A fast optical kinetic plate reader for assessing action potentials in human-induced pluripotent stem cell cardiomyocytes

2021 ◽  
Vol 111 ◽  
pp. 106967
Author(s):  
Stephen Smith ◽  
Joerg Oestreich ◽  
Jay Trautman ◽  
Andy Blatz
Keyword(s):  
Stem Cell ◽  
Action Potentials ◽  
Pluripotent Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
Plate Reader ◽  
Induced Pluripotent

Abstract 161: Induced Pluripotent Stem Cell-based Model of Cardiac Arrhythmia: New Platform for Drug Screen

2015 ◽  
Vol 117 (suppl_1) ◽  
Author(s):  
LouJin Song ◽  
Masayuki Yazawa
Keyword(s):  
Stem Cell ◽  
Action Potentials ◽  
Pluripotent Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
Calcium Handling ◽  
Drug Screen ◽  
Cardiac Diseases ◽  
Timothy Syndrome ◽  
Human Ipscs ◽  
Induced Pluripotent

Human induced pluripotent stem cell (iPSC)-based model of cardiac diseases has been proved to be useful and valuable for identifying new therapeutics. However, the use of human iPSC-based model of cardiac diseases for drug screen is hampered by the high-cost and complexity of methods used for reprogramming, in vitro differentiation, and phenotyping. To address the limitations, we first optimized a protocol for reprogramming of human fibroblasts and keratinocytes into pluripotency using single lipofection and the episomal vectors in a 24-well plate format. This method allowed us to generate multiple lines of integration-free and feeder-free iPSCs from seven patients with cardiac diseases and three controls. Second, we differentiated human iPSCs derived from Timothy syndrome patients into cardiomyocytes using a monolayer differentiation method. We found that Timothy syndrome cardiomyocytes showed slower, irregular contractions and abnormal calcium handling compared to controls, which were consistent with previous reports using a retroviral method for reprogramming and using an embryoid body-based method for cardiac differentiation. Third, we developed an efficient approach for recording action potentials and calcium transients simultaneously in control and patient cardiomyocytes using genetically encoded fluorescent indicators, ArcLight and R-GECO1. The dual optical recordings enabled us to observe prolonged action potentials and abnormal calcium handling in Timothy syndrome cardiomyocytes. We confirmed that roscovitine rescued the phenotypes in Timothy syndrome cardiomyocytes and these findings were consistent with previous studies using conventional electrophysiological recordings and calcium imaging with dyes. The approaches using our optimized methods and dual optical recordings will improve iPSC applicability for disease modeling to test potential therapeutics. With those new approaches in hand, next we plan to use the iPSC-based model of Timothy syndrome to investigate novel molecules involved in the pathogenesis of Timothy syndrome and to screen and identify new therapeutic compounds for Timothy syndrome patients.

scholarly journals Drug-Mediated Shortening of Action Potentials in LQTS2 Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

2017 ◽  
Vol 26 (23) ◽  
pp. 1695-1705 ◽  
Author(s):  
Gary Duncan ◽  
Karl Firth ◽  
Vinoj George ◽  
Minh Duc Hoang ◽  
Andrew Staniforth ◽  
...  
Keyword(s):  
Stem Cell ◽  
Action Potentials ◽  
Pluripotent Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
Induced Pluripotent

scholarly journals Dynamic Current Clamp Experiments Define the Functional Roles of IK1 and Ito,f in Human Induced Pluripotent Stem Cell Derived Cardiomyocytes

2017 ◽  
Author(s):  
Scott B. Marrus ◽  
Steven Springer ◽  
Eric Johnson ◽  
Rita J. Martinez ◽  
Edward J. Dranoff ◽  
...  
Keyword(s):  
Stem Cell ◽  
Action Potential ◽  
Action Potentials ◽  
Pluripotent Stem Cell ◽  
Phase 1 ◽  
Induced Pluripotent Stem Cell ◽  
Resting Membrane Potential ◽  
Current Clamp ◽  
Human Cardiomyocytes ◽  
Induced Pluripotent

AbstractThe transient outward potassium current (Ito) plays a key, albeit incompletely defined, role in cardiomyocyte physiology and pathophysiology. In light of the technical challenges of studying adult human cardiomyocytes, this study examines the use of induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) as a system which potentially preserves the native cellular milieu of human cardiomyocytes. ISPC-CMs express a robust Ito with slow recovery kinetics and fail to express the rapidly recovering Ito,f which is implicated in human disease. Overexpression of the accessory subunit KChIP2 (which is not expressed in iPSC-CMs) resulted in restoration of a rapid component of recovery. To define the functional role of Ito, dynamic current clamp was used to introduce computationally modeled currents into iPSC-CMs while recording action potentials. However, iPSC-CMs exhibit action potentials with multiple immature physiological properties, including slow upstroke velocity, heterogeneous action potential waveforms, and the absence of a phase 1 notch, thus potentially limiting the utility of these cells as a model of adult cardiomyocytes. Importantly, the introduction of modeled inwardly rectified current (IK1) ameliorated these immature properties by restoring a hyperpolarized resting membrane potential. In this context of normalized action potential morphologies, dynamic current clamp experiments introducing Ito,f demonstrated that there is significant cell-to-cell heterogeneity and that the functional effect of Ito,f is highly sensitive to the action potential plateau voltage in each cell.

Abstract 026: Optical Recording of Cardiac Action Potentials from Human Induced Pluripotent Stem Cell Derived Cardiomyocytes

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
B Alexander Yi ◽  
Joel M Kralj ◽  
Adam E Cohen
Keyword(s):  
Stem Cell ◽  
High Throughput ◽  
Action Potentials ◽  
Pluripotent Stem Cell ◽  
Gene Mutations ◽  
Induced Pluripotent Stem Cell ◽  
Patch Clamping ◽  
Cardiac Action ◽  
Or Gene ◽  
Induced Pluripotent

The electrically excitable properties of cardiomyocytes stem from the activity of ion channels that allow the coordinated entry of ions to generate cardiac action potentials. Disruptions in ion channel function either by drugs or gene mutations can lead to cardiac arrhythmias. The ability to screen drugs or gene mutations rapidly for effects on the cardiac action potential would be of interest for both drug discovery as well as for studies of ion channel function; however, the time-consuming and technically challenging nature of conventional patch clamping can limit the ability to perform high throughput screens. Archaerhodopsin3, or Arch, is an Archaebacterial variant of the membrane protein bacteriorhodopsin that binds a retinal fluorophore whose signal is rapidly responsive to changes in membrane potential. Here, we report the use of Arch to optically record action potentials from human induced pluripotent stem cell-derived cardiomyocytes. Human induced pluripotent stem cells that stably express Arch were generated and then differentiated into cardiomyocytes. As compared to patch clamping, Arch faithfully reproduces many of the key features of cardiac action potentials and may be a tool to be used for high throughput electrophysiological screens of cardiomyocytes.

scholarly journals Repolarization instability and arrhythmia by IKr block in single human-induced pluripotent stem cell-derived cardiomyocytes and 2D monolayers

EP Europace ◽  
2020 ◽  
Vol 22 (9) ◽  
pp. 1431-1441
Author(s):  
Cristina Altrocchi ◽  
Tessa de Korte ◽  
Joyce Bernardi ◽  
Roel L H M G Spätjens ◽  
Stefan R Braam ◽  
...  
Keyword(s):  
Stem Cell ◽  
Action Potential ◽  
Action Potentials ◽  
Pluripotent Stem Cell ◽  
Field Potential ◽  
Induced Pluripotent Stem Cell ◽  
Electrode Arrays ◽  
Cms Line ◽  
Induced Pluripotent

Abstract Aims Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have proven valuable for studies in drug discovery and safety, although limitations regarding their structural and electrophysiological characteristics persist. In this study, we investigated the electrophysiological properties of Pluricyte® CMs, a commercially available hiPSC-CMs line with a ventricular phenotype, and assessed arrhythmia incidence by IKr block at the single-cell and 2D monolayer level. Methods and results Action potentials were measured at different pacing frequencies, using dynamic clamp. Through voltage-clamp experiments, we determined the properties of INa, IKr, and ICaL. Intracellular Ca2+ measurements included Ca2+-transients at baseline and during caffeine perfusion. Effects of IKr block were assessed in single hiPSC-CMs and 2D monolayers (multi-electrode arrays). Action-potential duration (APD) and its rate dependence in Pluricyte® CMs were comparable to those reported for native human CMs. INa, IKr, and ICaL revealed amplitudes, kinetics, and voltage dependence of activation/inactivation similar to other hiPSC-CM lines and, to some extent, to native CMs. Near-physiological Ca2+-induced Ca2+ release, response to caffeine and excitation–contraction coupling gain characterized the cellular Ca2+-handling. Dofetilide prolonged the APD and field-potential duration, and induced early afterdepolarizations. Beat-to-beat variability of repolarization duration increased significantly before the first arrhythmic events in single Pluricyte® CMs and 2D monolayers, and predicted pending arrhythmias better than action-potential prolongation. Conclusion Taking their ion-current characteristics and Ca2+ handling into account, Pluricyte® CMs are suitable for in vitro studies on action potentials and field potentials. Beat-to-beat variability of repolarization duration proved useful to evaluate the dynamics of repolarization instability and demonstrated its significance as proarrhythmic marker in hiPSC-CMs during IKr block.

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