{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,22]],"date-time":"2026-10-22T07:15:35Z","timestamp":1792653335748,"version":"4.5.0"},"reference-count":41,"publisher":"Springer Science and Business Media LLC","issue":"1","license":[{"start":{"date-parts":[[2021,10,11]],"date-time":"2021-10-11T00:00:00Z","timestamp":1633910400000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"},{"start":{"date-parts":[[2021,10,11]],"date-time":"2021-10-11T00:00:00Z","timestamp":1633910400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"}],"funder":[{"DOI":"10.13039\/100009619","name":"Japan Agency for Medical Research and Development","doi-asserted-by":"publisher","award":["JP21bm0204001"],"award-info":[{"award-number":["JP21bm0204001"]}],"id":[{"id":"10.13039\/100009619","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100009619","name":"Japan Agency for Medical Research and Development","doi-asserted-by":"publisher","award":["JP21bm0804024"],"award-info":[{"award-number":["JP21bm0804024"]}],"id":[{"id":"10.13039\/100009619","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100009619","name":"Japan Agency for Medical Research and Development","doi-asserted-by":"publisher","award":["JP19bk0104077"],"award-info":[{"award-number":["JP19bk0104077"]}],"id":[{"id":"10.13039\/100009619","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001691","name":"Japan Society for the Promotion of Science","doi-asserted-by":"publisher","award":["JP18K08958"],"award-info":[{"award-number":["JP18K08958"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["Mol Brain"],"published-print":{"date-parts":[[2021,12]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p>The generation of mature synaptic structures using neurons differentiated from human-induced pluripotent stem cells (hiPSC-neurons) is expected to be applied to physiological studies of synapses in human cells and to pathological studies of diseases that cause abnormal synaptic function. Although it has been reported that synapses themselves change from an immature to a mature state as neurons mature, there are few reports that clearly show when and how human stem cell-derived neurons change to mature synaptic structures. This study was designed to elucidate the synapse formation process of hiPSC-neurons. We propagated hiPSC-derived neural progenitor cells (hiPSC-NPCs) that expressed localized markers of the ventral hindbrain as neurospheres by dual SMAD inhibition and then differentiated them into hiPSC-neurons in vitro. After 49 days of in vitro differentiation, hiPSC-neurons significantly expressed pre- and postsynaptic markers at both the transcript and protein levels. However, the expression of postsynaptic markers was lower than in normal human or normal rat brain tissues, and immunostaining analysis showed that it was relatively modest and was lower than that of presynaptic markers and that its localization in synaptic structures was insufficient. Neurophysiological analysis using a microelectrode array also revealed that no synaptic activity was generated on hiPSC-neurons at 49 days of differentiation. Analysis of subtype markers by immunostaining revealed that most hiPSC-neurons expressed vesicular glutamate transporter 2 (VGLUT2). The presence or absence of NGF, which is required for the survival of cholinergic neurons, had no effect on their cell fractionation. These results suggest that during the synaptogenesis of hiPSC-neurons, the formation of presynaptic structures is not the only requirement for the formation of postsynaptic structures and that the mRNA expression of postsynaptic markers does not correlate with the formation of their mature structures. Technically, we also confirmed a certain level of robustness and reproducibility of our neuronal differentiation method in a multicenter setting, which will be helpful for future research. Synapse formation with mature postsynaptic structures will remain an interesting issue for stem cell-derived neurons, and the present method can be used to obtain early and stable quality neuronal cultures from hiPSC-NPCs.<\/jats:p>","DOI":"10.1186\/s13041-021-00851-1","type":"journal-article","created":{"date-parts":[[2021,10,10]],"date-time":"2021-10-10T23:03:16Z","timestamp":1633906996000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":18,"title":["Postsynaptic structure formation of human iPS cell-derived neurons takes longer than presynaptic formation during neural differentiation in vitro"],"prefix":"10.1186","volume":"14","author":[{"given":"Kazuyuki","family":"Togo","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hayato","family":"Fukusumi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tomoko","family":"Shofuda","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hiroshi","family":"Ohnishi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hiroyuki","family":"Yamazaki","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mariko Kato","family":"Hayashi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nana","family":"Kawasaki","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nobuyuki","family":"Takei","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Takanobu","family":"Nakazawa","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yumiko","family":"Saito","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kousuke","family":"Baba","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hitoshi","family":"Hashimoto","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuko","family":"Sekino","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tomoaki","family":"Shirao","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hideki","family":"Mochizuki","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1894-1824","authenticated-orcid":false,"given":"Yonehiro","family":"Kanemura","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2021,10,11]]},"reference":[{"issue":"2","key":"851_CR1","doi-asserted-by":"publisher","first-page":"565","DOI":"10.1002\/cphy.c180025","volume":"9","author":"S Logan","year":"2019","unstructured":"Logan S, Arzua T, Canfield SG, Seminary ER, Sison SL, Ebert AD, et al. Studying human neurological disorders using induced pluripotent stem cells: from 2D monolayer to 3D organoid and blood brain barrier models. Compr Physiol. 2019;9(2):565\u2013611.","journal-title":"Compr Physiol"},{"issue":"3","key":"851_CR2","doi-asserted-by":"publisher","first-page":"166","DOI":"10.3390\/brainsci10030166","volume":"10","author":"E Cavalli","year":"2020","unstructured":"Cavalli E, Battaglia G, Basile MS, Bruno V, Petralia MC, Lombardo SD, et al. Exploratory analysis of iPSCS-derived neuronal cells as predictors of diagnosis and treatment of Alzheimer disease. Brain Sci. 2020;10(3):166.","journal-title":"Brain Sci"},{"issue":"4","key":"851_CR3","doi-asserted-by":"publisher","first-page":"331","DOI":"10.1016\/j.jphs.2019.08.002","volume":"140","author":"H Fukusumi","year":"2019","unstructured":"Fukusumi H, Handa Y, Shofuda T, Kanemura Y. Evaluation of the susceptibility of neurons and neural stem\/progenitor cells derived from human induced pluripotent stem cells to anticancer drugs. J Pharmacol Sci. 2019;140(4):331\u20136.","journal-title":"J Pharmacol Sci"},{"issue":"1","key":"851_CR4","doi-asserted-by":"publisher","first-page":"2225","DOI":"10.1038\/s41467-017-02330-5","volume":"8","author":"GE Hoffman","year":"2017","unstructured":"Hoffman GE, Hartley BJ, Flaherty E, Ladran I, Gochman P, Ruderfer DM, et al. Transcriptional signatures of schizophrenia in hiPSC-derived NPCs and neurons are concordant with post-mortem adult brains. Nat Commun. 2017;8(1):2225.","journal-title":"Nat Commun"},{"key":"851_CR5","doi-asserted-by":"publisher","first-page":"215","DOI":"10.1016\/j.neuro.2018.06.007","volume":"67","author":"AM Tukker","year":"2018","unstructured":"Tukker AM, Wijnolts FMJ, de Groot A, Westerink RHS. Human iPSC-derived neuronal models for in vitro neurotoxicity assessment. Neurotoxicology. 2018;67:215\u201325.","journal-title":"Neurotoxicology"},{"key":"851_CR6","doi-asserted-by":"crossref","unstructured":"Fukusumi H, Handa Y, Shofuda T, Kanemura Y. Small-scale screening of anticancer drugs acting specifically on neural stem\/progenitor cells derived from human-induced pluripotent stem cells using a time-course cytotoxicity test. PeerJ. 2018;6:e4187.","DOI":"10.7717\/peerj.4187"},{"issue":"10","key":"851_CR7","doi-asserted-by":"publisher","first-page":"72","DOI":"10.1007\/s11910-019-0995-y","volume":"19","author":"M Xylaki","year":"2019","unstructured":"Xylaki M, Atzler B, Outeiro TF. Epigenetics of the synapse in neurodegeneration. Curr Neurol Neurosci Rep. 2019;19(10):72.","journal-title":"Curr Neurol Neurosci Rep"},{"issue":"4","key":"851_CR8","doi-asserted-by":"publisher","first-page":"365","DOI":"10.1080\/15476278.2015.1011921","volume":"10","author":"L D'Aiuto","year":"2014","unstructured":"D\u2019Aiuto L, Zhi Y, Kumar Das D, Wilcox MR, Johnson JW, McClain L, et al. Large-scale generation of human iPSC-derived neural stem cells\/early neural progenitor cells and their neuronal differentiation. Organogenesis. 2014;10(4):365\u201377.","journal-title":"Organogenesis"},{"key":"851_CR9","doi-asserted-by":"crossref","unstructured":"Falk A, Koch P, Kesavan J, Takashima Y, Ladewig J, Alexander M, et al. Capture of neuroepithelial-like stem cells from pluripotent stem cells provides a versatile system for in vitro production of human neurons. PLoS ONE. 2012;7(1):e29597.","DOI":"10.1371\/journal.pone.0029597"},{"key":"851_CR10","doi-asserted-by":"crossref","unstructured":"Nadadhur AG, Emperador Melero J, Meijer M, Schut D, Jacobs G, Li KW, et al. Multi-level characterization of balanced inhibitory-excitatory cortical neuron network derived from human pluripotent stem cells. PLoS ONE. 2017;12(6):e0178533.","DOI":"10.1371\/journal.pone.0178533"},{"key":"851_CR11","doi-asserted-by":"publisher","first-page":"20033","DOI":"10.1038\/srep20033","volume":"6","author":"H Wang","year":"2016","unstructured":"Wang H, Hu L, Liu C, Su Z, Wang L, Pan G, et al. 5-HT2 receptors mediate functional modulation of GABAa receptors and inhibitory synaptic transmissions in human iPS-derived neurons. Sci Rep. 2016;6:20033.","journal-title":"Sci Rep"},{"issue":"7","key":"851_CR12","doi-asserted-by":"publisher","first-page":"2685","DOI":"10.1523\/JNEUROSCI.12-07-02685.1992","volume":"12","author":"KM Harris","year":"1992","unstructured":"Harris KM, Jensen FE, Tsao B. Three-dimensional structure of dendritic spines and synapses in rat hippocampus (CA1) at postnatal day 15 and adult ages: implications for the maturation of synaptic physiology and long-term potentiation. J Neurosci. 1992;12(7):2685\u2013705.","journal-title":"J Neurosci"},{"issue":"24","key":"851_CR13","doi-asserted-by":"publisher","first-page":"2913","DOI":"10.1091\/mbc.E18-04-0222","volume":"29","author":"ES Wilson","year":"2018","unstructured":"Wilson ES, Newell-Litwa K. Stem cell models of human synapse development and degeneration. Mol Biol Cell. 2018;29(24):2913\u201321.","journal-title":"Mol Biol Cell"},{"issue":"4","key":"851_CR14","doi-asserted-by":"publisher","first-page":"936","DOI":"10.3390\/ijms19040936","volume":"19","author":"BX Ho","year":"2018","unstructured":"Ho BX, Pek NMQ, Soh BS. Disease modeling using 3D organoids derived from human induced pluripotent stem cells. Int J Mol Sci. 2018;19(4):936.","journal-title":"Int J Mol Sci"},{"key":"851_CR15","doi-asserted-by":"publisher","first-page":"25","DOI":"10.1007\/978-4-431-56550-5_2","volume":"1006","author":"N Kojima","year":"2017","unstructured":"Kojima N. Molecular cloning of drebrin: progress and perspectives. Adv Exp Med Biol. 2017;1006:25\u201336.","journal-title":"Adv Exp Med Biol"},{"issue":"1","key":"851_CR16","doi-asserted-by":"publisher","first-page":"468","DOI":"10.1006\/bbrc.1993.2273","volume":"196","author":"M Toda","year":"1993","unstructured":"Toda M, Shirao T, Minoshima S, Shimizu N, Toya S, Uyemura K. Molecular cloning of cDNA encoding human drebrin E and chromosomal mapping of its gene. Biochem Biophys Res Commun. 1993;196(1):468\u201372.","journal-title":"Biochem Biophys Res Commun"},{"issue":"1\u20132","key":"851_CR17","doi-asserted-by":"publisher","first-page":"101","DOI":"10.1016\/0169-328X(93)90154-H","volume":"19","author":"N Kojima","year":"1993","unstructured":"Kojima N, Shirao T, Obata K. Molecular cloning of a developmentally regulated brain protein, chicken drebrin A and its expression by alternative splicing of the drebrin gene. Brain Res Mol Brain Res. 1993;19(1\u20132):101\u201314.","journal-title":"Brain Res Mol Brain Res"},{"issue":"16","key":"851_CR18","doi-asserted-by":"publisher","first-page":"6586","DOI":"10.1523\/JNEUROSCI.23-16-06586.2003","volume":"23","author":"H Takahashi","year":"2003","unstructured":"Takahashi H, Sekino Y, Tanaka S, Mizui T, Kishi S, Shirao T. Drebrin-dependent actin clustering in dendritic filopodia governs synaptic targeting of postsynaptic density-95 and dendritic spine morphogenesis. J Neurosci. 2003;23(16):6586\u201395.","journal-title":"J Neurosci"},{"issue":"10","key":"851_CR19","doi-asserted-by":"publisher","first-page":"1455","DOI":"10.2174\/1570159X16666180412092859","volume":"16","author":"ML Rocco","year":"2018","unstructured":"Rocco ML, Soligo M, Manni L, Aloe L. Nerve growth factor: early studies and recent clinical trials. Curr Neuropharmacol. 2018;16(10):1455\u201365.","journal-title":"Curr Neuropharmacol"},{"issue":"3","key":"851_CR20","doi-asserted-by":"publisher","first-page":"318","DOI":"10.1007\/BF00783131","volume":"113","author":"EG Markova","year":"1992","unstructured":"Markova EG, Isaev NK. Effects of nerve growth factor on the development of the dendritic system of cholinergic neurons in dissociated culture of the rat septum. Biull Eksp Biol Med. 1992;113(3):318\u201320.","journal-title":"Biull Eksp Biol Med"},{"key":"851_CR21","doi-asserted-by":"publisher","first-page":"62","DOI":"10.3389\/fnins.2019.00062","volume":"13","author":"AC Cuello","year":"2019","unstructured":"Cuello AC, Pentz R, Hall H. The brain NGF metabolic pathway in health and in Alzheimer\u2019s pathology. Front Neurosci. 2019;13:62.","journal-title":"Front Neurosci"},{"issue":"1","key":"851_CR22","doi-asserted-by":"publisher","first-page":"85","DOI":"10.1186\/s13041-016-0265-8","volume":"9","author":"K Sugai","year":"2016","unstructured":"Sugai K, Fukuzawa R, Shofuda T, Fukusumi H, Kawabata S, Nishiyama Y, et al. Pathological classification of human iPSC-derived neural stem\/progenitor cells towards safety assessment of transplantation therapy for CNS diseases. Mol Brain. 2016;9(1):85.","journal-title":"Mol Brain"},{"key":"851_CR23","doi-asserted-by":"publisher","first-page":"7235757","DOI":"10.1155\/2016\/7235757","volume":"2016","author":"H Fukusumi","year":"2016","unstructured":"Fukusumi H, Shofuda T, Bamba Y, Yamamoto A, Kanematsu D, Handa Y, et al. Establishment of human neural progenitor cells from human induced pluripotent stem cells with diverse tissue origins. Stem Cells Int. 2016;2016:7235757.","journal-title":"Stem Cells Int"},{"key":"851_CR24","doi-asserted-by":"publisher","first-page":"3594","DOI":"10.1038\/srep03594","volume":"4","author":"M Nakagawa","year":"2014","unstructured":"Nakagawa M, Taniguchi Y, Senda S, Takizawa N, Ichisaka T, Asano K, et al. A novel efficient feeder-free culture system for the derivation of human induced pluripotent stem cells. Sci Rep. 2014;4:3594.","journal-title":"Sci Rep"},{"issue":"3","key":"851_CR25","doi-asserted-by":"publisher","first-page":"458","DOI":"10.1002\/stem.1293","volume":"31","author":"K Okita","year":"2013","unstructured":"Okita K, Yamakawa T, Matsumura Y, Sato Y, Amano N, Watanabe A, et al. An efficient nonviral method to generate integration-free human-induced pluripotent stem cells from cord blood and peripheral blood cells. Stem Cells. 2013;31(3):458\u201366.","journal-title":"Stem Cells"},{"issue":"6","key":"851_CR26","doi-asserted-by":"publisher","first-page":"869","DOI":"10.1002\/jnr.10377","volume":"69","author":"Y Kanemura","year":"2002","unstructured":"Kanemura Y, Mori H, Kobayashi S, Islam O, Kodama E, Yamamoto A, et al. Evaluation of in vitro proliferative activity of human fetal neural stem\/progenitor cells using indirect measurements of viable cells based on cellular metabolic activity. J Neurosci Res. 2002;69(6):869\u201379.","journal-title":"J Neurosci Res"},{"issue":"2","key":"851_CR27","doi-asserted-by":"publisher","first-page":"84","DOI":"10.1097\/WNR.0b013e32835cb677","volume":"24","author":"T Shofuda","year":"2013","unstructured":"Shofuda T, Fukusumi H, Kanematsu D, Yamamoto A, Yamasaki M, Arita N, et al. A method for efficiently generating neurospheres from human-induced pluripotent stem cells using microsphere arrays. NeuroReport. 2013;24(2):84\u201390.","journal-title":"NeuroReport"},{"key":"851_CR28","doi-asserted-by":"publisher","first-page":"104902","DOI":"10.1016\/j.neuint.2020.104902","volume":"142","author":"Y Kobayashi","year":"2021","unstructured":"Kobayashi Y, Okada T, Miki D, Sekino Y, Koganezawa N, Shirao T, et al. Properties of primary cilia in melanin-concentrating hormone receptor 1-bearing hippocampal neurons in vivo and in vitro. Neurochem Int. 2021;142:104902.","journal-title":"Neurochem Int"},{"key":"851_CR29","doi-asserted-by":"publisher","first-page":"106583","DOI":"10.1016\/j.vascn.2019.106583","volume":"99","author":"T Mitsuoka","year":"2019","unstructured":"Mitsuoka T, Hanamura K, Koganezawa N, Kikura-Hanajiri R, Sekino Y, Shirao T. Assessment of NMDA receptor inhibition of phencyclidine analogues using a high-throughput drebrin immunocytochemical assay. J Pharmacol Toxicol Methods. 2019;99:106583.","journal-title":"J Pharmacol Toxicol Methods"},{"key":"851_CR30","doi-asserted-by":"publisher","first-page":"18","DOI":"10.1016\/j.neures.2016.04.003","volume":"110","author":"M Isoda","year":"2016","unstructured":"Isoda M, Kohyama J, Iwanami A, Sanosaka T, Sugai K, Yamaguchi R, et al. Robust production of human neural cells by establishing neuroepithelial-like stem cells from peripheral blood mononuclear cell-derived feeder-free iPSCs under xeno-free conditions. Neurosci Res. 2016;110:18\u201328.","journal-title":"Neurosci Res"},{"issue":"2","key":"851_CR31","doi-asserted-by":"publisher","first-page":"306","DOI":"10.1152\/jn.00093.2016","volume":"116","author":"E Cotterill","year":"2016","unstructured":"Cotterill E, Charlesworth P, Thomas CW, Paulsen O, Eglen SJ. A comparison of computational methods for detecting bursts in neuronal spike trains and their application to human stem cell-derived neuronal networks. J Neurophysiol. 2016;116(2):306\u201321.","journal-title":"J Neurophysiol"},{"key":"851_CR32","doi-asserted-by":"publisher","first-page":"26181","DOI":"10.1038\/srep26181","volume":"6","author":"A Odawara","year":"2016","unstructured":"Odawara A, Katoh H, Matsuda N, Suzuki I. Physiological maturation and drug responses of human induced pluripotent stem cell-derived cortical neuronal networks in long-term culture. Sci Rep. 2016;6:26181.","journal-title":"Sci Rep"},{"issue":"1","key":"851_CR33","doi-asserted-by":"publisher","first-page":"149","DOI":"10.1016\/j.mcn.2005.06.008","volume":"30","author":"T Mizui","year":"2005","unstructured":"Mizui T, Takahashi H, Sekino Y, Shirao T. Overexpression of drebrin A in immature neurons induces the accumulation of F-actin and PSD-95 into dendritic filopodia, and the formation of large abnormal protrusions. Mol Cell Neurosci. 2005;30(1):149\u201357.","journal-title":"Mol Cell Neurosci"},{"issue":"5481","key":"851_CR34","doi-asserted-by":"publisher","first-page":"957","DOI":"10.1126\/science.289.5481.957","volume":"289","author":"EE Bellocchio","year":"2000","unstructured":"Bellocchio EE, Reimer RJ, Fremeau RT Jr, Edwards RH. Uptake of glutamate into synaptic vesicles by an inorganic phosphate transporter. Science. 2000;289(5481):957\u201360.","journal-title":"Science"},{"issue":"6","key":"851_CR35","doi-asserted-by":"publisher","first-page":"2622","DOI":"10.1046\/j.1471-4159.2000.0742622.x","volume":"74","author":"Y Aihara","year":"2000","unstructured":"Aihara Y, Mashima H, Onda H, Hisano S, Kasuya H, Hori T, et al. Molecular cloning of a novel brain-type Na(+)-dependent inorganic phosphate cotransporter. J Neurochem. 2000;74(6):2622\u20135.","journal-title":"J Neurochem"},{"issue":"2","key":"851_CR36","doi-asserted-by":"publisher","first-page":"98","DOI":"10.1016\/j.tins.2003.11.005","volume":"27","author":"RT Fremeau Jr","year":"2004","unstructured":"Fremeau RT Jr, Voglmaier S, Seal RP, Edwards RH. VGLUTs define subsets of excitatory neurons and suggest novel roles for glutamate. Trends Neurosci. 2004;27(2):98\u2013103.","journal-title":"Trends Neurosci"},{"issue":"47","key":"851_CR37","doi-asserted-by":"publisher","first-page":"12294","DOI":"10.1523\/JNEUROSCI.3855-06.2006","volume":"26","author":"A Wall\u00e9n-Mackenzie","year":"2006","unstructured":"Wall\u00e9n-Mackenzie A, Gezelius H, Thoby-Brisson M, Nyg\u00e5rd A, Enjin A, Fujiyama F, et al. Vesicular glutamate transporter 2 is required for central respiratory rhythm generation but not for locomotor central pattern generation. J Neurosci. 2006;26(47):12294\u2013307.","journal-title":"J Neurosci"},{"issue":"46","key":"851_CR38","doi-asserted-by":"publisher","first-page":"12055","DOI":"10.1523\/JNEUROSCI.2556-06.2006","volume":"26","author":"D Moechars","year":"2006","unstructured":"Moechars D, Weston MC, Leo S, Callaerts-Vegh Z, Goris I, Daneels G, et al. Vesicular glutamate transporter VGLUT2 expression levels control quantal size and neuropathic pain. J Neurosci. 2006;26(46):12055\u201366.","journal-title":"J Neurosci"},{"key":"851_CR39","doi-asserted-by":"crossref","unstructured":"Bilican B, Livesey MR, Haghi G, Qiu J, Burr K, Siller R, et al. Physiological normoxia and absence of EGF is required for the long-term propagation of anterior neural precursors from human pluripotent cells. PLoS ONE. 2014;9(1):e85932.","DOI":"10.1371\/journal.pone.0085932"},{"issue":"4","key":"851_CR40","doi-asserted-by":"publisher","first-page":"183","DOI":"10.1016\/j.diff.2016.06.002","volume":"92","author":"S Zhou","year":"2016","unstructured":"Zhou S, Ochalek A, Szczesna K, Avci HX, Kobol\u00e1k J, Varga E, et al. The positional identity of iPSC-derived neural progenitor cells along the anterior-posterior axis is controlled in a dosage-dependent manner by bFGF and EGF. Differentiation. 2016;92(4):183\u201394.","journal-title":"Differentiation"},{"issue":"2","key":"851_CR41","doi-asserted-by":"publisher","first-page":"92","DOI":"10.1038\/35053563","volume":"2","author":"CD Stern","year":"2001","unstructured":"Stern CD. Initial patterning of the central nervous system: how many organizers? Nat Rev Neurosci. 2001;2(2):92\u20138.","journal-title":"Nat Rev Neurosci"}],"container-title":["Molecular Brain"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/link.springer.com\/content\/pdf\/10.1186\/s13041-021-00851-1.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/link.springer.com\/article\/10.1186\/s13041-021-00851-1\/fulltext.html","content-type":"text\/html","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/link.springer.com\/content\/pdf\/10.1186\/s13041-021-00851-1.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2021,10,10]],"date-time":"2021-10-10T23:03:28Z","timestamp":1633907008000},"score":1,"resource":{"primary":{"URL":"https:\/\/molecularbrain.biomedcentral.com\/articles\/10.1186\/s13041-021-00851-1"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,10,11]]},"references-count":41,"journal-issue":{"issue":"1","published-print":{"date-parts":[[2021,12]]}},"alternative-id":["851"],"URL":"https:\/\/doi.org\/10.1186\/s13041-021-00851-1","relation":{},"ISSN":["1756-6606"],"issn-type":[{"value":"1756-6606","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,10,11]]},"assertion":[{"value":"11 February 2021","order":1,"name":"received","label":"Received","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"4 September 2021","order":2,"name":"accepted","label":"Accepted","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"11 October 2021","order":3,"name":"first_online","label":"First Online","group":{"name":"ArticleHistory","label":"Article History"}},{"order":1,"name":"Ethics","group":{"name":"EthicsHeading","label":"Declarations"}},{"value":"This study was conducted in accordance with the principles of the Declaration of Helsinki. The use of hiPSCs was approved by the Osaka National Hospital hN-NSPCs and hiPSCs ethics committee (Nos. 110, 120, and 146).","order":2,"name":"Ethics","group":{"name":"EthicsHeading","label":"Ethics approval and consent to participate"}},{"value":"Not applicable.","order":3,"name":"Ethics","group":{"name":"EthicsHeading","label":"Consent for publication"}},{"value":"The authors have no competing interests to declare.","order":4,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}],"article-number":"149"}}