Postsynaptic structure formation of human iPS cell-derived neurons takes longer than presynaptic formation during neural differentiation in vitro.

Autor: Togo K; Department of Neurology, Graduate School of Medicine, Osaka University, Suita, Osaka, 565-0871, Japan.; Division of Stem Cell Research, Department of Biomedical Research and Innovation, Institute for Clinical Research, National Hospital Organization Osaka National Hospital, Osaka, Osaka, 540-0006, Japan., Fukusumi H; Division of Stem Cell Research, Department of Biomedical Research and Innovation, Institute for Clinical Research, National Hospital Organization Osaka National Hospital, Osaka, Osaka, 540-0006, Japan., Shofuda T; Division of Stem Cell Research, Department of Biomedical Research and Innovation, Institute for Clinical Research, National Hospital Organization Osaka National Hospital, Osaka, Osaka, 540-0006, Japan., Ohnishi H; Department of Laboratory Sciences, Gunma University Graduate School of Health Sciences, Maebashi, Gunma, 371-8514, Japan., Yamazaki H; Department of Neurobiology and Behavior, Gunma University Graduate School of Medicine, Maebashi, Gunma, 371-8511, Japan.; Faculty of Social Welfare, Gunma University of Health and Welfare, Maebashi, Gunma, 371-0823, Japan., Hayashi MK; School of Medicine, International University of Health and Welfare, Narita, Chiba, 286-8686, Japan.; Department of Food Science and Nutrition, Faculty of Food and Health Sciences, Showa Women's University, Setagaya-ku, Tokyo, 154-8533, Japan., Kawasaki N; Laboratory of Biopharmaceutical and Regenerative Sciences, Graduate School of Medical Life Science, Yokohama City University, Yokohama, Kanagawa, 230-0045, Japan., Takei N; Department of Brain Tumor Biology, Brain Research Institute, Niigata University, Niigata, Niigata, 951-8585, Japan., Nakazawa T; Laboratory of Molecular Neuropharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka, 565-0871, Japan.; Department of Bioscience, Faculty of Life Sciences, Tokyo University of Agriculture, Setagaya-ku, Tokyo, 156-8502, Japan., Saito Y; Graduate School of Integrated Sciences for Life, Hiroshima University, Higashi-Hiroshima, Hiroshima, 739-8521, Japan., Baba K; Department of Neurology, Graduate School of Medicine, Osaka University, Suita, Osaka, 565-0871, Japan., Hashimoto H; Laboratory of Molecular Neuropharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka, 565-0871, Japan.; Molecular Research Center for Children's Mental Development, United Graduate School of Child Development, Osaka University, Suita, Osaka, 565-0871, Japan.; Division of Bioscience, Institute for Datability Science, Osaka University, Suita, Osaka, 565-0871, Japan.; Open and Transdisciplinary Research Initiatives, Osaka University, Suita, Osaka, 565-0871, Japan.; Department of Molecular Pharmaceutical Sciences, Graduate School of Medicine, Osaka University, Suita, Osaka, 565-0871, Japan., Sekino Y; Endowed Laboratory of Human Cell-Based Drug Discovery, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, 113-0033, Japan., Shirao T; Department of Neurobiology and Behavior, Gunma University Graduate School of Medicine, Maebashi, Gunma, 371-8511, Japan., Mochizuki H; Department of Neurology, Graduate School of Medicine, Osaka University, Suita, Osaka, 565-0871, Japan., Kanemura Y; Division of Regenerative Medicine, Department of Biomedical Research and Innovation, Institute for Clinical Research, National Hospital Organization Osaka National Hospital, 2-1-14 Hoenzaka, Chuo-ku, Osaka, Osaka, 540-0006, Japan. kanemura.yonehiro.hk@mail.hosp.go.jp.; Department of Neurosurgery, National Hospital Organization Osaka National Hospital, Osaka, Osaka, 540-0006, Japan. kanemura.yonehiro.hk@mail.hosp.go.jp.
Jazyk: angličtina
Zdroj: Molecular brain [Mol Brain] 2021 Oct 11; Vol. 14 (1), pp. 149. Date of Electronic Publication: 2021 Oct 11.
DOI: 10.1186/s13041-021-00851-1
Abstrakt: 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.
(© 2021. The Author(s).)
Databáze: MEDLINE
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