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Stem Cells for Myocardial Regeneration pp 215–237Cite as

In Vitro Electrophysiological Mapping of Stem Cells

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 660))

Abstract

The use of stem cells for cardiac regeneration is a revolutionary, emerging research area. For proper function as replacement tissue, stem cell-derived cardiomyocytes (SC-CMs) must electrically couple with the host cardiac tissue. Electrophysiological mapping techniques, including microelectrode array (MEA) and optical mapping, have been developed to study cardiomyocytes and cardiac cell monolayers, and these can be applied to study stem cells and SC-CMs. MEA recordings take extracellular measurements at numerous points across a small area of cell cultures and are used to assess electrical propagation during cell culture. Optical mapping uses fluorescent dyes to monitor electrophysiological changes in cells, most commonly transmembrane potential and intracellular calcium, and can be easily scaled to areas of different sizes. The materials and methods for MEA and optical mapping are presented here, together with detailed notes on their use, design, and fabrication. We also provide examples of voltage and calcium maps of mouse embryonic stem cell-derived cardiomyocytes (mESC-CMs), obtained in our laboratory using optical mapping techniques.

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References

  1. Reppel, M., Pillekamp, F., Lu, Z. J., Halbach, M., Brockmeier, K., Fleischmann, B. K., and Hescheler, J. (2004) Microelectrode arrays: a new tool to measure embryonic heart activity. J Electrocardiol 37 Suppl, 104–9.

    Article  PubMed  Google Scholar 

  2. Egert, U., and Meyer, T. (2005) Heart on a chip – extracellular multielectrode recordings from cardiac myocytes in vitro. in Practical Methods in Cardiovascular Research (Dhein, S., Mohr, F. W., and Delmar, M., Eds.) pp 432–453, Springer, Berlin.

    Chapter  Google Scholar 

  3. Binah, O., Dolnikov, K., Sadan, O., Shilkrut, M., Zeevi-Levin, N., Amit, M., Danon, A., and Itskovitz-Eldor, J. (2007) Functional and developmental properties of human embryonic stem cells-derived cardiomyocytes J Electrocardiol 40, S192–6.

    Article  PubMed  Google Scholar 

  4. Igelmund, P., Fleischmann, B. K., Fischer, I. R., Soest, J., Gryshchenko, O., Bohm-Pinger, M. M., Sauer, H., Liu, Q., and Hescheler, J. (1999) Action potential propagation failures in long-term recordings from embryonic stem cell-derived cardiomyocytes in tissue culture Pflugers Arch 437, 669–79.

    Article  PubMed  CAS  Google Scholar 

  5. Kehat, I., Kenyagin-Karsenti, D., Snir, M., Segev, H., Amit, M., Gepstein, A., Livne, E., Binah, O., Itskovitz-Eldor, J., and Gepstein, L. (2001) Human embryonic stem cells can differentiate into myocytes with structural and functional properties of cardiomyocytes J Clin Invest 108, 407–14.

    PubMed  CAS  Google Scholar 

  6. Banach, K., Halbach, M. D., Hu, P., Hescheler, J., and Egert, U. (2003) Development of electrical activity in cardiac myocyte aggregates derived from mouse embryonic stem cells Am J Physiol Heart Circ Physiol 284, H2114–23.

    PubMed  CAS  Google Scholar 

  7. Beeres, S. L., Atsma, D. E., van der Laarse, A., Pijnappels, D. A., van Tuyn, J., Fibbe, W. E., de Vries, A. A., Ypey, D. L., van der Wall, E. E., and Schalij, M. J. (2005) Human adult bone marrow mesenchymal stem cells repair experimental conduction block in rat cardiomyocyte cultures J Am Coll Cardiol 46, 1943–52.

    Article  PubMed  Google Scholar 

  8. Kehat, I., Khimovich, L., Caspi, O., Gepstein, A., Shofti, R., Arbel, G., Huber, I., Satin, J., Itskovitz-Eldor, J., and Gepstein, L. (2004) Electromechanical integration of cardiomyocytes derived from human embryonic stem cells Nat Biotechnol 22, 1282–9.

    Article  PubMed  CAS  Google Scholar 

  9. Caspi, O., Itzhaki, I., Arbel, G., Kehat, I., Gepstien, A., Huber, I., Satin, J., and Gepstein, L. (2009) In vitro electrophysiological drug testing using human embryonic stem cell derived cardiomyocytes. Stem Cells Dev 18, 161-72.

    Article  PubMed  CAS  Google Scholar 

  10. Meyer, T., Boven, K. H., Gunther, E., and Fejtl, M. (2004) Micro-electrode arrays in cardiac safety pharmacology: a novel tool to study QT interval prolongation Drug Saf 27, 763–72.

    Article  PubMed  CAS  Google Scholar 

  11. Windisch, H., Ahammer, H., Schaffer, P., Muller, W., and Platzer, D. (1995) Optical multisite monitoring of cell excitation phenomena in isolated cardiomyocytes Pflugers Arch 430, 508–18.

    Article  PubMed  CAS  Google Scholar 

  12. Rohr, S., and Salzberg, B. M. (1994) Multiple site optical recording of transmembrane voltage (MSORTV) in patterned growth heart cell cultures: assessing electrical behavior, with microsecond resolution, on a cellular and subcellular scale Biophys J 67, 1301–15.

    Article  PubMed  CAS  Google Scholar 

  13. Fast, V. G., and Kleber, A. G. (1993) Microscopic conduction in cultured strands of neonatal rat heart cells measured with voltage-sensitive dyes Circ Res 73, 914–25.

    Article  PubMed  CAS  Google Scholar 

  14. Entcheva, E., Lu, S. N., Troppman, R. H., Sharma, V., and Tung, L. (2000) Contact fluorescence imaging of reentry in monolayers of cultured neonatal rat ventricular myocytes J Cardiovasc Electrophysiol 11, 665–76.

    Article  PubMed  CAS  Google Scholar 

  15. Efimov, I. R., Nikolski, V. P., and Salama, G. (2004) Optical imaging of the heart Circ Res 95, 21–33.

    Article  PubMed  CAS  Google Scholar 

  16. Salama, G., and Morad, M. (1976) Merocyanine 540 as an optical probe of transmembrane electrical activity in the heart. Science 191, 485–7.

    Article  PubMed  CAS  Google Scholar 

  17. Gray, R. A., Jalife, J., Panfilov, A., Baxter, W. T., Cabo, C., Davidenko, J. M., and Pertsov, A. M. (1995) Nonstationary vortexlike reentrant activity as a mechanism of polymorphic ventricular tachycardia in the isolated rabbit heart Circulation 91, 2454–69.

    Article  PubMed  CAS  Google Scholar 

  18. Dillon, S. M. (1991) Optical recordings in the rabbit heart show that defibrillation strength shocks prolong the duration of depolarization and the refractory period Circ Res 69, 842–56.

    Article  PubMed  CAS  Google Scholar 

  19. Morad, M., and Salama, G. (1979) Optical probes of membrane potential in heart muscle J Physiol 292, 267–95.

    PubMed  CAS  Google Scholar 

  20. Sato, D., Shiferaw, Y., Garfinkel, A., Weiss, J. N., Qu, Z., and Karma, A. (2006) Spatially discordant alternans in cardiac tissue: role of calcium cycling Circ Res 99, 520–7.

    Article  PubMed  CAS  Google Scholar 

  21. Hwang, S. M., Yea, K. H., and Lee, K. J. (2004) Regular and alternant spiral waves of contractile motion on rat ventricle cell cultures Phys Rev Lett 92, 198103.

    Article  PubMed  Google Scholar 

  22. Tung, L., and Zhang, Y. (2006) Optical ­imaging of arrhythmias in tissue culture J Electrocardiol 39, S2–6.

    Article  PubMed  Google Scholar 

  23. Entcheva, E., and Bien, H. (2006) Macroscopic optical mapping of excitation in cardiac cell networks with ultra-high spatiotemporal resolution Prog Biophys Mol Biol 92, 232–57.

    Article  PubMed  Google Scholar 

  24. Fast, V. G. (2005) Recording action potentials using voltage-sensitive dyes. in Practical Methods in Cardiovascular Research (Dhein, S., Mohr, F. W., and Delmar, M., Eds.) pp 233–255, Springer, Berlin.

    Chapter  Google Scholar 

  25. Lagostena, L., Avitabile, D., De Falco, E., Orlandi, A., Grassi, F., Iachininoto, M. G., Ragone, G., Fucile, S., Pompilio, G., Eusebi, F., Pesce, M., and Capogrossi, M. C. (2005) Electrophysiological properties of mouse bone marrow c-kit+ cells co-cultured onto neonatal cardiac myocytes Cardiovasc Res 66, 482–92.

    Article  PubMed  CAS  Google Scholar 

  26. Orlandi, A., Pagani, F., Avitabile, D., Bonanno, G., Scambia, G., Vigna, E., Grassi, F., Eusebi, F., Fucile, S., Pesce, M., and Capogrossi, M. C. (2008) Functional properties of cells obtained from human cord blood CD34+ stem cells and mouse cardiac myocytes in coculture Am J Physiol Heart Circ Physiol 294, H1541–9.

    Article  PubMed  CAS  Google Scholar 

  27. Dolnikov, K., Shilkrut, M., Zeevi-Levin, N., Gerecht-Nir, S., Amit, M., Danon, A., Itskovitz-Eldor, J., and Binah, O. (2006) Functional properties of human embryonic stem cell-derived cardiomyocytes: intracellular Ca2+ handling and the role of sarcoplasmic reticulum in the contraction Stem Cells 24, 236–45.

    Article  PubMed  CAS  Google Scholar 

  28. Kapur, N., Mignery, G. A., and Banach, K. (2007) Cell cycle-dependent calcium oscillations in mouse embryonic stem cells Am J Physiol Cell Physiol 292, C1510–8.

    Article  PubMed  CAS  Google Scholar 

  29. Sauer, H., Hofmann, C., Wartenberg, M., Wobus, A. M., and Hescheler, J. (1998) Spontaneous calcium oscillations in embryonic stem cell-derived primitive endodermal cells Exp Cell Res 238, 13–22.

    Article  PubMed  CAS  Google Scholar 

  30. Egert, U., Knott, T., Schwarz, C., Nawrot, M., Brandt, A., Rotter, S., and Diesmann, M. (2002) MEA-Tools: an open source toolbox for the analysis of multi-electrode data with MATLAB J Neurosci Methods 117, 33–42.

    Article  PubMed  CAS  Google Scholar 

  31. Multi Channel Systems (2006) MEA Application Note: Human Embryonic Stem Cell Derived Cardiac Myocytes (hESC-CM). Multi Channel Systems MCS GmbH.

    Google Scholar 

  32. Fast, V. G. (2005) Simultaneous optical imaging of membrane potential and intracellular calcium J Electrocardiol 38, 107–12.

    Article  PubMed  Google Scholar 

  33. Tritthart, H. A. (2005) Optical techniques for the recording of action potentials. in Practical Methods in Cardiovascular Research (Dhein, S., Mohr, F. W., and Delmar, M., Eds.) pp 215–232, Springer, Berlin.

    Chapter  Google Scholar 

  34. Ratzlaff, E. H., and Grinvald, A. (1991) A tandem-lens epifluorescence macroscope: hundred-fold brightness advantage for wide-field imaging J Neurosci Methods 36, 127–37.

    Article  PubMed  CAS  Google Scholar 

  35. Montana, V., Farkas, D. L., and Loew, L. M. (1989) Dual-wavelength ratiometric fluorescence measurements of membrane potential Biochemistry 28, 4536–9.

    Article  PubMed  CAS  Google Scholar 

  36. Muller, W., Windisch, H., and Tritthart, H. A. (1986) Fluorescent styryl dyes applied as fast optical probes of cardiac action potential Eur Biophys J 14, 103–11.

    Article  PubMed  CAS  Google Scholar 

  37. Knisley, S. B., Justice, R. K., Kong, W., and Johnson, P. L. (2000) Ratiometry of transmembrane voltage-sensitive fluorescent dye emission in hearts Am J Physiol Heart Circ Physiol 279, H1421–33.

    PubMed  CAS  Google Scholar 

  38. Beach, J. M., McGahren, E. D., Xia, J., and Duling, B. R. (1996) Ratiometric measurement of endothelial depolarization in arterioles with a potential-sensitive dye Am J Physiol 270, H2216–27.

    PubMed  CAS  Google Scholar 

  39. Takahashi, A., Camacho, P., Lechleiter, J. D., and Herman, B. (1999) Measurement of intracellular calcium Physiol Rev 79, 1089–125.

    PubMed  CAS  Google Scholar 

  40. Katra, R. P., Pruvot, E., and Laurita, K. R. (2004) Intracellular calcium handling heterogeneities in intact guinea pig hearts Am J Physiol Heart Circ Physiol 286, H648–56.

    Article  PubMed  CAS  Google Scholar 

  41. Field, M. L., Azzawi, A., Styles, P., Henderson, C., Seymour, A. M., and Radda, G. K. (1994) Intracellular Ca2+ transients in isolated ­perfused rat heart: measurement using the fluorescent indicator Fura-2/AM. Cell Calcium 16, 87–100.

    Article  PubMed  CAS  Google Scholar 

  42. Multi Channel Systems (2005) Microelectrode Array (MEA) User Manual. Multi Channel Systems MCS GmbH.

    Google Scholar 

  43. Potter, S. M., and DeMarse, T. B. (2001) A new approach to neural cell culture for long-term studies J Neurosci Methods 110, 17–24.

    Article  PubMed  CAS  Google Scholar 

  44. Yamamoto, M., Honjo, H., Niwa, R., and Kodama, I. (1998) Low-frequency extracellular potentials recorded from the sinoatrial node Cardiovasc Res 39, 360–72.

    Article  PubMed  CAS  Google Scholar 

  45. Fedorov, V. V., Lozinsky, I. T., Sosunov, E. A., Anyukhovsky, E. P., Rosen, M. R., Balke, C. W., and Efimov, I. R. (2007) Application of blebbistatin as an excitation-contraction uncoupler for electrophysiologic study of rat and rabbit hearts Heart Rhythm 4, 619–26.

    Article  PubMed  Google Scholar 

  46. Wu, J., Biermann, M., Rubart, M., and Zipes, D. P. (1998) Cytochalasin D as excitation-contraction uncoupler for optically mapping action potentials in wedges of ventricular myocardium J Cardiovasc Electrophysiol 9, 1336–47.

    Article  PubMed  CAS  Google Scholar 

  47. Cheng, Y., Mowrey, K., Efimov, I. R., Van Wagoner, D. R., Tchou, P. J., and Mazgalev, T. N. (1997) Effects of 2,3-butanedione monoxime on atrial-atrioventricular nodal conduction in isolated rabbit heart. J Cardiovasc Electrophysiol 8, 790–802.

    Article  PubMed  CAS  Google Scholar 

  48. Bursac, N., Loo, Y., Leong, K., and Tung, L. (2007) Novel anisotropic engineered cardiac tissues: studies of electrical propagation Biochem Biophys Res Commun 361, 847–53.

    Article  PubMed  CAS  Google Scholar 

  49. Schaffer, P., Ahammer, H., Muller, W., Koidl, B., and Windisch, H. (1994) Di-4-ANEPPS causes photodynamic damage to isolated cardiomyocytes Pflugers Arch 426, 548–51.

    Article  PubMed  CAS  Google Scholar 

  50. Windisch, H., Muller, W., and Tritthart, H. A. (1985) Fluorescence monitoring of rapid changes in membrane potential in heart muscle Biophys J 48, 877–84.

    Article  PubMed  CAS  Google Scholar 

  51. Boyett, M. R., and Jewell, B. R. (1980) Analysis of the effects of changes in rate and rhythm upon electrical activity in the heart Prog Biophys Mol Biol 36, 1–52.

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

This work was supported by NIH grants R01 HL066239 (L.T.) and T32-HL07581 (A. Shoukas), and grants from the Joint Technion-Hopkins Program for the Biomedical Sciences and Biomedical Engineering (L.T. and L. Gepstein) and from the Maryland Stem Cell Research Fund (L.T.). We thank Dr. Lior Gepstein for training E.L. in his lab on the use of MEAs.

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Authors and Affiliations

  1. Department of Biomedical Engineering, The Johns Hopkins University, Baltimore, MD, USA

    Seth Weinberg B.S & Leslie Tung PhD

  2. Department of Chemical Engineering, Auburn University, Auburn, AL, USA

    Elizabeth A. Lipke PhD

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  1. Seth Weinberg B.S
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  2. Elizabeth A. Lipke PhD
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  3. Leslie Tung PhD
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Correspondence to Leslie Tung PhD .

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Editors and Affiliations

  1. , Division of Cardiology, University of California, San Francisco, 500 Parnassus Avenue, San Francisco, 94143-1354, California, USA

    Randall J. Lee

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Weinberg, S., Lipke, E.A., Tung, L. (2010). In Vitro Electrophysiological Mapping of Stem Cells. In: Lee, R. (eds) Stem Cells for Myocardial Regeneration. Methods in Molecular Biology, vol 660. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-705-1_14

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  • DOI: https://doi.org/10.1007/978-1-60761-705-1_14

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  • Publisher Name: Humana Press, Totowa, NJ

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