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Biowire: a platform for maturation of human pluripotent stem cell–derived cardiomyocytes

Nature Methods volume 10, pages 781787 (2013) | Download Citation

Abstract

Directed differentiation protocols enable derivation of cardiomyocytes from human pluripotent stem cells (hPSCs) and permit engineering of human myocardium in vitro. However, hPSC-derived cardiomyocytes are reflective of very early human development, limiting their utility in the generation of in vitro models of mature myocardium. Here we describe a platform that combines three-dimensional cell cultivation with electrical stimulation to mature hPSC-derived cardiac tissues. We used quantitative structural, molecular and electrophysiological analyses to explain the responses of immature human myocardium to electrical stimulation and pacing. We demonstrated that the engineered platform allows for the generation of three-dimensional, aligned cardiac tissues (biowires) with frequent striations. Biowires submitted to electrical stimulation had markedly increased myofibril ultrastructural organization, elevated conduction velocity and improved both electrophysiological and Ca2+ handling properties compared to nonstimulated controls. These changes were in agreement with cardiomyocyte maturation and were dependent on the stimulation rate.

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Acknowledgements

We thank P. Lai, C. Laschinger, N. Dubois and B. Calvieri for technical assistance, C.C. Chang and L. Fu for assistance with biowire setup figure preparation. Funded by grants from Ontario Research Fund–Global Leadership Round 2 (ORF-GL2), National Sciences and Engineering Research Council of Canada (NSERC) Strategic Grant (STPGP 381002-09), Canadian Institutes of Health Research (CIHR) Operating Grant (MOP-126027 and MOP-62954), NSERC-CIHR Collaborative Health Research Grant (CHRPJ 385981-10), NSERC Discovery Grant (RGPIN 326982-10), and NSERC Discovery Accelerator Supplement (RGPAS 396125-10) and National Institutes of Health grant 2R01 HL076485.

Author information

Author notes

    • Jason W Miklas
    •  & Jie Liu

    These authors contributed equally to this work.

Affiliations

  1. Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada.

    • Sara S Nunes
    • , Jason W Miklas
    • , Yun Xiao
    • , Boyang Zhang
    • , Anne Hsieh
    • , Nimalan Thavandiran
    •  & Milica Radisic
  2. Toronto General Research Institute, University Health Network, Toronto, Ontario, Canada.

    • Sara S Nunes
  3. Department of Physiology and Medicine, University of Toronto, Toronto, Ontario, Canada.

    • Jie Liu
    • , Roozbeh Aschar-Sobbi
    •  & Peter H Backx
  4. Cardiology Division, Hospital for Sick Children, Toronto, Ontario, Canada.

    • Jiahua Jiang
    •  & Gil J Gross
  5. The Toby Hull Cardiac Fibrillation Management Laboratory, Toronto General Hospital, Toronto, Ontario, Canada.

    • Stéphane Massé
    •  & Kumaraswamy Nanthakumar
  6. McEwen Centre for Regenerative Medicine, University Health Network, Toronto, Ontario, Canada.

    • Mark Gagliardi
    •  & Gordon Keller
  7. Department of Pathology, University of Washington, Seattle, Washington, USA.

    • Michael A Laflamme
  8. Physiology and Experimental Medicine Program, Hospital for Sick Children Research Institute, Toronto, Ontario, Canada.

    • Gil J Gross
  9. Department of Pediatrics, University of Toronto, Toronto, Ontario, Canada.

    • Gil J Gross
  10. The Heart and Stroke/Richard Lewar Centre of Excellence, Toronto, Ontario, Canada.

    • Gil J Gross
    • , Peter H Backx
    •  & Milica Radisic
  11. Division of Cardiology, University Health Network, Toronto, Ontario, Canada.

    • Peter H Backx
  12. Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario, Canada.

    • Milica Radisic

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Contributions

S.S.N. developed biowire concept, designed and performed experiments, analyzed data and prepared the manuscript. J.W.M. performed experiments and analyzed data. J.L., R.A.-S. and P.H.B. performed patch clamping and microelectrode recordings. Y.X. designed and validated initial device. B.Z. designed and fabricated masters for device fabrication. J.J. and G.J.G. performed calcium transient measurement and analysis. S.M. and K.N. performed optical mapping measurements and analysis. M.G. and G.K. differentiated hESC-derived cardiomyocytes. A.H. designed primers. N.T. developed initial collagen gel mixture. M.A.L. provided training on hiPSC differentiation and cells. P.H.B. contributed to writing of the manuscript. M.R. envisioned the biowire concept and electrical stimulation protocol, supervised the work and wrote the manuscript.

Competing interests

M.A.L. is a cofounder and scientific advisor for BEAT BioTherapeutics Corp.

Corresponding author

Correspondence to Milica Radisic.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–14, Supplementary Tables 1–3 and Supplementary Results

Videos

  1. 1.

    Supplementary Video 1

    Human cardiac biowires started to beat synchronously and spontaneously between 2 d and 3 d after seeding, demonstrating that the biowire setup allows for electromechanical coupling of cells in the collagen type I matrix. Video shows biowire after 1 week of preculture, for Hes2 cell line hESC-derived cardiomyocytes.

  2. 2.

    Supplementary Video 2

    Impulse propagation of high frequency–stimulated (6 Hz) human cardiac biowires upon point stimulation at 3 Hz frequency. Hes2 cell line hESC-derived cardiomyocytes.

  3. 3.

    Supplementary Video 3

    Impulse propagation of high frequency–stimulated (6 Hz) human cardiac biowires upon point stimulation at 4 Hz frequency. Hes2 cell line hESC-derived cardiomyocytes.

  4. 4.

    Supplementary Video 4

    Impulse propagation of high frequency–stimulated (6 Hz) human cardiac biowires upon point stimulation at 5 Hz frequency. Hes2 cell line hESC-derived cardiomyocytes.

  5. 5.

    Supplementary Video 5

    Impulse propagation of high frequency–stimulated (6 Hz) human cardiac biowires upon point stimulation at 6 Hz frequency. Hes2 cell line hESC-derived cardiomyocytes.

  6. 6.

    Supplementary Video 6

    Impulse propagation of high frequency–stimulated (6 Hz) human cardiac biowires upon field stimulation at 3 Hz frequency. Hes2 cell line hESC-derived cardiomyocytes.

  7. 7.

    Supplementary Video 7

    Impulse propagation of high frequency–stimulated (6 Hz) human cardiac biowires upon field stimulation at 4 Hz frequency. Hes2 cell line hESC-derived cardiomyocytes.

  8. 8.

    Supplementary Video 8

    Impulse propagation of high frequency–stimulated (6 Hz) human cardiac biowires upon field stimulation at 5 Hz frequency. Hes2 cell line hESC-derived cardiomyocytes.

  9. 9.

    Supplementary Video 9

    Impulse propagation of high frequency–stimulated (6 Hz) human cardiac biowires upon field stimulation at 6 Hz frequency. Hes2 cell line hESC-derived cardiomyocytes.

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DOI

https://doi.org/10.1038/nmeth.2524

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