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

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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Figure 1: Generation of human cardiac biowires.
Figure 2: Cultured biowires in combination with electrical stimulation promoted physiological cell hypertrophy and improved cardiomyocyte phenotype.
Figure 3: Functional assessment of engineered biowires.
Figure 4: Electrical stimulation promoted improvement in Ca2+ handling properties.
Figure 5: Electrophysiological properties in single cardiomyocytes isolated from biowires or embryoid bodies and recorded with patch clamp.

References

  1. Kehat, I. et al. Human embryonic stem cells can differentiate into myocytes with structural and functional properties of cardiomyocytes. J. Clin. Invest. 108, 407–414 (2001).

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  3. Zhang, J. et al. Functional cardiomyocytes derived from human induced pluripotent stem cells. Circ. Res. 104, e30–e41 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. 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).

    CAS  Article  Google Scholar 

  5. Carvajal-Vergara, X. et al. Patient-specific induced pluripotent stem-cell-derived models of LEOPARD syndrome. Nature 465, 808–812 (2010).

    CAS  Article  Google Scholar 

  6. Laflamme, M.A. & Murry, C.E. Heart regeneration. Nature 473, 326–335 (2011).

    CAS  Article  Google Scholar 

  7. Snir, M. et al. Assessment of the ultrastructural and proliferative properties of human embryonic stem cell-derived cardiomyocytes. Am. J. Physiol. Heart Circ. Physiol. 285, H2355–H2363 (2003).

    CAS  Article  Google Scholar 

  8. McDevitt, T.C., Laflamme, M.A. & Murry, C.E. Proliferation of cardiomyocytes derived from human embryonic stem cells is mediated via the IGF/PI 3-kinase/Akt signaling pathway. J. Mol. Cell Cardiol. 39, 865–873 (2005).

    CAS  Article  Google Scholar 

  9. 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).

    CAS  Article  Google Scholar 

  10. Dolnikov, K. et al. Functional properties of human embryonic stem cell-derived cardiomyocytes: intracellular Ca2+ handling and the role of sarcoplasmic reticulum in the contraction. Stem Cells 24, 236–245 (2006).

    CAS  Article  Google Scholar 

  11. Doss, M.X. et al. Maximum diastolic potential of human induced pluripotent stem cell-derived cardiomyocytes depends critically on I(Kr). PLoS ONE 7, e40288 (2012).

    CAS  Article  Google Scholar 

  12. Liu, J., Fu, J.D., Siu, C.W. & Li, R.A. Functional sarcoplasmic reticulum for calcium handling of human embryonic stem cell-derived cardiomyocytes: insights for driven maturation. Stem Cells 25, 3038–3044 (2007).

    CAS  Article  Google Scholar 

  13. Satin, J. et al. Calcium handling in human embryonic stem cell-derived cardiomyocytes. Stem Cells 26, 1961–1972 (2008).

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  15. Caspi, O. et al. Tissue engineering of vascularized cardiac muscle from human embryonic stem cells. Circ. Res. 100, 263–272 (2007).

    CAS  Article  Google Scholar 

  16. Chien, K.R., Knowlton, K.U., Zhu, H. & Chien, S. Regulation of cardiac gene expression during myocardial growth and hypertrophy: molecular studies of an adaptive physiologic response. FASEB J. 5, 3037–3046 (1991).

    CAS  Article  Google Scholar 

  17. Frank, D. et al. Gene expression pattern in biomechanically stretched cardiomyocytes: evidence for a stretch-specific gene program. Hypertension 51, 309–318 (2008).

    CAS  Article  Google Scholar 

  18. Kuwahara, K. et al. NRSF regulates the fetal cardiac gene program and maintains normal cardiac structure and function. EMBO J. 22, 6310–6321 (2003).

    CAS  Article  Google Scholar 

  19. Nuccitelli, R. Endogenous ionic currents and DC electric fields in multicellular animal tissues. Bioelectromagnetics 1 (suppl.), 147–157 (1992).

    Article  Google Scholar 

  20. Henderson, D.J. & Chaudhry, B. Getting to the heart of planar cell polarity signaling. Birth Defects Res. A Clin. Mol. Teratol. 91, 460–467 (2011).

    CAS  Article  Google Scholar 

  21. Zhao, M., Forrester, J. & McCaig, C. A small, physiological electric field orients cell division. Proc. Natl. Acad. Sci. USA 96, 4942–4946 (1999).

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  23. Berger, H.J. et al. Continual electric field stimulation preserves contractile function of adult ventricular myocytes in primary culture. Am. J. Physiol. 266, H341–H349 (1994).

    CAS  PubMed  Google Scholar 

  24. Borg, T.K. et al. Specialization at the Z line of cardiac myocytes. Cardiovasc. Res. 46, 277–285 (2000).

    CAS  Article  Google Scholar 

  25. Bird, S.D. et al. The human adult cardiomyocyte phenotype. Cardiovasc. Res. 58, 423–434 (2003).

    CAS  Article  Google Scholar 

  26. Frey, N. & Olson, E.N. Cardiac hypertrophy: the good, the bad, and the ugly. Annu. Rev. Physiol. 65, 45–79 (2003).

    CAS  Article  Google Scholar 

  27. Wang, J., Huang, Y. & Ning, Q. Review on regulation of inwardly rectifying potassium channels. Crit. Rev. Eukaryot. Gene Expr. 21, 303–311 (2011).

    CAS  Article  Google Scholar 

  28. Dubois, N.C. et al. SIRPA is a specific cell-surface marker for isolating cardiomyocytes derived from human pluripotent stem cells. Nat. Biotechnol. 29, 1011–1018 (2011).

    CAS  Article  Google Scholar 

  29. Lieu, D.K. et al. Absence of transverse tubules contributes to non-uniform Ca2+ wavefronts in mouse and human embryonic stem cell-derived cardiomyocytes. Stem Cells Dev. 18, 1493–1500 (2009).

    CAS  Article  Google Scholar 

  30. Baharvand, H., Azarnia, M., Parivar, K. & Ashtiani, S.K. The effect of extracellular matrix on embryonic stem cell-derived cardiomyocytes. J. Mol. Cell Cardiol. 38, 495–503 (2005).

    CAS  Article  Google Scholar 

  31. De Weer, P., Gadsby, D.C. & Rakowski, R.F. Voltage dependence of the Na-K pump. Annu. Rev. Physiol. 50, 225–241 (1988).

    CAS  Article  Google Scholar 

  32. Sakai, R., Hagiwara, N., Matsuda, N., Kassanuki, H. & Hosoda, S. Sodium–potassium pump current in rabbit sino-atrial node cells. J. Physiol. (Lond.) 490, 51–62 (1996).

    CAS  Article  Google Scholar 

  33. Arduini, D. Fetal Cardiac Function (Parthenon Publishing Group, 1995).

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

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  36. McMullen, J.R. et al. The insulin-like growth factor 1 receptor induces physiological heart growth via the phosphoinositide 3-kinase(p110alpha) pathway. J. Biol. Chem. 279, 4782–4793 (2004).

    CAS  Article  Google Scholar 

  37. Seif-Naraghi, S.B. et al. Safety and efficacy of an injectable extracellular matrix hydrogel for treating myocardial infarction. Sci. Transl. Med. 5, 173ra125 (2013).

    Article  Google Scholar 

  38. Rodriguez, A.G., Han, S.J., Regnier, M. & Sniadecki, N.J. Substrate stiffness increases twitch power of neonatal cardiomyocytes in correlation with changes in myofibril structure and intracellular calcium. Biophys. J. 101, 2455–2464 (2011).

    CAS  Article  Google Scholar 

  39. Hazeltine, L.B. et al. Effects of substrate mechanics on contractility of cardiomyocytes generated from human pluripotent stem cells. Int. J. Cell Biol. 2012, 508294 (2012).

    Article  Google Scholar 

  40. Blazeski, A. et al. Electrophysiological and contractile function of cardiomyocytes derived from human embryonic stem cells. Prog. Biophys. Mol. Biol. 110, 178–195 (2012).

    CAS  Article  Google Scholar 

  41. Lundy, S.D., Zhu, W.Z., Regnier, M. & Laflamme, M. Structural and functional maturation of cardiomyocytes derived from human pluripotent stem cells. Stem Cells Dev. Advance online publication 6 March 2013 (10.1089/scd.2012.0490).

  42. Nanthakumar, K. et al. Optical mapping of Langendorff-perfused human hearts: establishing a model for the study of ventricular fibrillation in humans. Am. J. Physiol. Heart Circ. Physiol. 293, H875–H880 (2007).

    CAS  Article  Google Scholar 

  43. Witkowski, F.X., Clark, R.B., Larsen, T.S., Melnikov, A. & Giles, W.R. Voltage-sensitive dye recordings of electrophysiological activation in a Langendorff-perfused mouse heart. Can. J. Cardiol. 13, 1077–1082 (1997).

    CAS  PubMed  Google Scholar 

  44. Snyders, D.J. & Chaudhary, A. High affinity open channel block by dofetilide of HERG expressed in a human cell line. Mol. Pharmacol. 49, 949–955 (1996).

    CAS  PubMed  Google Scholar 

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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

Authors and Affiliations

Authors

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.

Corresponding author

Correspondence to Milica Radisic.

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Competing interests

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

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–14, Supplementary Tables 1–3 and Supplementary Results (PDF 2639 kb)

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. (MOV 64 kb)

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. (MOV 1940 kb)

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. (MOV 1504 kb)

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. (MOV 1539 kb)

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. (MOV 1429 kb)

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. (MOV 2358 kb)

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. (MOV 1674 kb)

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. (MOV 2202 kb)

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. (MOV 3407 kb)

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Nunes, S., Miklas, J., Liu, J. et al. Biowire: a platform for maturation of human pluripotent stem cell–derived cardiomyocytes. Nat Methods 10, 781–787 (2013). https://doi.org/10.1038/nmeth.2524

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