- Email: [email protected]
Force–frequency analysis of contraction using a novel laser-speckle contrast techniques in iPS-derived cardiomyocytes, Repro Cardio2™
Abstracts
OF
Human induced pluripotent stem cell (hIPSC) derived and purified cardiomyocytes (iCells®, CDI) form electrically coupled, spontaneously beating cell monolayers (syncytia) upon plating and continuous culture. In this study, we tested 20 compounds on the iCell syncytium to cast a wide safety net, probing not only ion channel related activity, but also cardiac receptors and signaling cascades known to affect cardiac automaticity, inotropy and rhythmicity. Ion channel and receptor modulators, electrogenic pump and enzyme inhibitors were tested in the label free impedance monitoring platform xCELLigence® Cardio. After 14 days in culture in 96-well E-plates, the spontaneous iCell beating rates vary minimally from well to well, with an average of about 0.5 Hz at 37 °C. Eighteen out-of 20 compounds tested had marked effects on the spontaneous beating of the myocytes. The effects on beating rate, impedance signal amplitude, impedance transient duration, as well as beat regularity reveal a beating parameter phenotype for each type of target activity. For example, adrenergic agonists (isoproterenol, phenylephrine, dobutamine) and Ca2 + channel blockers (diltiazem, nifedipine and verapamil) all increase the spontaneous beating rate at their respective target affinity concentrations. However, Ca2 + blockers also decrease the fluctuating impedance signal amplitude, while adrenergic agonists leave the signal amplitude relatively unchanged. Analogously, Na+/K+ pump inhibitors (digoxin and lanatoside), adrenergic antagonist (prazosin) and Na+ channel blocker (flecainide) first slow then cease spontaneous beating. Adenosine and carbachol also slow beating, but introduce beat rate variability, without cessation of beating. Effect phenotypes may be categorized by the type and extent of beat parameter perturbations.
RO
To assess pharmacological responsiveness of human iPS-derived cardiomyocytes, we developed a drug-screening system utilizing a field potential (FP) measurement with multi-array surface electrode recording. We measured drug effects on the rate-corrected field potential duration (FPDc) and the short term variability (STVc) of FPDc of the iPS-derived cardiomyocytes. Experiments were conducted under either spontaneously beating or electrically paced conditions. Pacing improved recordings of FP measurement and stabilized the amplitude of the 1st peak of FP, which reflects the depolarization phase mainly triggered by Na channels. Because of the low standard deviation (SD) of the amplitude of the 1st peak by pacing, a suppressing effect of disopyramide was sensitively detected at a lower dose compared with the spontaneous beating cells. Low SD value by pacing was also demonstrated in the 1st amplitude with E-4031, and in the FPDc with disopyramide, pilsicainide, diltiazem, verapamil, Bay K 8644, dl-sotalol and Chromanol 293B, and in the STVc with diltiazem. It was shown that our FP recording protocol of human iPS-derived cardiomyocytes predicts drug toxic effects on human hearts. Our challenge is to characterize appropriate indexes or their combinations, which will provide a reliable drug screening system on human hearts with good cost-performance and high throughput.
355
doi:10.1016/j.vascn.2014.03.155
DP
0151 Lack of clear effects of QT-shortening drugs in human pluripotent stem cell-derived cardiomyocytes: A mystery that still needs to be solved Hua Rong Lu, Jutta Rohrbacher, An N. Hermans, David J. Gallacher
0153
TE
Safety Pharmacology Research, Translational Sciences, Janssen Research & Development, Beerse, Belgium
doi:10.1016/j.vascn.2014.03.157
Force–frequency analysis of contraction using a novel laser-speckle contrast techniques in iPS-derived cardiomyocytes, Repro Cardio2™ Kohei Sawadaa, Atsuko Ojimaa, Tomohiko Taniguchia, Norimasa Miyamotoa, Hidenao Iwaib, Norikazu Sugiyamab, Takuji Kataokab, Shunsuke Yoshidac
UN
CO
RR
EC
Both human embryonic and induced pluripotent stem cell-derived cardiomyocytes (hES-CMs and hiPS-CMs) are increasingly used as new sources of cardiac cells for drug safety assessment. In the present study, we investigated the effects of IKr activators and a IKATP channel opener in hES-CMs and in hiPS-CMs using a microelectrode array system (MEA). These effects were compared with the results generated from the isolated rabbit left-ventricular wedge preparation (IWP). NS1643 and mallotoxin (IKr activators) did not shorten the field potential duration (fAPD) in either hES-CMs nor in hiPS-CMs. In addition, levcromakalim (IKATP channel opener) did not induce consistent shortening of fAPD in these CMs. On the other hand, all these 3 compounds markedly shortened QT-interval and elicited ventricular tachycardia/fibrillation in the IWP. The reasons why hES-CMs/hIPS-CMs do not show clear responses to these two types of drugs remain unknown. Conclusion: although hES-CMs and hiPS-CMs can be used to detect drug-induced long QT, the reason for the differences between the rabbit wedge (a currently used de-risking assay) and stem cell derived cardiomyocytes has still to be solved in future. doi:10.1016/j.vascn.2014.03.156
0152
Responses of iCell hIPSC-derived cardiomyocytes to cardiac modulators in the xCELLigence cardio label free impedance platform Bharathi Balasubramanian, Ted Lis, Zhenhua Wu, Cheryl Gretzula, Irene Gregan, Vic Uebele, Steve Fox, Armando Lagrutta, John Imredy, Frederick Sannajust Merck, Sharp, & Dohme, West Point, PA, USA
a
Eisai Co. Ltd., Tsukuba-shi, Ibaraki, Japan Hamamatsu Photonics K.K., Hamamatsu-shi, Shizuoka, Japan c ReproCell Inc., Yokohama-shi, Kanagawa, Japan b
It is important to control the beating rate to evaluate the effects of compounds on cardiac contractile function, especially for compounds affecting the beating rate, because force–frequency relationships in cardiac muscle contraction are known to differ between animal species, diseased states, or maturation stages. In the present study, effects of various stimulations on contractile properties of iPS cellderived cardiomyocytes were examined with novel analytical techniques of contractile function. A dome-shaped cluster of cardiomyocytes (Repro Cardio2™), which was re-constructed from cardiospheres and cultured for 7 days in adhesion culture conditions, was used. A novel laser-speckle contrast technique (Hamamatsu Photonics), was used to measure the contraction and to analyze contractile parameters (velocity, duration and amount of contraction and relaxation, etc.).To examine force–frequency relationships, electrical stimulation through extracellular electrodes was applied, and the effects of isoproterenol and verapamil, which affect both contraction and beating rates, were compared. The dome-shaped Repro Cardio2™ showed basal beating rates of 30–60 bpm and was driven at 1, 1.5 and 2 Hz. With an increase in stimulation frequency, the velocity of contraction decreased, suggesting a negative force–frequency relationship. Verapamil decreased the velocity of contraction and relaxation at concentrations higher than
Abstracts
doi:10.1016/j.vascn.2014.03.158
0154 Optimization and pharmacological characterization of iCell cardiomyocytes for electrophysiological assessment in a multi-electrode extracellular recording system Kohei Sawada, Tomohiko Taniguchi, Atsuko Ojima, Takkashi Yoshinaga, Norimasa Miyamoto Eisai Tsukuba Laboratories, Tsukuba-shi, Ibaraki, Japan
doi:10.1016/j.vascn.2014.03.160
DP
iPS cell derived cardiomyocytes have become a valuable tool for safety assessment in drug discovery. In order to translate the results obtained in these cells into in vivo effects in humans, basic physiological or pharmacological properties of these cells should be well characterized. Present studies examined the effects of temperature, cell density and representative ion channel modulators with MED64 in iCell cardiomyocytes. When the experimental temperature was changed from 35 to 38.5 °C, the corrected field potential duration (FPDc) decreased from 370 to 285 msec (24 msec/°C) almost linearly, but beating rate increased from 42 to 66 bpm at 37 °C and did not increase further even at 38.5 °C. The cell density of plating affected the response to Iks blockers. The concentration–response curves for 293B and HMR1556 shifted to the right when cell densities were decreased from 30,000, 15,000 and 7500 cells/well. In lower density conditions, cell sizes enlarged and expression of the KCNQ1 gene decreased. The hERG channel blocker, E4031, consistently prolonged the FPDc at concentrations higher than 3 nM regardless of the cell density. A calcium channel blocker, verapamil, decreased the FPDc concentration-dependently at concentrations 30 nM and above, but increased the beating rate significantly at the same concentrations. The If channel blocker, ZD7288 decreased the beating rate in a concentration-dependent manner at 0.3 mM and above. Sodium and T-type calcium channel blockers also had some effects. These results indicate that the basic information on experimental conditions and pharmacological effects is very important for translation of the data.
scarcity. Therefore other approaches have been pursued by researchers, including stem cell treatments. While cell based therapies are important and provide the future state of the art, there is also a need to develop new systems which more effectively prevent, diagnose and treat human liver injury and disease. Methods: A renewable stem cell resource was employed in these studies. Pluripotent stem cells were effectively driven through key developmental intermediates, using serum free components, to yield highly active and drug inducible human hepatocyte populations. Results: During the differentiation process, pluripotent stem cells displayed simultaneous down-regulation of pluripotent markers with up-regulation of key differentiation markers. In line with the human development, endodermal cell populations could be efficiently directed down the liver lineage, prior to mature hepatic differentiation. Of note, stem cell derived hepatocytes exhibited drug inducible CYP P450 metabolism and displayed superior predictive qualities to gold standard regulated human models used in a series of screening experiments. Conclusion: We have delivered a scalable human resource which faithfully models human liver development and biology in a dish. Such a resource has a direct application in safety testing, regenerative medicine and may play an important role in the development of future cell-based therapies.
OF
30 nM, but the beating rate increased and reached to about twice the basal rate at 300 nM. Isoproterenol increased both contraction and relaxation velocity and beating frequency. From these results, it is concluded that the laser-speckle contrast technique is useful for the analysis of the effects of compounds on contractile function.
RO
356
0156
TE
Efficient and cost-effective batch analysis of multi-electrode array (MEA) data from human iPS cardiomyocytes using Intelligent Waveform Service (IWS) Tyson Thomasa, Kate Harrisb, Arun Sridharb, Yi Cuib a
Neural ID, Redwood City, CA, USA GlaxoSmithKline, Ware, Hertfordshire, UK
0155
CO
doi:10.1016/j.vascn.2014.03.159
RR
EC
b
a
UN
Accurate prediction of drug induced liver injury using stem cell based approaches Dagmara Szkolnickaa, Sarah Farnwortha, Balta Lucendo-Villarina, Chris Storckb, Wenli Zhoua, John Iredalea, Oliver Flintb, David Haya University of Edinburgh, Edinburgh, UK Bristol-Myers Squibb, New Jersey, UK
b
Background: Despite major progress in the knowledge and management of human liver disease, there are in excess of 29 million people in the European Union estimated to suffer from chronic liver morbidities. Currently, the only cure for end-stage liver disease is orthotopic liver transplantation which is limited by donor organ
Human induced pluripotent stem cell cardiomyocytes (hiPSCCMs) are a potential source to develop assays for predictive electrophysiological safety screening. Published studies show relevant physiology, and recently we demonstrated strong translation between stem cell assays and other preclinical tests. Large volumes of multielectrode array (MEA) data are generated and analysis using existing tools is cumbersome. To improve timeliness and cost-effectiveness of data reporting, we demonstrate for the first time a scalable analysis platform and have tested its applicability compared to existing tools. Neural ID's Intelligent Waveform Service (IWS) with its patternbased methodology was used to analyze MEA data. Field potential duration (FPD), beat rate (RR interval), conduction time and total spike amplitude were measured with re-usable analysis templates that leverage machine learning. The easy set-up templates can be trained with example patterns and enable batch jobs to be run against multiple file sets and data formats, providing analysis standards that are shareable between researchers and groups. Results are written to a database and reporting is seamless with plug-ins for Microsoft Excel and IDBS BioBook using built-in queries (SQL) to generate relevant measurements. The accuracy of the data analysis using IWS was superior to existing tools and speed of analysis was dramatically improved; average analysis was five times faster compared to previous techniques. In conclusion, we show that analysis and reporting times can be reduced by up to five times and employing pattern recognition technology will improve accuracy and facilitate greater use of MEAs in mechanistic compound screening. doi:10.1016/j.vascn.2014.03.161
OF
Human induced pluripotent stem cell (hIPSC) derived and purified cardiomyocytes (iCells®, CDI) form electrically coupled, spontaneously beating cell monolayers (syncytia) upon plating and continuous culture. In this study, we tested 20 compounds on the iCell syncytium to cast a wide safety net, probing not only ion channel related activity, but also cardiac receptors and signaling cascades known to affect cardiac automaticity, inotropy and rhythmicity. Ion channel and receptor modulators, electrogenic pump and enzyme inhibitors were tested in the label free impedance monitoring platform xCELLigence® Cardio. After 14 days in culture in 96-well E-plates, the spontaneous iCell beating rates vary minimally from well to well, with an average of about 0.5 Hz at 37 °C. Eighteen out-of 20 compounds tested had marked effects on the spontaneous beating of the myocytes. The effects on beating rate, impedance signal amplitude, impedance transient duration, as well as beat regularity reveal a beating parameter phenotype for each type of target activity. For example, adrenergic agonists (isoproterenol, phenylephrine, dobutamine) and Ca2 + channel blockers (diltiazem, nifedipine and verapamil) all increase the spontaneous beating rate at their respective target affinity concentrations. However, Ca2 + blockers also decrease the fluctuating impedance signal amplitude, while adrenergic agonists leave the signal amplitude relatively unchanged. Analogously, Na+/K+ pump inhibitors (digoxin and lanatoside), adrenergic antagonist (prazosin) and Na+ channel blocker (flecainide) first slow then cease spontaneous beating. Adenosine and carbachol also slow beating, but introduce beat rate variability, without cessation of beating. Effect phenotypes may be categorized by the type and extent of beat parameter perturbations.
RO
To assess pharmacological responsiveness of human iPS-derived cardiomyocytes, we developed a drug-screening system utilizing a field potential (FP) measurement with multi-array surface electrode recording. We measured drug effects on the rate-corrected field potential duration (FPDc) and the short term variability (STVc) of FPDc of the iPS-derived cardiomyocytes. Experiments were conducted under either spontaneously beating or electrically paced conditions. Pacing improved recordings of FP measurement and stabilized the amplitude of the 1st peak of FP, which reflects the depolarization phase mainly triggered by Na channels. Because of the low standard deviation (SD) of the amplitude of the 1st peak by pacing, a suppressing effect of disopyramide was sensitively detected at a lower dose compared with the spontaneous beating cells. Low SD value by pacing was also demonstrated in the 1st amplitude with E-4031, and in the FPDc with disopyramide, pilsicainide, diltiazem, verapamil, Bay K 8644, dl-sotalol and Chromanol 293B, and in the STVc with diltiazem. It was shown that our FP recording protocol of human iPS-derived cardiomyocytes predicts drug toxic effects on human hearts. Our challenge is to characterize appropriate indexes or their combinations, which will provide a reliable drug screening system on human hearts with good cost-performance and high throughput.
355
doi:10.1016/j.vascn.2014.03.155
DP
0151 Lack of clear effects of QT-shortening drugs in human pluripotent stem cell-derived cardiomyocytes: A mystery that still needs to be solved Hua Rong Lu, Jutta Rohrbacher, An N. Hermans, David J. Gallacher
0153
TE
Safety Pharmacology Research, Translational Sciences, Janssen Research & Development, Beerse, Belgium
doi:10.1016/j.vascn.2014.03.157
Force–frequency analysis of contraction using a novel laser-speckle contrast techniques in iPS-derived cardiomyocytes, Repro Cardio2™ Kohei Sawadaa, Atsuko Ojimaa, Tomohiko Taniguchia, Norimasa Miyamotoa, Hidenao Iwaib, Norikazu Sugiyamab, Takuji Kataokab, Shunsuke Yoshidac
UN
CO
RR
EC
Both human embryonic and induced pluripotent stem cell-derived cardiomyocytes (hES-CMs and hiPS-CMs) are increasingly used as new sources of cardiac cells for drug safety assessment. In the present study, we investigated the effects of IKr activators and a IKATP channel opener in hES-CMs and in hiPS-CMs using a microelectrode array system (MEA). These effects were compared with the results generated from the isolated rabbit left-ventricular wedge preparation (IWP). NS1643 and mallotoxin (IKr activators) did not shorten the field potential duration (fAPD) in either hES-CMs nor in hiPS-CMs. In addition, levcromakalim (IKATP channel opener) did not induce consistent shortening of fAPD in these CMs. On the other hand, all these 3 compounds markedly shortened QT-interval and elicited ventricular tachycardia/fibrillation in the IWP. The reasons why hES-CMs/hIPS-CMs do not show clear responses to these two types of drugs remain unknown. Conclusion: although hES-CMs and hiPS-CMs can be used to detect drug-induced long QT, the reason for the differences between the rabbit wedge (a currently used de-risking assay) and stem cell derived cardiomyocytes has still to be solved in future. doi:10.1016/j.vascn.2014.03.156
0152
Responses of iCell hIPSC-derived cardiomyocytes to cardiac modulators in the xCELLigence cardio label free impedance platform Bharathi Balasubramanian, Ted Lis, Zhenhua Wu, Cheryl Gretzula, Irene Gregan, Vic Uebele, Steve Fox, Armando Lagrutta, John Imredy, Frederick Sannajust Merck, Sharp, & Dohme, West Point, PA, USA
a
Eisai Co. Ltd., Tsukuba-shi, Ibaraki, Japan Hamamatsu Photonics K.K., Hamamatsu-shi, Shizuoka, Japan c ReproCell Inc., Yokohama-shi, Kanagawa, Japan b
It is important to control the beating rate to evaluate the effects of compounds on cardiac contractile function, especially for compounds affecting the beating rate, because force–frequency relationships in cardiac muscle contraction are known to differ between animal species, diseased states, or maturation stages. In the present study, effects of various stimulations on contractile properties of iPS cellderived cardiomyocytes were examined with novel analytical techniques of contractile function. A dome-shaped cluster of cardiomyocytes (Repro Cardio2™), which was re-constructed from cardiospheres and cultured for 7 days in adhesion culture conditions, was used. A novel laser-speckle contrast technique (Hamamatsu Photonics), was used to measure the contraction and to analyze contractile parameters (velocity, duration and amount of contraction and relaxation, etc.).To examine force–frequency relationships, electrical stimulation through extracellular electrodes was applied, and the effects of isoproterenol and verapamil, which affect both contraction and beating rates, were compared. The dome-shaped Repro Cardio2™ showed basal beating rates of 30–60 bpm and was driven at 1, 1.5 and 2 Hz. With an increase in stimulation frequency, the velocity of contraction decreased, suggesting a negative force–frequency relationship. Verapamil decreased the velocity of contraction and relaxation at concentrations higher than
Abstracts
doi:10.1016/j.vascn.2014.03.158
0154 Optimization and pharmacological characterization of iCell cardiomyocytes for electrophysiological assessment in a multi-electrode extracellular recording system Kohei Sawada, Tomohiko Taniguchi, Atsuko Ojima, Takkashi Yoshinaga, Norimasa Miyamoto Eisai Tsukuba Laboratories, Tsukuba-shi, Ibaraki, Japan
doi:10.1016/j.vascn.2014.03.160
DP
iPS cell derived cardiomyocytes have become a valuable tool for safety assessment in drug discovery. In order to translate the results obtained in these cells into in vivo effects in humans, basic physiological or pharmacological properties of these cells should be well characterized. Present studies examined the effects of temperature, cell density and representative ion channel modulators with MED64 in iCell cardiomyocytes. When the experimental temperature was changed from 35 to 38.5 °C, the corrected field potential duration (FPDc) decreased from 370 to 285 msec (24 msec/°C) almost linearly, but beating rate increased from 42 to 66 bpm at 37 °C and did not increase further even at 38.5 °C. The cell density of plating affected the response to Iks blockers. The concentration–response curves for 293B and HMR1556 shifted to the right when cell densities were decreased from 30,000, 15,000 and 7500 cells/well. In lower density conditions, cell sizes enlarged and expression of the KCNQ1 gene decreased. The hERG channel blocker, E4031, consistently prolonged the FPDc at concentrations higher than 3 nM regardless of the cell density. A calcium channel blocker, verapamil, decreased the FPDc concentration-dependently at concentrations 30 nM and above, but increased the beating rate significantly at the same concentrations. The If channel blocker, ZD7288 decreased the beating rate in a concentration-dependent manner at 0.3 mM and above. Sodium and T-type calcium channel blockers also had some effects. These results indicate that the basic information on experimental conditions and pharmacological effects is very important for translation of the data.
scarcity. Therefore other approaches have been pursued by researchers, including stem cell treatments. While cell based therapies are important and provide the future state of the art, there is also a need to develop new systems which more effectively prevent, diagnose and treat human liver injury and disease. Methods: A renewable stem cell resource was employed in these studies. Pluripotent stem cells were effectively driven through key developmental intermediates, using serum free components, to yield highly active and drug inducible human hepatocyte populations. Results: During the differentiation process, pluripotent stem cells displayed simultaneous down-regulation of pluripotent markers with up-regulation of key differentiation markers. In line with the human development, endodermal cell populations could be efficiently directed down the liver lineage, prior to mature hepatic differentiation. Of note, stem cell derived hepatocytes exhibited drug inducible CYP P450 metabolism and displayed superior predictive qualities to gold standard regulated human models used in a series of screening experiments. Conclusion: We have delivered a scalable human resource which faithfully models human liver development and biology in a dish. Such a resource has a direct application in safety testing, regenerative medicine and may play an important role in the development of future cell-based therapies.
OF
30 nM, but the beating rate increased and reached to about twice the basal rate at 300 nM. Isoproterenol increased both contraction and relaxation velocity and beating frequency. From these results, it is concluded that the laser-speckle contrast technique is useful for the analysis of the effects of compounds on contractile function.
RO
356
0156
TE
Efficient and cost-effective batch analysis of multi-electrode array (MEA) data from human iPS cardiomyocytes using Intelligent Waveform Service (IWS) Tyson Thomasa, Kate Harrisb, Arun Sridharb, Yi Cuib a
Neural ID, Redwood City, CA, USA GlaxoSmithKline, Ware, Hertfordshire, UK
0155
CO
doi:10.1016/j.vascn.2014.03.159
RR
EC
b
a
UN
Accurate prediction of drug induced liver injury using stem cell based approaches Dagmara Szkolnickaa, Sarah Farnwortha, Balta Lucendo-Villarina, Chris Storckb, Wenli Zhoua, John Iredalea, Oliver Flintb, David Haya University of Edinburgh, Edinburgh, UK Bristol-Myers Squibb, New Jersey, UK
b
Background: Despite major progress in the knowledge and management of human liver disease, there are in excess of 29 million people in the European Union estimated to suffer from chronic liver morbidities. Currently, the only cure for end-stage liver disease is orthotopic liver transplantation which is limited by donor organ
Human induced pluripotent stem cell cardiomyocytes (hiPSCCMs) are a potential source to develop assays for predictive electrophysiological safety screening. Published studies show relevant physiology, and recently we demonstrated strong translation between stem cell assays and other preclinical tests. Large volumes of multielectrode array (MEA) data are generated and analysis using existing tools is cumbersome. To improve timeliness and cost-effectiveness of data reporting, we demonstrate for the first time a scalable analysis platform and have tested its applicability compared to existing tools. Neural ID's Intelligent Waveform Service (IWS) with its patternbased methodology was used to analyze MEA data. Field potential duration (FPD), beat rate (RR interval), conduction time and total spike amplitude were measured with re-usable analysis templates that leverage machine learning. The easy set-up templates can be trained with example patterns and enable batch jobs to be run against multiple file sets and data formats, providing analysis standards that are shareable between researchers and groups. Results are written to a database and reporting is seamless with plug-ins for Microsoft Excel and IDBS BioBook using built-in queries (SQL) to generate relevant measurements. The accuracy of the data analysis using IWS was superior to existing tools and speed of analysis was dramatically improved; average analysis was five times faster compared to previous techniques. In conclusion, we show that analysis and reporting times can be reduced by up to five times and employing pattern recognition technology will improve accuracy and facilitate greater use of MEAs in mechanistic compound screening. doi:10.1016/j.vascn.2014.03.161