Human iPS cell-Derived Cardiomyocytes ReproCardio2TM as an ... · Human iPS cell-derived...

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0 20 40 60 80 100 120 140 160 180 200 Human iPS cell-Derived Cardiomyocytes ReproCardio2 TM as an Analytic Tool for Large-scale Toxicity Screening Shunsuke Yoshida 1 , Zachary Yu-Ching Lin 1 , Joylynn Clark 2 , Mark Bryant 3 , Takashi Masai 1 , Mitsuru Inamura 1 ReproCell, Yokohama, Japan; 2 ReproCell, Boston, USA; 3 ReproCell, Crewe, UK Abstract Materials and Methods Result Conclusion & Discussion The force-frequency relationship in cardiac muscle contraction is different in developmental maturation and pathological states. Hence, cardiac contraction can be utilized for the evaluation of chemical compounds especially when these compounds affect the beating rate. In this study, various chemical stimulations on the contraction of human iPS cell- derived cardiomyocytes ReproCardio2 TM were examined with electrophysiological assay as well as calcium-imaging. ReproCardio2 TM possess stable BPM and regular QT interval with minimum differences between cell-lots after been thaw from cryopreservation. With the addition of chemical compounds, the extension or reduction of QT-interval can be obtained by both electrophysiological assay and calcium-imaging. Other novel utility of ReproCardio2 TM has also been developed. Taken together, ReproCardio2 TM is useful for analyzing the effects of chemical compounds on contractile function. Therefore, ReproCardio2 TM can be utilized in large-scale screening of large-scale of chemical compounds ReproCardio2 TM KIT (1 full kit for 96 well plate: ReproCELL # RCESD008) 1. 3.3×10 5 Single cells x 3 cryo-preserved vials 2. Empty Cryo-preserved Cell vial (dummy) 3. Maintenance culture medium (80mL) x 2 4. Coating solution (30mL) x 1 5. Low attachment plate for aggregated cells 6. Attachment plate for Thin-layer / Single cell Calcium-imaging Assay FDSS day 0 day 1-3 day 3 day 4-14 Testing compounds Electrophysiological assay day 0 day 1-3 day 3 day 4-14 Testing compounds Analyzer Step 0 1 2 3 4 9 14 1 Thawing 2 Making loose clump 3 Transfer into MEA assay probe 4 Making Thin layer 5 Drug evaluation 6 Analysis Step 0 1 2 3 4 9 14 1 Thawing 2 Making loose clump 3 Transfer into assay plate 4 Making Thin layer 5 Staining and Evaluate drugs 6 Analysis Immunocytochemistry Cells ・・・ 10,000 cells / well Equipment ・・・ FDSS Hamamatsu Ca indicator ・・・ Cal520 (AAT bioquest) Assay Medium ・・・ReproCardio assay medium (ReproCELL), Cremophor EL (Sigma) Cells ・・・ 15000 cells / well Equipment ・・・ Available (aMED, AXION, MCS) Coating ・・・ Fibronectin Assay Medium ・・・ ReproCardio assay medium (ReproCELL) or Culture medium (ReproCELL) with FBS Option ・・・ 5% CO2 gas/Air 1. Primary antibody: anti-Conexin 43, MYH6/7(Sigma); anti-MLC-2A/2V (Synaptic systems); anti-Troponin (A Bio-Rad company) and anti-Vimentin (Millipore). 2. Secondary antibody: Alexa Fluor 488 (Life Technologies) Characteristic of ReproCardio2™ MLC2V / DAPI αMHC / DAPI cTnT / DAPI CX43 / DAPI MLC2A / DAPI βMHC / DAPI Vimentin / DAPI Figure 1. ReproCardio2™ has various cardiac markers like human heart α-MHC and βMHC are the representative cardiac marker during the differentiation from iPS cells and expressed in heart. MLC-2A and MLC-2V are myosin specific marker for the atrium of the mammalian heart and for the ventricle of the mammalian heart, respectively. Cardiac troponin T (cTnT) is a specific marker in human heart and a thin filament protein which takes part in muscle contraction. Cx43 is conniving gap junction found in several organs and cell types, but is most often found in the heart. Those proteins were used as representative cardiac marker. Scale bar : 30 μm. Electrophysiological Assay utilizing ReproCardio2™ 1. MEA ASSAY Figure 2. Reproducibility of MEA assay: within the lot or between lot To prove there is no drastic difference within the cell lot, thin layers were made from cell clamps from the same vial of ReproCardio2 TM . The different clamp-derived thin layers were exposed to various concentration of E4031 from 300 pM to 30 μM. The data obtained from each thin layers were plotted together, and it shows that E4031 has similar prolonging QT interval affect in all thin layer from the same cell lot. To prove cell lots has no drastic difference, we exposed various concentration of E4031 from 300 pM to 30μM to 3 different cell lots. The graphs showed that 3 different cell lots exhibit similar prolonging QT interval that is known for. This demonstrates that ReproCardio2 TM has low production bias within lot as well as from lot to lot. Lot A [ , M ] E4031 0 20 40 60 80 100 120 140 160 180 200 Prolongation (%) 0 0 20 40 60 80 100 120 140 160 180 200 1.E-10 1.E-09 1.E-08 1.E-07 1.E-06 1.E-05 1.E-04 Lot B Lot C E401 concentration (M) QT Interval BPM 0 20 40 60 80 100 120 140 160 180 200 Na-K interval (%) 0 20 40 60 80 100 120 140 160 180 200 1.E-10 1.E-09 1.E-08 1.E-07 1.E-06 1.E-05 1.E-04 prolongation (%) Bazett (%) Fredericia (%) 0 20 40 60 80 100 120 140 160 180 200 Beat rate (%) 0 20 40 60 80 100 120 140 160 180 200 1.E-10 1.E-09 1.E-08 1.E-07 1.E-06 1.E-05 1.E-04 Aspirin: Non QT prolongation 0 20 40 60 80 100 120 140 160 180 200 Na-K interval (%) 0 20 40 60 80 100 120 140 160 180 200 prolongation (%) Bazett (%) Fredericia (%) 0 50 100 150 200 250 300 0 50 100 150 200 250 300 Beat rate (%) 0 0 Verapamil: Ca channel blocker 0 20 40 60 80 100 120 140 160 180 200 0 20 40 60 80 100 120 140 160 180 200 0 20 40 60 80 100 120 140 160 180 200 0 20 40 60 80 100 120 140 160 180 200 prolongation (%) Bazett (%) Fredericia (%) 0 Na-K interval (%) Beat rate (%) β-Adrenergic Agonist : Isoproterenol 0 50 100 150 200 prolongation (%) Bazett (%) Fredericia (%) 0 20 40 60 80 100 120 140 160 180 200 Beat rate (%) 0 50 100 150 200 K amplitude (%) K amplitude 0 20 40 60 80 100 120 140 160 180 200 0 50 100 150 200 0 50 100 150 200 1.E-10 1.E-09 1.E-08 1.E-07 1.E-06 1.E-05 1.E-04 QT Interval BPM I Kr Blocker : E-4031 0 Na-K interval (%) 0 20 40 60 80 100 120 140 160 180 200 Na-K interval (%) 0 20 40 60 80 100 120 140 160 180 200 prolongation (%) Bazett (%) Fredericia (%) 0 20 40 60 80 100 120 140 160 180 200 Beat rate (%) 0 20 40 60 80 100 120 140 160 180 200 0 20 40 60 80 100 120 140 160 180 200 1.E-13 1.E-12 Na amplitude (%) 0 50 100 150 200 1.E-10 1.E-09 1.E-08 1.E-07 1.E-06 1.E-05 1.E-04 Na amplitude QT Interval BPM Figure 3. The Effects of Various Chemical Compounds on ReproCardio2 TM by MEA. To see the affect of different drugs, thin layers were made from cell clamps. The thin layer ReproCardio2 TM was exposed to various concentration of drugs from 300 pM to 30 μM. The data was obtained from MCS system, with 2 minutes of drug exposure and 2 minutes of recording. 0 0 Na Blocker : Flecainide Na Blocker : Flecainide 2.Calcium imaging Contraction Relaxation Analysis points BPM AMP (a) Positive Slope (b) Negative Slope (c) PWD (10-90%) (d) etc. a b c d A.) Ikr antagonist (E-4031) Na channel blocker (Flecainide) 0 M (vehicle) Note. Arrows mean EAD (Arrhythmia) B.) BPM AMP Note. Conc. 0, 30, 100, 300, 3, 30, 100 μM Note. Each dot mean Average ± S.E. (n = 6) Positive slope /Negative slope PWD10/50/900 1E10 1E09 1E08 1E07 1E06 1E05 1E04 1E03 0 1E10 1E09 1E08 1E07 1E06 1E05 1E04 1E03 0 1E10 1E09 1E08 1E07 1E06 1E05 1E04 1E03 0 1E10 1E09 1E08 1E07 1E06 1E05 1E04 1E03 0 50 100 150 200 0 50 100 150 200 0 50 100 150 200 0 50 100 150 200 C.) Aspirin BPM AMP PWD10/50/900 50 100 150 200 0 1E10 1E09 1E08 1E07 1E06 1E05 1E04 1E03 0 50 100 150 200 0 1E10 1E09 1E08 1E07 1E06 1E05 1E04 1E03 0 50 100 150 200 0 1E10 1E09 1E08 1E07 1E06 1E05 1E04 1E03 0 1E10 1E09 1E08 1E07 1E06 1E05 1E04 1E03 0 50 100 150 200 Positive slope /Negative slope Verapamil BPM AMP Positive slope /Negative slope PWD10/50/900 1E10 1E09 1E08 1E07 1E06 1E05 1E04 1E03 0 1E10 1E09 1E08 1E07 1E06 1E05 1E04 1E03 0 1E10 1E09 1E08 1E07 1E06 1E05 1E04 1E03 0 1E10 1E09 1E08 1E07 1E06 1E05 1E04 1E03 0 50 100 150 200 0 50 100 150 200 0 50 100 150 200 0 50 100 150 200 Isoproterenol BPM AMP Positive slope /Negative slope PWD10/50/900 1E10 1E09 1E08 1E07 1E06 1E05 1E04 1E03 0 1E10 zz1E09 1E08 1E07 1E06 1E05 1E04 1E03 0 1E10 1E09 1E08 1E07 1E06 1E05 1E04 1E03 0 1E10 1E09 1E08 1E07 1E06 1E05 1E04 1E03 0 50 100 150 200 0 50 100 150 200 0 50 100 150 200 0 50 100 150 200 E4031 BPM AMP Positive slope /Negative slope PWD10/50/900 1E10 1E09 1E08 1E07 1E06 1E05 1E04 1E03 0 1E10 1E09 1E08 1E07 1E06 1E05 1E04 1E03 0 1E10 1E09 1E08 1E07 1E06 1E05 1E04 1E03 0 1E10 1E09 1E08 1E07 1E06 1E05 1E04 1E03 0 20 40 60 80 100 120 140 160 180 200 0 20 40 60 80 100 120 140 160 180 200 0 20 40 60 80 100 120 140 160 180 200 0 50 100 150 200 250 300 350 400 Flecainide Figure 4. The Effects of Various Chemical Compounds on ReproCardio2 TM by Calcium-imaging. A) The various parameters can be interpreted from Ca- imaging. B) The change of graph after admitting drugs. ReproCardio2 TM was exposed to various concentration of drugs from 300pM-30μM. Ca signal from ReproCardio2 TM can be detected for a hour. The data was obtained from 2 minutes of drug exposure and 2 minutes of recording. Human iPS cell-derived cardiomyocytes ReproCardio2 TM displays no differences between cell-lots , and exhibits constant affects of the drug screening even with different analyzing methods . The utility of ReproCardio2 TM is also expandable . This demonstrated that ReproCardio2 TM is suitable for the large-scale screening of chemical compounds. 0 E401 concentration (M) 0 20 40 60 80 100 120 140 160 180 200 Prolongation (%) 0 20 40 60 80 100 120 140 160 180 200 Prolongation (%) 0 0 20 40 60 80 100 120 140 160 180 200 1.E-10 1.E-09 1.E-08 1.E-07 1.E-06 1.E-05 1.E-04 prolongation (%) Bazett (%) Fredericia (%) E401 concentration (M)

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Page 1: Human iPS cell-Derived Cardiomyocytes ReproCardio2TM as an ... · Human iPS cell-derived cardiomyocytes ReproCardio2TM displays no differences between cell-lots, and exhibits constant

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Human iPS cell-Derived Cardiomyocytes ReproCardio2TM as an Analytic Tool for Large-scale Toxicity Screening

Shunsuke Yoshida1, Zachary Yu-Ching Lin1, Joylynn Clark2, Mark Bryant3, Takashi Masai1, Mitsuru Inamura1 1ReproCell, Yokohama, Japan; 2ReproCell, Boston, USA; 3ReproCell, Crewe, UK

Abstract

Materials and Methods

Result

Conclusion & Discussion

The force-frequency relationship in cardiac muscle contraction is different in developmental maturation and pathological states. Hence, cardiac contraction can be utilized for the evaluation of chemical compounds especially when these compounds affect the beating rate. In this study, various chemical stimulations on the contraction of human iPS cell-derived cardiomyocytes ReproCardio2TM were examined with electrophysiological assay as well as calcium-imaging. ReproCardio2TM possess stable BPM and regular QT interval with minimum differences between cell-lots after been thaw from cryopreservation. With the addition of chemical compounds, the extension or reduction of QT-interval can be obtained by both electrophysiological assay and calcium-imaging. Other novel utility of ReproCardio2TM has also been developed. Taken together, ReproCardio2TM is useful for analyzing the effects of chemical compounds on contractile function. Therefore, ReproCardio2TM can be utilized in large-scale screening of large-scale of chemical compounds

ReproCardio2TM KIT (1 full kit for 96 well plate: ReproCELL # RCESD008) 1. 3.3×105 Single cells x 3 cryo-preserved vials 2. Empty Cryo-preserved Cell vial (dummy) 3. Maintenance culture medium (80mL) x 2 4. Coating solution (30mL) x 1 5. Low attachment plate for aggregated cells 6. Attachment plate for Thin-layer / Single cell

Calcium-imaging Assay

FDSS day 0 day 1-3 day 3 day 4-14

Testing compounds

Electrophysiological assay

day 0 day 1-3 day 3 day 4-14

Testing compounds Analyzer

Step 0 1 2 3 4 … 9 … 14 1 Thawing 2 Making loose clump 3 Transfer into MEA assay probe 4 Making Thin layer 5 Drug evaluation 6 Analysis

Step 0 1 2 3 4 … 9 … 14 1 Thawing 2 Making  loose  clump 3 Transfer  into  assay  plate 4 Making  Thin  layer 5 Staining  and  Evaluate  drugs 6 Analysis

Immunocytochemistry

•  Cells ・・・ 10,000 cells / well •  Equipment ・・・ FDSS Hamamatsu •  Ca indicator ・・・ Cal520 (AAT bioquest) •  Assay Medium ・・・ReproCardio assay medium (ReproCELL), Cremophor EL (Sigma)

•  Cells ・・・ 15000 cells / well •  Equipment ・・・ Available (aMED, AXION, MCS) •  Coating ・・・ Fibronectin •  Assay Medium ・・・ ReproCardio assay medium (ReproCELL) or Culture medium (ReproCELL) with FBS •  Option ・・・ 5% CO2 gas/Air

1. Primary antibody: anti-Conexin 43, MYH6/7(Sigma); anti-MLC-2A/2V (Synaptic systems); anti-Troponin (A Bio-Rad company) and anti-Vimentin (Millipore). 2. Secondary antibody: Alexa Fluor 488 (Life Technologies)

Characteristic of ReproCardio2™ MLC2V / DAPI αMHC / DAPI cTnT / DAPI CX43 / DAPI MLC2A / DAPI βMHC / DAPI Vimentin / DAPI

Figure 1. ReproCardio2™ has various cardiac markers like human heart α-MHC and βMHC are the representative cardiac marker during the differentiation from iPS cells and expressed in heart. MLC-2A and MLC-2V are myosin specific marker for the atrium of the mammalian heart and for the ventricle of the mammalian heart, respectively. Cardiac troponin T (cTnT) is a specific marker in human heart and a thin filament protein which takes part in muscle contraction. Cx43 is conniving gap junction found in several organs and cell types, but is most often found in the heart. Those proteins were used as representative cardiac marker. Scale bar : 30 µm.

Electrophysiological Assay utilizing ReproCardio2™

1. MEA ASSAY

Figure 2. Reproducibility of MEA assay: within the lot or between lot To prove there is no drastic difference within the cell lot, thin layers were made from cell clamps from the same vial of ReproCardio2TM. The different clamp-derived thin layers were exposed to various concentration of E4031 from 300 pM to 30 µM. The data obtained from each thin layers were plotted together, and it shows that E4031 has similar prolonging QT interval affect in all thin layer from the same cell lot. To prove cell lots has no drastic difference, we exposed various concentration of E4031 from 300 pM to 30µM to 3 different cell lots. The graphs showed that 3 different cell lots exhibit similar prolonging QT interval that is known for. This demonstrates that ReproCardio2TM has low production bias within lot as well as from lot to lot.

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Figure 3. The Effects of Various Chemical Compounds on ReproCardio2TM by MEA. To see the affect of different drugs, thin layers were made from cell clamps. The thin layer ReproCardio2TM was exposed to various concentration of drugs from 300 pM to 30 µM. The data was obtained from MCS system, with 2 minutes of drug exposure and 2 minutes of recording.

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Figure 4. The Effects of Various Chemical Compounds on ReproCardio2TM by Calcium-imaging. A) The various parameters can be interpreted from Ca-imaging. B) The change of graph after admitting drugs. ReproCardio2TM was exposed to various concentration of drugs from 300pM-30µM. Ca signal from ReproCardio2TM

can be detected for a hour. The data was obtained from 2 minutes of drug exposure and 2 minutes of recording.

Human iPS cell-derived cardiomyocytes ReproCardio2TM displays no differences between cell-lots, and exhibits constant affects of the drug screening even with different analyzing methods. The utility of ReproCardio2TM is also expandable. This demonstrated that ReproCardio2TM is suitable for the large-scale screening of chemical compounds.

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