Engineering of human cardiac muscle electromechanically matured to an adult-like phenotype

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The application of tissue-engineering approaches to human induced pluripotent stem (hiPS) cells enables the development of physiologically relevant human tissue models for in vitro studies of development, regeneration, and disease. However, the immature phenotype of hiPS-derived cardiomyocytes (hiPS-CMs) limits their utility. We have developed a protocol to generate engineered cardiac tissues from hiPS cells and electromechanically mature them toward an adult-like phenotype. This protocol also provides optimized methods for analyzing these tissues’ functionality, ultrastructure, and cellular properties. The approach relies on biological adaptation of cultured tissues subjected to biomimetic cues, applied at an increasing intensity, to drive accelerated maturation. hiPS cells are differentiated into cardiomyocytes and used immediately after the first contractions are observed, when they still have developmental plasticity. This starting cell population is combined with human dermal fibroblasts, encapsulated in a fibrin hydrogel and allowed to compact under passive tension in a custom-designed bioreactor. After 7 d of tissue formation, the engineered tissues are matured for an additional 21 d by increasingly intense electromechanical stimulation. Tissue properties can be evaluated by measuring contractile function, responsiveness to electrical stimuli, ultrastructure properties (sarcomere length, mitochondrial density, networks of transverse tubules), force–frequency and force–length relationships, calcium handling, and responses to β-adrenergic agonists. Cell properties can be evaluated by monitoring gene/protein expression, oxidative metabolism, and electrophysiology. The protocol takes 4 weeks and requires experience in advanced cell culture and machining methods for bioreactor fabrication. We anticipate that this protocol will improve modeling of cardiac diseases and testing of drugs.

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Fig. 1: Stages involved in the generation and analysis of hiPS-derived cardiac tissues.
Fig. 2: Maturation of hiPS-cell-derived cardiac tissues to an adult-like phenotype.
Fig. 3: Enhanced isoproterenol response and ultrastructure of tissues formed from early-stage hiPS-CMs from different cell lines.
Fig. 4: Gene expression analysis of bioengineered heart tissues.
Fig. 5: Platforms for tissue formation and maturation.
Fig. 6: Setup of the intensity stimulation program in an Arduino electrical stimulator.
Fig. 7: Overview of cardiac tissue formation.

Data availability

Source data for the quantitative data shown in all figure panels are available without restrictions and can be accessed at The Genotype-Tissue Expression (GTEx) Project was supported by the Common Fund of the Office of the Director of the National Institutes of Health, and by NCI, NHGRI, NHLBI, NIDA, NIMH, and NINDS. The GTEx data used for the analyses described in this article were obtained from the dbGaP Portal ( using the following dbGaP accession number: phs000424.v7.p2.


  1. 1.

    Ronaldson-Bouchard, K. & Vunjak-Novakovic, G. Organs-on-a-chip: a fast track for engineered human tissues in drug development. Cell Stem Cell 22, 310–324 (2018).

  2. 2.

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

  3. 3.

    Ye, K. Y., Sullivan, K. E. & Black, L. D. Encapsulation of cardiomyocytes in a fibrin hydrogel for cardiac tissue engineering. J. Vis. Exp. 2011, e3251 (2011).

  4. 4.

    Burridge, P. W. et al. Chemically defined and small molecule-based generation of human cardiomyocytes. Nat. Methods 11, 855–860 (2014).

  5. 5.

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

  6. 6.

    Ronaldson-Bouchard, K. et al. Advanced maturation of human cardiac tissue grown from pluripotent stem cells. Nature 556, 239–243 (2018).

  7. 7.

    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).

  8. 8.

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

  9. 9.

    Ruan, J. L. et al. Mechanical stress conditioning and electrical stimulation promote contractility and force maturation of induced pluripotent stem cell-derived human cardiac tissue. Circulation 134, 1557–1567 (2016).

  10. 10.

    Schaaf, S. et al. Generation of strip-format fibrin-based engineered heart tissue (EHT). Methods Mol. Biol. 1181, 121–129 (2014).

  11. 11.

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

  12. 12.

    Mordwinkin, N. M., Burridge, P. W. & Wu, J. C. A review of human pluripotent stem cell-derived cardiomyocytes for high-throughput drug discovery, cardiotoxicity screening, and publication standards. J. Cardiovasc. Transl. Res. 6, 22–30 (2013).

  13. 13.

    Piccini, J. P. et al. Current challenges in the evaluation of cardiac safety during drug development: translational medicine meets the Critical Path Initiative. Am. Heart J. 158, 317–326 (2009).

  14. 14.

    Robertson, C., Tran, D. D. & George, S. C. Concise review: maturation phases of human pluripotent stem cell–derived cardiomyocytes. Stem Cells 31, 829–837 (2013).

  15. 15.

    Breckwoldt, K. et al. Differentiation of cardiomyocytes and generation of human engineered heart tissue. Nat. Protoc. 12, 1177–1197 (2017).

  16. 16.

    Eng, G. et al. Autonomous beating rate adaptation in human stem cell-derived cardiomyocytes. Nat. Commun. 7, 10312 (2016).

  17. 17.

    Radisic, M. et al. Functional assembly of engineered myocardium by electrical stimulation of cardiac myocytes cultured on scaffolds. Proc. Natl Acad. Sci. 101, 18129–18134 (2004).

  18. 18.

    Boudou, T. et al. A microfabricated platform to measure and manipulate the mechanics of engineered cardiac microtissues. Tissue Eng. Part A 18, 910–919 (2012).

  19. 19.

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

  20. 20.

    Robertson, C., Tran, D. D. & George, S. C. Concise review: maturation phases of human pluripotent stem cell-derived cardiomyocytes. Stem Cells 31, 829–837 (2013).

  21. 21.

    Feric, N. T. & Radisic, M. Maturing human pluripotent stem cell-derived cardiomyocytes in human engineered cardiac tissues. Adv. Drug Deliv. Rev. 96, 110–134 (2016).

  22. 22.

    Pervolaraki, E., Dachtler, J., Anderson, R. A. & Holden, A. V. The development transcriptome of the human heart. Sci. Rep. 8, 15362 (2018).

  23. 23.

    White, M. C., Pang, L. & Yang, X. MicroRNA-mediated maturation of human pluripotent stem cell-derived cardiomyocytes: towards a better model for cardiotoxicity? Food Chem. Toxicol. 98, 17–24 (2016).

  24. 24.

    Vandenburgh, H. et al. Automated drug screening with contractile muscle tissue engineered from dystrophic myoblasts. FASEB J. 23, 3325–3334 (2009).

  25. 25.

    Polacheck, W. J. & Chen, C. S. Measuring cell-generated forces: a guide to the available tools. Nat. Methods 13, 415–423 (2016).

  26. 26.

    Chattergoon, N. N. et al. Thyroid hormone drives fetal cardiomyocyte maturation. FASEB J. 26, 397–408 (2012).

  27. 27.

    Parikh, S. S. et al. Thyroid and glucocorticoid hormones promote functional T-tubule development in human-induced pluripotent stem cell-derived cardiomyocytes. Circ. Res. 121, 1323–1330 (2017).

  28. 28.

    Tandon, N. et al. Electrical stimulation systems for cardiac tissue engineering. Nat. Protoc. 4, 155–173 (2009).

  29. 29.

    Petriccione, M. et al. Reference gene selection for normalization of RT-qPCR gene expression data from Actinidia deliciosa leaves infected with Pseudomonas syringae pv. actinidiae. Sci. Rep. 5, 16961 (2015).

  30. 30.

    Godier-Furnemont, A. F. et al. Physiologic force-frequency response in engineered heart muscle by electromechanical stimulation. Biomaterials 60, 82–91 (2015).

  31. 31.

    Burke, M. A. et al. Clinical and mechanistic insights into the genetics of cardiomyopathy. J. Am. Coll. Cardiol. 68, 2871–2886 (2016).

  32. 32.

    Ronaldson-Bouchard, K. et al. Advanced maturation of human cardiac tissue grown from pluripotent stem cells. Nature 556, 239–243 (2018).

  33. 33.

    Subramanian, A. et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc. Natl Acad. Sci. 102, 15545–15550 (2005).

  34. 34.

    Hayakawa, T. et al. Noninvasive evaluation of contractile behavior of cardiomyocyte monolayers based on motion vector analysis. Tissue Eng. Part C Methods 18, 21–32 (2012).

  35. 35.

    Huebsch, N. et al. Automated video-based analysis of contractility and calcium flux in human-induced pluripotent stem cell-derived cardiomyocytes cultured over different spatial scales. Tissue Eng. Part C Methods 21, 467–479 (2015).

  36. 36.

    Sala, L. et al. MUSCLEMOTION: a versatile open software tool to quantify cardiomyocyte and cardiac muscle contraction in vitro and in vivo. Circ. Res. 122, e5–e16 (2018).

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The authors acknowledge funding support from the NIBIB and NCATS grant EB17103 (G.V.-N.); NIBIB, NCATS, NIAMS, NIDCR, and NIEHS grant EB025765 (G.V.-N.); NHLBI grants HL076485 (G.V.-N.) and HL138486 (M.Y.); NSF grant 16478 (G.V.-N.); the University of Minho MD/PhD program (D.T.); a Japan Society for the Promotion of Science fellowship (K.M.); and the Columbia University Stem Cell Initiative (L.S., M.Y.). The authors also acknowledge all co-authors of the original paper describing the methodology for bioengineering adult-like cardiac tissues for their initial support and contributions, S. Duncan (University of Wisconsin (C2A line)) and B. Conklin (Gladstone Institute (WTC-11 line)) for providing hiPS cells, M. B. Bouchard for assistance with image and video analysis, D. Sirabella at the Columbia Stem Cell Core for assistance with cell derivation, and A. Califano for expert help with gene expression analysis.

Author information

K.R.-B., K.Y., and G.V.-N. designed the methodological approach; K.R.-B., D.T., K.Y., and G.V.-N. designed the single-pillar molds and cardiac tissue formation and maturation platforms; K.R.-B., D.T., and L.S. cultured cell lines; K.R.-B., D.T., T.C., and S.M. formed cardiac tissues and performed maturity assessment experiments; L.S., K.M., and M.Y. conducted single-cell dissociation and electrophysiology readings; H.M.W. conducted flow cytometry analysis; A.V. and E.C.R. conducted the analysis of gene expression; K.R.-B., D.T., K.Y., M.Y., and G.V.-N interpreted the data and wrote the manuscript.

Correspondence to Gordana Vunjak-Novakovic.

Ethics declarations

Competing interests

G.V.-N. and K.R.-B. are co-founders of TARA Biosystems, a Columbia University spin-off that is commercializing the use of bioengineered human cardiac tissues for drug development.

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Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Related links

Key references using this protocol

Radisic, M. et al. Proc. Natl Acad. Sci. USA 101, 18129–18134 (2004):

Ronaldson-Bouchard, K. et al. Nature 556, 239–243 (2018):

Zhao, Y. et al. Cell 176, 913–927.E18 (2019):

Integrated supplementary information

Supplementary Figure 1 Contractility assessment of cardiac tissues.

A) Parameters evaluated by the custom contractility code. B) Baseline and peak threshold of an example of a contractility trace.

Supplementary Figure 2 Organ bath setup of the cardiac tissues.

A) Bring the hooks of the force transducer and tissue holder closer together until they are separated by a length similar to the length of the tissue from the inside on once PDMS pillar to the inside of the opposite PDMS pillar. B) Load the cardiac tissue by piercing the cardiac tissue on the inside of each PDMS pillar with the attachment hooks. C) Using an additional set of forceps to hold the tissue in place, remove the cardiac tissue from the PDMS pillars. D) Use forceps to ensure cardiac tissues are adequately attached to attachment hooks. E-F) Record the initial length of the cardiac tissue (E) and stretch the cardiac tissue by turning the positioning dial on the organ bath to find the optimal force-length relationship (F).

Supplementary Figure 3 Confocal imaging setup.

Confocal images of the whole tissues are difficult to obtain without having enough working distance to image the tissue portion, as opposed to the pillar regions. Subsequently, we found that laying the tissues on their side and keeping them hydrated in PBS during imaging enabled confocal images of the cardiac tissue to be obtained.

Supplementary Figure 4 Electrophysiological recording setup.

A glass-bottom dish containing cardiomyocytes was replaced in the dish holder with perfusion and temperature controllers on the microscopic stage. Eye pieces were used to find cells and computer monitor (~40-60x objective lens) was used to seal the cells with an electrode.

Supplementary Figure 5 Flow cytometry of early-stage hiPS-CMs.

A) Differentiation regimen used in the differentiation of C2A hiPS-CM shown below. B-C) Flow cytometry of an early-stage differentiation containing C2A cells labelled with cardiac troponin T(cTnT) reveals an efficiency greater than 80% (B) and myosin light chain ventricular isoform (MLC-2V) reveals an efficiency greater than 30% (C).

Supplementary Figure 6 Generation of cardiac tissues with electrophysiological activity, cell structure and mechanical activity closer to adult myocardium.

Schematic detailing the overall workflow to engineer adult-like cardiac tissues and the anticipated analysis of their functionality, calcium, force and drug response, metabolism, electrophysiology and ultrastructure that can be performed. Images used under a Creative Commons Attribution 3.0 license (

Supplementary information

Supplementary Information

Supplementary Figs. 1–6 and Supplementary Table 1

Reporting Summary

Supplementary Code 1

Code and libraries needed for the Arduino-based electrical stimulator detailed in Box 2.

Supplementary Code 2

Code and libraries needed for the MATLAB-based analysis detailed in Box 3.

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