ポスター

ポスター B. 発生・再生と可塑性 B. Development, Regeneration and Plasticity

2022年6月30日　13:00～14:00　沖縄コンベンションセンター展示棟　ポスター会場1 1P-014 哺乳類大脳皮質におけるサブプレートニューロンの発生起源 The origin of subplate neurons in the developing cerebral cortex 野口友理佳(1)、隈元拓馬(1)、丸山千秋(1) 1. 公益財団法人　東京都医学総合研究所 Yurika Noguchi(1), Takuma Kumamoto(1), Chiaki Ohtaka-Maruyama(1) 1. Tokyo Metropolitan Institute of Medical Science Keyword: Subplate neurons, Preplate, Cerebral cortex Subplate neurons (SpNs) are one of the first neurons born in the developing cerebral cortex and play a critical role in constructing the six-laminated neocortex in mammals. Several functional aspects have been reported. For instance, SpNs could affect the thalamocortical axon pathfinding into the cortical layer, establish the first neural circuit formation between the thalamus and layer IV cortical neurons, and regulate the radial migration of late-born neurons through the MP to BP transition. Moreover, accumulating studies highlight the possible roles of SpNs in adult brain functions and their involvement in psychiatric or other neurological disorders. Thus, SpNs perform multiple functions. However, the details of their subtypes and developmental origin remains unknown. It is known that SpNs form the preplate layer with the earliest born Cajal-Retzius (CR) neurons during the early neurogenic period around E10-12. To decipher the origin of SPNs, especially the molecular basis aspect, we examined the expression of markers in the cells that will become SpNs during the preplate period. Using recently published single-cell RNA seq data of cortical cells, we re-analyzed the data with Seurat to narrow down the genes specifically expressed in the preplate cells that will become subplate cells. We selected E10, E11, and E12 of mouse embryonic stages in the condition as Tbr1 + / Reelin- / Pax6- / Sox2-. Furthermore, we used the results of spatial transcriptome analysis performed at various stages of development in the mouse cortex. We identified several candidate genes as early SpN markers based on this analysis. We then confirmed the expression pattern of the candidate genes by in situ hybridization. In this presentation, we would like to discuss up-to-date candidate markers and their functions for cortical development.		2022年6月30日　13:00～14:00　沖縄コンベンションセンター展示棟　ポスター会場1 1P-015 プリン合成系タンパク質群の神経発達過程における発現・機能解析 Expression and functional analysis of purine synthetic proteins during neural development 水越智也(1)、山田晴也(1)、榊原伸一(1) 1. 早稲田大学人間科学研究科分子神経科学 Tomoya Mizukoshi(1), Seiya Yamada(1), Shin-ichi Sakakibara(1) 1. Lab Mol Neuro Bio, Grad Sch Hum Sci, Waseda Univ, Saitama, Japan Keyword: neurogenesis, purine synthesis, de novo pathway, salvage pathway Purines are essential molecules for the synthesis of DNA, RNA, and energy metabolism, and implicated in various cellular functions. There are two pathways for purine synthesis in mammals: de novo pathway and salvage pathway. The balance between these pathways is critical for normal brain development. Impairment in purine metabolism leads to various neurological diseases such as Lesch-Nyhan syndrome. At pesent, it remains to be elucidated how the deficiency in purine metabolism elicits the pathogenesis of these diseases. In addition, the spatiotemporal regulation of these purine synthesis pathways in the central nervous system remains unknown. Here, we investigated the expression profile and the functional significance of purine synthesis enzymes during mammalian brain development. Developmental immunoblot analysis of mice brain revealed that phosphoribosylaminoimidazole carboxylase phosphoribosylaminoimidazole succinocarboxamide synthetase (Paics) and formylglycin-amidine ribonucleotide synthase (Fgams), both of which catalyze de novo purine synthesis, are abundant in the embryonic stage and the expression level of these enzymes were downregulated toward the postnatal and adult stage. Conversely, the expression level of the salvage pathway enzyme, hypoxanthine-guanine phosphoribosyltransferase (Hgprt) is low in the embryonic brain and gradually increases in postnatal and adult stages. During the embryonic and early postnatal period, Paics, Fgams, and Hgprt immunoreactivities were broadly observed in the different brain regions. Noticeably, Hgprt was abundantly expressed in external germinal layer of neonatal cerebellum and blood vessels. In the adult brain, Paics and Fgams were strongly expressed in the brain stem nuclei including red nucleus, cerebellar nuclei, and facial nucleus, while the high level of expression of Hgprt was observed in forebrain and diencephalon, including arcuate hypothalamic nucleus, posterior intralaminar thalamic. Based on these observations, we speculate that the regulated driving balance of de novo and salvage pathways is essential for the proper function of distinct brain regions or developmental stages. To elucidate the functional significance of two pathways, we are currently estimating the effects of the selective inhibitors for de novo or salvage pathways on the embryonic brain neurogenesis. Our findings will contribute to better understanding of neurological diseases caused by abnormalities in purine metabolism.

2022年6月30日　13:00～14:00　沖縄コンベンションセンター展示棟　ポスター会場1 1P-016 Dab1はプルキンエ細胞に発現し生後小脳の細胞配置と葉形成を制御する Dab1 is expressed in Purkinje cells and regulates cell arrangement and lobule formation in the postnatal cerebellum 笠井拓登(1)、本田岳夫(1,2)、廣田ゆき(1)、仲嶋一範(1) 1. 慶應義塾大学医学部解剖学教室、2. 岐阜薬科大学生体機能解析学大講座分子生物学研究室 Takuto Kasai(1), Takao Honda(1,2), Yuki Hirota(1), Kazunori Nakajima(1) 1. Department of Anatomy, Keio University School of Medicine, 2. Laboratory of Molecular biology, Department of Biofunctional Analysis, Gifu Pharmaceutical University Keyword: Reelin, Dab1, Purkinje cell, Cerebellum Reelin and Dab1 play an important role in the development of the central nervous system. It is known that loss of Reelin and Dab1 results in marked cerebellar hypoplasia, but the reason why defect of these molecules causes cerebellar hypoplasia remains largely unknown. We injected Cre-expressing adeno-associated virus (AAV) into the lateral ventricles of Dab1-floxed mice on postnatal day 1 to knock out Dab1, and observed that AAV predominantly infected Purkinje cells, the major Dab1-expressing cells in the cerebellum. As a result, whereas the principal fissures were not affected, some non-principal fissures became extremely shallow compared to the control. In addition, the arrangement of Purkinje cells was greatly disrupted and many Purkinje cells were abnormally embedded in the inner granular layer (IGL). Furthermore, the thickness of the IGL, which should be thinner at the base of fissures compared to that at the crown of folia, became almost comparable between these two regions. The Bergmann glia were also abnormally aligned. Since the radial processes of Bergmann glia extending to the brain surface serve a scaffold for migration of granule cells, granule cell migration into the IGL occurs perpendicular to the brain surface. That means that the growth rate of the IGL would become different according to the surface curvature, with more granule cells invading the underlying IGL at more curved crowns of folia. In this model, defect in the radial migration of granule cells could explain the change in the cerebellar foliation pattern observed in AAV-injected Dab1-floxed mice. Because our results suggest that loss of Dab1 in Purkinje cells caused abnormal positioning of both Bergmann glia and granule cells, we consider the possibility that Purkinje cells regulate the direction of granule cell migration in a Dab1-dependent manner and promote cerebellar lobule formation.		2022年6月30日　13:00～14:00　沖縄コンベンションセンター展示棟　ポスター会場1 1P-017 脳の発生過程における前障神経細胞の移動様式の解析 Analyses of migration profiles of the claustral neurons during brain development 大島鴻太(1)、吉永怜史(1,2)、北澤彩子(1)、仲嶋一範(1)、久保健一郎(1,2) 1. 慶應義塾大学医学部、2. 東京慈恵会医科大学 Kota Oshima(1), Satoshi Yoshinaga(1,2), Ayako Kitazawa(1), Kazunori Nakajima(1), Ken-ichiro Kubo(1,2) 1. Keio University School of Medicine, Tokyo, Japan, 2. The Jikei University School of Medicine, Tokyo, Japan Keyword: claustrum, development, neuronal migration The claustrum is a cluster of neurons located between the insular cortex and the striatum. Many studies have shown that the claustrum plays important roles in higher brain functions. Additionally, there is a growing body of evidence that dysfunctions of the claustrum are associated with neuropsychological symptoms. However, analyses of the development of the claustrum have not been performed extensively. For instance, migration profiles of the claustral neurons are largely unknown. In the present study, we tried to analyze the development of the mouse claustrum, especially focusing on elucidating migration profiles of the claustral neurons. First, we analyzed migration profiles of the claustral neurons by taking advantage of the FlashTag technology, in which fluorescent dyes were injected into the ventricle of the developing forebrains. Our analyses showed that the claustral neurons were mainly generated between embryonic day (E) 10.5 and E12.5 and that some claustral neurons migrated radially outward and then inward after they reached the surface. Next, we confirmed these unique migration profiles by performing time-lapse imaging of GFP-labeled cells. We considered that these migration behaviors of the claustral neurons showed a sharp contrast to those of neurons in the insular cortex, which migrate just outward. Interestingly, during the inward migrations of the claustral neurons, they transiently directed their major process inward and seemed to contact with fibers. Lastly, we will discuss molecular mechanisms that underlie migration profiles of the claustral neurons.

2022年6月30日　13:00～14:00　沖縄コンベンションセンター展示棟　ポスター会場1 1P-018 大脳皮質における神経細胞微小核の局在領域と放出メカニズムの解析 Analysis of the area and the secretory mechanism of neuronal micronuclei in the cerebral cortex 浅見奈都(1)、矢野更紗(2)、鶴田文憲(3) 1. 筑波大学生命環境学群生物学類、2. 筑波大学大学院生命環境科学研究科、3. 筑波大学生命環境系 Natsu Asami(1), Sarasa Yano(2), Fuminori Tsuruta(3) 1. Col of Biol Sci, Sch of Life and Env Sci, Univ of Tsukuba, Tsukuba, Japan, 2. Grad Sch of Life and Env Sci, Univ of Tsukuba, Tsukuba, Japan, 3. Faculty of Life and Env Sci, Univ of Tsukuba, Tsukuba, Japan Keyword: micronuclei, extracellular vesicle, neuronal migration, Rab proteins During the embryonic stage, neurons are generated from neural stem cells in the ventricular zone and migrate to the surface. Previously, we found that migrating neurons produce micronuclei through passing narrow spaces and release micronuclei to the extracellular space. After releasing to the extracellular spaces, micronuclei are incorporated into microglia in Layer 1 and regulate microglial maturation during the early postnatal stages. Thus, it is likely that extracellular neuronal micronuclei serve as novel mediators for acquiring proper characteristics of microglia. However, little is not known about the mechanism of the secretion of micronuclei in Layer 1. In this study, we report the regional specificity of the micronuclear appearance and potential candidates regulating micronuclear secretion. We first identified the cortical region with a high frequency of neuronal micronuclei secretion. Using NexCre; Sun1-GFP mice, which can label the neuronal nuclear envelope, we analyzed 60 slices from bregma and quantified micronuclei in Layer 1. We found that neuronal micronuclei were frequently observed in Layer 1 of the somatomotor area and the somatosensory area. To investigate the mechanism of neuronal micronuclei secretion, we transfected Neuro-2a cells with the expression plasmids encoding Rab2A and Rab3A, which have been identified as components of the extracellular micronuclei by our previous analysis. This study shows that RAB2A and RAB3A were colocalized with micronuclei.　These results suggest that the colocalization of RAB2A and RAB3A with micronuclei regulates the secretion of neuronal micronuclei to the extracellular space. Because RAB2A regulates the formation of phagophores in autophagy and RAB3A is related to the control of docking of secretory vesicles to the plasma membrane, these proteins are potential regulators that facilitate micronuclei secretion. Taken together, our data provide the possibility that these Rab proteins involve in the secretory of neuronal micronuclei and regulate microglial mature in the neocortex.		2022年6月30日　13:00～14:00　沖縄コンベンションセンター展示棟　ポスター会場1 1P-019 両耳間時差検出を担う軸索配線パターンの発達期形態解析 Developmental morphometry of the axonal wiring pattern underlying interaural time difference detection 宮田花梨(1,2)、古道万喜(1,2)、江川遼(2)、久場博司(2) 1. 名古屋大学医学部、2. 名古屋大学大学院医学系研究科細胞生理学 Karin Miyata(1,2), Kazuki Furumichi(1,2), Ryo Egawa(2), Hiroshi Kuba(2) 1. Dept Med, Nagoya Univ, Nagoya, Japan, 2. Cell Physiol, Grad Sch Med, Nagoya Univ, Nagoya, Japan Keyword: avian brainstem auditory circuits, single axon tracing, light sheet fluorescence microscopy, sound localization Wiring patterns of neural circuits underlie complex information processing in the brain. Avian brainstem auditory circuits play a major role in sound localization by detecting interaural time difference (ITD) through their unique wiring patterns. In birds, sound information from the cochlea is first transmitted to the ipsilateral nucleus magnocellularis (NM), and then to the bilateral nucleus laminaris (NL). NM axons projecting to the contralateral NL have a sequential branching structure called the delay line, which produces a delay in the timing of axonal output from each branch according to the conduction length. NL neurons are excited strongly when the ITD is balanced by the conduction delay and the neurons receive the coincident inputs from NM axons of both sides, enabling the circuits to encode the ITD as the position of neurons. However, the principle of how NM axons form such precise wiring patterns is still largely unclear. In this study, we established an efficient pipeline for 3-D morphometry of single NM axons and quantitatively evaluated the developmental process of the wiring pattern. NM neurons were sparsely labeled with fluorescent proteins by in ovo electroporation method in the chick embryos at embryonic day 1.5 (E1.5). The brainstem was dissected between E6 and E20, cleared using CUBIC method, and imaged using light-sheet microscopy. The axon morphology was traced with Neurolucida software. Developmental analysis showed that NM axons first elongated toward the cerebellar primordium by E6, and then began to extend one collateral branch toward the ipsilateral NL at E7 and several collateral branches toward the contralateral NL at E9. The number of the primary branch of the delay line remained almost constant at six between E10 and E20. Inhibiting Rac signaling by co-expression of a dominant-negative mutant of Rac1 eliminated the ipsilateral collateral branch, while increased the number of contralateral collateral branches for the delay line. These results indicated that the collateral branches for contralateral and ipsilateral NLs are induced by independent mechanisms in a single NM axon.

2022年6月30日　13:00～14:00　沖縄コンベンションセンター展示棟　ポスター会場1 1P-020 両耳間時差検出の基盤となる軸索分岐パターンの形成に関わる分子スクリーニング Molecular screening for the formation of axonal branching pattern underlying interaural time difference detection 古道万喜(1,2)、宮田花梨(1,2)、江川遼(2)、久場博司(2) 1. 名古屋大学医学部、2. 名古屋大学大学院医学系研究科細胞生理学 Kazuki Furumichi(1,2), Karin Miyata(1,2), Ryo Egawa(2), Hiroshi Kuba(2) 1. Dept Med, Nagoya Univ, Nagoya, Japan, 2. Cell Physiol, Grad Sch Med, Nagoya Univ, Nagoya, Japan Keyword: Rho family small G protein, single axon morphometry, light-sheet fluorescence microscopy, avian brainstem auditory circuits Integration of sound information from each ear is fundamental to calculation of interaural time differences for sound localization. In avian species, this integration takes place in a brainstem auditory circuit composed of nucleus magnocellularis (NM) and nucleus laminaris (NL); NL neurons receive projections from ipsilateral and contralateral sides of NM separately onto dorsal and ventral dendritic layers, respectively. Interestingly, an ipsilateral projection arises as a single collateral branch, whereas a contralateral projection forms multiple collateral branches, called delay line. In addition, the collateral branches of these projections are induced at different time points during development. However, the mechanisms that regulate the formation of these branches are poorly understood. In this study, we screened for molecules involved in the collateral branch formation of NM axons in the brainstem auditory circuit of the chicken by combining single axon morphometry and perturbation of signaling pathways. We introduced plasmid vectors which co-express fluorescent proteins and genes-of-interest under Cre recombinase into NM progenitor cells at embryonic days 1.5 (E1.5) by in-ovo electroporation. The brainstem was isolated at E13, sliced to 3-mm thickness, cleared with CUBIC method and imaged three-dimensionally with light-sheet microscopy. Screening experiments using dominant-negative mutants showed that multiple signaling molecules related to Rho family small G proteins contribute in a complex manner to the collateral branch formation. The ipsilateral projection was abolished by inhibition of Rac1 or PAK1 pathways. In the contralateral projection, primary branch formation was promoted by inhibition of Rac1, RhoA, or PAK1 pathways and slightly suppressed by inhibition of Cdc42 pathway. These results suggested that signaling pathways for the collateral branch formation are highly distinct for each side in a single NM axon, providing important insights into the mechanism of circuit formation underlying interaural time difference detection.		2022年6月30日　13:00～14:00　沖縄コンベンションセンター展示棟　ポスター会場1 1P-021 網膜層構造形成と細胞運命決定におけるaPKCλの役割 The functional role of aPKCλ in the retinal lamination and cell fate determination 澤田綾(1)、井上祐介(2)、西村勇輝(1)、前嶋千瀬都(1)、秋葉唯(1)、秋本和憲(3)、大野茂男(4)、小池千恵子(1,5) 1. 立命館大学薬学部、2. 立命館大学大学院薬学研究科、3. 東京理科大学薬学部、4. 順天堂大学大学院医学研究科老人性疾患病態・治療研究センター、5. 立命館大学総合技術研究機構システム視覚科学研究センター Rin Sawada(1), Yusuke Inoue(2), Yuki Nishimura(1), Chiseto Maezima(1), Yui Akiba(1), Kazunori Akimoto(3), Shigeo Ono(4), Chieko Koike(1,5) 1. Col Pharm Sci, Ritsumeikan Univ, Shiga, Japan, 2. Grad Sch Pharm, Ritsumeikan Univ, Shiga, Japan, 3. Faculty Pharm Sci, Tokyo Univ of Sci, Tokyo, Japan , 4. Lab Cancer Biol, Res Inst for Diseases of Old Age, Grad Sch Med, Juntendo Univ, Tokyo, Japan, 5. Ctr Syst Vision Sci, Res Org Sci Tech, Ritsumeikan Univ, Shiga, Japan Keyword: retina, aPKCλ, Cre-loxP Atypical protein kinase C lambda (aPKC lambda) plays a critical role in the establishment of epithelial and neuronal polarity. We had previously investigated the mechanisms of polarity formation of the photoreceptor cells by generating conditionally knocked out aPKC lambda in differentiating photoreceptor cells using the Crx-Cre-loxP system. In aPKC lambda conditional knock-out (CKO) mice, the photoreceptor cells displayed morphological defects and failed to form ribbon synapses. Intriguingly, the lack of aPKC lambda in differentiating photoreceptors led to severe laminar disorganization not only in the photoreceptor layer but also in the entire retina. We indicated the ectopic formation of adherens junctions may lead to entire retinal laminar disruption. To investigate the function of aPKC lambda in the earlier stage of retinal lamination, we generated conditionally knocked out aPKC lambda in retinal epithelial cells using the Cre-loxP system using DKK3-Cre mouse line. We observed that retinal lamination was severely disrupted in the CKO mouse retinas that lack aPKC lambda in retinal epithelial cells, including photoreceptor layers. Unlikely CKO mouse retina lacks aPKC lambda only in differentiating photoreceptor cells, cell fate determination was affected along with laminar disorganization. Interestingly, the number of photoreceptors was significantly reduced, but bipolars were increased in the CKO retina; the numbers of horizontal, amacrine, Müller cells, and ganglion cells were unchanged. Our data suggest that the lack of aPKC lambda in retinal progenitors changes cell fate determination along with disruption of total retinal lamination. Our data suggest that the lack of aPKC lambda in retinal progenitors changes cell fate determination along with disruption of total retinal lamination. We are currently studying the mechanisms aPKC lambda modifies fate in retinal cells by using RNA-seq analysis.

2022年6月30日　13:00～14:00　沖縄コンベンションセンター展示棟　ポスター会場1 1P-022 PcdhγC4のホモフィリック接着活性を失ったγC4ΔEC2マウスの解析 The analysis of PcdhgC4 specific homophilic interaction in vivo from molecular to behavior 梅澤遥香(1)、樋口流音(1)、渡辺雅彦(2)、三宝誠(3)、平林真澄(3)、八木健(1) 1. 大阪大学大学院生命機能研究科、2. 北海道大学大学院医学研究院、3. 生理学研究所　行動・代謝分子解析センター Haruka Umezawa(1), Ryuon Higuchi(1), Masahiko Watanabe(2), Makoto Sanbo(3), Masumi Hirabayashi(3), Takeshi Yagi(1) 1. Graduate School of Frontier Biosciences, Osaka University, Suita, Japan, 2. Graduate School of Medicine, Hokkaido University, Sapporo, Japan, 3. Center for Genetic Analysis of Behavior, National Institute for Physiological Sciences, Okazaki, Japan Keyword: apoptosis, clustered protocadherin, homophilic interaction The formation of proper neural circuits leads to higher functions such as locomotion, memory, and learning, but the details of the diverse molecular mechanisms involved in neural circuit formation are unknown. In mammals, the candidate of gene diversity we are focusing on is clustered protocadherins (cPcdh). One of the major features of cPcdh isoforms is its trans homophilic interaction via EC1-4 at the cell surface. This activity is thought to be important for and the appropriate neural circuit formation through the self-recognition and non-self distinction in neurons. But the concrete significance of cPcdh homophilic interaction is not unclear. We revealed that only PcdhgC4 among 58 cPcdh isoforms knock out (∆γC4) mice died within one day of birth. This result indicates that the PcdhgC4 is essential for mouse survival. However, the detail function of the Pcdh-γC4 protein is unknown. In this study, we hypothesized that homophilic interaction of PcdhgC4 is particularly important for mouse survival. To reveal this hypothesis, we generated and analyzed only EC2 domain of PcdhgC4 deletion (ΔEC2) mice. As a result, we found that despite expressing Pcdh-γC4-∆EC2 protein, ΔEC2 mice died within one day of birth. In addition, the degree of cell death in embryonic day 18.5was increased compared to ∆γC4 mice especially in the midbrain and reticular formation. We also analyzed ΔEC2/+ heterozygous mice, because the western blot analysis showed that ΔEC2/+ mice expressed both native-Pcdh-γC4 protein and Pcdh-γC4-∆EC2 protein. We observed cell death and found that there was no significant difference between ΔEC2/+ mice and WT. Next, to investigate the effect of Pcdh-γC4-∆EC2 protein in ΔEC2/+ mice for the higher brain function, we performed some behavioral test. These results indicate that the specific homophilic cell-cell interaction via PcdhgC4 EC2 domain is essential for mouse survival and neural circuit formation. In this presentation, we will discuss the unique features of homophilic interaction derived PcdhgC4.		2022年6月30日　13:00～14:00　沖縄コンベンションセンター展示棟　ポスター会場1 1P-023 ゲノム編集によるヒトiPS細胞由来抑制性神経細胞のサブタイプ特異的イメージング Subtype-specific imaging of hiPSC-derived GABAergic interneurons by CRISPR/Cas9-mediated genome editing 銭映美(1)、石川充(1)、吉松祥(1)、森本悟(1)、岡野栄之(1) 1. 慶應義塾大学医学部生理学教室 Emi Qian(1), Mitsuru Ishikawa(1), Sho Yoshimatsu(1), Satoru Morimoto(1), Hideyuki Okano(1) 1. Dept Physiol, Sch Med, Keio Univ, Tokyo, Japan Keyword: iPS cells, GABAergic interneuron, Neuronal differentiation, CRISPR/Cas9 Cortical interneurons (cINs) are heterogeneous with diverse morphological and electrophysiological properties and different neuronal circuit formation in the neocortex, and thus they are classified into various subtypes including Parvalbumin-positive (PV) and Somatostatin-positive (SST) neurons. While the defects of cINs in neuropsychiatry disorders are extensively studied using patient-derived iPSCs, the neuronal vulnerability can be different depending on IN subtypes among diseases, indicating the necessity of study that precisely segregates the cell lineages and function of subtype-specific cINs. Developmentally, PV and SST neurons are both cIN-subtypes derived from progenitor cells in Medial ganglionic eminence (MGE), but it is poorly understood how the distinct subtypes are emerged and separated from the same origin. Therefore, our study is aimed to distinguish cIN-subtypes derived from hiPSCs and analyze them individually for developmental and pathophysiological research. First, we established a hiPSC line harboring PV and SST double reporters for subtype-specific visualization. The mNeonGreen and tdTomato fluorescent reporter genes were respectively inserted into the direct downstream of PV and SST coding regions with self-cleaving 2A peptide sequences by CRISPR/Cas9-mediated genome editing. The reporter functionality was verified by a dCas9-mediated endogenous gene activation system and fluorescence expressions. Next, we employed a regional patterning method to generate cINs using the reporter hiPSC line by modulating neuronal identity-inducing signals. As a result, we successfully detected the fluorescence in the living state, and we sorted the differentiated cells based on fluorescence expressions. In summary, we established a double reporter knock-in hiPSC line, and generated cINs with evident fluorescence expressions along with differentiation. Transcriptomic analysis of subtype-specific neurons purified by FACS would be effective for further study on subtype-specific characterization. The reporter hiPSC line would serve as a valuable tool to recapitulate various molecular features of cINs in the developing human brain.

2022年6月30日　13:00～14:00　沖縄コンベンションセンター展示棟　ポスター会場1 1P-024 細胞外マトリックスによる傷害大脳皮質における新生ニューロンの移動促進 Promotion of postnatal neuroblast migration is promoted by scaffold material containing extracellular matrix (ECM)-containing biomaterial 山本悟暁(1)、中嶋智佳子(1)、中村春野(1)、島田直樹(3)、中村耕一郎(4)、上杉昭二(4)、田畑泰彦(5)、澤本和延(1,2) 1. 名古屋市立大学大学院医学研究科脳神経科学研究所神経発達・再生医学分野、2. 生理学研究所神経発達・再生機構研究部門、3. （株）日本毛織株式会社　研究開発センター、4. （株）ニッケ・メディカル　医療機器事業部、5. 京都大学再生医科学研究所生体組織工学研究部門生体材料学分野 Satoaki Yamamoto(1), Chikako Nakajima(1), Haruno Nakamura(1), Naoki Shimada(3), Koichiro Nakamura(4), Shoji Uesugi(4), Yasuhiko Tabata(5), Kazunobu Sawamoto(1,2) 1. Dept Dev Regen Neurobiol, Inst Brain Sci, Nagoya City Univ Grad Sch Med Sci, Nagoya, Japan, 2. Div Neural Dev Regen, NIPS, Okazaki, Japan, 3. Research & Development Center, The Japan Wool Textile Co., Ltd., Japan , 4. Medical Device Department, Nikke Medical Co., Ltd., Japan, 5. Dept Biomat, Field Tissue Eng, Inst Front Med Sci, Kyoto Univ, Kyoto, Japan Keyword: neuronal migration, extracellular matrix, biomaterial, neonatal brain injury Neural stem cells in the ventricular-subventricular zone (V-SVZ) generate neuroblasts throughout life. The neuroblasts of postnatal mice migrate through the rostral migratory stream (RMS) toward the olfactory bulb (OB) forming chain-like aggregates, differentiate into mature interneurons, and integrate functionally into the OB neuronal networks. In pathological brain conditions, e.g., strokes, neuroblasts not only migrate toward the OB, but also toward lesion sites along scaffold cells. The molecular interplay between neuroblasts and the scaffold cells regulates neuroblast migration and is indispensable. Successful delivery of neuroblasts to the lesion sites is required for functional recovery. Therefore, it is a promising approach to promote the neuroblast migration toward the injured site by introducing biomaterials as an alternative to the damaged scaffold cells. In this study, we aimed at evaluating the potential of several ECMs to promote neuroblast migration. First, we analyzed time-lapse images of V-SVZ-derived neuroblasts cultured on ECM-plated dishes. Interestingly, ECMs, known to promote axonal elongation, significantly increased the migration velocity of neuroblasts. Second, to test whether the neuroblast migration in the brain can also be promoted, we prepared gelatin-fiber-lattice materials containing the selected ECMs. We implanted the materials in cryo-injured cortex of neonatal mice and analyzed the neuroblast distribution in the brain. The implant of ECM-containing material resulted in more neuroblasts distribution along the gelatin fibers and in the upper cortical areas, compared to that of the control vehicle-treated material. The neuroblasts observed extending along the gelatin fibers resembled those migrating on the radial glial cells, the endogenous scaffold cells. Finally, we verified one of the ECMs was expressed in astrocytes both in the RMS and injured cortex, suggesting the functional relevance of these ECMs in promoting neuroblast migrations. Taken together, these results suggest that the ECMs promote neuronal migration in the postnatal injured brain.