Journal of Cardiovascular Translational Research

, Volume 6, Issue 6, pp 989–999 | Cite as

Electrical Stimulation Promotes Maturation of Cardiomyocytes Derived from Human Embryonic Stem Cells

  • Yau-Chi Chan
  • Sherwin Ting
  • Yee-Ki Lee
  • Kwong-Man Ng
  • Jiao Zhang
  • Zi Chen
  • Chung-Wah Siu
  • Steve K. W. Oh
  • Hung-Fat TseEmail author


While human embryonic stem cells (hESCs) can differentiate into functional cardiomyocytes, their immature phenotypes limit their therapeutic application for myocardial regeneration. We sought to determine whether electrical stimulation could enhance the differentiation and maturation of hESC-derived cardiomyocytes. Cardiac differentiation was induced in a HES3 hESC line via embryoid bodies formation treated with a p38 MAP kinase inhibitor. Detailed molecular and functional analysis were performed in those hESC-derived cardiomyocytes cultured for 4 days in the absence or presence of electrical field stimulation (6.6 V/cm, 1 Hz, and 2 ms pulses) using an eight-channel C-Pace stimulator (Ion-Optics Co., MA). Upon electrical stimulation, quantitative polymerase chain reaction demonstrated significant upregulation of cardiac-specific gene expression including HCN1, MLC2V, SCN5A, SERCA, Kv4.3, and GATA4; immunostaining and flow cytometry analysis revealed cellular elongation and an increased proportion of troponin-T positive cells (6.3 ± 1.2 % vs. 15.8 ± 2.1 %; n = 3, P < 0.01). Electrophysiological studies showed an increase in the proportion of ventricular-like hESC-derived cardiomyocytes (48 vs. 29 %, P < 0.05) with lengthening of their action potential duration at 90 % repolarization (387.7 ± 35.35; n = 11 vs. 291.8 ± 20.82; n = 10, P < 0.05) and 50 % repolarization (313.9 ± 27.94; n = 11 vs. 234.0 ± 16.10; n = 10, P < 0.05) after electrical stimulation. Nonetheless, the membrane diastolic potentials and action potential upstrokes of different hESC-derived cardiomyocyte phenotypes, and the overall beating rate remained unchanged (all P > 0.05). Fluorescence confocal imaging revealed that electrical stimulation significantly increased both spontaneous and caffeine-induced calcium flux in the hESC-derived cardiomyocytes (approximately 1.6-fold for both cases; P < 0.01). In conclusion, electrical field stimulation increased the expression of cardiac-specific genes and the yield of differentiation, promoted ventricular-like phenotypes, and improved the calcium handling of hESC-derived cardiomyocytes.


Human embryonic stem cells Cardiomyocytes Electrical stimulation 



This study was supported by Hong Kong Research Grant Council (HKU 8/CRF/09, HKU 8/CRF/10, HKU 780110 M to H.F.T), Theme-based Research Scheme (T12-705/11 to C.W.S and H.F.T), and CRCG Small Project Funding of University of Hong Kong (Y.C.C); Agency for Science Technology and Research (S.T. and S.K.O).

Disclosure Statement

The authors have nothing to declare.


  1. 1.
    Senyo, S. E., Steinhauser, M. L., Pizzimenti, C. L., Yang, V. K., Cai, L., Wang, M., et al. (2013). Mammalian heart renewal by pre-existing cardiomyocytes. Nature, 493(7432), 433–436.PubMedCrossRefGoogle Scholar
  2. 2.
    Siu, C. W., & Tse, H. F. (2012). Cardiac regeneration: messages from CADUCEUS. Lancet, 379(9819), 870–871.PubMedCrossRefGoogle Scholar
  3. 3.
    Liao, S. Y., Liu, Y., Siu, C. W., Zhang, Y., Lai, W. H., Au, K. W., et al. (2010). Proarrhythmic risk of embryonic stem cell-derived cardiomyocyte transplantation in infarcted myocardium. Heart Rhythm, 7(12), 1852–1859.PubMedCrossRefGoogle Scholar
  4. 4.
    Liao, S. Y., Tse, H. F., Chan, Y. C., Mei-Chu Yip, P., Zhang, Y., Liu, Y., et al. (2013). Overexpression of Kir2.1 channel in embryonic stem cell-derived cardiomyocytes attenuates posttransplantation proarrhythmic risk in myocardial infarction. Heart Rhythm, 10(2), 273–282.PubMedCrossRefGoogle Scholar
  5. 5.
    Ng, K. M., Lee, Y. K., Chan, Y. C., Lai, W. H., Fung, M. L., Li, R. A., et al. (2010). Exogenous expression of HIF-1 alpha promotes cardiac differentiation of embryonic stem cells. Journal of Molecular and Cellular Cardiology, 48(6), 1129–1137.PubMedCrossRefGoogle Scholar
  6. 6.
    Ng, K. M., Chan, Y. C., Lee, Y. K., Lai, W. H., Au, K. W., Fung, M. L., et al. (2011). Cobalt chloride pretreatment promotes cardiac differentiation of human embryonic stem cells under atmospheric oxygen level. Cellular Reprogramming, 13(6), 527–537.PubMedGoogle Scholar
  7. 7.
    Cameron, I. L., Hardman, W. E., Winters, W. D., Zimmerman, S., & Zimmerman, A. M. (1993). Environmental magnetic fields: influences on early embryogenesis. Journal of Cellular Biochemistry, 51(4), 417–425.PubMedGoogle Scholar
  8. 8.
    Robinson, K. R. (1985). The responses of cells to electrical fields: a review. The Journal of Cell Biology, 101(6), 2023–2027.PubMedCrossRefGoogle Scholar
  9. 9.
    Chen, M. Q., Xie, X., Hollis Whittington, R., Kovacs, G. T., Wu, J. C., & Giovangrandi, L. (2008). Cardiac differentiation of embryonic stem cells with point-source electrical stimulation. Conference Proceedings, IEEE Engineering in Medicine and Biology Society, 2008, 1729–1732.Google Scholar
  10. 10.
    Sauer, H., Rahimi, G., Hescheler, J., & Wartenberg, M. (1999). Effects of electrical fields on cardiomyocyte differentiation of embryonic stem cells. Journal of Cellular Biochemistry, 75(4), 710–723.PubMedCrossRefGoogle Scholar
  11. 11.
    Serena, E., Figallo, E., Tandon, N., Cannizzaro, C., Gerecht, S., Elvassore, N., et al. (2009). Electrical stimulation of human embryonic stem cells: cardiac differentiation and the generation of reactive oxygen species. Experimental Cell Research, 315(20), 3611–3619.PubMedCrossRefGoogle Scholar
  12. 12.
    Choo, A., Padmanabhan, J., Chin, A., Fong, W. J., & Oh, S. K. (2006). Immortalized feeders for the scale-up of human embryonic stem cells in feeder and feeder-free conditions. Journal of Biotechnology, 122(1), 130–141.PubMedCrossRefGoogle Scholar
  13. 13.
    Ting, S., Lecina, M., Reuveny, S., & Oh, S. (2012). Differentiation of human embryonic stem cells to cardiomyocytes on microcarrier cultures. Current Protocols in Stem Cell Biology, Chapter 1, Unit1D 7.Google Scholar
  14. 14.
    Lecina, M., Ting, S., Choo, A., Reuveny, S., & Oh, S. (2010). Scalable platform for human embryonic stem cell differentiation to cardiomyocytes in suspended microcarrier cultures. Tissue Engineering. Part C, Methods, 16(6), 1609–1619.PubMedCrossRefGoogle Scholar
  15. 15.
    Chan, Y. C., Siu, C. W., Lau, Y. M., Lau, C. P., Li, R. A., & Tse, H. F. (2009). Synergistic effects of inward rectifier (I) and pacemaker (I) currents on the induction of bioengineered cardiac automaticity. Journal of Cardiovascular Electrophysiology, 20(9), 1048–1054.PubMedCrossRefGoogle Scholar
  16. 16.
    Tandon, N., Cannizzaro, C., Chao, P. H., Maidhof, R., Marsano, A., Au, H. T., et al. (2009). Electrical stimulation systems for cardiac tissue engineering. Nature Protocols, 4(2), 155–173.PubMedCrossRefGoogle Scholar
  17. 17.
    Pu, W. T., Ishiwata, T., Juraszek, A. L., Ma, Q., & Izumo, S. (2004). GATA4 is a dosage-sensitive regulator of cardiac morphogenesis. Biology, 275(1), 235–244.Google Scholar
  18. 18.
    Zeisberg, E. M., Ma, Q., Juraszek, A. L., Moses, K., Schwartz, R. J., Izumo, S., et al. (2005). Morphogenesis of the right ventricle requires myocardial expression of Gata4. Journal of Clinical Investigation, 115(6), 1522–1531.PubMedCrossRefGoogle Scholar
  19. 19.
    Charpentier, F., Merot, J., Loussouarn, G., & Baro, I. (2010). Delayed rectifier K(+) currents and cardiac repolarization. Journal of Molecular and Cellular Cardiology, 48(1), 37–44.PubMedCrossRefGoogle Scholar
  20. 20.
    Ravens, U., & Wettwer, E. (1998). Electrophysiological aspects of changes in heart rate. Basic Research in Cardiology, 93(Suppl 1), 60–65.PubMedCrossRefGoogle Scholar
  21. 21.
    Qu, Z., & Chung, D. (2012). Mechanisms and determinants of ultralong action potential duration and slow rate-dependence in cardiac myocytes. PLoS One, 7(8), e43587.PubMedCrossRefGoogle Scholar
  22. 22.
    Genovese, J. A., Spadaccio, C., Chachques, E., Schussler, O., Carpentier, A., Chachques, J. C., et al. (2009). Cardiac pre-differentiation of human mesenchymal stem cells by electrostimulation. Frontiers in Bioscience, 14, 2996–3002.CrossRefGoogle Scholar
  23. 23.
    Genovese, J. A., Spadaccio, C., Langer, J., Habe, J., Jackson, J., & Patel, A. N. (2008). Electrostimulation induces cardiomyocyte predifferentiation of fibroblasts. Biochemical and Biophysical Research Communications, 370(3), 450–455.PubMedCrossRefGoogle Scholar
  24. 24.
    Gamry Instruments. (2005). Electrochemical impedance spectroscopy theory: a primer.Google Scholar
  25. 25.
    Setsukinai, K., Urano, Y., Kakinuma, K., Majima, H. J., & Nagano, T. (2003). Development of novel fluorescence probes that can reliably detect reactive oxygen species and distinguish specific species. Journal of Biological Chemistry, 278(5), 3170–3175.PubMedCrossRefGoogle Scholar
  26. 26.
    Sauer, H., & Wartenberg, M. (2005). Reactive oxygen species as signaling molecules in cardiovascular differentiation of embryonic stem cells and tumor-induced angiogenesis. Antioxidants and Redox Signaling, 7(11–12), 1423–1434.PubMedCrossRefGoogle Scholar
  27. 27.
    Puceat, M., Travo, P., Quinn, M. T., & Fort, P. (2003). A dual role of the GTPase Rac in cardiac differentiation of stem cells. Molecular Biology of the Cell, 14(7), 2781–2792.PubMedCrossRefGoogle Scholar
  28. 28.
    Puceat, M. (2005). Role of Rac-GTPase and reactive oxygen species in cardiac differentiation of stem cells. Antioxidants and Redox Signaling, 7(11–12), 1435–1439.PubMedCrossRefGoogle Scholar
  29. 29.
    Cannizzaro, C., Tandon, N., Figallo, E., Park, H., Gerecht, S., Radisic, M., et al. (2007). Practical aspects of cardiac tissue engineering with electrical stimulation. Methods in Molecular Medicine, 140, 291–307.PubMedCrossRefGoogle Scholar
  30. 30.
    Huang, J. Z. C., Zhang, W., & Zhou, X. (1997). Application of a platinum dual-disk microelectrode to measurement of the electron transfer number of dioxygen reduction. Journal of Electroanalytical Chemistry, 433, 33–39.CrossRefGoogle Scholar
  31. 31.
    Li, J., Stouffs, M., Serrander, L., Banfi, B., Bettiol, E., Charnay, Y., et al. (2006). The NADPH oxidase NOX4 drives cardiac differentiation: role in regulating cardiac transcription factors and MAP kinase activation. Molecular Biology of the Cell, 17(9), 3978–3988.PubMedCrossRefGoogle Scholar
  32. 32.
    Radisic, M., Park, H., Shing, H., Consi, T., Schoen, F. J., Langer, R., et al. (2004). Functional assembly of engineered myocardium by electrical stimulation of cardiac myocytes cultured on scaffolds. Proceedings of the National Academy of Sciences of the United States of America, 101(52), 18129–18134.PubMedCrossRefGoogle Scholar
  33. 33.
    Satin, J., Itzhaki, I., Rapoport, S., Schroder, E. A., Izu, L., Arbel, G., et al. (2008). Calcium handling in human embryonic stem cell-derived cardiomyocytes. Stem Cells, 26(8), 1961–1972.PubMedCrossRefGoogle Scholar
  34. 34.
    Burridge, P. W., Keller, G., Gold, J. D., & Wu, J. C. (2012). Production of de novo cardiomyocytes: human pluripotent stem cell differentiation and direct reprogramming. Cell Stem Cell, 10(1), 16–28.PubMedCrossRefGoogle Scholar
  35. 35.
    Loeb, G. E., Zamin, C. J., Schulman, J. H., & Troyk, P. R. (1991). Injectable microstimulator for functional electrical stimulation. Medical & Biological Engineering & Computing, 29(6), NS13–19.CrossRefGoogle Scholar
  36. 36.
    Tandon, N., Goh, B., Marsano, A., Chao, P. H., Montouri-Sorrentino, C., Gimble, J., et al. (2009). Alignment and elongation of human adipose-derived stem cells in response to direct-current electrical stimulation. Conference Proceedings, IEEE Engineering in Medicine and Biology Society, 2009, 6517–6521.Google Scholar
  37. 37.
    Levin, M. (2003). Motor protein control of ion flux is an early step in embryonic left-right asymmetry. Bioessays, 25(10), 1002–1010.PubMedCrossRefGoogle Scholar
  38. 38.
    Donaldson, N. D., & Donaldson, P. E. (1986). When are actively balanced biphasic (‘lilly’) stimulating pulses necessary in a neurological prosthesis? I. Historical background; Pt resting potential; Q studies. Medical & Biological Engineering & Computing, 24, 41–49.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Yau-Chi Chan
    • 1
  • Sherwin Ting
    • 2
  • Yee-Ki Lee
    • 1
  • Kwong-Man Ng
    • 1
  • Jiao Zhang
    • 1
  • Zi Chen
    • 1
  • Chung-Wah Siu
    • 1
    • 3
  • Steve K. W. Oh
    • 2
    • 4
  • Hung-Fat Tse
    • 1
    • 3
    Email author
  1. 1.Cardiology Division, Department of Medicine, Queen Mary HospitalThe University of Hong KongHong KongChina
  2. 2.Bioprocessing Technology InstituteA*STAR (Agency for Science, Technology and Research)SingaporeSingapore
  3. 3.Research Centre of Heart, Brain, Hormone and Healthy AgeingThe University of Hong KongHong KongChina
  4. 4.Stem Cell Group, Bioprocessing Technology InstituteAgency for Science, Technology and Research (A*STAR)SingaporeSingapore

Personalised recommendations