title: Improvement in protocol to generate homogeneous glutamatergic neurons from mouse embryonic stem cells reduced apoptosis.
abstract: Obtaining a homogenous population of central nervous system neurons has been a significant challenge in neuroscience research; however, a recent study established a retinoic acid-treated embryoid bodies-based differentiation protocol that permits the effective generation of highly homogeneous glutamatergic cortical pyramidal neurons from embryonic stem cells. We were able to reproduce this protocol regarding the purity of glutamatergic neurons, but these neurons were not sufficiently healthy for long-term observation under the same conditions that were originally described. Here, we achieved a substantial improvement in cell survival by applying a simple technique: We changed the medium for glutamatergic neurons from the original complete medium to commercially available SBM (the Nerve-Cell Culture Medium manufactured by Sumitomo Bakelite Co. Ltd.) and finally succeeded in maintaining healthy neurons for at least 3 weeks without decreasing their purity. Because SBM contains glial conditioned medium, we postulated that brain-derived neurotrophic factor or basic fibroblast growth factor is the key components responsible for pro-survival effect of SBM on neurons, and examined their effects by adding them to CM. As a result, neither of them had pro-survival effect on pure glutamatergic neuronal population.
authors.affiliation: Department of Molecular Neuroscience, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Ryosuke
authors.initials: R
authors.lastname: Fujiki
authors.forename: Akinobu
authors.initials: A
authors.lastname: Sato
authors.forename: Katsuhiko
authors.initials: K
authors.lastname: Hata
authors.forename: Fumi
authors.initials: F
authors.lastname: Tashiro
authors.forename: Noriko
authors.initials: N
authors.lastname: Yasuhara
authors.forename: Jun-Ichi
authors.initials: J
authors.lastname: Miyazaki
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Yoneda
authors.forename: Masashi
authors.initials: M
authors.lastname: Fujitani
authors.forename: Toshihide
authors.initials: T
authors.lastname: Yamashita
mesh_terms: D000818:Animals; D017209:Apoptosis; D019208:Brain-Derived Neurotrophic Factor / Q000494:pharmacology; D053148:Caspase 3 / Q000378:metabolism; D018929:Cell Culture Techniques; D002470:Cell Survival; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000187:drug effects; D005346:Fibroblast Growth Factors / Q000494:pharmacology; D018698:Glutamic Acid / Q000378:metabolism*; D051379:Mice; D055495:Neurogenesis; D009474:Neurons / Q000166:cytology*; D014404:Tubulin / Q000378:metabolism
chemical_list: D019208:Brain-Derived Neurotrophic Factor; D014404:Tubulin; C462941:beta3 tubulin, mouse; C108111:fibroblast growth factor 13; D018698:Glutamic Acid; D005346:Fibroblast Growth Factors; D053148:Caspase 3