title: Dual optical recordings for action potentials and calcium handling in induced pluripotent stem cell models of cardiac arrhythmias using genetically encoded fluorescent indicators.
abstract: 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.
authors.affiliation: Columbia University Stem Cell Initiative, Department of Rehabilitation and Regenerative Medicine, Department of Pharmacology, and Department of Pediatrics and Medicine, College of Physicians and Surgeons, Columbia University, New York, New York, USA; Barnard College, New York, New York, USA.
authors.forename: LouJin
authors.initials: L
authors.lastname: Song
authors.affiliation: Columbia University Stem Cell Initiative, Department of Rehabilitation and Regenerative Medicine, Department of Pharmacology, and Department of Pediatrics and Medicine, College of Physicians and Surgeons, Columbia University, New York, New York, USA; Barnard College, New York, New York, USA.
authors.forename: Daniel W
authors.initials: DW
authors.lastname: Awari
authors.affiliation: Columbia University Stem Cell Initiative, Department of Rehabilitation and Regenerative Medicine, Department of Pharmacology, and Department of Pediatrics and Medicine, College of Physicians and Surgeons, Columbia University, New York, New York, USA; Barnard College, New York, New York, USA.
authors.forename: Elizabeth Y
authors.initials: EY
authors.lastname: Han
authors.affiliation: Columbia University Stem Cell Initiative, Department of Rehabilitation and Regenerative Medicine, Department of Pharmacology, and Department of Pediatrics and Medicine, College of Physicians and Surgeons, Columbia University, New York, New York, USA; Barnard College, New York, New York, USA.
authors.forename: Eugenia
authors.initials: E
authors.lastname: Uche-Anya
authors.affiliation: Columbia University Stem Cell Initiative, Department of Rehabilitation and Regenerative Medicine, Department of Pharmacology, and Department of Pediatrics and Medicine, College of Physicians and Surgeons, Columbia University, New York, New York, USA; Barnard College, New York, New York, USA.
authors.forename: Seon-Hye E
authors.initials: SH
authors.lastname: Park
authors.affiliation: Columbia University Stem Cell Initiative, Department of Rehabilitation and Regenerative Medicine, Department of Pharmacology, and Department of Pediatrics and Medicine, College of Physicians and Surgeons, Columbia University, New York, New York, USA; Barnard College, New York, New York, USA.
authors.forename: Yoko A
authors.initials: YA
authors.lastname: Yabe
authors.affiliation: Columbia University Stem Cell Initiative, Department of Rehabilitation and Regenerative Medicine, Department of Pharmacology, and Department of Pediatrics and Medicine, College of Physicians and Surgeons, Columbia University, New York, New York, USA; Barnard College, New York, New York, USA.
authors.forename: Wendy K
authors.initials: WK
authors.lastname: Chung
authors.affiliation: Columbia University Stem Cell Initiative, Department of Rehabilitation and Regenerative Medicine, Department of Pharmacology, and Department of Pediatrics and Medicine, College of Physicians and Surgeons, Columbia University, New York, New York, USA; Barnard College, New York, New York, USA my2387@columbia.edu masayuki.yazawa@gmail.com.
authors.forename: Masayuki
authors.initials: M
authors.lastname: Yazawa
mesh_terms: D000200:Action Potentials / Q000235:genetics; D001145:Arrhythmias, Cardiac / Q000473:pathology*; D001321:Autistic Disorder; D002118:Calcium / Q000378:metabolism; D002454:Cell Differentiation / Q000235:genetics*; D065150:Cellular Reprogramming / Q000235:genetics*; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism; D015603:Keratinocytes / Q000166:cytology; D008133:Long QT Syndrome / Q000473:pathology; D008164:Luminescent Proteins / Q000737:chemistry; D032383:Myocytes, Cardiac / Q000166:cytology*; D010641:Phenotype; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013576:Syndactyly / Q000473:pathology
chemical_list: C576393:Arclight fusion protein; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D002118:Calcium
keywords: Cardiac arrhythmia; Disease modeling; Drug test; Human induced pluripotent stem cells; Optical recording