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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References
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).
Yang, L. et al. Human cardiovascular progenitor cells develop from a KDR+ embryonic-stem-cell-derived population. Nature 453, 524–528 (2008).
Zhang, J. et al. Functional cardiomyocytes derived from human induced pluripotent stem cells. Circ. Res. 104, e30–e41 (2009).
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).
Carvajal-Vergara, X. et al. Patient-specific induced pluripotent stem-cell-derived models of LEOPARD syndrome. Nature 465, 808–812 (2010).
Laflamme, M.A. & Murry, C.E. Heart regeneration. Nature 473, 326–335 (2011).
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).
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).
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).
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).
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).
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).
Satin, J. et al. Calcium handling in human embryonic stem cell-derived cardiomyocytes. Stem Cells 26, 1961–1972 (2008).
Tulloch, N.L. et al. Growth of engineered human myocardium with mechanical loading and vascular coculture. Circ. Res. 109, 47–59 (2011).
Caspi, O. et al. Tissue engineering of vascularized cardiac muscle from human embryonic stem cells. Circ. Res. 100, 263–272 (2007).
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).
Frank, D. et al. Gene expression pattern in biomechanically stretched cardiomyocytes: evidence for a stretch-specific gene program. Hypertension 51, 309–318 (2008).
Kuwahara, K. et al. NRSF regulates the fetal cardiac gene program and maintains normal cardiac structure and function. EMBO J. 22, 6310–6321 (2003).
Nuccitelli, R. Endogenous ionic currents and DC electric fields in multicellular animal tissues. Bioelectromagnetics 1 (suppl.), 147–157 (1992).
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).
Zhao, M., Forrester, J. & McCaig, C. A small, physiological electric field orients cell division. Proc. Natl. Acad. Sci. USA 96, 4942–4946 (1999).
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).
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).
Borg, T.K. et al. Specialization at the Z line of cardiac myocytes. Cardiovasc. Res. 46, 277–285 (2000).
Bird, S.D. et al. The human adult cardiomyocyte phenotype. Cardiovasc. Res. 58, 423–434 (2003).
Frey, N. & Olson, E.N. Cardiac hypertrophy: the good, the bad, and the ugly. Annu. Rev. Physiol. 65, 45–79 (2003).
Wang, J., Huang, Y. & Ning, Q. Review on regulation of inwardly rectifying potassium channels. Crit. Rev. Eukaryot. Gene Expr. 21, 303–311 (2011).
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).
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).
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).
De Weer, P., Gadsby, D.C. & Rakowski, R.F. Voltage dependence of the Na-K pump. Annu. Rev. Physiol. 50, 225–241 (1988).
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).
Arduini, D. Fetal Cardiac Function (Parthenon Publishing Group, 1995).
Schaaf, S. et al. Human engineered heart tissue as a versatile tool in basic research and preclinical toxicology. PLoS ONE 6, e26397 (2011).
Chattergoon, N.N. et al. Thyroid hormone drives fetal cardiomyocyte maturation. FASEB J. 26, 397–408 (2012).
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).
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).
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).
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).
Blazeski, A. et al. Electrophysiological and contractile function of cardiomyocytes derived from human embryonic stem cells. Prog. Biophys. Mol. Biol. 110, 178–195 (2012).
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).
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).
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).
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).
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.
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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.
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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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DOI: https://doi.org/10.1038/nmeth.2524
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