- Email: [email protected]
HeJ mouse
Journal of Molecular and Cellular Cardiology 128 (2019) 90–95
Contents lists available at ScienceDirect
Journal of Molecular and Cellular Cardiology journal homepage: www.elsevier.com/locate/yjmcc
Short communication
Characterization of a small molecule that promotes cell cycle activation of human induced pluripotent stem cell-derived cardiomyocytes
T
Masamichi Itoa,1, Hironori Haraa,1, Norifumi Takedaa, Atsuhiko T. Naitob, Seitaro Nomuraa, ⁎ Masaki Kondoc, Yutaka Hatad, Masanobu Uchiyamac,e, Hiroyuki Moritaa, Issei Komuroa, a
Department of Cardiovascular Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, Japan Department of Pharmacology, Faculty of Medicine, Toho University, 5-21-16 Omori-nishi, Ohta-ku, Tokyo, Japan c Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, Tokyo, Japan d Department of Medical Biochemistry, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo, Japan e Advanced Elements Chemistry Research Team, RIKEN Center for Sustainable Resource Science, and Elements Chemistry Laboratory, RIKEN, 2-1, Hirosawa, Wako-city, Saitama, Japan b
A R T I C LE I N FO
A B S T R A C T
Keywords: Cell cycle activation Induced pluripotent stem cell-derived cardiomyocyte Hippo pathway Regeneration Small molecule Regenerative medicine
Background: Since regenerative capacity of adult mammalian myocardium is limited, activation of the endogenous proliferative capacity of existing cardiomyocytes is a potential therapeutic strategy for treating heart diseases accompanied by cardiomyocyte loss. Recently, we performed a compound screening and developed a new drug named TT-10 (C11H10FN3OS2) which promotes the proliferation of murine cardiomyocytes via enhancement of YES-associated protein (YAP)-transcriptional enhancer factor domain (TEAD) activity and improves cardiac function after myocardial infarction in adult mice. Methods and results: To test whether TT-10 can also promote the proliferative capacity of human cardiomyocytes, we investigated the efficacy of TT-10 on human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (hiPSCMs). The hiPSCs were established from monocytes obtained from healthy donors and cardiac differentiation was performed using a chemically defined protocol. As was observed in murine cardiomyocytes, TT-10 markedly promoted cell cycle activation and increased cell division of hiPSCMs. We then evaluated other effects of TT-10 on the functional properties of hiPSCMs by gene expression and cell motion analyses. We observed that TT-10 had no unfavorable effects on the expression of functional and structural genes or the contractile properties of hiPSCMs. Conclusions: Our results suggest that the novel drug TT-10 effectively activated the cell cycle of hiPSCMs without apparent functional impairment of myocardium, suggesting the potential of clinical usefulness of this drug.
1. Introduction The cell division capacity of mammalian cardiomyocytes (CMs) diminishes dramatically soon after birth, and it was once widely believed that adult myocardium does not regenerate after injury. However, a previous study demonstrated that CMs are replenished in the adult human heart, though the rate is very low [1]. A number of studies have focused on the underlying molecular mechanisms of CM
proliferation, with low oxygen environments, micro RNAs, extracellular matrix, and paracrine factors from non-CMs reported as key factors that stimulate CMs for cell-cycle reentry [2]. It would therefore be reasonable to activate the endogenous proliferation capacity of CMs by manipulating these factors in the treatment of heart disease caused by CM loss, including myocardial infarction (MI). The Hippo-YES-associated protein-transcriptional enhancer factor domain (Hippo-YAP-TEAD) signaling pathway regulates cell
Abbreviations: YAP, YES-associated protein; TEAD, transcriptional enhancer factor domain; hiPSC, human induced pluripotent stem cell; hiPSCM, human induced pluripotent stem cell-derived cardiomyocyte; CM, cardiomyocyte; MI, myocardial infarction; iPSCMs, induced pluripotent stem cell-derived cardiomyocytes; iPSC, induced pluripotent stem cell; IMDM, Iscove's modified Dulbecco's medium; FCS, fetal calf serum; EdU, 5-ethynyl-2′-deoxyuridine; ROI, region of interest; CV, contraction velocity; RV, relaxation velocity; DD, deformation distance; GSK3β, glycogen synthase kinase 3 beta; BIO, (2′Z, 3′E)-6-bromoindirubin-3′-oxime; FGF, fibroblast growth factor; NRG, neuregulin; GO, Gene Ontology; NRF2, nuclear factor erythroid 2-related factor 2 ⁎ Corresponding author at: Department of Cardiovascular Medicine, The University of Tokyo Graduate School of Medicine, 7-3-1 Hongo, Tokyo 113-8655, Japan. E-mail address: [email protected] (I. Komuro). 1 These authors equally contributed to the work. https://doi.org/10.1016/j.yjmcc.2019.01.020 Received 18 September 2018; Received in revised form 18 January 2019; Accepted 23 January 2019 Available online 23 January 2019 0022-2828/ © 2019 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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Fig. 1. TT-10 promotes cell division of hiPSCMs. (A) Chemical structures of TAZ-12 and TT-10. (B) Schematic outline of cardiac differentiation of hiPSCs and schedule of proliferation assay timeline. EB, embryoid body; hiPSC, human induced pluripotent stem cell. (C) A representative image of the EdU-positive hiPSCMs after treatment with TT-10. The (−) image shows a control treated with vehicle. Arrowheads indicate positive hiPSCMs. Scale bar: 100 μm. (D) Percentages of EdUpositive hiPSCMs after treatment with the indicated reagents. The percentage of EdU-positive hiPSCMs was analyzed 48 h after compound administration. (n = 5). (−) indicates the control, with vehicle alone administered (DMSO). The concentrations of the drugs are as follows: FCS, 2%; TAZ-12, 10 μmol/l; TT-10, 10 μmol/l; BIO, 1 μmol/l; NRG1, 100 ng/ml; and FGF1, 100 ng/ml. *; p < .05, ***; p < .001 vs. control (vehicle only). EdU, 5-ethynyl-2′-deoxyuridine; hiPSCM, human induced pluripotent stem cell-derived cardiomyocyte; DMSO, dimethyl sulfoxide; CMs, cardiomyocytes; FCS, fetal calf serum; BIO, (2′Z, 3′E)-6-bromoindirubin-3′oxime; NRG1, neuregulin-1; FGF1, fibroblast growth factor-1. (E) A representative image of Aurora B-positive hiPSCMs after TT-10 treatment. The (−) image shows a control without any drug treatment. Arrowheads indicate positive staining of hiPSCMs. Scale bars: 50 μm and 25 μm (in inset). (F) Percentages of Aurora B-positive hiPSCMs after treatment with 10 μmol/l TT-10. The percentages of Aurora B-positive hiPSCMs were analyzed 48 h after compound administration (n = 4). *; p < .05 vs. control (vehicle only). (G) Relative cell number of CMs 48 h after treatment with TT-10 (10 μmol/l). In the EdU assay, the absolute cell number of hiPSCMs was counted and normalized to the number of untreated CMs. n = 4; **; p < .01 vs. control (vehicle only). (H) MTS cell viability assay. Cell viability was analyzed after a 48-h incubation with TT-10. The viability was normalized to the vehicle control. The positive control was 1 mmol/l H2O2. n = 12 per group; **; p < .01, ***; p < .001 vs. control (vehicle only).
Cambridge, UK) and Aurora B kinase (BD Biosciences, Franklin Lakes, NJ). In this study, hiPSCMs were defined as troponin positive cells. Cytokinesis was defined by the presence of an Aurora B-stained spindleshaped structure in the middle of the intercellular bridge. Stained cells were visualized and evaluated using the Operetta High-Content Imaging System (PerkinElmer, Waltham, MA). Brightness and contrast were adjusted linearly across the entirety of each image.
proliferation, apoptosis, and stem cell self-renewal and expansion. Activated nuclear YAP in CMs promotes cardiac regeneration after injury [3]. We recently performed a large-scale cell-based compound screening using a cell-based reporter assay for YAP-TEAD activators and a proliferation assay using neonatal rat CMs. We identified a small molecule, TAZ-12, developed its fluorine substituent TT-10 (Fig. 1A) and found that TT-10 strongly promoted the proliferation of rat neonatal CMs and activated CM cell division and that it could also potently activate CM cell division in murine hearts after MI [4]. Before clinical application of this compound, its efficacy and safety must be assessed using human CMs. Today, human induced pluripotent stem cell-derived cardiomyocytes (hiPSCMs) are the only available source of human CMs and a promising tool for regenerative medicine. They have been shown to reproduce molecular mechanisms observed in diseased human hearts [5]. Furthermore, hiPSCMs are expected to be a complementary cellular source for injured heart tissue, considering transplantation of allogenic induced pluripotent stem cell-derived cardiomyocytes (iPSCMs) has been shown to improve the function of infarcted hearts in non-human primates [6]. To advance the translational research using TT-10, we examined whether the compound can activate the cell-cycle and cell division in hiPSCMs as well as murine CMs, and we also characterized the effect of TT-10 on the molecular and physiological properties of hiPSCMs.
2.3. Motion analysis of hiPSCMs To evaluate the contractile function of hiPSCMs, we used a motion vector analysis system, namely the SI8000 Cell Motion Imaging System® (SONY, Tokyo, Japan). This system enables quantitative analysis of the motion of synchronously beating hiPSCMs through video capture. The motion of each detection point was converted into a motion vector. The motion velocity within each region of interest (ROI) was calculated based on the sum of the vector magnitudes. The maximum contraction velocity (CV) is considered to correspond to the contractile function of the myocardium. Similarly, the maximum relaxation velocity (RV) is considered to correspond to the diastolic function, and the deformation distance (DD) is assumed to reflect the distance the myocardium moved within one beating cycle. Video images were taken 2 days after drug administration, and the relative changes in CV, RV, and DD were analyzed.
2. Materials and methods 3. Results 2.1. Preparation of hiPSCMs 3.1. TT-10 promotes the cell cycle of hiPSCMs Induced pluripotent stem cell (iPSC) lines were established from the peripheral blood of healthy male volunteers as previously described [7]. For cardiac differentiation, we adopted a chemically defined protocol. CMs were enriched based on the cellular density as previously described [8]. The hiPSCMs were once frozen for storage at day 28. Then, the stocks were thawed and 2 days later were passaged for assays. Four days after thawing, the medium was exchanged with Iscove's modified Dulbecco's medium (IMDM) with 0.1% fetal calf serum (FCS). On day 6, the medium was replaced with IMDM that contained test compounds. The cells were analyzed 48 h after drug administration. Thus, hiPSCMs were prepared and used for analysis around 35 days after differentiation (Fig. 1B). Details are described in the Supplementary Materials.
We first evaluated the effect of TT-10 on the proliferation of hiPSCMs using the EdU incorporation assay. The ratio of hiPSCMs that entered S phase was markedly enhanced after incubation with TT-10 for 48 h (Fig. 1C). This effect was observed among all hiPSCM lines produced from different donors (Supplementary Fig. 1). We also compared the effect of TT-10 with those of other compounds that have been reported to promote the proliferation of CMs, including glycogen synthase kinase 3 beta (GSK3β) inhibitor (2′Z, 3′E)-6-bromoindirubin-3′oxime (BIO) [9], fibroblast growth factor (FGF) -1 [10], and neuregulin (NRG) -1 [11]. BIO increased the ratio of EdU-positive hiPSCMs to a level that is comparable to that under TT-10, whereas NRG-1 and FGF-1 did not affect the proliferation of hiPSCMs (Fig. 1D). The extent of DNA synthesis promotion by TT-10 was more prominent compared to TAZ12 in one hiPSCM clone (Supplementary Fig. 1(a)) and was similar to TAZ-12 in three other hiPSCM lines (Supplementary Fig. 1(b)-(d)). We also evaluated the EdU positivity after TT-10 treatment by flow cytometry, finding that the percentage of EdU-positive CMs was increased from 6.4 ± 0.85% to 28.2% ± 3.3% after treatment with TT-10 (Supplementary Fig. 2). We next checked the effect of TT-10 on cytokinesis of hiPSCMs by staining Aurora B. The percentage of Aurora Bpositive hiPSCMs increased approximately threefold after TT-10 treatment (Fig. 1E, F). Indeed, TT-10 increased the absolute number of
2.2. Cell proliferation assay To detect cells in S phase, cells were labeled with 5 μmol/l 5ethynyl-2′-deoxyuridine (EdU) in IMDM containing 0.1% FCS for 48 h. After incubation, EdU was detected using Click-iT® EdU detection reagent (Thermo Fisher Scientific, Waltham, MA) according to the manufacture's protocol and cells were immunostained with cardiac troponin T antibody (Thermo Fisher Scientific) to label CMs. To detect CMs in M phase, cells were stained with antibodies against troponin I (Abcam, 92
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Fig. 2. The effect of TT-10 on other properties of hiPSCMs. (A) Gene expression analysis of hiPSCMs after the drug treatment. hiPSCMs were treated with 10 μmol/l TAZ-12 or TT-10 or 1 μmol/l BIO, and RNA was extracted 48 h after drug administration. RNA expression was analyzed by quantitative real time-PCR (n = 4). *; p < .05, **; p < .01 vs. control (vehicle only). The primer sequences are listed in Supplementary Table 1. (B) (Left) Scatter plot showing the correlation between the control and TT-10 treatment in average expression levels. Upregulated, downregulated, and structural and functional genes are colored red, blue, and orange, respectively. An analysis of these structural and functional genes is shown in Fig. 2A. (Right) List of the most enriched GO terms among upregulated and downregulated genes. (C) Expression of cell cycle-related genes. hiPSCMs were treated with vehicle or 10 μmol/l TT-10, and RNA was extracted 48 h after the drug administration. The RNA expression was analyzed by quantitative real-time PCR (n = 4). **; p < .01 vs. control (vehicle only). (D) Motion analysis of hiPSCMs using the SI8000 Cell Motion Imaging System. (Upper left); hiPSCMs forming a beating sheet. The beating was captured as video. (Upper right); Motion vectors were detected, and the magnitude of each dot is displayed in color. (Lower); Analysis of beating cycle and quantification of parameters. CV, contraction velocity (μm/s); RV, relaxation velocity (μm/s); DD, deformation distance (μm). (E) The effect of TT-10 on the contractile properties of hiPSCMs. hiPSCMs were treated with 10 μmol/ l TAZ-12 or TT-10 or 1 μmol/l BIO, and contractile properties were analyzed using the SI8000 Cell Motion Imager. Measurement was performed before and 30 min after the drug administration. Each parameter was normalized by the value of the control well (n = 3–6). NS; no significant difference. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
not show significant impairment of the CV and DD. These results support the notion that TT-10 does not unfavorably affect the contractile function of hiPSCMs.
hiPSCMs after the treatment (Fig. 1G). In addition, we also performed MTS cell viability assays and confirmed that TT-10 did not show cytotoxicity; conversely, it revealed an increase in cellular viability (Fig. 1H), which may result from cell proliferation. These results collectively indicate that TT-10 can activate the cell cycle and promote the proliferation of hiPSCMs. In order to clarify the mechanism by which TT-10 promoted the cell cycle of hiPSCMs, we evaluated changes in the YAP-TEAD pathway after treatment. TT-10 enhanced TEAD promoter activities (Supplementary Fig. 3A) and promoted the nuclear translocation of YAP in hiPSCMs (Supplementary Fig. 3B). These results collectively indicate that TT-10 enhances hiPSCM proliferation, at least in part, via activation of the YAP-TEAD pathway.
4. Discussion
We next examined the gene expression of hiPSCMs after the 48-h treatments with TAZ-12, TT-10, or BIO. We first compared the expression levels of sarcomeric genes, including MYH6, MYH7, TNNI1, TNNI3, and MYL2. The expression of these genes did not show significant responses to TT-10 treatment (Fig. 2A). As TNNI1 (the slow skeletal type of troponin I) and TNNI3 (a cardiac isoform of troponin I) are the markers that are preferentially expressed in fetal and mature CMs respectively [12], our findings suggest that TT-10 would not affect the maturity of hiPSCMs. In contrast, the expression level of MYH7 was significantly reduced after the treatment with BIO, suggesting that BIO would induce immaturity of CMs. We also checked the expression levels of ATP2A2, CACNA1C, and RYR2, which are all important endoplasmic reticulum components. The expression levels of these genes were also not affected by treatment with TT-10 (Fig. 2A). Importantly, BIO significantly reduced the expression level of these genes (Fig. 2A), suggesting the toxicity of BIO to hiPSCMs. These results indicate that TT-10 does not show unfavorable effects on the expression of structural and functional molecules in CMs. To further analyze the effect of TT-10, we performed transcriptome analysis using hiPSCMs after treatment with TT-10 (Fig. 2B). Gene Ontology (GO) enrichment analysis revealed that genes related to cell division were most significantly upregulated. Quantitative real timePCR of genes related to the cell cycle (AURKA, CCNA2, and CCNB1) consistently showed that TT-10 enhanced their expression levels (Fig. 2C). These results support the cell-proliferative effect of TT-10.
The induction of hiPSCM cell cycles via gene transfection techniques has already been investigated by several research groups. Dietz-Cunado et al. [14] screened miRNAs that could increase proliferation of hiPSCMs and found that those identified miRNAs targeted Hippo pathway genes. Zhu et al. [15] also demonstrated that overexpression of cyclin D2 in hiPSCMs enhanced the regenerative capabilities when engrafted in infarct myocardium of mice. However, it is preferable to use small molecules for translational purposes as they can be produced inexpensively with high reproducibility and under xeno-free conditions, which conforms to good pharmaceutical production practices. Few studies have focused on functional and physiological effects other than the promotion of proliferative capacity of identified cell cycle stimulators. Bassat et al. [16] demonstrated that the extra cellular matrix protein Agrin can promote the division of hiPSCMs; however, Agrin also decreased the conduction velocity of CMs and reduced the expression of functional and maturation genes. Our results highlight a translational advantage, in that unfavorable effects on function or maturation were not observed after TT-10 treatment, though longerterm effects of the compound should be analyzed. The precise mechanisms by which TT-10 promoted the hiPSCM proliferation were not analyzed in the present study. In a previous study [4], we revealed that TT-10 not only activated the YAP-TEAD axis, but also had crosstalk with Wnt signaling via mild inhibition of GSK3β. Furthermore, TT-10 upregulated nuclear factor erythroid 2-related factor 2 (NRF-2) and enhanced antioxidant responses in vitro. Further studies are necessary to reveal the effects of TT-10 on Wnt/β-catenin and Hippo signaling pathways in hiPSCMs. In our previous work [4], we demonstrated that TT-10 promoted the cell cycle of adult CMs and had a protective effect on cardiac function after MI in an in vivo mice model. Taken together, the findings of the previous and current study indicate that TT-10 is a highly promising compound for applications in clinical setting for human disease. The present study advances translational research utilizing the novel TT-10 molecule and serves as the basis for its potential clinical application in MI treatment.
3.3. The effect of TT-10 on the contractile function of hiPSCMs
Disclosures
3.2. The effect of TT-10 on gene expression level in hiPSCMs
None.
Promotion of the cell cycle does not necessarily improve cardiac function. We next tested the effect of TT-10 and BIO on the contractile function of hiPSCMs. We created homogeneously beating hiPSCM sheets and analyzed their contractile function using a Cell Motion Analyzer® (SONY) after TAZ-12, TT-10, or BIO treatment (Fig. 2D). Recently, this device has been more frequently used to analyze contractile properties of iPSCMs [13]. The beating rate, CV, RV, and DD were all unaffected by the treatment with TT-10 (Fig. 2E). BIO also did
Funding This work was supported by grants-in-aid for Scientific Research, Japan (17K15987 to H.H., 15K09132 to N.T.); a grant-in-aid from the Kanae Foundation for the Promotion of Medical Science, Japan (to N.T.); grants-in-aid from the SENSHIN Medical Research Foundation, 94
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Japan (to H.H. and N.T.); grants-in-aid from the Japan Foundation for Applied Enzymology, Japan (to M.I., H.H., A.T.N., and N.T.); a grant-inaid from the Japan Heart Foundation, Japan (to A.T.N. and N.T.); a grant-in-aid from The Tokyo Society of Medical Sciences, Japan (to N.T.); a grant-in-aid from the Takeda Science Foundation, Japan; a grant-in-aid from The Fugaku Trust for Medical Research, Japan (to N.T.); a Sakakibara Memorial Research Grant from The Japan Research Promotion Society for Cardiovascular Diseases, Japan (to H.H.); a grant-in-aid from the Agency for Medical Research and Development, Japan, AMED (JP16bm0609004 to A.T.N. and I.K.; JP17am0101122 to A.T.N.).
[5]
[6]
[7]
[8]
Declarations of interest None.
[9]
Acknowledgments [10]
We would like to thank Takizawa R, Yokota Y, Matsuyama K, and Naito M for their technical assistance.
[11]
Appendix A. Supplementary data
[12]
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.yjmcc.2019.01.020.
[13]
References [14]
[1] O. Bergmann, R.D. Bhardwaj, S. Bernard, S. Zdunek, F. Barnabe-Heider, S. Walsh, et al., Evidence for cardiomyocyte renewal in humans, Science (New York, N.Y.) 324 (2009) 98–102. [2] H. Hara, N. Takeda, I. Komuro, Pathophysiology and therapeutic potential of cardiac fibrosis, Inflamm. Regen. 37 (2017) 13. [3] M. Xin, Y. Kim, L.B. Sutherland, M. Murakami, X. Qi, J. McAnally, et al., Hippo pathway effector Yap promotes cardiac regeneration, Proc. Natl. Acad. Sci. U. S. A. 110 (2013) 13839–13844. [4] H. Hara, N. Takeda, M. Konodo, M. Kubota, T. Saito, J. Maruyama, T. Fujiwara,
[15]
[16]
95
S. Maemura, M. Ito, A.T. Naito, M. Harada, H. Toko, S. Nomura, H. Kumagai, Y. Ikeda, H. Ueno, E. Takimoto, H. Akazawa, H. Morita, H. Aburatani, Y. Hata, M. Uchiyama, I. Komuro, Discovery of a small molecule to increase cardiomyocytes and rotect the heart after ischemic injury, JACC: Basic Trans. Sci. 3 (2018) 639–653. A.S. Smith, J. Macadangdang, W. Leung, M.A. Laflamme, D.H. Kim, Human iPSCderived cardiomyocytes and tissue engineering strategies for disease modeling and drug screening, Biotechnol. Adv. 35 (2017) 77–94. Y. Shiba, T. Gomibuchi, T. Seto, Y. Wada, H. Ichimura, Y. Tanaka, et al., Allogeneic transplantation of iPS cell-derived cardiomyocytes regenerates primate hearts, Nature 538 (2016) 388–391. K. Okita, T. Yamakawa, Y. Matsumura, Y. Sato, N. Amano, A. Watanabe, et al., An efficient nonviral method to generate integration-free human-induced pluripotent stem cells from cord blood and peripheral blood cells, Stem Cells 31 (2013) 458–466 (Dayton, Ohio). M. Boerma, C.G. van der Wees, J. Wondergem, A. van der Laarse, M. Persoon, A.A. van Zeeland, et al., Separation of neonatal rat ventricular myocytes and nonmyocytes by centrifugal elutriation, Pflugers Arch. - Eur. J. Physiol. 444 (2002) 452–456. H. Uosaki, A. Magadum, K. Seo, H. Fukushima, A. Takeuchi, Y. Nakagawa, et al., Identification of chemicals inducing cardiomyocyte proliferation in developmental stage-specific manner with pluripotent stem cells, Circ. Cardiovasc. Genet. 6 (2013) 624–633. T. Novoyatleva, A. Sajjad, D. Pogoryelov, C. Patra, R.T. Schermuly, F.B. Engel, FGF1-mediated cardiomyocyte cell cycle reentry depends on the interaction of FGFR-1 and Fn14, FASEB J. 28 (2014) 2492–2503. K. Bersell, S. Arab, B. Haring, B. Kuhn, Neuregulin1/ErbB4 signaling induces cardiomyocyte proliferation and repair of heart injury, Cell 138 (2009) 257–270. F.B. Bedada, S.S. Chan, S.K. Metzger, L. Zhang, J. Zhang, D.J. Garry, et al., Acquisition of a quantitative, stoichiometrically conserved ratiometric marker of maturation status in stem cell-derived cardiac myocytes, Stem Cell Rep. 3 (2014) 594–605. T. Hayakawa, T. Kunihiro, T. Ando, S. Kobayashi, E. Matsui, H. Yada, et al., Imagebased evaluation of contraction-relaxation kinetics of human-induced pluripotent stem cell-derived cardiomyocytes: correlation and complementarity with extracellular electrophysiology, J. Mol. Cell. Cardiol. 77 (2014) 178–191. M. Diez-Cunado, K. Wei, P.J. Bushway, M.R. Maurya, R. Perera, S. Subramaniam, et al., miRNAs that induce human cardiomyocyte proliferation converge on the Hippo pathway, Cell Rep. 23 (2018) 2168–2174. W. Zhu, M. Zhao, S. Mattapally, S. Chen, J. Zhang, CCND2 overexpression enhances the regenerative potency of human induced pluripotent stem cell-derived cardiomyocytes: remuscularization of injured ventricle, Circ. Res. 122 (2018) 88–96. E. Bassat, Y.E. Mutlak, A. Genzelinakh, I.Y. Shadrin, K. Baruch Umansky, O. Yifa, et al., The extracellular matrix protein agrin promotes heart regeneration in mice, Nature 547 (2017) 179–184.
Contents lists available at ScienceDirect
Journal of Molecular and Cellular Cardiology journal homepage: www.elsevier.com/locate/yjmcc
Short communication
Characterization of a small molecule that promotes cell cycle activation of human induced pluripotent stem cell-derived cardiomyocytes
T
Masamichi Itoa,1, Hironori Haraa,1, Norifumi Takedaa, Atsuhiko T. Naitob, Seitaro Nomuraa, ⁎ Masaki Kondoc, Yutaka Hatad, Masanobu Uchiyamac,e, Hiroyuki Moritaa, Issei Komuroa, a
Department of Cardiovascular Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, Japan Department of Pharmacology, Faculty of Medicine, Toho University, 5-21-16 Omori-nishi, Ohta-ku, Tokyo, Japan c Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, Tokyo, Japan d Department of Medical Biochemistry, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo, Japan e Advanced Elements Chemistry Research Team, RIKEN Center for Sustainable Resource Science, and Elements Chemistry Laboratory, RIKEN, 2-1, Hirosawa, Wako-city, Saitama, Japan b
A R T I C LE I N FO
A B S T R A C T
Keywords: Cell cycle activation Induced pluripotent stem cell-derived cardiomyocyte Hippo pathway Regeneration Small molecule Regenerative medicine
Background: Since regenerative capacity of adult mammalian myocardium is limited, activation of the endogenous proliferative capacity of existing cardiomyocytes is a potential therapeutic strategy for treating heart diseases accompanied by cardiomyocyte loss. Recently, we performed a compound screening and developed a new drug named TT-10 (C11H10FN3OS2) which promotes the proliferation of murine cardiomyocytes via enhancement of YES-associated protein (YAP)-transcriptional enhancer factor domain (TEAD) activity and improves cardiac function after myocardial infarction in adult mice. Methods and results: To test whether TT-10 can also promote the proliferative capacity of human cardiomyocytes, we investigated the efficacy of TT-10 on human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (hiPSCMs). The hiPSCs were established from monocytes obtained from healthy donors and cardiac differentiation was performed using a chemically defined protocol. As was observed in murine cardiomyocytes, TT-10 markedly promoted cell cycle activation and increased cell division of hiPSCMs. We then evaluated other effects of TT-10 on the functional properties of hiPSCMs by gene expression and cell motion analyses. We observed that TT-10 had no unfavorable effects on the expression of functional and structural genes or the contractile properties of hiPSCMs. Conclusions: Our results suggest that the novel drug TT-10 effectively activated the cell cycle of hiPSCMs without apparent functional impairment of myocardium, suggesting the potential of clinical usefulness of this drug.
1. Introduction The cell division capacity of mammalian cardiomyocytes (CMs) diminishes dramatically soon after birth, and it was once widely believed that adult myocardium does not regenerate after injury. However, a previous study demonstrated that CMs are replenished in the adult human heart, though the rate is very low [1]. A number of studies have focused on the underlying molecular mechanisms of CM
proliferation, with low oxygen environments, micro RNAs, extracellular matrix, and paracrine factors from non-CMs reported as key factors that stimulate CMs for cell-cycle reentry [2]. It would therefore be reasonable to activate the endogenous proliferation capacity of CMs by manipulating these factors in the treatment of heart disease caused by CM loss, including myocardial infarction (MI). The Hippo-YES-associated protein-transcriptional enhancer factor domain (Hippo-YAP-TEAD) signaling pathway regulates cell
Abbreviations: YAP, YES-associated protein; TEAD, transcriptional enhancer factor domain; hiPSC, human induced pluripotent stem cell; hiPSCM, human induced pluripotent stem cell-derived cardiomyocyte; CM, cardiomyocyte; MI, myocardial infarction; iPSCMs, induced pluripotent stem cell-derived cardiomyocytes; iPSC, induced pluripotent stem cell; IMDM, Iscove's modified Dulbecco's medium; FCS, fetal calf serum; EdU, 5-ethynyl-2′-deoxyuridine; ROI, region of interest; CV, contraction velocity; RV, relaxation velocity; DD, deformation distance; GSK3β, glycogen synthase kinase 3 beta; BIO, (2′Z, 3′E)-6-bromoindirubin-3′-oxime; FGF, fibroblast growth factor; NRG, neuregulin; GO, Gene Ontology; NRF2, nuclear factor erythroid 2-related factor 2 ⁎ Corresponding author at: Department of Cardiovascular Medicine, The University of Tokyo Graduate School of Medicine, 7-3-1 Hongo, Tokyo 113-8655, Japan. E-mail address: [email protected] (I. Komuro). 1 These authors equally contributed to the work. https://doi.org/10.1016/j.yjmcc.2019.01.020 Received 18 September 2018; Received in revised form 18 January 2019; Accepted 23 January 2019 Available online 23 January 2019 0022-2828/ © 2019 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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Fig. 1. TT-10 promotes cell division of hiPSCMs. (A) Chemical structures of TAZ-12 and TT-10. (B) Schematic outline of cardiac differentiation of hiPSCs and schedule of proliferation assay timeline. EB, embryoid body; hiPSC, human induced pluripotent stem cell. (C) A representative image of the EdU-positive hiPSCMs after treatment with TT-10. The (−) image shows a control treated with vehicle. Arrowheads indicate positive hiPSCMs. Scale bar: 100 μm. (D) Percentages of EdUpositive hiPSCMs after treatment with the indicated reagents. The percentage of EdU-positive hiPSCMs was analyzed 48 h after compound administration. (n = 5). (−) indicates the control, with vehicle alone administered (DMSO). The concentrations of the drugs are as follows: FCS, 2%; TAZ-12, 10 μmol/l; TT-10, 10 μmol/l; BIO, 1 μmol/l; NRG1, 100 ng/ml; and FGF1, 100 ng/ml. *; p < .05, ***; p < .001 vs. control (vehicle only). EdU, 5-ethynyl-2′-deoxyuridine; hiPSCM, human induced pluripotent stem cell-derived cardiomyocyte; DMSO, dimethyl sulfoxide; CMs, cardiomyocytes; FCS, fetal calf serum; BIO, (2′Z, 3′E)-6-bromoindirubin-3′oxime; NRG1, neuregulin-1; FGF1, fibroblast growth factor-1. (E) A representative image of Aurora B-positive hiPSCMs after TT-10 treatment. The (−) image shows a control without any drug treatment. Arrowheads indicate positive staining of hiPSCMs. Scale bars: 50 μm and 25 μm (in inset). (F) Percentages of Aurora B-positive hiPSCMs after treatment with 10 μmol/l TT-10. The percentages of Aurora B-positive hiPSCMs were analyzed 48 h after compound administration (n = 4). *; p < .05 vs. control (vehicle only). (G) Relative cell number of CMs 48 h after treatment with TT-10 (10 μmol/l). In the EdU assay, the absolute cell number of hiPSCMs was counted and normalized to the number of untreated CMs. n = 4; **; p < .01 vs. control (vehicle only). (H) MTS cell viability assay. Cell viability was analyzed after a 48-h incubation with TT-10. The viability was normalized to the vehicle control. The positive control was 1 mmol/l H2O2. n = 12 per group; **; p < .01, ***; p < .001 vs. control (vehicle only).
Cambridge, UK) and Aurora B kinase (BD Biosciences, Franklin Lakes, NJ). In this study, hiPSCMs were defined as troponin positive cells. Cytokinesis was defined by the presence of an Aurora B-stained spindleshaped structure in the middle of the intercellular bridge. Stained cells were visualized and evaluated using the Operetta High-Content Imaging System (PerkinElmer, Waltham, MA). Brightness and contrast were adjusted linearly across the entirety of each image.
proliferation, apoptosis, and stem cell self-renewal and expansion. Activated nuclear YAP in CMs promotes cardiac regeneration after injury [3]. We recently performed a large-scale cell-based compound screening using a cell-based reporter assay for YAP-TEAD activators and a proliferation assay using neonatal rat CMs. We identified a small molecule, TAZ-12, developed its fluorine substituent TT-10 (Fig. 1A) and found that TT-10 strongly promoted the proliferation of rat neonatal CMs and activated CM cell division and that it could also potently activate CM cell division in murine hearts after MI [4]. Before clinical application of this compound, its efficacy and safety must be assessed using human CMs. Today, human induced pluripotent stem cell-derived cardiomyocytes (hiPSCMs) are the only available source of human CMs and a promising tool for regenerative medicine. They have been shown to reproduce molecular mechanisms observed in diseased human hearts [5]. Furthermore, hiPSCMs are expected to be a complementary cellular source for injured heart tissue, considering transplantation of allogenic induced pluripotent stem cell-derived cardiomyocytes (iPSCMs) has been shown to improve the function of infarcted hearts in non-human primates [6]. To advance the translational research using TT-10, we examined whether the compound can activate the cell-cycle and cell division in hiPSCMs as well as murine CMs, and we also characterized the effect of TT-10 on the molecular and physiological properties of hiPSCMs.
2.3. Motion analysis of hiPSCMs To evaluate the contractile function of hiPSCMs, we used a motion vector analysis system, namely the SI8000 Cell Motion Imaging System® (SONY, Tokyo, Japan). This system enables quantitative analysis of the motion of synchronously beating hiPSCMs through video capture. The motion of each detection point was converted into a motion vector. The motion velocity within each region of interest (ROI) was calculated based on the sum of the vector magnitudes. The maximum contraction velocity (CV) is considered to correspond to the contractile function of the myocardium. Similarly, the maximum relaxation velocity (RV) is considered to correspond to the diastolic function, and the deformation distance (DD) is assumed to reflect the distance the myocardium moved within one beating cycle. Video images were taken 2 days after drug administration, and the relative changes in CV, RV, and DD were analyzed.
2. Materials and methods 3. Results 2.1. Preparation of hiPSCMs 3.1. TT-10 promotes the cell cycle of hiPSCMs Induced pluripotent stem cell (iPSC) lines were established from the peripheral blood of healthy male volunteers as previously described [7]. For cardiac differentiation, we adopted a chemically defined protocol. CMs were enriched based on the cellular density as previously described [8]. The hiPSCMs were once frozen for storage at day 28. Then, the stocks were thawed and 2 days later were passaged for assays. Four days after thawing, the medium was exchanged with Iscove's modified Dulbecco's medium (IMDM) with 0.1% fetal calf serum (FCS). On day 6, the medium was replaced with IMDM that contained test compounds. The cells were analyzed 48 h after drug administration. Thus, hiPSCMs were prepared and used for analysis around 35 days after differentiation (Fig. 1B). Details are described in the Supplementary Materials.
We first evaluated the effect of TT-10 on the proliferation of hiPSCMs using the EdU incorporation assay. The ratio of hiPSCMs that entered S phase was markedly enhanced after incubation with TT-10 for 48 h (Fig. 1C). This effect was observed among all hiPSCM lines produced from different donors (Supplementary Fig. 1). We also compared the effect of TT-10 with those of other compounds that have been reported to promote the proliferation of CMs, including glycogen synthase kinase 3 beta (GSK3β) inhibitor (2′Z, 3′E)-6-bromoindirubin-3′oxime (BIO) [9], fibroblast growth factor (FGF) -1 [10], and neuregulin (NRG) -1 [11]. BIO increased the ratio of EdU-positive hiPSCMs to a level that is comparable to that under TT-10, whereas NRG-1 and FGF-1 did not affect the proliferation of hiPSCMs (Fig. 1D). The extent of DNA synthesis promotion by TT-10 was more prominent compared to TAZ12 in one hiPSCM clone (Supplementary Fig. 1(a)) and was similar to TAZ-12 in three other hiPSCM lines (Supplementary Fig. 1(b)-(d)). We also evaluated the EdU positivity after TT-10 treatment by flow cytometry, finding that the percentage of EdU-positive CMs was increased from 6.4 ± 0.85% to 28.2% ± 3.3% after treatment with TT-10 (Supplementary Fig. 2). We next checked the effect of TT-10 on cytokinesis of hiPSCMs by staining Aurora B. The percentage of Aurora Bpositive hiPSCMs increased approximately threefold after TT-10 treatment (Fig. 1E, F). Indeed, TT-10 increased the absolute number of
2.2. Cell proliferation assay To detect cells in S phase, cells were labeled with 5 μmol/l 5ethynyl-2′-deoxyuridine (EdU) in IMDM containing 0.1% FCS for 48 h. After incubation, EdU was detected using Click-iT® EdU detection reagent (Thermo Fisher Scientific, Waltham, MA) according to the manufacture's protocol and cells were immunostained with cardiac troponin T antibody (Thermo Fisher Scientific) to label CMs. To detect CMs in M phase, cells were stained with antibodies against troponin I (Abcam, 92
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Fig. 2. The effect of TT-10 on other properties of hiPSCMs. (A) Gene expression analysis of hiPSCMs after the drug treatment. hiPSCMs were treated with 10 μmol/l TAZ-12 or TT-10 or 1 μmol/l BIO, and RNA was extracted 48 h after drug administration. RNA expression was analyzed by quantitative real time-PCR (n = 4). *; p < .05, **; p < .01 vs. control (vehicle only). The primer sequences are listed in Supplementary Table 1. (B) (Left) Scatter plot showing the correlation between the control and TT-10 treatment in average expression levels. Upregulated, downregulated, and structural and functional genes are colored red, blue, and orange, respectively. An analysis of these structural and functional genes is shown in Fig. 2A. (Right) List of the most enriched GO terms among upregulated and downregulated genes. (C) Expression of cell cycle-related genes. hiPSCMs were treated with vehicle or 10 μmol/l TT-10, and RNA was extracted 48 h after the drug administration. The RNA expression was analyzed by quantitative real-time PCR (n = 4). **; p < .01 vs. control (vehicle only). (D) Motion analysis of hiPSCMs using the SI8000 Cell Motion Imaging System. (Upper left); hiPSCMs forming a beating sheet. The beating was captured as video. (Upper right); Motion vectors were detected, and the magnitude of each dot is displayed in color. (Lower); Analysis of beating cycle and quantification of parameters. CV, contraction velocity (μm/s); RV, relaxation velocity (μm/s); DD, deformation distance (μm). (E) The effect of TT-10 on the contractile properties of hiPSCMs. hiPSCMs were treated with 10 μmol/ l TAZ-12 or TT-10 or 1 μmol/l BIO, and contractile properties were analyzed using the SI8000 Cell Motion Imager. Measurement was performed before and 30 min after the drug administration. Each parameter was normalized by the value of the control well (n = 3–6). NS; no significant difference. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
not show significant impairment of the CV and DD. These results support the notion that TT-10 does not unfavorably affect the contractile function of hiPSCMs.
hiPSCMs after the treatment (Fig. 1G). In addition, we also performed MTS cell viability assays and confirmed that TT-10 did not show cytotoxicity; conversely, it revealed an increase in cellular viability (Fig. 1H), which may result from cell proliferation. These results collectively indicate that TT-10 can activate the cell cycle and promote the proliferation of hiPSCMs. In order to clarify the mechanism by which TT-10 promoted the cell cycle of hiPSCMs, we evaluated changes in the YAP-TEAD pathway after treatment. TT-10 enhanced TEAD promoter activities (Supplementary Fig. 3A) and promoted the nuclear translocation of YAP in hiPSCMs (Supplementary Fig. 3B). These results collectively indicate that TT-10 enhances hiPSCM proliferation, at least in part, via activation of the YAP-TEAD pathway.
4. Discussion
We next examined the gene expression of hiPSCMs after the 48-h treatments with TAZ-12, TT-10, or BIO. We first compared the expression levels of sarcomeric genes, including MYH6, MYH7, TNNI1, TNNI3, and MYL2. The expression of these genes did not show significant responses to TT-10 treatment (Fig. 2A). As TNNI1 (the slow skeletal type of troponin I) and TNNI3 (a cardiac isoform of troponin I) are the markers that are preferentially expressed in fetal and mature CMs respectively [12], our findings suggest that TT-10 would not affect the maturity of hiPSCMs. In contrast, the expression level of MYH7 was significantly reduced after the treatment with BIO, suggesting that BIO would induce immaturity of CMs. We also checked the expression levels of ATP2A2, CACNA1C, and RYR2, which are all important endoplasmic reticulum components. The expression levels of these genes were also not affected by treatment with TT-10 (Fig. 2A). Importantly, BIO significantly reduced the expression level of these genes (Fig. 2A), suggesting the toxicity of BIO to hiPSCMs. These results indicate that TT-10 does not show unfavorable effects on the expression of structural and functional molecules in CMs. To further analyze the effect of TT-10, we performed transcriptome analysis using hiPSCMs after treatment with TT-10 (Fig. 2B). Gene Ontology (GO) enrichment analysis revealed that genes related to cell division were most significantly upregulated. Quantitative real timePCR of genes related to the cell cycle (AURKA, CCNA2, and CCNB1) consistently showed that TT-10 enhanced their expression levels (Fig. 2C). These results support the cell-proliferative effect of TT-10.
The induction of hiPSCM cell cycles via gene transfection techniques has already been investigated by several research groups. Dietz-Cunado et al. [14] screened miRNAs that could increase proliferation of hiPSCMs and found that those identified miRNAs targeted Hippo pathway genes. Zhu et al. [15] also demonstrated that overexpression of cyclin D2 in hiPSCMs enhanced the regenerative capabilities when engrafted in infarct myocardium of mice. However, it is preferable to use small molecules for translational purposes as they can be produced inexpensively with high reproducibility and under xeno-free conditions, which conforms to good pharmaceutical production practices. Few studies have focused on functional and physiological effects other than the promotion of proliferative capacity of identified cell cycle stimulators. Bassat et al. [16] demonstrated that the extra cellular matrix protein Agrin can promote the division of hiPSCMs; however, Agrin also decreased the conduction velocity of CMs and reduced the expression of functional and maturation genes. Our results highlight a translational advantage, in that unfavorable effects on function or maturation were not observed after TT-10 treatment, though longerterm effects of the compound should be analyzed. The precise mechanisms by which TT-10 promoted the hiPSCM proliferation were not analyzed in the present study. In a previous study [4], we revealed that TT-10 not only activated the YAP-TEAD axis, but also had crosstalk with Wnt signaling via mild inhibition of GSK3β. Furthermore, TT-10 upregulated nuclear factor erythroid 2-related factor 2 (NRF-2) and enhanced antioxidant responses in vitro. Further studies are necessary to reveal the effects of TT-10 on Wnt/β-catenin and Hippo signaling pathways in hiPSCMs. In our previous work [4], we demonstrated that TT-10 promoted the cell cycle of adult CMs and had a protective effect on cardiac function after MI in an in vivo mice model. Taken together, the findings of the previous and current study indicate that TT-10 is a highly promising compound for applications in clinical setting for human disease. The present study advances translational research utilizing the novel TT-10 molecule and serves as the basis for its potential clinical application in MI treatment.
3.3. The effect of TT-10 on the contractile function of hiPSCMs
Disclosures
3.2. The effect of TT-10 on gene expression level in hiPSCMs
None.
Promotion of the cell cycle does not necessarily improve cardiac function. We next tested the effect of TT-10 and BIO on the contractile function of hiPSCMs. We created homogeneously beating hiPSCM sheets and analyzed their contractile function using a Cell Motion Analyzer® (SONY) after TAZ-12, TT-10, or BIO treatment (Fig. 2D). Recently, this device has been more frequently used to analyze contractile properties of iPSCMs [13]. The beating rate, CV, RV, and DD were all unaffected by the treatment with TT-10 (Fig. 2E). BIO also did
Funding This work was supported by grants-in-aid for Scientific Research, Japan (17K15987 to H.H., 15K09132 to N.T.); a grant-in-aid from the Kanae Foundation for the Promotion of Medical Science, Japan (to N.T.); grants-in-aid from the SENSHIN Medical Research Foundation, 94
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Japan (to H.H. and N.T.); grants-in-aid from the Japan Foundation for Applied Enzymology, Japan (to M.I., H.H., A.T.N., and N.T.); a grant-inaid from the Japan Heart Foundation, Japan (to A.T.N. and N.T.); a grant-in-aid from The Tokyo Society of Medical Sciences, Japan (to N.T.); a grant-in-aid from the Takeda Science Foundation, Japan; a grant-in-aid from The Fugaku Trust for Medical Research, Japan (to N.T.); a Sakakibara Memorial Research Grant from The Japan Research Promotion Society for Cardiovascular Diseases, Japan (to H.H.); a grant-in-aid from the Agency for Medical Research and Development, Japan, AMED (JP16bm0609004 to A.T.N. and I.K.; JP17am0101122 to A.T.N.).
[5]
[6]
[7]
[8]
Declarations of interest None.
[9]
Acknowledgments [10]
We would like to thank Takizawa R, Yokota Y, Matsuyama K, and Naito M for their technical assistance.
[11]
Appendix A. Supplementary data
[12]
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.yjmcc.2019.01.020.
[13]
References [14]
[1] O. Bergmann, R.D. Bhardwaj, S. Bernard, S. Zdunek, F. Barnabe-Heider, S. Walsh, et al., Evidence for cardiomyocyte renewal in humans, Science (New York, N.Y.) 324 (2009) 98–102. [2] H. Hara, N. Takeda, I. Komuro, Pathophysiology and therapeutic potential of cardiac fibrosis, Inflamm. Regen. 37 (2017) 13. [3] M. Xin, Y. Kim, L.B. Sutherland, M. Murakami, X. Qi, J. McAnally, et al., Hippo pathway effector Yap promotes cardiac regeneration, Proc. Natl. Acad. Sci. U. S. A. 110 (2013) 13839–13844. [4] H. Hara, N. Takeda, M. Konodo, M. Kubota, T. Saito, J. Maruyama, T. Fujiwara,
[15]
[16]
95
S. Maemura, M. Ito, A.T. Naito, M. Harada, H. Toko, S. Nomura, H. Kumagai, Y. Ikeda, H. Ueno, E. Takimoto, H. Akazawa, H. Morita, H. Aburatani, Y. Hata, M. Uchiyama, I. Komuro, Discovery of a small molecule to increase cardiomyocytes and rotect the heart after ischemic injury, JACC: Basic Trans. Sci. 3 (2018) 639–653. A.S. Smith, J. Macadangdang, W. Leung, M.A. Laflamme, D.H. Kim, Human iPSCderived cardiomyocytes and tissue engineering strategies for disease modeling and drug screening, Biotechnol. Adv. 35 (2017) 77–94. Y. Shiba, T. Gomibuchi, T. Seto, Y. Wada, H. Ichimura, Y. Tanaka, et al., Allogeneic transplantation of iPS cell-derived cardiomyocytes regenerates primate hearts, Nature 538 (2016) 388–391. K. Okita, T. Yamakawa, Y. Matsumura, Y. Sato, N. Amano, A. Watanabe, et al., An efficient nonviral method to generate integration-free human-induced pluripotent stem cells from cord blood and peripheral blood cells, Stem Cells 31 (2013) 458–466 (Dayton, Ohio). M. Boerma, C.G. van der Wees, J. Wondergem, A. van der Laarse, M. Persoon, A.A. van Zeeland, et al., Separation of neonatal rat ventricular myocytes and nonmyocytes by centrifugal elutriation, Pflugers Arch. - Eur. J. Physiol. 444 (2002) 452–456. H. Uosaki, A. Magadum, K. Seo, H. Fukushima, A. Takeuchi, Y. Nakagawa, et al., Identification of chemicals inducing cardiomyocyte proliferation in developmental stage-specific manner with pluripotent stem cells, Circ. Cardiovasc. Genet. 6 (2013) 624–633. T. Novoyatleva, A. Sajjad, D. Pogoryelov, C. Patra, R.T. Schermuly, F.B. Engel, FGF1-mediated cardiomyocyte cell cycle reentry depends on the interaction of FGFR-1 and Fn14, FASEB J. 28 (2014) 2492–2503. K. Bersell, S. Arab, B. Haring, B. Kuhn, Neuregulin1/ErbB4 signaling induces cardiomyocyte proliferation and repair of heart injury, Cell 138 (2009) 257–270. F.B. Bedada, S.S. Chan, S.K. Metzger, L. Zhang, J. Zhang, D.J. Garry, et al., Acquisition of a quantitative, stoichiometrically conserved ratiometric marker of maturation status in stem cell-derived cardiac myocytes, Stem Cell Rep. 3 (2014) 594–605. T. Hayakawa, T. Kunihiro, T. Ando, S. Kobayashi, E. Matsui, H. Yada, et al., Imagebased evaluation of contraction-relaxation kinetics of human-induced pluripotent stem cell-derived cardiomyocytes: correlation and complementarity with extracellular electrophysiology, J. Mol. Cell. Cardiol. 77 (2014) 178–191. M. Diez-Cunado, K. Wei, P.J. Bushway, M.R. Maurya, R. Perera, S. Subramaniam, et al., miRNAs that induce human cardiomyocyte proliferation converge on the Hippo pathway, Cell Rep. 23 (2018) 2168–2174. W. Zhu, M. Zhao, S. Mattapally, S. Chen, J. Zhang, CCND2 overexpression enhances the regenerative potency of human induced pluripotent stem cell-derived cardiomyocytes: remuscularization of injured ventricle, Circ. Res. 122 (2018) 88–96. E. Bassat, Y.E. Mutlak, A. Genzelinakh, I.Y. Shadrin, K. Baruch Umansky, O. Yifa, et al., The extracellular matrix protein agrin promotes heart regeneration in mice, Nature 547 (2017) 179–184.