Poster

Poster 16 Development 3 ポスター 16 発生・分化3

P16-1 Differentiation of human iPSCs into cortex modified by combination of FGF2 and ambient oxygen ヒトiPS細胞から大脳皮質への分化誘導法におけるFGF2の効果 Eguchi Noriomi（江口典臣）¹，曽良一郎¹，六車恵子² ¹Dept. of Psychiatry, Univ. of Kobe ²Laboratory for Cell Asymmetry, RIKEN Center for Developmental Biology ヒトiPS細胞を利用した研究の発展により、ヒトの脳の発生を実験的に再現することが可能となり、新たな生物モデルとして利用されている。これにより、精神神経疾患の患者に由来する脳の神経組織を得ることが可能となり、自閉スペクトラム症（ASD）や注意欠如多動性障害（ADHD）をはじめとする神経発達障害の病態解明や創薬への応用が期待されている。iPS細胞から神経細胞を得るための分化誘導法は複数の報告があるが、特別な機器や複雑な操作が必要なものが少なくない。このことはiPS細胞を利用した精神疾患研究の発展を妨げる一因となっていた。発表者らは、分化誘導法の更なる効率化と簡略化を目指し、iPS細胞から大脳の神経細胞を効率的に得ることが可能であるSFEBq法（Kadoshimaら、2013年）を基に、実験条件の検討を行った。その結果、（1）塩基性線維芽細胞増殖因子（FGF2）を添加することで、ほぼ全てのiPS細胞の細胞塊が生存し大脳の神経へと分化すること、（2）この方法では従来の分化誘導法で必要であった高濃度酸素（40% O₂）が必要ないことを見出した。また、この条件で70日間培養された組織中には、脳室帯（VZ）、脳室下帯（SVZ）、皮質板（CP）からなる層構造が認められ、大脳皮質の発生を実験的に再現していると考えられた。以上により、この分化誘導法は、脳の神経細胞を得るのに十分な効率を有していると考えられた。我々の報告した分化誘導法は、iPS細胞から大脳の神経細胞を得る実験をより容易にするものであり、これからの精神神経疾患研究の発展に貢献し得るものと考える。		P16-2 Characterization of neural networks of human induced pluripotent stem cell-derived neurons ヒトiPS細胞由来神経細胞による神経回路形成とその特性解析 Takahashi Kanako（高橋華奈子），中條かおり，諫田泰成，佐藤薫 Div. of Pharmacology, Nat. Inst. Hlth. Sci., Kanagawa, Japan Neural networks generated from human induced pluripotent stem cell-derived neurons (hiPSCN) have great potential in drug development. However, reports concerning stable hiPSCN network activities are still scarce. In this study, we cultured hiPSCN at high density for long period and characterized these cells to see their availability in drug development. XCell neurons (XCell Science) are commercially available hiPSCN comprised of glutamatergic excitatory neurons and GABAergic inhibitory neurons. XCell neurons were seeded at 3.0×10⁵ cells/cm² and cultured for 3 months. We first characterized these cells pharmacologically by Ca²⁺ imaging. Most cells with neuron-like shape showed the responsiveness to L-glutamate (L-Glu, 100 μM) from 14 DIV. L-Glu induced Ca²⁺ increases were suppressed by NMDAR antagonist AP5 (100 μM) at 28 DIV. Spontaneous activity appeared at 14 DIV and thereafter. These activities were blocked by Na⁺ channel blocker tetrodotoxin (1 μM) and were enhanced by GABA_A antagonist picrotoxin (100 μM). At 90 DIV, 80.3 % hiPSCN were responsive to picrotoxin. Synaptogenesis were confirmed immunohistochemically as well. We used Scales, optical clearing methods, because of the poor transparency of the samples. The presynaptic marker synapsin1 and postsynaptic marker PSD95 were co-localized on MAP2-positive fiber at DIV 42. The number of synapsin1/PSD95 co-localized clusters was increased along with the development of network activities. These data suggest that we can stably reproduce hiPSCN neural networks by the above protocols and the obtained human in vitro networks are promising tools for the drug development.

P16-3 Characterization of primary cilia in human induced pluripotent stem cell-derived cortical neurons ヒトiPSC細胞由来神経細胞における非シナプスマーカー一次繊毛の検出 Miki Daisuke（三木大輔）¹，小林勇喜¹，宮本達雄²，小金澤紀子³，武井延之⁴，関野祐子⁵，白尾智明³，斎藤祐見子¹ ¹Graduate School of Integrated Arts and Sciences, Hiroshima University, Hiroshima, Japan ²Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, Japan ³Department of Neurobiology and Behavior, Gunma University Graduate School of Medicine, Gunma, Japan ⁴Brain Research Institute, Niigata University, Niigata, Japan ⁵Graduate School of Pharmaceutical Sciences, The University of Tokyo Department of Pharmacology, University of Tokyo, Tokyo, Japan The primary cilium is a non-motile singular cellular structure that extends from the surface of nearly every cell in the body. Defective cilia function leads to a range of severe disorders affecting many tissues including neuronal systems. Because signaling molecules are selectively and highly expressed in tiny structure, primary cilium considered as a higher-level neuronal signaling node in addition to synapse. To clarify neurodegenerative disease mechanisms, rodent neuronal cultures have shown to be indispensable, yet it is hard to translate new findings into new medicines to human. Commercially available human iPSC-derived neurons might represent a useful model for drug screening and toxicity studies. However, few efforts have been previously described concerning primary cilia as a signaling platform in human iPSC-derived neurons. In the present study, we assessed the morphological features and functionality of primary cilia in human iPSC-derived cortical neurons, iCell GlutaNeuron (iGlu) which is a mixture of post-mitotic neuronal subtypes composed predominantly of cortical glutamatergic neurons. To characterize neuronal cilia, the cells were initially cultured for various periods and subjected to immunohistochemistry by co-staining with antibodies against a variety kind of cilium markers and against MAP2. Primary cilia were first detected on neurons on Day 4, and their lengths were elongated with cultivation days to 4.60 μm on Day 18. Further, by using Day 11-18 neurons, we confirmed the effects of several pharmacological drugs that have been previously shown to regulate cilia length. We conclude that iGlu expresses functional primary cilia and could serve as a valuable system for development and validation of pharmaceutical agents.		P16-4 Examination of the effects of carbonyl stress on neuronal differentiation and development 神経分化・発達におけるカルボニルストレスの影響 Toyoshima Manabu（豊島学）¹，大西哲生¹，新井誠²，糸川昌成²，岡野栄之³ ¹RIKEN Center for Brain Science, Lab. for Molecular Psychiatry ²Department of Psychiatry and Behavioral Sciences, Tokyo Metropolitan Institute of Medical Science ³Department of Physiology, School of Medicine, Keio University Carbonyl stress is a central mediator of advanced glycation end product (AGE) formation. Accumulation of such reactive carbonyl compounds, referred to as carbonyl stress, results in the modification of proteins by AGEs like pentosidine. Cellular removal of AGEs hinges largely on the activity of GLO1. Recent studies have revealed that carbonyl stress, along with dysfunction of GLO1, plays an important role not only in systemic diseases such as diabetes mellitus, but also in neuropsychiatric disorders including schizophrenia, mood disorder, autism and Alzheimer disease. Regarding schizophrenia, it has been reported that subset of patients showed increases in plasma AGEs and that offspring from diabetic mothers showed 7-fold increased risk for schizophrenia, suggesting that carbonyl stress in neurodevelopment could be future schizophrenia risk. To obtain insight into abnormalities in neurodevelopmental trajectories of schizophrenia by carbonyl stress, analyses of human induced pluripotent stem cells (hiPSCs) should be informative. Here, we established hiPSCs from schizophrenia patients with the GLO1 frameshift mutation and GLO1 KO hiPSCs from control iPSC lines by gene editing using CRISPR-Cas9. Neurosphere size, neurosphere number, neural differentiation efficiency and expression of neural stem cell marker were significantly reduced in the patient-derived iPSCs and GLO1 KO hiPSCs. Furthermore, these iPSCs showed increase of carboxymethyllysine (CML) modification of a certain protein. Reduction of carbonyl stress by adding piridoxamine to patient-derived iPSCs and GLO1 KO hiPSCs partially restored neural differentiation efficiency and decreased CML modification. These results suggest that carbonyl stress may affect neurodevelopmental process.

P16-5 Mechanisms of effects of antidepressant SSRI during development of zebrafish brain ゼブラフィッシュ脳の発生過程における抗うつ剤SSRIの作用機構 Sato Tomomi（佐藤智美）¹，梶原健²，半田宏³，永島雅文¹ ¹Dept. of Anatomy,, Sch. of Med., Saitama Med. Univ. ²Dept. of Obs. Gyn., Sch. of Med., Saitama Med. Univ. ³Dept. of Nanoparticle Transl. Res., Tokyo Med. Univ. For risk management between maternal mental health and fetal development, it is important to understand effects on embryos exposed to antidepressant, selective serotonin reuptake inhibitor (SSRI). Recently, ingestion of SSRI is reported to increase the risk of autism. However, it remains to be elucidated the role of serotonin transporter (SERT), the target of SSRI during development of the brain. Placenta-derived serotonin is transiently localized in the forebrain of early mouse embryos, suggesting a fundamental role of serotonin in early development of the brain. To understand the role of SERT in early brain development, we investigated the effects of SSRI on brain development using zebrafish embryos as a vertebrate model system. Immunohistochemistry with anti-serotonin antibody showed that serotonergic neurons were developed in the raphe nuclei of zebrafish embryos from 2 days post fertilization (dpf). Treatment with SSRI from early stages when the head primordium is developed, produced embryos with a small head and decreased neurons at 2 dpf. We found that SERT was localized in the apical surface of radial glial cells in the ventricular region at 1 dpf. Knockdown of SERT showed a small head and decreased radial glial cells at 1 dpf, although phospho-histone H3 (pH3)-positive mitotic cells were obviously increased in the brain. Thereafter, the head was slightly enlarged and retinotectal projection areas were also expanded in knockdown embryos at 3 dpf. These results suggest that transient knockdown of SERT at early developmental stages causes a decrease of neural stem/progenitor cells in the brain, consequently generates abnormal development of neural circuitry, implicating a possibility that exposure of early embryos to SSRI is a risk factor for autism.		P16-6 Mechanism of neuron specific activation of Factor XIII-A after optic nerve injury for wound healing 神経特異的なFactor FXIII-Aの活性化と創傷治癒への関与 Sugitani Kayo（杉谷加代）¹，郡山恵樹²，大貝和裕³，加藤聖³ ¹Dept. Clinical Laboratory Sci., Grad. Sch. Med. Sci., Kanazawa Univ. Japan ²Grad. Sch. Pharm Sci, Suzuka University of Med Sci, Suzuka, Japan. ³Wellness Promotion Science Center, Institute of Medical, Pharmaceutical and Health Sci., Kanazawa Univ., Japan Fish central nervous system (CNS) neurons can regrow their axons and restore their function even after nerve lesion. Especially, the zebrafish visual system have widely used as a CNS nerve regeneration model because of the ability to exploit the physiological, histological, behavioral and genetic analysis. In the process of zebrafish optic nerve regeneration, a large number of genes are upregulated as regeneration-associated molecules. Coagulation factor XIII A subunit (FXIII-A), a cross-linking enzyme, was identified as one of the regeneration-associated molecules after fish optic nerve injury. FXIII-A shows very rapid activation at the lesion site of optic nerve and injured retina. However, this rapid activation mechanism of FXIII-A and its physiological roles are not well known. Blood FXIII-A is activated by thrombin to cleave the activation peptide on the N-terminus. However, thrombin mRNA was undetectable in zebrafish optic nerve and retina both before and after optic nerve injury. Sequence analysis of FXIII-A 5’RACE analysis indicated that many clones derived from injured optic nerve showed short sequences of FXIII-A which lacked exon 1-2 region whereas intact control optic nerve showed full length of FXIII-A cDNA. Furthermore, this short type of FXIII-A had heat shock factors binding site just before the start regions. These results suggest that the expression of heat shock factor allows for the directly production of activated FXIII-A protein in zebrafish retina and optic nerve after nerve injury.

P16-7 Fork cell-like neurons in the mouse insular cortex マウス島皮質におけるFork cell類似形態を示す神経細胞の同定 Hattori Misaki（服部美咲）¹，佐藤真^1,2 ¹Department of Anatomy and Neuroscience, Graduate School of Medicine, Osaka University, Japan ²United Graduate School of Child Development, Osaka University, Kanazawa University, Hamamatsu University School of Medicine, Chiba University and University of Fukui, Osaka, Japan The human insular cortex receives a variety of sensory information such as taste and visceral information and reciprocally connects with the amygdala and association cortices, serving as a sense-integration center in the cortex. Because of its dramatic changes in the structure during evolution, neuronal circuits responsible for the integration are not fully elucidated. Such progressive evolution is supported by the fact that von Economo neurons (VENs) and Fork cells, which are morphologically-defined layer 5 neurons in the insular cortex, are only found in higher animals. VENs and Fork cells have attracted attention, since their involvement in frontotemporal dementia (FTD) as well as autism is suggested. Yet, if the analogous cells exist in the experimental animals, it would present a unique platform to study the hominid insular cortex.VENs are originally defined with their bipolar and spindle shape, whereas Fork cells are with their two apical dendrites in Y shape. Up to now, several characteristic molecules of VENs and Fork cells have been reported in human and great apes. We first asked whether such specific molecules for VENs and/or Fork cells are expressed in the mouse insular cortex using in situ hybridization histochemistry and immunohistochemistry. Here, we found that some layer 5 neurons express some of such molecules, such as neuromedin B, gastrin releasing peptide, and Fezf2. Interestingly, some of such neurons exhibited a fork cell like shape.