2100) for prediction of drug-induced proarrhythmia using human iPS cell-derived cardiomyocytes: Assessment of reference compounds and comparison with non-clinical studies and clinical information

Regulatory Toxicology and Pharmacology 88 (2017) 238e251

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CSAHi study-2: Validation of multi-electrode array systems (MEA60/ 2100) for prediction of drug-induced proarrhythmia using human iPS cell-derived cardiomyocytes: Assessment of reference compounds and comparison with non-clinical studies and clinical information Yumiko Nozaki a, Yayoi Honda a, Hitoshi Watanabe a, Shota Saiki b, i, Kiyotaka Koyabu b, Tetsuji Itoh b, Chiho Nagasawa c, i, Chiaki Nakamori c, Chiaki Nakayama c, Hiroshi Iwasaki c, Shinobu Suzuki d, Kohji Tanaka d, Etsushi Takahashi e, i, Kaori Miyamoto e, Kaoru Morimura e, Atsuhiro Yamanishi f, i, Hiroko Endo f, Junko Shinozaki f, Hisashi Nogawa f, Tadahiro Shinozawa g, i, j, Fumiyo Saito h, i, Takeshi Kunimatsu a, i, j, * a

Preclinical Research Laboratories, Sumitomo Dainippon Pharma Co., Ltd., 3-1-98 Kasugade-naka, Konohana-ku, Osaka 554-0022, Japan Research Laboratory for Development, Shionogi & Co., Ltd., 3-1-1, Futaba-cho, Toyonaka, Osaka 561-0825, Japan c Drug Safety, Taisho Pharmaceutical Co., Ltd., 1-403, Yoshino-cho, Kita-ku, Saitama-shi, Saitama 331-9530, Japan d Nippon Boehringer Ingelheim Co., Ltd., 6-7-5, Minatojima-Minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan e Research Laboratories, Toyama Chemical Co., Ltd., 4-1, Shimookui 2-chome, Toyama 930-8508, Japan f Toxicology Research Laboratory, Kyorin Pharmaceutical Co., Ltd., 1848, Nogi, Nogi-machi, Shimotsuga-gun, Tochigi 329-0114, Japan g Drug Safety Research Laboratories, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-chome Fujisawa, Kanagawa 251-8555, Japan h Chemicals Assessment and Research Center, Chemicals Evaluation and Research Institute, Japan (CERI), 1600, Shimotakano, Sugito-machi, Kitakatsushikagun, Saitama 345-0043, Japan i Consortium for Safety Assessment using Human iPS Cells (CSAHi), Japan j Japan Pharmaceutical Manufacturers Association, Drug Evaluation Committee, Non-Clinical Evaluation Expert Committee, Japan b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 15 November 2016 Received in revised form 15 June 2017 Accepted 15 June 2017 Available online 17 June 2017

With the aim of reconsidering ICH S7B and E14 guidelines, a new in vitro assay system has been subjected to worldwide validation to establish a better prediction platform for potential drug-induced QT prolongation and the consequent TdP in clinical practice. In Japan, CSAHi HEART team has been working on hiPS-CMs in the MEA (hiPS-CMs/MEA) under a standardized protocol and found no inter-facility or lotto-lot variability for proarrhythmic risk assessment of 7 reference compounds. In this study, we evaluated the responses of hiPS-CMs/MEA to another 31 reference compounds associated with cardiac toxicities, and gene expression to further clarify the electrophysiological characteristics over the course of culture period. The hiPS-CMs/MEA assay accurately predicted reference compounds potential for arrhythmogenesis, and yielded results that showed better correlation with target concentrations of QTc prolongation or TdP in clinical setting than other current in vitro and in vivo assays. Gene expression analyses revealed consistent profiles in all samples within and among the testing facilities. This report would provide CiPA with informative guidance on the use of the hiPS-CMs/MEA assay, and promote the establishment of a new paradigm, beyond conventional in vitro and in vivo assays for cardiac safety assessment of new drugs. © 2017 Elsevier Inc. All rights reserved.

Keywords: CSAHi CiPA hiPS-CMs FPD TdP QT prolongation EAD or TA Arrest

Abbreviations: APD, Action potential duration; CSAHi, Consortium for Safety Assessment using Human iPS Cells; CiPA, Comprehensive in vitro Proarrhythmia Assay; EAD, Early after depolarization; FPD, Field potential duration; hiPS-CMs, human induced pluripotent stem cells-derived cardiomyocytes; MEA, Multi-electrode array; TA, Triggered activity; TdP, Torsade de Pointes; VF, Ventricular fibrillation. * Corresponding author. Preclinical Research Laboratories, Sumitomo Dainippon Pharma Co., Ltd., 3-1-98 Kasugade-naka, Konohana-ku, Osaka 554-0022, Japan. E-mail addresses: [email protected] (Y. Nozaki), [email protected] (T. Kunimatsu). http://dx.doi.org/10.1016/j.yrtph.2017.06.006 0273-2300/© 2017 Elsevier Inc. All rights reserved.

Y. Nozaki et al. / Regulatory Toxicology and Pharmacology 88 (2017) 238e251

1. Introduction Drug proarrhythmic risk assessment constitutes a critical safety pharmacology evaluation tool, because proarrhythmia accompanied by Torsade de Pointes (TdP) may lead to sudden death. Determinants of clinical proarrhythmic risk will be proposed in the upcoming revised ICH S7B and E14 guidelines based on scores from 1) functional effects on multiple cardiac ion channel currents, 2) in silico cellular simulations and 3) integrated human cellular studies (PT Sager et al., 2014). In Japan, ‘the Consortium for Safety Assessment using Human iPS Cells (CSAHi)’, which was established by the Non-Clinical Evaluation Expert Committee of Japan Pharmaceutical Manufacturers Association, has previously reported that assessment of field potentials (FPs) obtained from human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) using multi-electrode array (hiPS-CMs/MEA) can potentially be replaced with current in vitro and in vivo assay in new drug cardiac safety assessment (Y Nozaki et al., 2016). The hiPS-CMs/MEA assay reveals no apparent inter-facility or lot-to-lot variability when conducted under the standardized protocol for proarrhythmic risk assessment of representative hERG, KvLQT1, Na, Ca and multi-ion channel blockers. In view of a subsequent validation for the hiPS-CMs/MEA assay, 5 Japanese pharmaceutical companies in the CSAHi additionally assessed the effects of 31 reference compounds on multiple endpoints including beat rate, corrected field potential duration (FPDc), 10% prolongation of FPDc (FPDc10) and generation of arrhythmialike waveforms. Reference compounds that induce various effects on cardiac multi-ion channels, cardiac contraction and cardiac morphology were selected to achieve comprehensive validation. Furthermore, to characterize possible variability in electrophysiological profiles over the course of the culture period or among the test facilities, genes expression profiles were determined using microarray analysis on Day 1, and Days 13, 14 or 15 after thawing hiPS-CMs in 3 testing facilities. In this set of the 2nd validation studies, we concluded that the use of hiPS-CMs/MEA assay allows effective detection of arrhythmogenesis potential. Thus, an overall validation study of hiPS-CMs/ MEA assay that evaluates a total of 38 compounds, including the 7 compounds reported in our previous report (Y Nozaki et al., 2016), would provide informative guidance to CiPA initiative for establishment of new paradigm in non-clinical safety assessment of new drugs cardiac liability. 2. Materials and methods 2.1. Test compounds and vehicle The effects on FP were examined for a total of 31 compounds (Table 1). These compounds were selected on the basis of their ability to evoke cardiac liabilities through cardiac ion channels, autonomic nervous systems and other cardiac mechanisms. Those with effects on cardiac ion channels were domperidone, NS-1643, mibefradil, ranolazine, alfuzosin, mexiletine, cisapride, astemizole, pimozide, chlorpromazine, clozapine, dl-sotalol, azimilide, bepridil, amiodarone (Sigma-Aldrich Co. LLC., USA), Bay K 8644, ZD 7288, levcromakalim (Abcam, Japan), thioridazine (MP biomedicals, USA), quinidine (Wako Pure Chemicals Industries, Ltd., Japan), dofetilide, crizotinib (Tocris Bioscience, BK), ibutilide, loratadine (Tokyo Chemical Industry Co., Ltd., Japan), and sunitinib (Cayman Chemicals, USA). Those with effects on autonomic nervous systems were propranolol (Sigma-Aldrich Co. LLC.), isoproterenol (LKT Laboratories, Inc, USA), and carbachol (Tokyo Chemical Industry Co., Ltd.). Those with effects on other cardiac mechanisms were ouabain octahydrate, glycyrrhizic acid (Wako), and

239

blebbistatin (Tronto Research Chemicals, Canada). To prepare stock solutions, each compound was dissolved in dimethyl sulfoxide (DMSO) at 1000-fold the testing concentration. 2.2. hiPS-CMs/MEA assay 2.2.1. Cell culture and plating Cells culture and re-plating onto MEA dishes were performed as previously reported (Y Nozaki et al., 2016). Briefly, commercially available iCell® Cardiomyocytes [Cellular Dynamics International (CDI), USA] were cultured according to a modified protocol from that provided by CDI. The cells were thawed in Plating Medium (CDI), seeded onto 6-well tissue culture plates coated with 0.1% Gelatin (Sigma-Aldrich Co. LLC.), and incubated in a humidified incubator with 5% CO2 at 37
C for 48 h. After carefully washing the cells with a Maintenance Medium (CDI) to rinse off debris, the plated cells were maintained for an additional 5 days during which the Maintenance Medium was replaced every 2 or 3 days. The cells were then dissociated from the 6-well plates using TrypLE™ Select (Life technologies, Japan), and reseeded onto 50 mg/ mL fibronectin-coated wells of MEA dishes (60MEA200/30iR-Ti or 60-6wellMEA200/30iR-Ti-tcr, Multi Channel Systems, Germany) at a density of 2.5  104 cells in a 2 mL bead of cell suspension per well. After incubation at 37
C (5% CO2) for 1e3 h, each well was filled with the Maintenance Medium, which was subsequently renewed every other day throughout the 7e10 days culture period. 2.2.2. FP recording Following an equilibration period of about 20 min, FPs were recorded on spontaneously beating iCell® Cardiomyocytes incubated at 37
C (5% CO2) using an MEA60 or MEA2100 data acquisition system (MC-Rack, Multi Channel Systems) with second lowpass filter set at 3000e3500 Hz. Each test solution or vehicle (0.1% DMSO) was directly added into each well, and the medium was gently stirred with a pipette to avoid uneven distribution. Test solutions were applied every 10 min to each recording well in a graded concentration manner. The final concentration of DMSO in the Maintenance Medium reached up to 0.5%. Each compound was tested in at least 4 wells. 2.2.3. Data analyses Data acquisition system was standardized with a 0.1 Hz high pass filter. The digital data obtained from hiPS-CMs/MEA assay were filtered over the 0.1 Hz filter to eliminate electrical noise, and the waveform was generated for analysis with LabChart® (AD instruments, USA). As same as our previous report, FPDs were measured for the vehicle and all test compounds at various concentrations from the last 30 beats of a 10 min exposure and corrected with inter-spike interval (ISI) according to Fridericia's formula (FPDc ¼ FPD/ISI1/3) (Y Nozaki et al., 2016). The concentration inducing FPDc10 was calculated by logistic analysis with GraphPad Prism (GraphPad Software, USA). Incidences of arrhythmia-like waveforms, including arrhythmia waveform (EAD: Early after depolarization or TA: Triggered activity) and beating arrest, were also obtained. 2.3. Global gene expression analysis 2.3.1. RNA isolation In total, 11 RNA samples were extracted from iCell® Cardiomyocytes 1 day after thawing (Day 1) and just before FP measurement (Day 13, 14 or 15) in three laboratories. Total RNA was purified using miRNeasy Micro Kit (Qiagen, Germany) according to the manufacturer's protocol. Total RNA yield was assessed spectrophotometrically, and RNA quality was determined by automated

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Table 1 Effects of reference compounds on beat rate, FPDc and the incidence of arrhythmia-like waveforms. Drug

Mechanism

Ion channel information

Concentration (nM)

Beat rate (%)

FPDc (%)

FPDc10 (nM)

Arrhythmia-like waveforms (type and incidence)

hERG channel blocker or activator

Domperidone

Gastrointestinal prokinetic agent

hERG channel block

10 30 100 300

3.13 ± 1.99 5.64 ± 1.95 9.43 ± 5.17 14.85

5.10 ± 1.43 10.84 ± 2.34 24.51 ± 4.10 36.22

27.33

e e e EAD or TA 3/4

NS-1643

hERG channel activator

hERG channel activation

3 (mM) 10 30 100

1.92 ± 2.64 8.95 ± 11.50 17.23 ± 10.23 20.25

1.40 ± 1.06 0.76 ± 2.92 3.03 ± 5.65 14.19

e

e e e Arrest 2/5 Unclear T 1/5

Ranolazine

Antianginal drug

late Na current inhibition and hERG channel block

0.3 (mM) 1 3 10

1.35 ± 0.65 1.95 ± 1.24 1.73 ± 2.65 2.70 ± 2.03

1.41 ± 0.68 4.06 ± 1.47 9.40 ± 1.63 19.47 ± 3.38

3.63 (mM)

e e e e

Mexiletine

Anti-arrhythmic drug (Class Ib)

late Na current inhibition and hERG channel block

1 (mM) 3 10 30

3.37 ± 3.31 4.73 ± 4.91 8.92 ± 2.78 24.74

2.61 ± 1.00 6.02 ± 1.42 11.73 ± 2.55 19.42

6.77 (mM)

e e e Arrest 3/4

Alfuzosin

Medicine for prostate hypertrophy

late Na current enhancement and hERG channel block

30 100 300 1000

3.02 ± 3.20 3.91 ± 6.20 2.71 ± 5.79 1.77

3.88 ± 1.02 6.43 ± 2.35 14.07 ± 3.77 25.57

190.31

e e e EAD or TA 3/5

Ca channel activator

Bay K 8644

L-type calcium channel agonist

Ca (L-type) channel activation

0.1 0.3 1 3

4.24 ± 5.61 4.44 ± 7.96 2.62 ± 8.95 3.04 ± 10.48

2.38 ± 1.46 6.52 ± 1.95 15.17 ± 3.86 29.10 ± 7.43

0.63

e e e e

Multi-ion channel blockers

Mibefradil

Antianginal drug

T-type Ca > L-type Ca  hERG  Na j KvLQT1 channel block

0.3 (mM)

3.13 ± 2.49

1.07 ± 1.50

e

e

1 3 10

15.44 ± 8.65 41.57 ± 9.39 52.54

6.38 ± 7.47 28.93 ± 16.33 59.89

hERG >> Ca >> Na channel block

10

5.45 ± 7.37

5.28 ± 7.27

30 100 300

15.59 ± 8.26 28.85 ± 22.21 e

25.12 ± 13.90 33.19 ± 4.49 e

late Na current inhibitors and enhancer

Cisapride

Gastrointestinal prokinetic agent

e e Arrest 4/6 22.55

e e EAD or TA 4/7 EAD or TA 3/3

Astemizole

Anti-allergic drug

hERG >> Ca  Na channel block

3 10 30 100

2.38 ± 2.51 4.16 ± 2.61 9.82 ± 4.91 16.63

5.10 ± 1.50 13.80 ± 4.74 33.06 ± 11.96 54.39

8.03

e e e EAD or TA 8/10

Thioridazine

Antipsychotic

hERG  Na  Ca channel block

100 300 1000 3000

2.16 ± 2.17 3.06 ± 4.26 0.35 ± 5.22 2.13 ± 2.61

1.88 ± 1.67 4.70 ± 4.00 14.89 ± 4.20 28.40 ± 7.72

701.18

e e e EAD or TA 1/5

Pimozide

Antipsychotic

hERG > Ca  Na channel block

3 10 30 100

2.98 ± 2.47 8.26 ± 2.83 12.44 ± 7.90 23.62 ± 2.81

4.66 ± 2.57 10.84 ± 4.72 22.85 ± 1.77 37.96

9.41

e e EAD or TA 1/5 EAD or TA 3/4

Y. Nozaki et al. / Regulatory Toxicology and Pharmacology 88 (2017) 238e251

Category

Antipsychotic

hERG j Na j Ca channel block

0.3 (mM) 1 3 10

1.17 ± 1.50 3.45 ± 3.12 8.78 ± 2.71 e

2.40 ± 0.57 7.03 ± 3.18 3.57 ± 6.68 e

e

e e EAD or TA 2/6 Arrest 4/4

Clozapine

Antipsychotic

hERG j Ca  Na channel block

30 100 300 1000

2.96 ± 2.85 7.96 ± 8.42 20.18 ± 8.74 38.65 ± 8.47

0.14 ± 1.65 0.08 ± 3.66 5.11 ± 3.77 17.12 ± 3.03

e

e e e e

dl-Sotalol

Anti-arrhythmic drug (Class III)

hERG j Ca >> Na channel block

100 300 1000 3000

0.27 ± 1.08 1.14 ± 2.14 0.35 ± 1.27 3.87 ± 2.66

1.97 ± 1.33 3.99 ± 1.50 9.73 ± 2.87 24.36 ± 6.11

1017.61

e e e e

Azimilide

Anti-arrhythmic drug (Class III)

hERG and KvLQT1 channel block

30 100 300 1000

12.14 ± 6.79 17.20 ± 8.70 15.48 ± 8.97 18.07

5.39 ± 3.83 13.26 ± 6.86 15.71 ± 6.24 17.62

129.11

e e EAD or TA 3/6 EAD or TA 2/3

Quinidine

Anti-arrhythmic drug (Class Ia)

hERG > Ca j Na channel block

30 100 300 1000

0.34 ± 2.51 0.30 ± 4.79 3.04 e

4.29 ± 2.00 11.94 ± 4.08 27.35 e

84.92

e e EAD or TA 2/4 EAD or TA 2/2

Bepridil

Anti-arrhythmic drug (Class IV)

hERG > Ca j Na channel block

100 300 1000 3000

0.42 ± 1.36 1.55 ± 2.29 5.20 ± 3.05 11.00 ± 5.42

2.94 ± 1.15 6.95 ± 3.14 17.52 ± 6.51 37.11 ± 8.39

552.80

e e e e

Amiodarone

Anti-arrhythmic drug (Class III)

hERG j Ca > Na channel block

100 300 1000 3000

1.01 ± 2.15 1.54 ± 1.73 0.70 ± 3.93 7.92 ± 11.56

3.63 ± 1.69 9.71 ± 3.60 18.00 ± 8.69 18.91 ± 13.46

444.28

e e e e

Dofetilide

Anti-arrhythmic drug (Class III)

hERG >> Ca > Na channel block

0.1 0.3 1 3

1.36 ± 0.84 4.06 ± 2.61 6.68 ± 2.93 10.18 ± 5.28

4.12 ± 0.58 10.92 ± 2.52 26.50 ± 6.37 47.59 ± 9.89

0.3011

e e e EAD or TA 1/4

Ibutilide

Anti-arrhythmic drug (Class III)

hERG >> Na j Ca channel block

0.1 0.3 1 3

0.28 1.01 4.49 7.67

4.03 ± 1.00 8.94 ± 1.12 22.23 ± 2.34 44.59 ± 15.66

0.3481

e e e EAD or TA 1/4

Loratadine

Anti-allergic drug

hERG j Ca  Na channel block

100 300 1000 3000

5.53 ± 1.31 9.08 ± 3.56 18.97 ± 4.61 30.81 ± 4.68

0.32 ± 1.14 2.68 ± 1.56 1.68 ± 2.18 3.51 ± 4.96

e

e e e e

Crizotinib

Anticancer drug

hERG j Ca j Na channel block

0.3 (mM) 1 3 10

3.54 ± 1.02 9.69 ± 2.14 24.16 ± 2.18 e

1.17 ± 1.03 0.51 ± 1.02 10.38 ± 2.17 e

e

e e e Arrest 3/4 1st peak disappearance 1/4

Sunitinib

Anticancer drug

hERG >> Na j Ca channel block

0.3 (mM) 1 3 10

1.18 ± 1.64 5.84 ± 8.49 7.19 ± 12.78 e

6.22 ± 3.31 14.31 ± 3.73 24.16 ± 3.19 e

0.6688 (mM)

e e EAD or TA 2/5 EAD or TA 3/3

± ± ± ±

1.68 0.99 2.29 7.59

Y. Nozaki et al. / Regulatory Toxicology and Pharmacology 88 (2017) 238e251

Chlorpromazine

(continued on next page) 241

242

Table 1 (continued ) Drug

Mechanism

Ion channel information

Concentration (nM)

Beat rate (%)

FPDc (%)

FPDc10 (nM)

Arrhythmia-like waveforms (type and incidence)

Other ion channel blocker and opener

ZD 7288

HCN blocker

HCN channel block

3 10 30 100

4.50 ± 4.11 17.15 ± 7.26 31.36 ± 6.57 42.21 ± 7.99

1.91 ± 1.10 6.56 ± 1.26 12.09 ± 2.88 18.73 ± 4.65

29.94

e e e e

Levcromakalim

KATP channel opener

KATP channel activation

100 300 1000 3000

1.26 ± 1.24 3.92 ± 2.71 18.68 ± 2.99 e

1.04 ± 4.97 6.46 ± 8.96 30.21 ± 6.41 e

e

e e Arrest 3/6 Arrest 3/3

Ouabain octahydrate

Cardiac glycoside

Naþ, Kþ-ATPase inhibition

10 30 100 300

1.53 ± 1.50 3.16 ± 2.44 3.28 ± 5.05 e

1.76 ± 2.92 8.71 ± 6.73 26.35 ± 9.57 e

e

e e e Arrest 1/5 Unclear T 4/5

Isoproterenol

Bronchodilator

No information

3 10 30 100

19.22 25.45 34.79 53.21

7.50 ± 5.59 9.31 ± 6.29 12.40 ± 6.26 20.27 ± 5.39

e

e e e e

Propranolol

Anti-arrhythmic drug (Class II)

Na > hERG channel block

0.3 (mM) 1 3 10

0.98 ± 2.20 5.02 ± 3.52 15.76 ± 3.17 e

0.73 ± 1.58 2.20 ± 1.03 7.82 ± 2.82 e

e

e e e EAD or TA and arrest 4/7 EAD or TA 2/7 Arrest 1/7

Carbachol

Gastrointestinal prokinetic agent and glaucoma

No information

10 30 100 300

2.01 ± 1.52 10.66 ± 6.60 20.92 ± 2.83 32.30 ± 11.27

2.35 ± 3.01 6.23 ± 9.39 11.69 ± 11.17 19.08 ± 12.35

116.93

e e e e

Glycyrrhizic acid

liquorice extract

gap junction inhibition

30 (mM) 100 300 1000

0.86 ± 2.16 1.99 ± 3.09 4.68 ± 3.12 12.33 ± 4.75

3.62 ± 2.50 5.78 ± 3.85 9.27 ± 3.77 13.69

373.39 (mM)

e e e e

Blebbistatin

Myosin II ATPase specific inhibitor

No information

1 (mM) 3 10 30

9.28 ± 2.44 11.71 ± 2.44 17.21 ± 3.18 20.36 ± 4.72

5.71 6.32 8.96 9.91

e

e e e Arrest 2/7

Compounds with other mechanisms

± ± ± ±

14.80 18.10 19.01 15.29

± ± ± ±

2.12 1.90 2.56 1.90

Y. Nozaki et al. / Regulatory Toxicology and Pharmacology 88 (2017) 238e251

Category

Y. Nozaki et al. / Regulatory Toxicology and Pharmacology 88 (2017) 238e251

gel electrophoresis (Bioanalyzer 2100; Agilent Technologies, USA). Samples with RNA Integrity Numbers 7.0 were considered to be of sufficient quality. 2.3.2. Quality analyses For DNA microarray analysis, cyanine dye (Cy)-labeled cRNA was prepared and fragmented using Low Input Amp Labeling Kit and Expression Hybridization Kit (Agilent Technologies) according to the manufacturer's protocols. Briefly, total RNAs (10e100 ng) isolated from hiPS-CMs were reverse-transcribed into doublestranded cDNAs, and then fluorescence-labeled cRNA samples were fragmented randomly. After hybridization to the SurePrint G3 Human Gene Expression v2 Microarray (8  60 K; Agilent Technologies) by incubation at 65
C for 17 h in a rotisserie oven set at 10 rpm, the microarrays were washed using Agilent's wash buffers according to the wash protocol and scanned using the Agilent Scanner. Array quality was assessed through the use of control features as well as spike-in controls (One-Color Spike Mix Kit; Agilent Technologies). 2.3.3. Data analyses Text files extracted from the Feature Extraction software were imported into GeneSpring GX software (version 12.0; Agilent Technologies) for further analysis. Background intensity was cut off before normalization. Microarray data sets were normalized in GeneSpring GX using the Agilent FE one-color scenario (mainly 75% normalization), and the normalized signals were logarithmically (base 2) transformed for hierarchical clustering analysis of strain differences in the vehicle control groups. Detected-CompromisedNot detected flags were used to identify and omit genes with probes of poor quality or with low expression values. Correlation coefficients of signal intensity among the three laboratories were calculated by JMP 5.5 (SAS Institute, USA). Microarray data were statistically analyzed using GeneSpring GX software, and the results are reported as mean based on 4 independent replications. Differentially expressed genes on Day1, and Days 14 or 15 in a laboratory were selected based on a  4-fold change cut-off using Welch's t-test (p < 0.05), and functional analyses of the differentially expressed genes, in accordance with the culture period, were carried out using Gene Ontology (GO) annotations. PCA (principal component analysis), an unsupervised dimension reduction method commonly used in the context of high-dimensional data analyses, was performed using GeneSpring GX. In gene clusters related to cardiac system development and function, genes of well-known functional mechanisms and those altered in cardiomyocytes were selected for validation of expression changes by real-time RT-PCR. 3. Results 3.1. Effects of reference compounds on FPs in iCell® cardiomyocytes (Table 1) 3.1.1. hERG channel blocker and activator Domperidone, a gastrointestinal prokinetic drug and a known hERG channel blocker (S Claassen and Zunkler, 2005), prolonged FPDc with FPDc10 of 27.33 nM, decreased beat rate in a concentration-dependent manner, and provoked EAD or TA at 300 nM in 3 out of 4 wells. NS-1643, a hERG channel activator (RS Hansen et al., 2006), shortened FPDc at 100 mM and increased beat rate at a concentration of 30 mM and above. NS-1643 was also associated with arrest and unclear T waveform (deformation of positive deflection following to the sharp spike) at 100 mM in 2 and 1 out of 5 wells, respectively.

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3.1.2. Late Na current inhibitors and enhancer with hERG channel blocking activity Ranolazine, an antianginal drug and a known late Na current inhibitor with hERG channel blocking activity (BR Chaitman, 2006), prolonged FPDc in a concentration-dependent manner with FPDc10 of 3.63 mM, but had no effect on beat rate or no arrhythmia-like waveform at concentrations up to 10 mM. Mexiletine, an anti-arrhythmic drug and a known late Na current inhibitor with hERG channel blocking activity (R Gualdani et al., 2015), prolonged FPDc in a concentration-dependent manner with FPDc10 of 6.77 mM and decreased beat rate at a concentration of 30 mM. Although Mexiletine was associated with arrest at 30 mM in 3 out of 4 wells, neither EAD nor TA was induced at any concentration. Alfuzosin, a drug for prostate hypertrophy and a known late Na current enhancer with hERG channel blocking activity (P Liang et al., 2013), prolonged FPDc in a concentrationedependent manner with FPDc10 of 190.31 nM. EAD or TA was also induced at 1 mM in 3 out of 5 wells. Beat rate was unchanged at all concentrations. 3.1.3. L-type Ca channel activator Bay K 8644, a known L-type Ca channel activator (CT January et al., 1988), prolonged FPDc in a concentrationedependent manner with FPDc10 of 0.63 nM. Beat rate was unaffected by treatment with Bay K 8644, and neither EAD nor TA was evoked at any concentration. 3.1.4. Multi-ion channel blockers Mibefradil, a T-type Ca channel blocker with L-type Ca channel, hERG channel, KvLQT1 channel and Na channel blocking activity (A Benardeau et al., 2000; C Chouabe et al., 1998; MM McNulty et al., 2006; R SoRelle, 1998), shortened FPDc in a concentrationdependent manner and increased beat rate. Arrest was also observed at 10 mM in 4 out of 6 wells. Cisapride, a gastrointestinal prokinetic drug, and astemizole, an anti-allergic drug, are known as potent hERG channel blockers with weak to mild Na and Ca channel blocking activity (J Kramer et al., 2013). Both cisapride and astemizole prolonged FPDc with FPDc10 of 22.55 nM and 8.03 nM, respectively, and decreased beat rate at concentrations of 30 nM and above, and 100 nM, respectively. In addition, EAD or TA was observed following treatment with cisapride at 100 and 300 nM in 4 out of 7 wells and all wells, and astemizole at 100 nM in 8 out of 10 wells. Thioridazine and pimozide, two antipsychotic drugs, are known as potent hERG channel blockers with mild to potent Na and Ca channel blocking activity (J Kramer et al., 2013). Both thioridazine and pimozide prolonged FPDc in a concentration-dependent manner with FPDc10 of 701.18 nM and 9.41 nM, respectively. Beat rate was unaffected by thioridazine at concentrations up to 3 mM, while pimozide resulted in a decrease in beat rate at 30 nM and above. EAD or TA was evoked by thioridazine at 3 mM in 1 out of 5 wells, and by pimozide at 30 and 100 nM in 1 out of 5 wells and 3 out of 4 wells, respectively. Chlorpromazine and clozapine, another two antipsychotic drugs, inhibit hERG channel, Na channel and Ca channel at an equal potency (J Kramer et al., 2013). FPDc and beat rate were unaffected by treatment with chlorpromazine at concentrations up to 3 mM. Yet, EAD or TA and arrest were evoked at 3 mM in 2 out of 6 wells and at 10 mM in all wells, respectively. Clozapine shortened FPDc at 1 mM and increased beat rate at 300 nM and above, although no arrhythmia-like waveforms were observed at concentrations up to 1 mM. dl-Sotalol, an anti-arrhythmic drug and a known weak hERG and Ca channel blocker (J Kramer et al., 2013), prolonged FPDc in a

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concentration-dependent manner with FPDc10 of 1.02 mM. Beat rate was unchanged following treatment with dl-sotalol, and EAD or TA was not induced at concentrations up to 3 mM. Azimilide, an anti-arrhythmic drug and a known hERG and KvLQT1 channel blocker (J Miake et al., 2004; BD Walker et al., 2000), prolonged FPDc with an FPDc10 of 129.11 nM and decreased beat rate at 30 nM and above. EAD or TA was produced at 300 and 1000 nM in 3 out of 6 wells and 2 out of 3 wells, respectively. Quinidine, bepridil and amiodarone are anti-arrhythmic drugs known as hERG channel blockers with mild to weak Na and Ca channel blocking activity (J Kramer et al., 2013; WS Redfern et al., 2003). All three drugs prolonged FPDc in a concentrationdependent manner with FPDc10 of 84.92 nM, 0.55 mM and 0.44 mM, respectively. Bepridil decreased beat rate in a concentration-dependent manner, though quinidine and amiodarone had no effect on beat rate at any concentration. In addition, quinidine evoked EAD or TA at 300 and 1000 nM in 2 out of 4 wells and all wells, respectively. In contrast, bepridil and amiodarone did not induce EAD or TA at any concentration. Dofetilide and ibutilide, yet another two anti-arrhythmic drugs known as potent hERG channel blockers with weak Na and Ca channel blocking activity (J Kramer et al., 2013; WS Redfern et al., 2003), prolonged FPDc in a concentration-dependent manner with FPDc10 of 0.30 nM and 0.35 nM, respectively. Dofetilide also decreased beat rate in a concentration-dependent manner. EAD or TA was evoked with both compounds at 3 nM in 1 out of 4 wells. Loratadine, an anti-allergic drug and a known hERG channel blocker with Ca and Na channel blocking activity (WJ Crumb, Jr., 2000; J Kramer et al., 2013), increased beat rate in a concentration-dependent manner. However, FPDc was unaffected, and arrhythmia-like waveforms were not produced at any concentration. Crizotinib and sunitinib are two anticancer drugs known as hERG channel blockers with Ca and Na channel blocking activity (KR Doherty et al., 2013). Crizotinib shortened FPDc at 3 mM, whereas sunitinib prolonged FPDc in a concentration-dependent manner with FPDc10 of 0.67 mM. Beat rate was clearly decreased by crizotinib at 3 mM, but it was not changed by sunitinib. Arrest and disappearance of the 1st positive peak were evoked by crizotinib at 10 mM in 3 and 1 out of 4 wells, respectively. For sunitinib, EAD or TA was induced at 3 and 10 mM in 2 out of 5 and all wells, respectively. 3.1.5. Other ion channel blocker and opener ZD 7288, a widely used blocker of hyperpolarization-activated cyclic nucleotide-gated channels (HCN channels) (X Wu et al., 2012), prolonged FPDc with FPDc10 of 29.94 nM and decreased beat rate in a concentration-dependent manner (concentrations up to 100 nM). EAD, TA, or arrest was not observed at any concentration. Levcromakalim, an ATP-sensitive Kþ channel opener that causes membrane hyperpolarization (Y Ohya et al., 1996), shortened FPDc and decreased beat rate in a concentration-dependent manner. In addition, levcromakalim evoked arrests at 1 and 3 mM in 3 out of 6 wells and all wells, respectively. 3.1.6. Compounds with other pharmacological mechanisms of action Ouabain octahydrate, a cardiac glycoside and an Naþ-Kþ-ATPase inhibitor (JC Louie et al., 2016), shortened FPDc in a concentrationdependent manner (concentrations up to 100 nM). Beat rate was unaffected by this glycoside, although arrest and unclear T waveform were evoked at 300 nM in 1 and 4 out of 5 wells, respectively. Isoproterenol, a beta stimulant bronchodilator (JR Stratton et al.,

1992) with no available data on its effects on cardiac ion channels, shortened FPDc in a concentration-dependent manner and increased beat rate at concentrations up to 100 nM. EAD or TA was not induced at any concentration. Propranolol, an anti-arrhythmic beta blocker with Na current inhibitory activity (DW Wang et al., 2010) had no effect on FPDc at any concentration, but evoked EAD or TA and/or arrest at 10 mM in all 7 wells. Beat rate was decreased in a concentration-dependent manner. Carbachol, a gastrointestinal prokinetic agent, is a cholinergic agonist that binds and activates the acetylcholine receptor (R Matucci et al., 2016). No information is available on the effects of this agonist on cardiac ion channels. Carbachol prolonged FPDc and decreased beat rate in a concentration-dependent manner with FPDc10 of 116.93 nM. Arrhythmia-like waveforms were not produced at any concentration. Glycyrrhizic acid, a liquorice extract, is a known inhibitor of gap junction (D Zhao et al., 2015). No information is available on the effects of this inhibitor on cardiac ion channels. Glycyrrhizic acid prolonged FPDc and decreased beat rate in a concentrationdependent manner with FPDc10 of 373.39 mM. EAD or TA was not induced at concentrations up to 1000 mM. Blebbistatin is a myosin II ATPase specific inhibitor (HH Wang et al., 2008). No information is available on the effects of this inhibitor on cardiac ion channels. Blebbistatin had no effect on FPDc, but increased beat rate in a concentration-dependent manner. Arrest was induced only at 30 mM in 2 out of 7 wells. 3.2. Effects of culture period on genes expression Prior to investigation of distinctive changes in gene expression profiles association with culture period, inter-facility variability was assessed to ensure that the data set obtained from 3 testing facilities was scientifically warranted in the context of equivalency. Gene samples were collected from 3 testing facilities on Day 1, and Day 13, 14 or 15 of thawing the iCell® Cardiomyocytes. All samples revealed high correlation among the testing facility, resulting in coefficients of determination greater than 0.95 [0.962e0.998]. The samples from all 3 testing facilities also showed good correlation in scatter plot analysis with high coefficient of determination [0.942e0.984] (data not shown). Thus, the data set of genes expression obtained from these samples was confirmed to be valid for subsequent gene profiling. To examine whether gene characteristics were altered by the culture period, gene ontology analysis (GO analysis) was conducted on the collected samples on Day 1, and Day 14 or 15. Among the genes involved in voltage-gated ion channel activity, cardiac muscle contraction and regulation of muscle contraction, the expression levels of 13, 8 and 11 genes increased by over 4-fold, although those of 8, 1 and 3 genes decreased below one fourth on Day 14 or 15 as compared to Day 1 (Fig. 1). The gene that increased most in relation to voltage-gated ion channel activity was KCNV1 (potassium channel, subfamily V), resulting in 18-fold increase. SCN2B, CACNA2D2 and KCNT1 increased 12e16 fold in this gene family. The genes that showed statistically significant increase on Day 14 or 15 were KCNJ2, which is related to inward-rectifier potassium current (IK1), KCNQ1, which is related to the slow activating delayedrectifier potassium current (IKs) and SCN10A, which is related to Nav1.8. Regarding genes involved in cardiac muscle contraction or its regulation, CASQ2, calsequestrin 2, was the mostly increased gene showing 47-fold increase on Day 14 or 15 of the culture period. ATP1A2, Naþ-Kþ ATPase alpha2 polypeptide involved in regulation of cardiac muscle contraction, was also increased by up to 41-fold. On the other hand, the gene that decreased most in relation to

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Fig. 1. GO analysis of gene samples on Day 1, and Day14 or 15. Genes indicating up (a) and down (b) regulation among those involved in voltage-gated ion channel activity, cardiac muscle contraction and regulation of cardiac muscle contraction were extracted from a total of 50,599 genes.

regulation of cardiac muscle contraction was RGS2, a regulator of Gprotein signaling 2. The decrease in this gene was below one tenth of the original expression level on Day 14 or 15. As for genes associated with IKs current, KCNE1L (KCNE1-like genes), unlike KCNQ1, decreased to one fifth of the original level on Day 14 or 15.

4. Discussion 4.1. Effects of reference compounds on FP in iCell® cardiomyocytes 4.1.1. hERG channel blockers and activator Comparing the effects of hERG channel blockers (domperidone

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and E-4031 (Y Nozaki et al., 2016)) to those of hERG channel activator (NS-1643), the resultant beat rate and FPDc were as expected at the other end of the spectrum. Beat rates were decreased by hERG channel blockers, but increased by hERG channel activator (Fig. 2). Similarly, FPDcs were prolonged by hERG channel blockers, but shortened by hERG channel activator. Of note is the fact that EADs were associated with hERG channel blockers, whereas arrest and unclear T waveform were evoked by hERG channel activator. NS-1643 is reported to elicit shortening in action potential duration (APD) in ventricular cardiomyocytes isolated from guinea pigs (RS Hansen et al., 2006). This finding is consistent with the results of the present study related to hiPS-CMs tested on MEA. Recently, short QT syndrome has been considered to constitute a new primary cardiac electrical abnormality with high incidence of sudden death. Short QT syndrome may not result in TdPs, but could lead to ventricular fibrillation (VF) (HR Lu et al., 2008b). Therefore, arrest accompanied by FPDc shortening may be represented by an electrocardiogram reflecting VF in humans, and thus, the hiPS-CMs/ MEA system may be useful to elucidate drugs modes of action and predict short QT syndrome in humans. Overall, hiPS-CMs/MEA assay would be a system that can detect not only the effects of hERG channel blockers but also hERG channel activators.

4.1.2. Late Na current inhibitors and enhancer with hERG channel blocking activity Ranolazine, equipotentially inhibits late Na current and hERG channel at the low end of the therapeutic dose range, and more potently inhibits late Na current at the high end (BR Chaitman, 2006). Although ranolazine prolonged FPDc, no arrhythmia-like waveforms were detected at concentrations up to 10 mM (Fig. 2). QTc prolongation is reported in patients with chronic angina (BR Chaitman, 2006); however, arrhythmia has not been detected. The results of the present study are in well accordance with clinical data. However, evaluation on proarrhythmia of ranolazine might require higher concentrations taking into account the hERG IC50 of 8.3 mM. Mexiletine, a class Ib anti-arrhythmic drug that increases upstroke velocity and shortens APD, is used predominantly for the treatment of ventricular arrhythmia, and is known to preferentially block late Na current at therapeutic unbound concentrations in clinical setting [4e11 mM] (WS Redfern et al., 2003). These unique features of mexiletine are expected to ameliorate QT prolongation in humans. Indeed, mexiletine reduces QT prolongation magnitude induced by dofetilide in dogs (H Todt et al., 1994). On the contrary, an inexplicable concentration-dependent FPDc prolongation and arrests were observed in the hiPS-CMs/MEA system. This inconsistency between the results of in vivo studies and our results encourages us to proceed to the next step where electrophysiological profiles are investigated for compounds such as mexiletine using hiPS-CMs derived from patients showing QT prolongation, or those exhibiting prolonged FPDc by pre-treatment. Alfuzosin is a selective antagonist of postsynaptic a1-adrenergic receptors marketed for the treatment for benign prostatic hyperplasia. Alfuzosin has a weak potential for hERG inhibition, although it increases Na current, which is known to cause QT prolongation in humans (P Liang et al., 2013). Nonclinical studies clearly showed that alfuzosin prolonged APD in Purkinje fibers of rabbit ventricles and QT in isolated rabbit heart, the results being consistent with QT prolongation in clinical setting (AE Lacerda et al., 2008). In the hiPSCMs/MEA system, FPDc was prolonged in a concentrationdependent manner with FPDc10 of 190.31 nM. In addition, arrhythmia was evident at 1 mM. The FPDc prolongation of alfuzosin was likely the consequence of the delay of cardiac repolarization together with increasing Na current.

4.1.3. L-type Ca channel blocker and activator Verapamil, an L-type Ca channel blocker, was found to shorten FPDc in a previous study (Y Nozaki et al., 2016). For comparison, Bay K 8644, an L-type Ca channel activator, was evaluated in the present study and found to prolong FPDc (Fig. 2). Neither arrhythmia-like waveform nor arrest was observed at any concentration with either verapamil or Bay K 8644. As all these findings reflect clinical data, the hiPS-CMs/MEA system is likely to be appropriate when predicting arrhythmia for both L-type Ca channel blockers and activators. However, the results on beat rate were inconsistent with the pharmacological effects. Verapamil increased beat rate (Y Nozaki et al., 2016) and Bay K 8644 did not alter beat rate in the hiPS-CMs/MEA system. Unlike human cardiomyocytes in vivo, beat rate in hiPS-CMs may not be controlled by beat impulse generated in nodal-like cells (G Stark et al., 1988). 4.1.4. Multi-ion channel blockers Most multi-ion channel blockers with hERG channel blocking activity (cisapride, astemizole, thioridazine, pimozide, azimilide, quinidine, dofetilide, ibutilide and sunitinib) prolonged FPDc, and were associated with a decrease in beat rate and arrhythmia-like waveforms (Table 1). However, there were exceptions. Mibefradil, a multi-ion channel blocker that primarily inhibits Ttype Ca channel, slightly reduces heart rate in humans (R SoRelle, 1998). Like other L-type Ca channel blockers, mibefradil shortened FPDc, leading to increase in beat rate (see section 4.1.3). These findings are most likely due to stronger inhibitory effect on Ca channel rather than on hERG channel. Mibefradil at 10 mM was associated with arrest in which the prominent FPDc shortening evident at this concentration may have played a role. There are only few reports on mibefradil associated TdP in humans (S Glaser et al., 2001). Although the root cause for EAD or TA and arrest that appeared in the hiPS-CMs/MEA system remain unknown, these waveforms may provide new insights in predicting the proarrhythmic potential of mibefradil in humans. Chlorpromazine, loratadine, clozapine and crizotinib can potentially prolong QT and arrhythmogenic activity, including TdP in humans (I Berling and Isbister, 2015; M Bruggisser et al., 2009; KR Doherty et al., 2013; H Ochiai et al., 1990; SH Ou et al., 2011; E Poluzzi et al., 2015; K Wenzel-Seifert et al., 2011). However, these drugs produced either a shortening or no effect on FPDc in the hiPSCMs/MEA system. Unlike other drugs in this group, clozapine and loratadine evoked neither EAD or TA nor arrest even at maximum concentrations. As for the inhibitory effect on Ca channel and hERG channel, it is reported to be equipotent among all drugs in this group (J Kramer et al., 2013). Drug inhibitory potential on Ca channel may be developed to a greater degree in hiPS-CMs and might counteract or cancel FPDc prolongation induced through hERG channel blocking activity, thereby, rendering hiPS-CMs resistant to arrhythmogenesis. Further investigation is needed to elucidate the mechanisms associated with these findings, which are contradictory to those in clinical setting. dl-Sotalol, amiodarone and bepridil were not associated with arrhythmia-like waveforms at any concentration in spite of prolonging FPDc in the hiPS-CMs/MEA system. All these compounds have been shown to have arrhythmogenic activity in few clinical cases, although arrhythmia could not be reproduced in non-clinical in vivo assays (T Omata et al., 2005; JM van Opstal et al., 2001; M Yasuda et al., 2006). Meanwhile, dl-sotalol induced EADs at 3 mM in a preliminary study. Therefore, arrhythmia-like waveforms may appear at higher concentrations of these compounds. 4.1.5. Other ion channel blockers and opener ZD 7288 blocks HCN channels in the sinoatrial node and decreases heart rate (X Wu et al., 2012). HCN channels density is

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Fig. 2. Effects of ion channel blockers and activators on FPDc and beat rate.

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comparable between sinoatrial nodal cells and human embryonic stem cells-derived cardiomyocytes (hESC-CMs). In addition, HCN channels contribute to the spontaneous beating activity of hESCCM. Moreover, zatebradine, a HCN channel blocker, is reported to prolong APD90 in hESC-CMs (MK Jonsson et al., 2012). In the hiPSCMs/MEA system, ZD 7288 was associated with prolonged FPDc accompanied by a decrease in beat rate, although no EAD or TA was evoked. These findings indicate that HCN channels are sufficiently expressed and play a role in regulating beat rate in hiPS-CMs. IK,Ach underlying the ATP-sensitive Kþ channel, is activated by stretch of ventricular cells and can slow pacemaker activity (ME Mangoni and Nargeot, 2008). Levcromakalim, an ATP-sensitive Kþ channel opener, shortened FPDc in the hiPS-CMs/MEA system. This finding is well in accord with APD shortening in Purkinje fibers, papillary muscles and ventricular trabecula in rabbits, as well as QT shortening in isolated heart in rabbits (HR Lu, et al., 2008a; HR Lu et al., 2008b). Levcromakalim is known to be associated with VF at an incidence of 87.5% in isolated hearts (HR Lu et al., 2008b). The hiPS-CMs/MEA system revealed a decrease in beat rate and arrest associated with levcromakalim. Overall, these findings indicate that ATP-sensitive Kþ channels are expressed in hiPS-CMs and that they play a critical role in heart automaticity. Thus, the hiPS-CMs/MEA system is a valid tool for detecting the proarrhythmic risk of various ion channel blockers and activators that have the potential to induce not only QT prolongation but also QT shortening in clinical practice. 4.1.6. Compounds with other pharmacological mechanisms of action Ouabain inhibits Naþ-Kþ ATPase, which is followed by an increase in intracellular Ca2þ by the Naþ-Ca2þ exchanger (JC Louie et al., 2016). The finding that ouabain shortened FPDc in a concentration-dependent manner in the hiPS-CMs/MEA system may infer an increase in intracellular Ca2þ. Isoproterenol, a b agonist, increased beat rate and shortened FPDc in a concentration-dependent manner (Fig. 2). The increased beat rate is likely the result of an effect on If current. b-adrenergic receptors are involved in automaticity, which is regulated by If current, and activation of b-adrenergic receptors is known to potentiate If current (e.g. HCN channel) via increase in cAMP (ME Mangoni and Nargeot, 2008). In sharp contrast, propranolol, a b antagonist, decreased beat rate and had no significant effect on FPDc. In addition, EAD or TA, and/or arrest were evoked at 10 mM in all wells though propranolol is not known as arrhythmogenic in clinical practice. EAD or TA, and arrest resulted from indirect negative effect on If current. The effects of b agonist and antagonist on FPDc, and the subsequent arrhythmia-like waveforms indicate that the hiPS-CMs/MEA assay could detect indirect effects on automaticity. Carbachol produces parasympathetic stimulation and may induce bradycardia in clinical use. It prolonged FPDc and decreased beat rate in hiPS-CMs, indicating that the effects of muscarinic agents can be monitored in the hiPS-CMs/MEA assay. Overall, the hiPS-CMs/MEA system is believed to be of great value when evaluating the proarrhythmic risk and QT prolongation potential of drugs that may interact with or modify the functions of various cardiac ion channels, cardiac receptors and ion-exchange pumps. 4.2. Comparison of hiPS-CMs/MEA assay with conventional in vitro and in vivo assays 4.2.1. Comparison with hERG assay To compare the results of the hiPS-CMs/MEA assay to those of hERG assay, hERG IC50 data were collected from pertinent

publications for 24 compounds with diverse modes of pharmacological action. The results obtained for FPDc10 in the hiPS-CMs/MEA assay correlated well with hERG IC50 data within a range of 10-fold in 18 out of 24 compounds (Fig. 3a). Among the remaining 6 out of 24 compounds, greater than 50-fold differences were shown by dlsotalol and alfuzosin in the FPDc10s from hERG IC50s. This is likely the result from weak potential of hERG inhibition in these compounds in spite of the fact that both compounds are known to induce TdP in clinical setting. In this regard, the hiPS-CMs/MEA assay can better predict the potential for arrhythmogenicity. As described in Fig. 3e, the FPDc10 and human EC10 of QTc or TdP in dlsotalol is highly correlated. In the case of verapamil, known to be more potent in blocking Ca channel than hERG channel, the positive results of hERG assay may be misleading on assessment of potential for QT prolongation in human. The hiPS-CMs/MEA assay resulted in unequivocal negative results for FPDc, demonstrating a high specificity for evaluation of potential for QT prolongation and TdP stemmed from comprehensive assessment of cardiac ion currents. 4.2.2. Comparison with guinea pig APD EC10 (effective concentration at 10% prolongation of APD90, unbound) in APD assay using guinea pig cardiomyocytes was relatively higher than FPDc10 in the hiPS-CMs/MEA assay (Fig. 3b). Guinea pig APD assay cannot detect potential for QT prolongation in some compounds, such as astemizole, bepridil, pimozide and terfenadine (T Omata et al., 2005). This is likely due to the low expression of hERG channels and no expression of Ito in cardiomyocytes of guinea pigs (Z Lu et al., 2001). These findings strongly support the notion that the hiPS-CMs/MEA assay is highly sensitive and superior to guinea pig APD assay in predicting QT prolongation, especially for multi-ion channels blockers. 4.2.3. Comparison with in vivo telemetry assays E-4031 and cisapride are strong hERG channel blockers. In comparison with the EC10 (effective plasma concentration at 10% prolongation of QTc, unbound) of these compounds obtained in telemetry studies using conscious monkeys and dogs, the FPDc10 obtained in the hiPS-CMs/MEA assay showed high correlation within the range of 10-fold (Fig. 3c and d). Although only limited telemetry data have been reported for cardiotoxic compounds and conclusive assessment of in vivo-in vitro correlation is currently not possible, the hiPS-CMs/MEA assay is considered to be a system at least equivalent to telemetry in investigating cardiotoxicity. 4.2.4. Comparison with clinically established data on QT prolongation and TdP FPDc10 and MCEAD/TA (the lowest concentration at which EAD or TA was evoked) in the hiPS-CMs/MEA assay highly correlated with unbound concentrations at which QTc prolongation or TdP were evoked in humans (Fig. 3e). Yet, arrhythmia-like waveforms were found at higher concentrations in the hiPS-CMs/MEA assay than in clinical settings. Multi-ion channel blockers, such as terfenadine, pimozide and bepridil, and late Na current enhancers with hERG channel blocking activity, such as alfuzosin, resulted in FPDc10 and arrhythmia-like waveforms appeared at concentrations higher than those at which QT prolongation or TdP were evoked in humans. The hiPS-CMs present characteristics similar to those of human fetal cardiomyocytes, but different expression levels for each ion channel (L Guo et al., 2011). These differences in expression levels of ion channels would influence the outcome of the hiPS-CMs/MEA assay, and the biological characteristics of hiPS-CMs may need to be further improved for better assessment of new drugs cardiac liabilities. All the results above warrant the quality, reliability and predictability of the hiPS-CMs/MEA assay in evaluating drugs risk for

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Fig. 3. Comparison of the hiPS-CMs/MEA assay with current in vitro and in vivo assays; a) hERG IC50, b) EC10 in guinea pig APD90, c) EC10 of QTc in monkey telemetry, d) EC10 of QTc in dog telemetry, e) Human EC10 of QTc or TdP with concentrations for FPDc10 (circle) and MCEAD/TA (square) in the hiPS-CMs/MEA assay. References: (RE BoSmith et al., 1993; BR Chaitman, 2006; HC Cheng and Incardona, 2009; J Kramer et al., 2013; R Krapf and Gertsch, 1985; AE Lacerda et al., 2008; P Morissette et al., 2013; T Omata et al., 2005; WS Redfern et al., 2003; S Toyoshima et al., 2005).

QT prolongation and TdP. Considering the fact that EC10 in the in vivo telemetry assay, IC50 in the hERG assay and EC10 in the APD assay may overestimate or underestimate drugs potential for arrhythmia, the FPDc10 and the concentrations yielding arrhythmia-like waveforms in the hiPS-CMs/MEA assay are concluded to be better predictors with high sensitivity and specificity. 4.3. Global gene expression analyses The protocols and procedures were precisely standardized in the CSAHi study, and the hiPS-CMs/MEA data obtained from the 3

testing facilities turned to be highly homogenous, showing analogous gene expression patterns. In the GO analysis, genes in three categories were extracted: voltage-gated ion channel activity, cardiac muscle contraction and regulation of cardiac muscle contraction. The genes encoding Kþ, Naþ and Ca2þ channels, including SCN2B, CACNA2D2, SCN10A, KCNJ2 and KCNQ1, were strikingly upregulated along the culture period, indicating that hiPS-CMs may still be in the process of cell maturation with regards to ion channels (C Mummery et al., 2007; L Sartiani et al., 2007). Though some gene expressions such as KCNE1L which is associated with IKs current similar to KCNQ1 was down-regulated along the culture

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period, drug responses on FPs were evident in the hiPS-CMs/MEA, suggesting that the hiPS-CMs cultured for 14 or 15 days were applicable to electrophysiological assessment. CASQ2 and ATP1A2 were the most upregulated genes in relation to cardiac muscle contraction. CASQ2 encodes a calcium-binding protein in the sarcoplasmic reticulum, and is involved in storage and transport of the Ca2þ (DO Kryshtal et al., 2015). ATP1A2 indirectly regulates the concentration of Ca2þ by maintaining the gradients of Naþ and Kþ (S Despa et al., 2012). Thus, the hiPS-CMs were likely under development of contractile function during the culture period. RGS2 was the most down-regulated gene in GO analysis (Day 14 or 15). RGS2 is known to inhibit the a1 receptor, which is related to hypertrophy via attenuation of Gq-mediated signals (MX Zou et al., 2006). Low density cultured hiPS-CMs become enlarged, indicating significant changes in expression levels of cardiac hypertrophy related genes (M Uesugi et al., 2013). In line with these reports, we have previously shown that hiPS-CMs cultured at low density were larger than those cultured at high density (Y Nozaki et al., 2016). Our optimized protocol adopted high density hiPS-CMs, and no cell enlargement was observed. The down-regulation of RGS2 expression well supports the lack of hypertrophy of hiPS-CMs. 5. Conclusion Based on the findings of this study, it is believed that the hiPSCMs/MEA assay will likely constitute a core new platform for cardiac safety assessment where drug-induced arrhythmogenesis may be evaluated using human iPS-derived cardiomyocytes under nonclinical setting. Following a feasibility study where 7 compounds were evaluated, this communication attempts to further validate the hiPS-CMs/MEA system using a total 31 compounds known to modify the functions of cardiac multi-ion channels, cardiac receptors and ion-exchange pumps. Cell characteristic analysis of hiPS-CMs indicated that gene expression profiles were well maintained under the standardized protocols in all 3 testing facilities. The results clearly demonstrate that primary endpoints were consistent with those reported for conventional in vitro and in vivo assays, and clinical practice. Based on the findings of this study, the hiPS-CMs/MEA system can be warranted in context of quality, reliability, accuracy and predictability of drug-induced arrhythmogenesis. However, caution should be taken in overestimating the proarrhythmic risk of compounds showing hERG-positive and TdPnegative results, and in not being applicable to compounds showing QT shortening (K Takasuna et al., 2017). Further refinement may be required in the hiPS-CMs/MEA system in context of overall system maturation as a new evaluation platform for cardiotoxicity. Compared to conventional non-clinical in vitro and in vivo assays, the following advantages were found for the hiPS-CMs/MEA system; 1) being capable to test multiple compounds in one assay, 2) being capable to evaluate compounds with diverse pharmacological modes of cardiac action, including multiple cardiac ion channel blockage, 3) being capable to provide data with superior accuracy than current assays, 4) requiring only a small amount of test compounds, and most importantly 5) being based on the use of human derived cells which can overcome the species differences. Thus, we recommend that the hiPS-CMs/MEA system should be implemented as new paradigm for non-clinical testing of new drugs cardiac liabilities. Acknowledgement We thank iPS PORTAL, Inc. for the provision of iCell®s, Bio Research Center Co., Ltd. for the technical assistance, and Chemicals

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