title: Functional Comparison of Neuronal Cells Differentiated from Human Induced Pluripotent Stem Cell-Derived Neural Stem Cells under Different Oxygen and Medium Conditions.
abstract: Because neurons are difficult to obtain from humans, generating functional neurons from human induced pluripotent stem cells (hiPSCs) is important for establishing physiological or disease-relevant screening systems for drug discovery. To examine the culture conditions leading to efficient differentiation of functional neural cells, we investigated the effects of oxygen stress (2% or 20% O2) and differentiation medium (DMEM/F12:Neurobasal-based [DN] or commercial [PhoenixSongs Biologicals; PS]) on the expression of genes related to neural differentiation, glutamate receptor function, and the formation of networks of neurons differentiated from hiPSCs (201B7) via long-term self-renewing neuroepithelial-like stem (lt-NES) cells. Expression of genes related to neural differentiation occurred more quickly in PS and/or 2% O2than in DN and/or 20% O2, resulting in high responsiveness of neural cells to glutamate, N-methyl-d-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), and ( S)-3,5-dihydroxyphenylglycine (an agonist for mGluR1/5), as revealed by calcium imaging assays. NMDA receptors, AMPA receptors, mGluR1, and mGluR5were functionally validated by using the specific antagonists MK-801, NBQX, JNJ16259685, and 2-methyl-6-(phenylethynyl)-pyridine, respectively. Multielectrode array analysis showed that spontaneous firing occurred earlier in cells cultured in 2% O2than in 20% O2. Optimization of O2tension and culture medium for neural differentiation of hiPSCs can efficiently generate physiologically relevant cells for screening systems.
authors.affiliation: 1 Next Generation Systems CFU, Eisai Product Creation Systems, Tokodai, Tsukuba, Japan.
authors.forename: Kazuto
authors.initials: K
authors.lastname: Yamazaki
authors.affiliation: 1 Next Generation Systems CFU, Eisai Product Creation Systems, Tokodai, Tsukuba, Japan.
authors.forename: Kazuyuki
authors.initials: K
authors.lastname: Fukushima
authors.affiliation: 1 Next Generation Systems CFU, Eisai Product Creation Systems, Tokodai, Tsukuba, Japan.|2 Department of Physiology, School of Medicine, Keio University, Shinjuku-ku, Tokyo, Japan.
authors.forename: Michiko
authors.initials: M
authors.lastname: Sugawara
authors.affiliation: 1 Next Generation Systems CFU, Eisai Product Creation Systems, Tokodai, Tsukuba, Japan.|2 Department of Physiology, School of Medicine, Keio University, Shinjuku-ku, Tokyo, Japan.
authors.forename: Yoshikuni
authors.initials: Y
authors.lastname: Tabata
authors.affiliation: 1 Next Generation Systems CFU, Eisai Product Creation Systems, Tokodai, Tsukuba, Japan.
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Imaizumi
authors.affiliation: 1 Next Generation Systems CFU, Eisai Product Creation Systems, Tokodai, Tsukuba, Japan.
authors.forename: Yasuharu
authors.initials: Y
authors.lastname: Ishihara
authors.affiliation: 1 Next Generation Systems CFU, Eisai Product Creation Systems, Tokodai, Tsukuba, Japan.
authors.forename: Masashi
authors.initials: M
authors.lastname: Ito
authors.affiliation: 1 Next Generation Systems CFU, Eisai Product Creation Systems, Tokodai, Tsukuba, Japan.
authors.forename: Kappei
authors.initials: K
authors.lastname: Tsukahara
authors.affiliation: 2 Department of Physiology, School of Medicine, Keio University, Shinjuku-ku, Tokyo, Japan.
authors.forename: Jun
authors.initials: J
authors.lastname: Kohyama
authors.affiliation: 2 Department of Physiology, School of Medicine, Keio University, Shinjuku-ku, Tokyo, Japan.
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Okano
mesh_terms: D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000187:drug effects*; D017077:Culture Media, Conditioned / Q000494:pharmacology; D016291:Dizocilpine Maleate / Q000737:chemistry; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000187:drug effects; D016202:N-Methylaspartate / Q000737:chemistry; D058953:Neural Stem Cells / Q000166:cytology* / Q000187:drug effects; D009474:Neurons / Q000166:cytology / Q000187:drug effects; D018384:Oxidative Stress / Q000187:drug effects; D010100:Oxygen / Q000378:metabolism; D010101:Oxygen Consumption / Q000235:genetics
chemical_list: D017077:Culture Media, Conditioned; D016202:N-Methylaspartate; D016291:Dizocilpine Maleate; D010100:Oxygen
keywords: functional neurons; human induced pluripotent stem cell–derived neural stem cells; medium; neural differentiation; oxygen tension