Summary and Discussion
Heart development is a complex process with temporal and spatial gradients of myriad molecules. Since the critical stages of cardiac specification have been recapitulated through the sequential addition of cytokines, PSCs efficiently give rise to CMs, PSC-CMs. However, as this is the very first phase to be fully functional matured CMs, the PSC-CMs simply display immature phenotypes and limit their use for clinical translations. Previous study has shown that ECMs could enhance CM maturation48. However, the maturation degrees of the PSC-CMs with those ECMs remain unknown and no appropriate assessment method to determine the effects of ECMs to CM maturation. Here, I successfully developed qualitative and quantitative methods to assess CM maturation. For qualitative method, I generated a novel Myom2-RFP reporter line, using CRISPR/Cas9 system. After cardiac differentiation, RFP+ cells were observed and increased throughout the experimental process, which is a corresponding increase in the maturity of PSC-CMs. RFP+ cells were improved their morphology and physiology towards adult-like mature CMs. Morphologically, RFP+ cells had long sarcomere length, increased cell area and aspect ratio, and showed higher proportion of binuclear cells compared to gelatin. For physiology, RFP+ cells had improved calcium handling property by which they had higher peak amplitude and reduced time to decay. In addition to calcium transients, these cells also displayed contraction steadily and sarcomere shortening ~6.8%. For quantitative method, transcriptome data from PSC-CMs were compared to mouse heart counterparts. The results revealed that RFP+ cells closely resemble relatively mature CMs. These RFP+ cells co-expressed sarcomere genes (Myh7, Myl2, Myl3, Myoz2, and Mypn), calcium-handling gene (Casq2), and ion transporter at sarcolemma (Kcna4). RFP+ cells also expressed the cardiac muscle gene (ACTC1). However, it is well known from previous studies, PSC-CMs are immature compared to adult CMs1,30. Using qualitative and quantitative methods, I identified laminin-511/521 as enhancers for CM maturation.
During heart development, it is well known that CMs undergo structural changes, leading to their adult phenotypes. The growth of the embryonic heart relies on CM proliferation95. Postnatally, the endogenous proliferative capacity diminishes, and physiological hypertrophic growth becomes dominant, leading to an increase in
cell size (around 30- to 40-fold in both rodents and human) and binuclear cells31,44. In mice, the proportion of binuclear cells increases after birth to adult hearts (1.5% to 91.5%, respectively)32. Consistently, PSC-CMs plated on laminin-511/521 were increased cell size, and also showed higher proportions of binuclear cells than gelatin (gelatin, 7.77%; laminin-511, 38.68%; laminin-521, 54.72%). Moreover, PSC-CMs cultured on laminin-511/521 were also increased sarcomere length (gelatin, ~1.91 μm;
laminin-511, ~1.99 μm; laminin-521, ~1.97 μm). In agreement with this result, the sarcomere length of CMs is increased from fetal to adult stage (fetal CMs, ~1.6 μm;
adult CMs, ~2.2 μm)30. In addition, Cx43 localization also considered as a hallmark for CM maturation. In embryonic, Cx43 diffusely expresses in the cytoplasm, whereas it localizes to the lateral cell-axis in neonate stage. Since the CMs reach to mature stage, Cx43 mostly localizes to intercalated disk11. In this study, the result showed that PSC-CMs plated on laminin-511/521 promoted localization of Cx43 to lateral cell-axis.
Along with transcriptome analysis, the maturation scores of the PSC-CMs plated on laminin-511/521 were comparable to P7 and P14 heart. Altogether, laminin-511/521 enhanced the maturity of PSC-CMs equivalent to postnatal heart.
Laminins consist of three chains termed α, β, and γ. The laminin proteins are named in accordance with their chain compositions. For instance, laminin-511 composes of α5, β1, and γ1 chains96. Laminin E8 fragments are minimal forms of intact laminins, which contain C-terminal regions of the α, β, and γ chains. These minimal fragments contain the active integrin-binding site, which consists of laminin globular 1-3 domains of α subunit and the glutamate residue at C-terminal tail of the γ chain97, but absent other parts such as heparin/heparan sulphate-binding region98. Thus, the laminin E8 fragments retain the full capacity of binding to integrins similar to intact laminins99. Integrins are transmembrane receptors, contributing to cell-ECM proteins and cell-cell adhesion. Integrin-binding ECMs are able to activate several intracellular signaling cascades, which relate to cell survival, proliferation, motility, and differentiation100–103. Previous study has shown that the interactions between laminin-511/521 E8 fragments are primarily α6β1 integrin-dependent, by which blocking of α6β1 integrin suppressed adhesion of cells to those laminins104. Moreover, a mutant laminin-511 E8 fragment lost their binding affinity to α6β1 integrin, even though a single amino-acid substitution97. Thus, I believe that the interactions of
laminin-511/521 with α6β1 integrin are the important route for cell-ECM binding and enhancing PSC-CMs maturation.
Although PSC-CMs displayed more mature phenotypes on laminin-511/521, these cells were still immature compared to adult CMs. As developing CM are exposed to combinatorial effects of several environmental cues such as hormones, substrate stiffness, and electrical stimulation, combinations of these environmental cues would likely lead the PSC-CMs mature like adult CMs.
Acknowledgments
I gratefully acknowledge technical supports and helpful discussions from all members in Regenerative Medicine including Prof.Yutaka Hanazono, Hideki Uosaki, Tomoyuki Abe, Hiroaki Shibata, Hiromasa Hara, Suvd Byambaa, Rie Ishihara. Mari Karube, and Eri Noguchi.
Funding supports
This work was supported by The Program for Technological Innovation of Regenerative Medicine, Research Center Network for Realization of Regenerative Medicine from Japan Agency for Medical Research and Development (AMED, 18bm0704012h0003), Grant-in-Aid for Early-Career Scientists (19K17613) and Fund for the Promotion of Joint International Research (Fostering Joint International Research (B), 19KK0219) from Japan Society for the Promotion of Science, Novartis Research Grant, Sakakibara Memorial Research Grant from Japan Research Promotion Society for Cardiovascular Diseases, Takeda Science Foundation, The Uehara Memorial Foundation, SENSHIN Medical Research Foundation, and the Grant for Basic Research of the Japanese Circulation Society (to Hideki Uosaki). This work was also partly supported by JMU start-up award and JMU graduate student research award from Jichi Medical University (to me, Nawin Chanthra).
References
1. Uosaki, H., Cahan, P., Lee, D. I., Wang, S., Miyamoto, M., Fernandez, L., Kass, D.
A. & Kwon, C. Transcriptional Landscape of Cardiomyocyte Maturation. Cell Rep.
13, 1705–1716 (2015).
2. Till, J. E. & McCULLOCH, E. A. A direct measurement of the radiation sensitivity of normal mouse bone marrow cells. Radiat. Res. 14, 213–222 (1961).
3. Wobus, A. M. & Boheler, K. R. Embryonic stem cells: prospects for developmental biology and cell therapy. Physiol. Rev. 85, 635–678 (2005).
4. Evans, M. J. & Kaufman, M. H. Establishment in culture of pluripotential cells from mouse embryos. Nature 292, 154–156 (1981).
5. Thomson, J. A., Itskovitz-Eldor, J., Shapiro, S. S., Waknitz, M. A., Swiergiel, J. J., Marshall, V. S. & Jones, J. M. Embryonic stem cell lines derived from human blastocysts. Science 282, 1145–1147 (1998).
6. Takahashi, K. & Yamanaka, S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663–676 (2006).
7. Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K. &
Yamanaka, S. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861–872 (2007).
8. Chong, J. J. H. & Murry, C. E. Cardiac regeneration using pluripotent stem cells--progression to large animal models. Stem Cell Res. 13, 654–665 (2014).
9. Uosaki, H., Fukushima, H., Takeuchi, A., Matsuoka, S., Nakatsuji, N., Yamanaka, S. & Yamashita, J. K. Efficient and scalable purification of cardiomyocytes from human embryonic and induced pluripotent stem cells by VCAM1 surface expression. PloS One 6, e23657 (2011).
10. Denning, C., Borgdorff, V., Crutchley, J., Firth, K. S. A., George, V., Kalra, S., Kondrashov, A., Hoang, M. D., Mosqueira, D., Patel, A., Prodanov, L., Rajamohan, D., Skarnes, W. C., Smith, J. G. W. & Young, L. E. Cardiomyocytes from human pluripotent stem cells: From laboratory curiosity to industrial biomedical platform.
Biochim. Biophys. Acta 1863, 1728–1748 (2016).
11. Vreeker, A., van Stuijvenberg, L., Hund, T. J., Mohler, P. J., Nikkels, P. G. J. & van Veen, T. A. B. Assembly of the cardiac intercalated disk during pre- and postnatal development of the human heart. PloS One 9, e94722 (2014).
12. Zhang, J., Wilson, G. F., Soerens, A. G., Koonce, C. H., Yu, J., Palecek, S. P., Thomson, J. A. & Kamp, T. J. Functional cardiomyocytes derived from human induced pluripotent stem cells. Circ. Res. 104, e30-41 (2009).
13. Itzhaki, I., Rapoport, S., Huber, I., Mizrahi, I., Zwi-Dantsis, L., Arbel, G., Schiller, J.
& Gepstein, L. Calcium handling in human induced pluripotent stem cell derived cardiomyocytes. PloS One 6, e18037 (2011).
14. Germanguz, I., Sedan, O., Zeevi-Levin, N., Shtrichman, R., Barak, E., Ziskind, A., Eliyahu, S., Meiry, G., Amit, M., Itskovitz-Eldor, J. & Binah, O. Molecular characterization and functional properties of cardiomyocytes derived from human inducible pluripotent stem cells. J. Cell. Mol. Med. 15, 38–51 (2011).
15. Gherghiceanu, M., Barad, L., Novak, A., Reiter, I., Itskovitz-Eldor, J., Binah, O. &
Popescu, L. M. Cardiomyocytes derived from human embryonic and induced pluripotent stem cells: comparative ultrastructure. J. Cell. Mol. Med. 15, 2539–
2551 (2011).
16. Snir, M., Kehat, I., Gepstein, A., Coleman, R., Itskovitz-Eldor, J., Livne, E. &
Gepstein, L. Assessment of the ultrastructural and proliferative properties of human embryonic stem cell-derived cardiomyocytes. Am. J. Physiol. Heart Circ.
Physiol. 285, H2355-2363 (2003).
17. Karakikes, I., Ameen, M., Termglinchan, V. & Wu, J. C. Human induced pluripotent stem cell-derived cardiomyocytes: insights into molecular, cellular, and functional phenotypes. Circ. Res. 117, 80–88 (2015).
18. Lopaschuk, G. D. & Jaswal, J. S. Energy metabolic phenotype of the cardiomyocyte during development, differentiation, and postnatal maturation. J.
Cardiovasc. Pharmacol. 56, 130–140 (2010).
19. Kim, C., Wong, J., Wen, J., Wang, S., Wang, C., Spiering, S., Kan, N. G., Forcales, S., Puri, P. L., Leone, T. C., Marine, J. E., Calkins, H., Kelly, D. P., Judge, D. P. &
Chen, H.-S. V. Studying arrhythmogenic right ventricular dysplasia with patient-specific iPSCs. Nature 494, 105–110 (2013).
20. Zhu, W.-Z., Santana, L. F. & Laflamme, M. A. Local control of excitation-contraction coupling in human embryonic stem cell-derived cardiomyocytes. PloS One 4, e5407 (2009).
21. Satin, J., Itzhaki, I., Rapoport, S., Schroder, E. A., Izu, L., Arbel, G., Beyar, R., Balke, C. W., Schiller, J. & Gepstein, L. Calcium handling in human embryonic stem cell-derived cardiomyocytes. Stem Cells 26, 1961–1972 (2008).
22. 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).
23. Liu, J., Lieu, D. K., Siu, C. W., Fu, J.-D., Tse, H.-F. & Li, R. A. Facilitated maturation of Ca2+ handling properties of human embryonic stem cell-derived cardiomyocytes by calsequestrin expression. Am. J. Physiol. Cell Physiol. 297, C152-159 (2009).
24. Janowski, E., Cleemann, L., Sasse, P. & Morad, M. Diversity of Ca2+ signaling in developing cardiac cells. Ann. N. Y. Acad. Sci. 1080, 154–164 (2006).
25. Liu, W., Yasui, K., Opthof, T., Ishiki, R., Lee, J.-K., Kamiya, K., Yokota, M. &
Kodama, I. Developmental changes of Ca(2+) handling in mouse ventricular cells from early embryo to adulthood. Life Sci. 71, 1279–1292 (2002).
26. Escobar, A. L., Ribeiro-Costa, R., Villalba-Galea, C., Zoghbi, M. E., Pérez, C. G.
& Mejía-Alvarez, R. Developmental changes of intracellular Ca2+ transients in beating rat hearts. Am. J. Physiol. Heart Circ. Physiol. 286, H971-978 (2004).
27. Tanaka, H. & Shigenobu, K. Effect of ryanodine on neonatal and adult rat heart:
developmental increase in sarcoplasmic reticulum function. J. Mol. Cell. Cardiol.
21, 1305–1313 (1989).
28. Huang, J., Hove-Madsen, L. & Tibbits, G. F. Ontogeny of Ca2+-induced Ca2+
release in rabbit ventricular myocytes. Am. J. Physiol. Cell Physiol. 294, C516-525 (2008).
29. Veerman, C. C., Kosmidis, G., Mummery, C. L., Casini, S., Verkerk, A. O. & Bellin, M. Immaturity of human stem-cell-derived cardiomyocytes in culture: fatal flaw or soluble problem? Stem Cells Dev. 24, 1035–1052 (2015).
30. Yang, X., Pabon, L. & Murry, C. E. Engineering adolescence: maturation of human pluripotent stem cell-derived cardiomyocytes. Circ. Res. 114, 511–523 (2014).
31. Mollova, M., Bersell, K., Walsh, S., Savla, J., Das, L. T., Park, S.-Y., Silberstein, L.
E., Dos Remedios, C. G., Graham, D., Colan, S. & Kühn, B. Cardiomyocyte proliferation contributes to heart growth in young humans. Proc. Natl. Acad. Sci. U.
S. A. 110, 1446–1451 (2013).
32. Soonpaa, M. H., Kim, K. K., Pajak, L., Franklin, M. & Field, L. J. Cardiomyocyte DNA synthesis and binucleation during murine development. Am. J. Physiol. 271, H2183-2189 (1996).
33. Lundy, S. D., Zhu, W.-Z., Regnier, M. & Laflamme, M. A. Structural and functional maturation of cardiomyocytes derived from human pluripotent stem cells. Stem Cells Dev. 22, 1991–2002 (2013).
34. Porter, G. A., Hom, J., Hoffman, D., Quintanilla, R., de Mesy Bentley, K. & Sheu, S.-S. Bioenergetics, mitochondria, and cardiac myocyte differentiation. Prog.
Pediatr. Cardiol. 31, 75–81 (2011).
35. Feric, N. T. & Radisic, M. Maturing human pluripotent stem cell-derived cardiomyocytes in human engineered cardiac tissues. Adv. Drug Deliv. Rev. 96, 110–134 (2016).
36. Jiang, Y., Park, P., Hong, S.-M. & Ban, K. Maturation of Cardiomyocytes Derived from Human Pluripotent Stem Cells: Current Strategies and Limitations. Mol. Cells 41, 613–621 (2018).
37. Lompre, A. M., Schwartz, K., d’Albis, A., Lacombe, G., Van Thiem, N. &
Swynghedauw, B. Myosin isoenzyme redistribution in chronic heart overload.
Nature 282, 105–107 (1979).
38. Zhu, H., Scharnhorst, K. S., Stieg, A. Z., Gimzewski, J. K., Minami, I., Nakatsuji, N., Nakano, H. & Nakano, A. Two dimensional electrophysiological characterization of human pluripotent stem cell-derived cardiomyocyte system. Sci.
Rep. 7, 43210 (2017).
39. Kadota, S., Minami, I., Morone, N., Heuser, J. E., Agladze, K. & Nakatsuji, N.
Development of a reentrant arrhythmia model in human pluripotent stem cell-derived cardiac cell sheets. Eur. Heart J. 34, 1147–1156 (2013).
40. Drouin, E., Charpentier, F., Gauthier, C., Laurent, K. & Le Marec, H.
Electrophysiologic characteristics of cells spanning the left ventricular wall of human heart: evidence for presence of M cells. J. Am. Coll. Cardiol. 26, 185–192 (1995).
41. Peters, N. S., Green, C. R., Poole-Wilson, P. A. & Severs, N. J. Reduced content of connexin43 gap junctions in ventricular myocardium from hypertrophied and ischemic human hearts. Circulation 88, 864–875 (1993).
42. Cho, G.-S., Lee, D. I., Tampakakis, E., Murphy, S., Andersen, P., Uosaki, H., Chelko, S., Chakir, K., Hong, I., Seo, K., Chen, H.-S. V., Chen, X., Basso, C., Houser, S. R., Tomaselli, G. F., O’Rourke, B., Judge, D. P., Kass, D. A. & Kwon, C. Neonatal Transplantation Confers Maturation of PSC-Derived Cardiomyocytes Conducive to Modeling Cardiomyopathy. Cell Rep. 18, 571–582 (2017).
43. Peters, N. S., Severs, N. J., Rothery, S. M., Lincoln, C., Yacoub, M. H. & Green, C. R. Spatiotemporal relation between gap junctions and fascia adherens junctions during postnatal development of human ventricular myocardium. Circulation 90, 713–725 (1994).
44. Laflamme, M. A. & Murry, C. E. Heart regeneration. Nature 473, 326–335 (2011).
45. Kamakura, T., Makiyama, T., Sasaki, K., Yoshida, Y., Wuriyanghai, Y., Chen, J., Hattori, T., Ohno, S., Kita, T., Horie, M., Yamanaka, S. & Kimura, T. Ultrastructural maturation of human-induced pluripotent stem cell-derived cardiomyocytes in a long-term culture. Circ. J. 77, 1307–1314 (2013).
46. Sartiani, L., Bettiol, E., Stillitano, F., Mugelli, A., Cerbai, E. & Jaconi, M. E.
Developmental changes in cardiomyocytes differentiated from human embryonic stem cells: a molecular and electrophysiological approach. Stem Cells 25, 1136–
1144 (2007).
47. Herron, T. J., Rocha, A. M. D., Campbell, K. F., Ponce-Balbuena, D., Willis, B. C., Guerrero-Serna, G., Liu, Q., Klos, M., Musa, H., Zarzoso, M., Bizy, A., Furness, J., Anumonwo, J., Mironov, S. & Jalife, J. Extracellular Matrix-Mediated Maturation of Human Pluripotent Stem Cell-Derived Cardiac Monolayer Structure and Electrophysiological Function. Circ. Arrhythm. Electrophysiol. 9, e003638 (2016).
48. VanWinkle, W. B., Snuggs, M. B. & Buja, L. M. Cardiogel: a biosynthetic extracellular matrix for cardiomyocyte culture. In Vitro Cell. Dev. Biol. Anim. 32, 478–485 (1996).
49. Zhang, J., Klos, M., Wilson, G. F., Herman, A. M., Lian, X., Raval, K. K., Barron, M. R., Hou, L., Soerens, A. G., Yu, J., Palecek, S. P., Lyons, G. E., Thomson, J.
A., Herron, T. J., Jalife, J. & Kamp, T. J. Extracellular matrix promotes highly efficient cardiac differentiation of human pluripotent stem cells: the matrix sandwich method. Circ. Res. 111, 1125–1136 (2012).
50. Yang, X., Rodriguez, M., Pabon, L., Fischer, K. A., Reinecke, H., Regnier, M., Sniadecki, N. J., Ruohola-Baker, H. & Murry, C. E. Tri-iodo-l-thyronine promotes the maturation of human cardiomyocytes-derived from induced pluripotent stem cells. J. Mol. Cell. Cardiol. 72, 296–304 (2014).
51. Krüger, M., Sachse, C., Zimmermann, W. H., Eschenhagen, T., Klede, S. & Linke, W. A. Thyroid hormone regulates developmental titin isoform transitions via the phosphatidylinositol-3-kinase/ AKT pathway. Circ. Res. 102, 439–447 (2008).
52. Kosmidis, G., Bellin, M., Ribeiro, M. C., van Meer, B., Ward-van Oostwaard, D., Passier, R., Tertoolen, L. G. J., Mummery, C. L. & Casini, S. Altered calcium handling and increased contraction force in human embryonic stem cell derived cardiomyocytes following short term dexamethasone exposure. Biochem. Biophys.
Res. Commun. 467, 998–1005 (2015).
53. Parikh, S. S., Blackwell, D. J., Gomez-Hurtado, N., Frisk, M., Wang, L., Kim, K., Dahl, C. P., Fiane, A., Tønnessen, T., Kryshtal, D. O., Louch, W. E. & Knollmann, B. C. Thyroid and Glucocorticoid Hormones Promote Functional T-Tubule Development in Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes.
Circ. Res. 121, 1323–1330 (2017).
54. Jacot, J. G., Martin, J. C. & Hunt, D. L. Mechanobiology of cardiomyocyte development. J. Biomech. 43, 93–98 (2010).
55. Prakash, Y. S., Cody, M. J., Housmans, P. R., Hannon, J. D. & Sieck, G. C.
Comparison of cross-bridge cycling kinetics in neonatal vs. adult rat ventricular muscle. J. Muscle Res. Cell Motil. 20, 717–723 (1999).
56. Bajaj, P., Tang, X., Saif, T. A. & Bashir, R. Stiffness of the substrate influences the phenotype of embryonic chicken cardiac myocytes. J. Biomed. Mater. Res. A 95, 1261–1269 (2010).
57. Jacot, J. G., McCulloch, A. D. & Omens, J. H. Substrate stiffness affects the functional maturation of neonatal rat ventricular myocytes. Biophys. J. 95, 3479–
3487 (2008).
58. Berry, M. F., Engler, A. J., Woo, Y. J., Pirolli, T. J., Bish, L. T., Jayasankar, V., Morine, K. J., Gardner, T. J., Discher, D. E. & Sweeney, H. L. Mesenchymal stem cell injection after myocardial infarction improves myocardial compliance. Am. J.
Physiol. Heart Circ. Physiol. 290, H2196-2203 (2006).
59. McCain, M. L. & Parker, K. K. Mechanotransduction: the role of mechanical stress, myocyte shape, and cytoskeletal architecture on cardiac function. Pflugers Arch.
462, 89–104 (2011).
60. 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).
61. Engler, A. J., Carag-Krieger, C., Johnson, C. P., Raab, M., Tang, H.-Y., Speicher, D. W., Sanger, J. W., Sanger, J. M. & Discher, D. E. Embryonic cardiomyocytes
beat best on a matrix with heart-like elasticity: scar-like rigidity inhibits beating. J.
Cell Sci. 121, 3794–3802 (2008).
62.Asahi, M., Otsu, K., Nakayama, H., Hikoso, S., Takeda, T., Gramolini, A. O., Trivieri, M. G., Oudit, G. Y., Morita, T., Kusakari, Y., Hirano, S., Hongo, K., Hirotani, S., Yamaguchi, O., Peterson, A., Backx, P. H., Kurihara, S., Hori, M. & MacLennan, D. H. Cardiac-specific overexpression of sarcolipin inhibits sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA2a) activity and impairs cardiac function in mice.
Proc. Natl. Acad. Sci. U. S. A. 101, 9199–9204 (2004).
63. Radisic, M., Park, H., Shing, H., Consi, T., Schoen, F. J., Langer, R., Freed, L. E.
& Vunjak-Novakovic, G. Functional assembly of engineered myocardium by electrical stimulation of cardiac myocytes cultured on scaffolds. Proc. Natl. Acad.
Sci. U. S. A. 101, 18129–18134 (2004).
64. Sathaye, A., Bursac, N., Sheehy, S. & Tung, L. Electrical pacing counteracts intrinsic shortening of action potential duration of neonatal rat ventricular cells in culture. J. Mol. Cell. Cardiol. 41, 633–641 (2006).
65. Xia, Y., Buja, L. M., Scarpulla, R. C. & McMillin, J. B. Electrical stimulation of neonatal cardiomyocytes results in the sequential activation of nuclear genes governing mitochondrial proliferation and differentiation. Proc. Natl. Acad. Sci. U.
S. A. 94, 11399–11404 (1997).
66. Nunes, S. S., Miklas, J. W., Liu, J., Aschar-Sobbi, R., Xiao, Y., Zhang, B., Jiang, J., Massé, S., Gagliardi, M., Hsieh, A., Thavandiran, N., Laflamme, M. A., Nanthakumar, K., Gross, G. J., Backx, P. H., Keller, G. & Radisic, M. Biowire: a platform for maturation of human pluripotent stem cell-derived cardiomyocytes.
Nat. Methods 10, 781–787 (2013).
67. You, J.-O., Rafat, M., Ye, G. J. C. & Auguste, D. T. Nanoengineering the heart:
conductive scaffolds enhance connexin 43 expression. Nano Lett. 11, 3643–3648 (2011).
68. Zhou, P. & Pu, W. T. Recounting Cardiac Cellular Composition. Circ. Res. 118, 368–370 (2016).
69. Kim, C., Majdi, M., Xia, P., Wei, K. A., Talantova, M., Spiering, S., Nelson, B., Mercola, M. & Chen, H.-S. V. Non-cardiomyocytes influence the electrophysiological maturation of human embryonic stem cell-derived cardiomyocytes during differentiation. Stem Cells Dev. 19, 783–795 (2010).
70. Gao, L., Gregorich, Z. R., Zhu, W., Mattapally, S., Oduk, Y., Lou, X., Kannappan, R., Borovjagin, A. V., Walcott, G. P., Pollard, A. E., Fast, V. G., Hu, X., Lloyd, S.
G., Ge, Y. & Zhang, J. Large Cardiac Muscle Patches Engineered From Human Induced-Pluripotent Stem Cell-Derived Cardiac Cells Improve Recovery From Myocardial Infarction in Swine. Circulation 137, 1712–1730 (2018).
71. Ruan, J.-L., Tulloch, N. L., Razumova, M. V., Saiget, M., Muskheli, V., Pabon, L., Reinecke, H., Regnier, M. & Murry, C. E. Mechanical Stress Conditioning and Electrical Stimulation Promote Contractility and Force Maturation of Induced Pluripotent Stem Cell-Derived Human Cardiac Tissue. Circulation 134, 1557–1567 (2016).
72. Shadrin, I. Y., Allen, B. W., Qian, Y., Jackman, C. P., Carlson, A. L., Juhas, M. E.
& Bursac, N. Cardiopatch platform enables maturation and scale-up of human pluripotent stem cell-derived engineered heart tissues. Nat. Commun. 8, 1825 (2017).
73. Chan, H. Y. S., Keung, W., Li, R. A., Miller, A. L. & Webb, S. E. Morphometric Analysis of Human Embryonic Stem Cell-Derived Ventricular Cardiomyocytes:
Determining the Maturation State of a Population by Quantifying Parameters in Individual Cells. Stem Cells Int. 2015, 586908 (2015).
74. Hayes, H. B., Nicolini, A. M., Arrowood, C. A., Chvatal, S. A., Wolfson, D. W., Cho, H. C., Sullivan, D. D., Chal, J., Fermini, B., Clements, M., Ross, J. D. & Millard, D.
C. Novel method for action potential measurements from intact cardiac monolayers with multiwell microelectrode array technology. Sci. Rep. 9, 11893 (2019).
75. Sasaki, D., Matsuura, K., Seta, H., Haraguchi, Y., Okano, T. & Shimizu, T.
Contractile force measurement of human induced pluripotent stem cell-derived cardiac cell sheet-tissue. PloS One 13, e0198026 (2018).
76. Horikoshi, Y., Yan, Y., Terashvili, M., Wells, C., Horikoshi, H., Fujita, S., Bosnjak, Z. J. & Bai, X. Fatty Acid-Treated Induced Pluripotent Stem Cell-Derived Human Cardiomyocytes Exhibit Adult Cardiomyocyte-Like Energy Metabolism Phenotypes. Cells 8, (2019).
77. Den Hartogh, S. C. & Passier, R. Concise Review: Fluorescent Reporters in Human Pluripotent Stem Cells: Contributions to Cardiac Differentiation and Their Applications in Cardiac Disease and Toxicity. Stem Cells 34, 13–26 (2016).
78. Yamanaka, S., Zahanich, I., Wersto, R. P. & Boheler, K. R. Enhanced proliferation of monolayer cultures of embryonic stem (ES) cell-derived cardiomyocytes following acute loss of retinoblastoma. PloS One 3, e3896 (2008).
79. Uosaki, H., Andersen, P., Shenje, L. T., Fernandez, L., Christiansen, S. L. & Kwon, C. Direct contact with endoderm-like cells efficiently induces cardiac progenitors from mouse and human pluripotent stem cells. PloS One 7, e46413 (2012).
80. Kattman, S. J., Witty, A. D., Gagliardi, M., Dubois, N. C., Niapour, M., Hotta, A., Ellis, J. & Keller, G. 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).
81. Ran, F. A., Hsu, P. D., Wright, J., Agarwala, V., Scott, D. A. & Zhang, F. Genome engineering using the CRISPR-Cas9 system. Nat. Protoc. 8, 2281–2308 (2013).
82. Dobin, A., Davis, C. A., Schlesinger, F., Drenkow, J., Zaleski, C., Jha, S., Batut, P., Chaisson, M. & Gingeras, T. R. STAR: ultrafast universal RNA-seq aligner.
Bioinformatics 29, 15–21 (2013).
83. Liao, Y., Smyth, G. K. & Shi, W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 30, 923–930 (2014).
84. Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014).
85. Yu, G., Wang, L.-G., Han, Y. & He, Q.-Y. clusterProfiler: an R package for comparing biological themes among gene clusters. Omics 16, 284–287 (2012).
86. Muto, A., Ohkura, M., Abe, G., Nakai, J. & Kawakami, K. Real-time visualization of neuronal activity during perception. Curr. Biol. 23, 307–311 (2013).
87. Pasqualin, C., Gannier, F., Yu, A., Malécot, C. O., Bredeloux, P. & Maupoil, V.
SarcOptiM for ImageJ: high-frequency online sarcomere length computing on stimulated cardiomyocytes. Am. J. Physiol. Cell Physiol. 311, C277-283 (2016).
88. Tirosh-Finkel, L., Zeisel, A., Brodt-Ivenshitz, M., Shamai, A., Yao, Z., Seger, R., Domany, E. & Tzahor, E. BMP-mediated inhibition of FGF signaling promotes cardiomyocyte differentiation of anterior heart field progenitors. Development 137, 2989–3000 (2010).
89. Wang, J., Greene, S. B., Bonilla-Claudio, M., Tao, Y., Zhang, J., Bai, Y., Huang, Z., Black, B. L., Wang, F. & Martin, J. F. Bmp signaling regulates myocardial
differentiation from cardiac progenitors through a MicroRNA-mediated mechanism.
Dev. Cell 19, 903–912 (2010).
90. Masaki, T. & Takaiti, O. M-protein. J. Biochem. (Tokyo) 75, 367–380 (1974).
91. Noguchi, J., Yanagisawa, M., Imamura, M., Kasuya, Y., Sakurai, T., Tanaka, T. &
Masaki, T. Complete primary structure and tissue expression of chicken pectoralis M-protein. J. Biol. Chem. 267, 20302–20310 (1992).
92. Obermann, W. M., Gautel, M., Steiner, F., van der Ven, P. F., Weber, K. & Fürst, D. O. The structure of the sarcomeric M band: localization of defined domains of myomesin, M-protein, and the 250-kD carboxy-terminal region of titin by immunoelectron microscopy. J. Cell Biol. 134, 1441–1453 (1996).
93. Steiner, F., Weber, K. & Fürst, D. O. Structure and expression of the gene encoding murine M-protein, a sarcomere-specific member of the immunoglobulin superfamily. Genomics 49, 83–95 (1998).
94. Zemljic-Harpf, A. E., Godoy, J. C., Platoshyn, O., Asfaw, E. K., Busija, A. R., Domenighetti, A. A. & Ross, R. S. Vinculin directly binds zonula occludens-1 and is essential for stabilizing connexin-43-containing gap junctions in cardiac myocytes. J. Cell Sci. 127, 1104–1116 (2014).
95. Li, F., Wang, X., Capasso, J. M. & Gerdes, A. M. Rapid transition of cardiac myocytes from hyperplasia to hypertrophy during postnatal development. J. Mol.
Cell. Cardiol. 28, 1737–1746 (1996).
96. Aumailley, M., Bruckner-Tuderman, L., Carter, W. G., Deutzmann, R., Edgar, D., Ekblom, P., Engel, J., Engvall, E., Hohenester, E., Jones, J. C. R., Kleinman, H.
K., Marinkovich, M. P., Martin, G. R., Mayer, U., Meneguzzi, G., Miner, J. H., Miyazaki, K., Patarroyo, M., Paulsson, M., Quaranta, V., Sanes, J. R., Sasaki, T., Sekiguchi, K., Sorokin, L. M., Talts, J. F., Tryggvason, K., Uitto, J., Virtanen, I., von der Mark, K., Wewer, U. M., Yamada, Y. & Yurchenco, P. D. A simplified laminin nomenclature. Matrix Biol. J. Int. Soc. Matrix Biol. 24, 326–332 (2005).
97. Ido, H., Nakamura, A., Kobayashi, R., Ito, S., Li, S., Futaki, S. & Sekiguchi, K. The requirement of the glutamic acid residue at the third position from the carboxyl termini of the laminin gamma chains in integrin binding by laminins. J. Biol. Chem.
282, 11144–11154 (2007).
98. Ido, H., Harada, K., Futaki, S., Hayashi, Y., Nishiuchi, R., Natsuka, Y., Li, S., Wada, Y., Combs, A. C., Ervasti, J. M. & Sekiguchi, K. Molecular dissection of the