title: Purification of small molecule-induced cardiomyocytes from human induced pluripotent stem cells using a reporter system.
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.
authors.affiliation: College of Pharmacy, Research Institute of Pharmaceutical Sciences, Kyungpook National University, Daegu, Korea.
authors.forename: Geun Hye
authors.initials: GH
authors.lastname: Hwang
authors.affiliation: College of Pharmacy, Research Institute of Pharmaceutical Sciences, Kyungpook National University, Daegu, Korea.
authors.forename: So Mi
authors.initials: SM
authors.lastname: Park
authors.affiliation: College of Veterinary Medicine, Seoul National University, Seoul, Korea.
authors.forename: Ho Jae
authors.initials: HJ
authors.lastname: Han
authors.affiliation: Research Center, Dongnam Institute of Radiological & Medical Sciences (DIRAMS), Busan, Korea.
authors.forename: Joong Sun
authors.initials: JS
authors.lastname: Kim
authors.affiliation: Neuroregeneration and Stem Cell Programs, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland.
authors.forename: Seung Pil
authors.initials: SP
authors.lastname: Yun
authors.affiliation: Department of Veterinary Physiology, College of Veterinary Medicine, Chonnam National University, Gwangju, Korea.
authors.forename: Jung Min
authors.initials: JM
authors.lastname: Ryu
authors.affiliation: Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut.
authors.forename: Ho Jin
authors.initials: HJ
authors.lastname: Lee
authors.affiliation: Department of Biology Education, College of Education, Pusan National University, Busan, Korea.
authors.forename: Woochul
authors.initials: W
authors.lastname: Chang
authors.affiliation: College of Pharmacy, The Catholic University of Korea, Bucheon, Gyeonggi-do, Korea.
authors.forename: Su-Jin
authors.initials: SJ
authors.lastname: Lee
authors.affiliation: College of Pharmacy, The Catholic University of Korea, Bucheon, Gyeonggi-do, Korea.
authors.forename: Jeong-Hee
authors.initials: JH
authors.lastname: Choi
authors.affiliation: College of Pharmacy, The Catholic University of Korea, Bucheon, Gyeonggi-do, Korea.
authors.forename: Jin-Sung
authors.initials: JS
authors.lastname: Choi
authors.affiliation: College of Pharmacy, Research Institute of Pharmaceutical Sciences, Kyungpook National University, Daegu, Korea.
authors.forename: Min Young
authors.initials: MY
authors.lastname: Lee
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D002460:Cell Line; D002469:Cell Separation; D017930:Genes, Reporter; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism; D051379:Mice; D032383:Myocytes, Cardiac / Q000166:cytology* / Q000378:metabolism; D060449:Wnt Signaling Pathway
keywords: cardiac differentiation; human induced pluripotent stem cells; purification of cardiomyocytes; reporter system; small molecule