, I.C. et al. Advancing functional engineered cardiac tissues toward a preclinical model of human myocardium. FASEB J. 28, 644–654 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Hinson, J.T. et al. HEART DISEASE. Titin mutations in iPS cells define sarcomere insufficiency as a cause of dilated cardiomyopathy. Science 349, 982–986 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Kuppusamy, K.T. et al. Let-7 family of microRNA is required for maturation and adult-like metabolism in stem cell-derived cardiomyocytes. Proc. Natl. Acad. Sci. USA 112, E2785–2794 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Stillitano, F. et al. Genomic correction of familial cardiomyopathy in human engineered cardiac tissues. Eur. Heart J. 37, 3282–3284 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Riegler, J. et al. Human engineered heart muscles engraft and survive long term in a rodent myocardial infarction model. Circ. Res. 117, 720–730 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Zhang, D. et al. Tissue-engineered cardiac patch for advanced functional maturation of human ESC-derived cardiomyocytes. Biomaterials 34, 5813–5820 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Wang, G. et al. Modeling the mitochondrial cardiomyopathy of Barth syndrome with induced pluripotent stem cell and heart-on-chip technologies. Nat. Med. 20, 616–623 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Eschenhagen, T. et al. Three-dimensional reconstitution of embryonic cardiomyocytes in a collagen matrix: a new heart muscle model system. FASEB J. 11, 683–694 (1997).

    Article  CAS  PubMed  Google Scholar 

  47. Zimmermann, W.H. et al. Three-dimensional engineered heart tissue from neonatal rat cardiac myocytes. Biotechnol. Bioeng. 68, 106–114 (2000).

    Article  CAS  PubMed  Google Scholar 

  48. Zimmermann, W.H. et al. Tissue engineering of a differentiated cardiac muscle construct. Circ. Res. 90, 223–230 (2002).

    Article  CAS  PubMed  Google Scholar 

  49. Neuber, C. et al. Paradoxical effects on force generation after efficient β1-adrenoceptor knockdown in reconstituted heart tissue. J. Pharmacol. Exp. Ther. 349, 39–46 (2014).

    Article  PubMed  CAS  Google Scholar 

  50. Stohr, A. et al. Contractile abnormalities and altered drug response in engineered heart tissue from Mybpc3-targeted knock-in mice. J. Mol. Cell. Cardiol. 63, 189–198 (2013).

    Article  PubMed  CAS  Google Scholar 

  51. Dussurget, O. & Roulland-Dussoix, D. Rapid, sensitive PCR-based detection of mycoplasmas in simulated samples of animal sera. Appl. Environ. Microbiol. 60, 953–959 (1994).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Ungrin, M.D., Joshi, C., Nica, A., Bauwens, C. & Zandstra, P.W. Reproducible, ultra high-throughput formation of multicellular organization from single cell suspension-derived human embryonic stem cell aggregates. PLoS One 3, e1565 (2008).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  53. Kempf, H. et al. Bulk cell density and Wnt/TGFbeta signalling regulate mesendodermal patterning of human pluripotent stem cells. Nat. Commun. 7, 13602 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Lian, X., Zhang, J., Zhu, K., Kamp, T.J. & Palecek, S.P. Insulin inhibits cardiac mesoderm, not mesendoderm, formation during cardiac differentiation of human pluripotent stem cells and modulation of canonical Wnt signaling can rescue this inhibition. Stem Cells 31, 447–457 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Tran, T.H. et al. Wnt3a-induced mesoderm formation and cardiomyogenesis in human embryonic stem cells. Stem Cells 27, 1869–1878 (2009).

    Article  CAS  PubMed  Google Scholar 

  56. Freund, C. et al. Insulin redirects differentiation from cardiogenic mesoderm and endoderm to neuroectoderm in differentiating human embryonic stem cells. Stem Cells 26, 724–733 (2008).

    Article  CAS  PubMed  Google Scholar 

  57. Moretti, A. et al. Patient-specific induced pluripotent stem-cell models for long-QT syndrome. N. Engl. J. Med. 363, 1397–1409 (2010).

    Article  CAS  PubMed  Google Scholar 

  58. Takahashi, K. et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861–872 (2007).

    Article  CAS  PubMed  Google Scholar 

  59. Eschenhagen, T., Eder, A., Vollert, I. & Hansen, A. Physiological aspects of cardiac tissue engineering. Am. J. Physiol. Heart Circ. Physiol. 303, H133–H143 (2012).

    Article  CAS  PubMed  Google Scholar 

  60. Lotz, S. et al. Sustained levels of FGF2 maintain undifferentiated stem cell cultures with biweekly feeding. PLoS One 8, e56289 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

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).

Author information

Authors and Affiliations

  1. Department of Experimental Pharmacology and Toxicology, Cardiovascular Research Center, University Medical Center Hamburg-Eppendorf, and DZHK (German Center for Cardiovascular Research), Partner Site Hamburg/Kiel/Lübeck, Hamburg, Germany

    Kaja Breckwoldt, David Letuffe-Brenière, Ingra Mannhardt, Thomas Schulze, Bärbel Ulmer, Tessa Werner, Anika Benzin, Birgit Klampe, Marina C Reinsch, Maksymilian Prondzynski, Giulia Mearini, Christiane Neuber, Elisabeth Krämer, Umber Saleem, Mirja L Schulze, Marita L Rodriguez, Thomas Eschenhagen & Arne Hansen

  2. HEXT-Stem Cell Facility, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

    Sandra Laufer & Aya Shibamiya

  3. Faculty of Chemistry and Chemical Biology, TU Dortmund University, Dortmund, Germany

    Dennis Schade

  4. Department of Human Genetics, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

    Sigrid Fuchs

Authors
  1. Kaja Breckwoldt
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. David Letuffe-Brenière
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ingra Mannhardt
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Thomas Schulze
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Bärbel Ulmer
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Tessa Werner
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Anika Benzin
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Birgit Klampe
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Marina C Reinsch
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Sandra Laufer
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Aya Shibamiya
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Maksymilian Prondzynski
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Giulia Mearini
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Dennis Schade
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Sigrid Fuchs
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Christiane Neuber
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. Elisabeth Krämer
    View author publications

    You can also search for this author in PubMed Google Scholar

  18. Umber Saleem
    View author publications

    You can also search for this author in PubMed Google Scholar

  19. Mirja L Schulze
    View author publications

    You can also search for this author in PubMed Google Scholar

  20. Marita L Rodriguez
    View author publications

    You can also search for this author in PubMed Google Scholar

  21. Thomas Eschenhagen
    View author publications

    You can also search for this author in PubMed Google Scholar

  22. Arne Hansen
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

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.

Corresponding author

Correspondence to Arne Hansen.

Ethics declarations

Competing interests

I.M., T.E. and A.H. are cofounders of EHT Technologies.

Integrated supplementary information

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.

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2017.033

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research