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J Cell Physiol. 2017 Jan 7. doi: 10.1002/jcp.25783. [Epub ahead of print]

Purification of Small Molecule-induced Cardiomyocytes from Human Induced Pluripotent Stem Cells Using a Reporter System.

Author information

1
College of Pharmacy, Research Institute of Pharmaceutical Sciences, Kyungpook National University, Daegu, Korea.
2
College of Veterinary Medicine, Seoul National University, Seoul, Korea.
3
Research Center, Dongnam Institute of Radiological & Medical Sciences (DIRAMS), Busan, Korea.
4
Neuroregeneration and Stem Cell Programs, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD.
5
Department of Veterinary Physiology, College of Veterinary Medicine, Chonnam National University, Gwangju, Korea.
6
Department of Pharmacology, Yale University School of Medicine, New Haven, CT.
7
Department of Biology Education, College of Education, Pusan National University, Busan, Korea.
8
College of Pharmacy, The Catholic University of Korea, Bucheon, Gyeonggi-do, Korea.

Abstract

In order to realize the practical use of human pluripotent stem cell (hPSC)-derived cardiomyocytes for the purpose of clinical use or cardiovascular research, the generation of large numbers of highly purified cardiomyocytes should be achieved. Here, we show an efficient method for cardiac differentiation of human induced pluripotent stem cells (hiPSCs) in chemically defined conditions and purification of hiPSC-derived cardiomyocytes using a reporter system. Regulation of the Wnt/β-catenin signaling pathway is implicated in the induction of the cardiac differentiation of hPSCs. We increased cardiac differentiation efficiency of hiPSCs in chemically defined conditions through combined treatment with XAV939, a tankyrase inhibitor and IWP2, a porcupine inhibitor and optimized concentrations. Although cardiac differentiation efficiency was high (>80%), it was difficult to suppress differentiation into non-cardiac cells, Therefore, we applied a lentiviral reporter system, wherein green fluorescence protein (GFP) and Zeocin-resistant gene are driven by promoter activation of a gene (TNNT2) encoding cardiac troponin T (cTnT), a cardiac-specific protein, to exclude non-cardiomyocytes from differentiated cell populations. We transduced this reporter construct into differentiated cells using a lentiviral vector and then obtained highly purified hiPSC-derived cardiomyocytes by treatment with the lowest effective dose of Zeocin. We significantly increased transgenic efficiency through manipulation of the cells in which the differentiated cells were simultaneously infected with virus and re-plated after single-cell dissociation. Purified cells specifically expressed GFP, cTnT, displayed typical properties of cardiomyocytes. This study provides an efficient strategy for obtaining large quantities of highly purified hPSC-derived cardiomyocytes for application in regenerative medicine and biomedical research. This article is protected by copyright. All rights reserved.

KEYWORDS:

Cardiac differentiation; Human induced pluripotent stem cells; Purification of cardiomyocytes; Reporter system; Small molecule

PMID:
28063225
DOI:
10.1002/jcp.25783
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