title: A massive suspension culture system with metabolic purification for human pluripotent stem cell-derived cardiomyocytes.
abstract: Cardiac regenerative therapy with human pluripotent stem cells (hPSCs), such as human embryonic stem cells and induced pluripotent stem cells, has been hampered by the lack of efficient strategies for expanding functional cardiomyocytes (CMs) to clinically relevant numbers. The development of the massive suspension culture system (MSCS) has shed light on this critical issue, although it remains unclear how hPSCs could differentiate into functional CMs using a MSCS. The proliferative rate of differentiating hPSCs in the MSCS was equivalent to that in suspension cultures using nonadherent culture dishes, although the MSCS provided more homogeneous embryoid bodies (EBs), eventually reducing apoptosis. However, pluripotent markers such as Oct3/4 and Tra-1-60 were still expressed in EBs 2 weeks after differentiation, even in the MSCS. The remaining undifferentiated stem cells in such cultures could retain a strong potential for teratoma formation, which is the worst scenario for clinical applications of hPSC-derived CMs. The metabolic purification of CMs in glucose-depleted and lactate-enriched medium successfully eliminated the residual undifferentiated stem cells, resulting in a refined hPSC-derived CM population. In colony formation assays, no Tra-1-60-positive colonies appeared after purification. The nonpurified CMs in the MSCS produced teratomas at a rate of 60%. However, purified CMs never induced teratomas, and enriched CMs showed proper electrophysiological properties and calcium transients. Overall, the combination of a MSCS and metabolic selection is a highly effective and practical approach to purify and enrich massive numbers of functional CMs and provides an essential technique for cardiac regenerative therapy with hPSC-derived CMs.
authors.affiliation: Departments of Cardiology and Cardiovascular Surgery, Keio University School of Medicine, Tokyo, Japan; Japan Society for the Promotion of Science, Tokyo, Japan; Department of Cardiovascular Research, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan.
authors.forename: Natsuko
authors.initials: N
authors.lastname: Hemmi
authors.affiliation: Departments of Cardiology and Cardiovascular Surgery, Keio University School of Medicine, Tokyo, Japan; Japan Society for the Promotion of Science, Tokyo, Japan; Department of Cardiovascular Research, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan.
authors.forename: Shugo
authors.initials: S
authors.lastname: Tohyama
authors.affiliation: Departments of Cardiology and Cardiovascular Surgery, Keio University School of Medicine, Tokyo, Japan; Japan Society for the Promotion of Science, Tokyo, Japan; Department of Cardiovascular Research, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan.
authors.forename: Kazuaki
authors.initials: K
authors.lastname: Nakajima
authors.affiliation: Departments of Cardiology and Cardiovascular Surgery, Keio University School of Medicine, Tokyo, Japan; Japan Society for the Promotion of Science, Tokyo, Japan; Department of Cardiovascular Research, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan.
authors.forename: Hideaki
authors.initials: H
authors.lastname: Kanazawa
authors.affiliation: Departments of Cardiology and Cardiovascular Surgery, Keio University School of Medicine, Tokyo, Japan; Japan Society for the Promotion of Science, Tokyo, Japan; Department of Cardiovascular Research, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan.
authors.forename: Tomoyuki
authors.initials: T
authors.lastname: Suzuki
authors.affiliation: Departments of Cardiology and Cardiovascular Surgery, Keio University School of Medicine, Tokyo, Japan; Japan Society for the Promotion of Science, Tokyo, Japan; Department of Cardiovascular Research, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan.
authors.forename: Fumiyuki
authors.initials: F
authors.lastname: Hattori
authors.affiliation: Departments of Cardiology and Cardiovascular Surgery, Keio University School of Medicine, Tokyo, Japan; Japan Society for the Promotion of Science, Tokyo, Japan; Department of Cardiovascular Research, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan.
authors.forename: Tomohisa
authors.initials: T
authors.lastname: Seki
authors.affiliation: Departments of Cardiology and Cardiovascular Surgery, Keio University School of Medicine, Tokyo, Japan; Japan Society for the Promotion of Science, Tokyo, Japan; Department of Cardiovascular Research, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan.
authors.forename: Yoshikazu
authors.initials: Y
authors.lastname: Kishino
authors.affiliation: Departments of Cardiology and Cardiovascular Surgery, Keio University School of Medicine, Tokyo, Japan; Japan Society for the Promotion of Science, Tokyo, Japan; Department of Cardiovascular Research, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan.
authors.forename: Akinori
authors.initials: A
authors.lastname: Hirano
authors.affiliation: Departments of Cardiology and Cardiovascular Surgery, Keio University School of Medicine, Tokyo, Japan; Japan Society for the Promotion of Science, Tokyo, Japan; Department of Cardiovascular Research, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan.
authors.forename: Marina
authors.initials: M
authors.lastname: Okada
authors.affiliation: Departments of Cardiology and Cardiovascular Surgery, Keio University School of Medicine, Tokyo, Japan; Japan Society for the Promotion of Science, Tokyo, Japan; Department of Cardiovascular Research, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan.
authors.forename: Ryota
authors.initials: R
authors.lastname: Tabei
authors.affiliation: Departments of Cardiology and Cardiovascular Surgery, Keio University School of Medicine, Tokyo, Japan; Japan Society for the Promotion of Science, Tokyo, Japan; Department of Cardiovascular Research, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan.
authors.forename: Rei
authors.initials: R
authors.lastname: Ohno
authors.affiliation: Departments of Cardiology and Cardiovascular Surgery, Keio University School of Medicine, Tokyo, Japan; Japan Society for the Promotion of Science, Tokyo, Japan; Department of Cardiovascular Research, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan.
authors.forename: Chihana
authors.initials: C
authors.lastname: Fujita
authors.affiliation: Departments of Cardiology and Cardiovascular Surgery, Keio University School of Medicine, Tokyo, Japan; Japan Society for the Promotion of Science, Tokyo, Japan; Department of Cardiovascular Research, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan.
authors.forename: Tomoko
authors.initials: T
authors.lastname: Haruna
authors.affiliation: Departments of Cardiology and Cardiovascular Surgery, Keio University School of Medicine, Tokyo, Japan; Japan Society for the Promotion of Science, Tokyo, Japan; Department of Cardiovascular Research, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan.
authors.forename: Shinsuke
authors.initials: S
authors.lastname: Yuasa
authors.affiliation: Departments of Cardiology and Cardiovascular Surgery, Keio University School of Medicine, Tokyo, Japan; Japan Society for the Promotion of Science, Tokyo, Japan; Department of Cardiovascular Research, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan.
authors.forename: Motoaki
authors.initials: M
authors.lastname: Sano
authors.affiliation: Departments of Cardiology and Cardiovascular Surgery, Keio University School of Medicine, Tokyo, Japan; Japan Society for the Promotion of Science, Tokyo, Japan; Department of Cardiovascular Research, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan jfujita@a6.keio.jp.
authors.forename: Jun
authors.initials: J
authors.lastname: Fujita
authors.affiliation: Departments of Cardiology and Cardiovascular Surgery, Keio University School of Medicine, Tokyo, Japan; Japan Society for the Promotion of Science, Tokyo, Japan; Department of Cardiovascular Research, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan.
authors.forename: Keiichi
authors.initials: K
authors.lastname: Fukuda
mesh_terms: D000818:Animals; D000943:Antigens, Differentiation / Q000378:metabolism; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D002469:Cell Separation / Q000379:methods; D006801:Humans; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D032383:Myocytes, Cardiac / Q000166:cytology* / Q000378:metabolism*; D039904:Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism*
chemical_list: D000943:Antigens, Differentiation
keywords: Cardiac; Cell culture; Cellular therapy; Differentiation; Embryonic stem cells; Induced pluripotent stem cells; Stem cell transplantation