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

Nature Protocols volume 14, pages 2781–2817 (2019)Cite this article

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Abstract

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.

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Data availability

Source data for the quantitative data shown in all figure panels are available without restrictions and can be accessed at https://doi.org/10.6084/m9.figshare.5765559. 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 (https://www.ncbi.nlm.nih.gov/gap/) using the following dbGaP accession number: phs000424.v7.p2.

Change history

  • 20 December 2019

    In the supplementary information initally published online to accompany this article, the zip file for Supplementary Code 2 (“Code and libraries needed for the MATLAB-based analysis detailed in Box 3”) was empty. The file is now complete.

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Acknowledgements

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.

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Authors and Affiliations

  1. Department of Biomedical Engineering, Columbia University, New York, NY, USA

    Kacey Ronaldson-Bouchard, Keith Yeager, Diogo Teles, Timothy Chen, Stephen Ma, Holly M. Wobma, Edward C. Ruiz & Gordana Vunjak-Novakovic

  2. Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Braga, Portugal

    Diogo Teles

  3. ICVS/3B’s, PT Government Associate Laboratory, Braga/Guimarães, Portugal

    Diogo Teles

  4. Departments of Rehabilitation and Regenerative Medicine, and of Pharmacology, College of Physicians and Surgeons, Columbia University, New York, NY, USA

    LouJin Song, Kumi Morikawa & Masayuki Yazawa

  5. Department of Systems Biology, Columbia University, New York, NY, USA

    Alessandro Vasciaveo & Edward C. Ruiz

  6. Department of Medicine, Columbia University, New York, NY, USA

    Gordana Vunjak-Novakovic

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Contributions

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.

Corresponding author

Correspondence to Gordana Vunjak-Novakovic.

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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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Key references using this protocol

Radisic, M. et al. Proc. Natl Acad. Sci. USA 101, 18129–18134 (2004): https://doi.org/10.1073/pnas.0407817101

Ronaldson-Bouchard, K. et al. Nature 556, 239–243 (2018): https://doi.org/10.1038/s41586-018-0016-3

Zhao, Y. et al. Cell 176, 913–927.E18 (2019): https://doi.org/10.1016/j.cell.2018.11.042

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 (https://creativecommons.org/licenses/by/3.0/legalcode).

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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Ronaldson-Bouchard, K., Yeager, K., Teles, D. et al. Engineering of human cardiac muscle electromechanically matured to an adult-like phenotype. Nat Protoc 14, 2781–2817 (2019). https://doi.org/10.1038/s41596-019-0189-8

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