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Human iPS cell-derived cardiomyocytes ReproCardio 2™ as an analytic tool for large-scale toxicity screening
Abstracts
which allows investigations of both the extracellular field potential (EFP) and the total impedance in High Throughput format. In addition, the instrument is capable of electrical stimulation of the cellular monolayer, while the rapid acquisition rate of 1 ms allows a more accurate description of signal features and nuances stemming from pharmacological effects. This technology addresses the lack of easy-to-use HighThroughput in vitro assays, and permits the reliable investigation of short- and long-term pharmacological effects including compounds such as the hERG trafficking inhibitor Pentamidin. A broad range of cell lines was successfully validated for use with the CardioExcyte 96, such as stem cell-derived cardiomyocytes from Axiogenesis (human: Cor4U®, murine:CorAt®), GE Healthcare (Cytiva), Cellectis (human, 3D-clusters: hES-CMC™) and CDI (iCell®). doi:10.1016/j.vascn.2015.08.117
0120 Pluricyte® cardiomyocytes: Benefits and characteristics of improving the maturity of stem cell-derived cardiomyocytes with a fully-defined culture medium Stefan Braam, Rob Towart Pluriomics BV, Leiden, The Netherlands Human pluripotent stem cell (hPSC)-derived cardiomyocytes are providing an attractive opportunity for front-loading cardiac safety pharmacology and toxicology. These cardiomyocytes express most relevant ion channels and demonstrate beating and action potentials similar to primary cardiac cells. However, when compared with primary adult cardiomyocytes, hPSC-CMs until now have remained immature, based on their membrane potentials, action potential characteristics and ill-defined ultra-structural organization. A new initiative (Comprehensive in vitro Proarrhythmia Assay (CiPA)) is a novel cardiac safety screening proposal, partly relying on hPSC-derived cardiomyocytes. Intended to replace the existing regulatory strategy (ICH S7B), implementation of this initiative will replace the ICH S7B guidelines worldwide. Until now, almost all laboratories producing hPSC-derived cardiomyocytes have used — have had to use — culture systems containing serum. Two disadvantages are that 1) the cells remain immature even after long periods of culture, and 2) undefined growth hormones and/or tissue factors in the serum can cause variable characteristics. To solve these problems, Pluriomics has now developed novel cell culture systems, which are free of serum and other undefined components. These systems support efficient differentiation and maturation of cardiomyocytes. The Pluricyte® cardiomyocytes generated and maintained in this new culture system show enhanced sarcomeric organization, a more negative membrane resting potential and increased upstroke velocities. Pluricyte® cardiomyocytes functionally resemble human primary cells, with the additional benefit of stability in culture. In particular the avoidance of serum enables long term assays, without commonly-encountered problems such as compound plasma protein binding.
doi:10.1016/j.vascn.2015.08.118
0121 Human iPS cell-derived cardiomyocytes ReproCardio 2™ as an analytic tool for large-scale toxicity screening Shunsuke Yoshidaa, Yu Ching Lina, Joylynn Clarkb, Mark Bryantc, Takashi Masaia, Mitsuru Inamuraa
193
a
ReproCELL, Yokohama, Japan ReproCELL, Boston, MA, USA c ReproCELL, Crewe, UK b
Force–frequency relationships in cardiac muscle contraction vary between animal species, as well as in different pathological states and developmental maturation stages. Hence, cardiac contraction analysis allows the evaluation of chemical compounds which affect the beating rate, in particular. In the present study, various effects of chemical stimulations on the contractile properties of human iPS cell-derived cardiomyocytes were examined with electrophysiological assay (MEA assay) as well as calcium-imaging. Both MEA assay and calcium-imaging were used to measure the contraction and to analyze contractile parameters (beating per minute; sodium and potassium amplitudes as well as the QT interval). To examine force– frequency relationships, various chemical compounds such as Aspirin, Verapamil, Isoproterenol, E-4031 and Flecainide were applied to ReproCardio 2™. ReproCardio 2™ possesses stable beat rate and regular QT interval with minimum variability between celllots. Upon the addition of chemical compounds, the prolongation or reduction of the QT interval was observed. Similar results were obtained by calcium-image analysis. Taken together, these results show that ReproCardio 2™ is useful for analyzing the effects of chemical compounds on contractile function. Hence, ReproCardio 2™ can be utilized in the evaluation of drug toxicity screening.
doi:10.1016/j.vascn.2015.08.119
0122 Measurement of optical action potentials and calcium transients in hIPSC-derived cardiomyocytes using the novel Optopatch© fluorescent protein platform Khuram W. Chaudharya, Graham Dempseyb, Joel Kraljb, Cuong Nguyenb, Nicholas Atwaterb, Barry S. Browna, Dennis Murphya, John McNeisha a
GlaxoSmithKline, King of Prussia, PA, USA Q-STATE Biosciences, Cambridge, MA, USA
b
The CIPA initiative suggests the adoption of a hIPSC-derived cardiomyocyte (hIPSC) assay to identify proarrhythmic potential of novel pharmaceutics. Because calcium-triggered early after depolarizations (EADs) are an electrical phenomenon, the measurement of field potentials and action potentials (APs) have been identified as appropriate endpoints for CIPA. Objective: To identify compound-dependent effects on APs and calcium transients (CTs) with the novel Optopatch© technology; utilizing fluorescent proteins for stimulation of cardiomyocytes and AP/CT recording. Methods: The “CheRiff”, “QuasAr2” and/or “CaViar” proteins were expressed in hIPSCs, allowing for pacing and reporting of fluorescent APs and/or CTs, respectively. Electrophysiological (AP rise time, APD50, APD90, beat rate) and/or CT effects of 13 compounds were tested after two weeks of hIPSC culture (as prescribed by cell provider — CDI) in spontaneously beating and paced cultures (1 Hz and 2 Hz). Results: Quinidine, flecainide, E-4031, digoxin and cisapride prolonged, while verapamil and nifedipine shortened APs. Unexpectedly, azithromycin and clarithromycin shortened, while lidocaine prolonged APs. EADs were elicited by quinidine, flecainide and cisapride, but not by E-4031. All but 2 compounds (amiodarone, chromanol) prolonged AP rise time and cessation of beating was observed for lidocaine, flecainide, terfenadine, azithromycin, nifedipine,
which allows investigations of both the extracellular field potential (EFP) and the total impedance in High Throughput format. In addition, the instrument is capable of electrical stimulation of the cellular monolayer, while the rapid acquisition rate of 1 ms allows a more accurate description of signal features and nuances stemming from pharmacological effects. This technology addresses the lack of easy-to-use HighThroughput in vitro assays, and permits the reliable investigation of short- and long-term pharmacological effects including compounds such as the hERG trafficking inhibitor Pentamidin. A broad range of cell lines was successfully validated for use with the CardioExcyte 96, such as stem cell-derived cardiomyocytes from Axiogenesis (human: Cor4U®, murine:CorAt®), GE Healthcare (Cytiva), Cellectis (human, 3D-clusters: hES-CMC™) and CDI (iCell®). doi:10.1016/j.vascn.2015.08.117
0120 Pluricyte® cardiomyocytes: Benefits and characteristics of improving the maturity of stem cell-derived cardiomyocytes with a fully-defined culture medium Stefan Braam, Rob Towart Pluriomics BV, Leiden, The Netherlands Human pluripotent stem cell (hPSC)-derived cardiomyocytes are providing an attractive opportunity for front-loading cardiac safety pharmacology and toxicology. These cardiomyocytes express most relevant ion channels and demonstrate beating and action potentials similar to primary cardiac cells. However, when compared with primary adult cardiomyocytes, hPSC-CMs until now have remained immature, based on their membrane potentials, action potential characteristics and ill-defined ultra-structural organization. A new initiative (Comprehensive in vitro Proarrhythmia Assay (CiPA)) is a novel cardiac safety screening proposal, partly relying on hPSC-derived cardiomyocytes. Intended to replace the existing regulatory strategy (ICH S7B), implementation of this initiative will replace the ICH S7B guidelines worldwide. Until now, almost all laboratories producing hPSC-derived cardiomyocytes have used — have had to use — culture systems containing serum. Two disadvantages are that 1) the cells remain immature even after long periods of culture, and 2) undefined growth hormones and/or tissue factors in the serum can cause variable characteristics. To solve these problems, Pluriomics has now developed novel cell culture systems, which are free of serum and other undefined components. These systems support efficient differentiation and maturation of cardiomyocytes. The Pluricyte® cardiomyocytes generated and maintained in this new culture system show enhanced sarcomeric organization, a more negative membrane resting potential and increased upstroke velocities. Pluricyte® cardiomyocytes functionally resemble human primary cells, with the additional benefit of stability in culture. In particular the avoidance of serum enables long term assays, without commonly-encountered problems such as compound plasma protein binding.
doi:10.1016/j.vascn.2015.08.118
0121 Human iPS cell-derived cardiomyocytes ReproCardio 2™ as an analytic tool for large-scale toxicity screening Shunsuke Yoshidaa, Yu Ching Lina, Joylynn Clarkb, Mark Bryantc, Takashi Masaia, Mitsuru Inamuraa
193
a
ReproCELL, Yokohama, Japan ReproCELL, Boston, MA, USA c ReproCELL, Crewe, UK b
Force–frequency relationships in cardiac muscle contraction vary between animal species, as well as in different pathological states and developmental maturation stages. Hence, cardiac contraction analysis allows the evaluation of chemical compounds which affect the beating rate, in particular. In the present study, various effects of chemical stimulations on the contractile properties of human iPS cell-derived cardiomyocytes were examined with electrophysiological assay (MEA assay) as well as calcium-imaging. Both MEA assay and calcium-imaging were used to measure the contraction and to analyze contractile parameters (beating per minute; sodium and potassium amplitudes as well as the QT interval). To examine force– frequency relationships, various chemical compounds such as Aspirin, Verapamil, Isoproterenol, E-4031 and Flecainide were applied to ReproCardio 2™. ReproCardio 2™ possesses stable beat rate and regular QT interval with minimum variability between celllots. Upon the addition of chemical compounds, the prolongation or reduction of the QT interval was observed. Similar results were obtained by calcium-image analysis. Taken together, these results show that ReproCardio 2™ is useful for analyzing the effects of chemical compounds on contractile function. Hence, ReproCardio 2™ can be utilized in the evaluation of drug toxicity screening.
doi:10.1016/j.vascn.2015.08.119
0122 Measurement of optical action potentials and calcium transients in hIPSC-derived cardiomyocytes using the novel Optopatch© fluorescent protein platform Khuram W. Chaudharya, Graham Dempseyb, Joel Kraljb, Cuong Nguyenb, Nicholas Atwaterb, Barry S. Browna, Dennis Murphya, John McNeisha a
GlaxoSmithKline, King of Prussia, PA, USA Q-STATE Biosciences, Cambridge, MA, USA
b
The CIPA initiative suggests the adoption of a hIPSC-derived cardiomyocyte (hIPSC) assay to identify proarrhythmic potential of novel pharmaceutics. Because calcium-triggered early after depolarizations (EADs) are an electrical phenomenon, the measurement of field potentials and action potentials (APs) have been identified as appropriate endpoints for CIPA. Objective: To identify compound-dependent effects on APs and calcium transients (CTs) with the novel Optopatch© technology; utilizing fluorescent proteins for stimulation of cardiomyocytes and AP/CT recording. Methods: The “CheRiff”, “QuasAr2” and/or “CaViar” proteins were expressed in hIPSCs, allowing for pacing and reporting of fluorescent APs and/or CTs, respectively. Electrophysiological (AP rise time, APD50, APD90, beat rate) and/or CT effects of 13 compounds were tested after two weeks of hIPSC culture (as prescribed by cell provider — CDI) in spontaneously beating and paced cultures (1 Hz and 2 Hz). Results: Quinidine, flecainide, E-4031, digoxin and cisapride prolonged, while verapamil and nifedipine shortened APs. Unexpectedly, azithromycin and clarithromycin shortened, while lidocaine prolonged APs. EADs were elicited by quinidine, flecainide and cisapride, but not by E-4031. All but 2 compounds (amiodarone, chromanol) prolonged AP rise time and cessation of beating was observed for lidocaine, flecainide, terfenadine, azithromycin, nifedipine,