Yongming Wang1, Ping Liang2, Feng Lan2, Haodi Wu2, Leszek Lisowski3, Mingxia Gu2, Shijun Hu2, Mark A Kay3, Fyodor D Urnov4, Rami Shinnawi5, Joseph D Gold6, Lior Gepstein5, Joseph C Wu7. 1. Department of Medicine, Division of Cardiology, Stanford University School of Medicine, Stanford, California; Stanford Cardiovascular Institute, Stanford University School of Medicine, Stanford, California; State Key Laboratory of Genetic Engineering, Fudan University, Shanghai, China. 2. Department of Medicine, Division of Cardiology, Stanford University School of Medicine, Stanford, California; Stanford Cardiovascular Institute, Stanford University School of Medicine, Stanford, California; Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, California. 3. Departments of Pediatrics and Genetics, Stanford University School of Medicine, Stanford, California. 4. Sangamo Biosciences, Richmond, California. 5. Rambam Medical Center, Technion, Israel Institute of Technology, Haifa, Israel. 6. Department of Medicine, Division of Cardiology, Stanford University School of Medicine, Stanford, California; Stanford Cardiovascular Institute, Stanford University School of Medicine, Stanford, California. 7. Department of Medicine, Division of Cardiology, Stanford University School of Medicine, Stanford, California; Stanford Cardiovascular Institute, Stanford University School of Medicine, Stanford, California; Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, California. Electronic address: joewu@stanford.edu.
Abstract
BACKGROUND: Human induced pluripotent stem cells (iPSCs) play an important role in disease modeling and drug testing. However, the current methods are time-consuming and lack an isogenic control. OBJECTIVES: This study sought to establish an efficient technology to generate human PSC-based disease models with isogenic control. METHODS: The ion channel genes KCNQ1 and KCNH2 with dominant negative mutations causing long QT syndrome types 1 and 2, respectively, were stably integrated into a safe harbor AAVS1 locus using zinc finger nuclease technology. RESULTS: Patch-clamp recording revealed that the edited iPSC-derived cardiomyocytes (iPSC-CMs) displayed characteristic long QT syndrome phenotype and significant prolongation of the action potential duration compared with the unedited control cells. Finally, addition of nifedipine (L-type calcium channel blocker) or pinacidil (KATP-channel opener) shortened the action potential duration of iPSC-CMs, confirming the validity of isogenic iPSC lines for drug testing in the future. CONCLUSIONS: Our study demonstrates that iPSC-CM-based disease models can be rapidly generated by overexpression of dominant negative gene mutants.
BACKGROUND:Human induced pluripotent stem cells (iPSCs) play an important role in disease modeling and drug testing. However, the current methods are time-consuming and lack an isogenic control. OBJECTIVES: This study sought to establish an efficient technology to generate human PSC-based disease models with isogenic control. METHODS: The ion channel genes KCNQ1 and KCNH2 with dominant negative mutations causing long QT syndrome types 1 and 2, respectively, were stably integrated into a safe harbor AAVS1 locus using zinc finger nuclease technology. RESULTS: Patch-clamp recording revealed that the edited iPSC-derived cardiomyocytes (iPSC-CMs) displayed characteristic long QT syndrome phenotype and significant prolongation of the action potential duration compared with the unedited control cells. Finally, addition of nifedipine (L-type calcium channel blocker) or pinacidil (KATP-channel opener) shortened the action potential duration of iPSC-CMs, confirming the validity of isogenic iPSC lines for drug testing in the future. CONCLUSIONS: Our study demonstrates that iPSC-CM-based disease models can be rapidly generated by overexpression of dominant negative gene mutants.
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