Dual Optical Recordings for Action Potentials and Calcium Handling in Induced Pluripotent Stem Cell Models of Cardiac Arrhythmias Using Genetically Encoded Fluorescent Indicators

• Published in: Stem cells translational medicine Vol. 4; no. 5; pp. 468 - 475
• Main Authors: Song, LouJin, Awari, Daniel W., Han, Elizabeth Y., Uche-Anya, Eugenia, Park, Seon-Hye E., Yabe, Yoko A., Chung, Wendy K., Yazawa, Masayuki
• Format: Journal Article
• Language: English
• Published: Durham, NC, USA AlphaMed Press 01.05.2015; Oxford University Press
• Subjects: Action Potentials - genetics; Arrhythmia; Arrhythmias, Cardiac - pathology; Autistic Disorder; Calcium - metabolism; Calcium imaging; Cardiac arrhythmia; Cardiomyocytes; Cardiovascular disease; Cell culture; Cell Differentiation - genetics; Cell-Based Drug Development, Screening, and Toxicology; Cellular Reprogramming - genetics; Congenital diseases; Coronary artery disease; Data analysis; Deoxyribonucleic acid; Disease modeling; DNA; Drug test; Drug testing; Efficiency; Fibroblasts; Fluorescent indicators; Heart diseases; Human induced pluripotent stem cells; Humans; Hypoxia; Induced Pluripotent Stem Cells - cytology; Induced Pluripotent Stem Cells - metabolism; Keratinocytes; Keratinocytes - cytology; Long QT syndrome; Long QT Syndrome - pathology; Luminescent Proteins - chemistry; Medicine; Myocytes, Cardiac - cytology; Optical recording; Phenotype; Phenotypes; Phenotyping; Pluripotency; Recombinant Fusion Proteins - chemistry; Roscovitine; Somatic cells; Stem cells; Studies; Syndactyly - pathology; Human induced pluripotent stem cells; Cardiac arrhythmia; Drug test; Disease modeling; Optical recording
• ISSN: 2157-6564; 2157-6580
• DOI: 10.5966/sctm.2014-0245

Reprogramming of human somatic cells to pluripotency has been used to investigate disease mechanisms and to identify potential therapeutics. However, the methods used for reprogramming, in vitro differentiation, and phenotyping are still complicated, expensive, and time‐consuming. 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 induced pluripotent stem cells (iPSCs) from seven patients with cardiac diseases and three controls. Second, we differentiated human iPSCs derived from patients with Timothy syndrome into cardiomyocytes using a monolayer differentiation method. We found that Timothy syndrome cardiomyocytes showed slower, irregular contractions and abnormal calcium handling compared with the controls. The results are consistent with previous reports using a retroviral method for reprogramming and an embryoid body‐based method for cardiac differentiation. Third, we developed an efficient approach for recording the 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 that 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 investigate mechanisms underlying cardiac arrhythmias and to test potential therapeutics. An optimized protocol enabled efficient reprogramming that can be applied to a variety of patients for developing induced pluripotent stem cell (iPSC)‐based models of cardiac diseases. The dual optical recording using R‐GECO1 and ArcLight fluorescent indicators is a robust approach to examine physiological phenotypes and to test drug candidates in long QT syndrome iPSC‐derived cardiomyocytes.