幹細胞、ニューロンとグリアの分化 Stem Cells, Neuronal and Glial Production/Differentiation
P1-2-33 ヒトiPS細胞からアストロサイトの分化誘導 Efficient derivation of functional astrocytes from human induced pluripotent stem cells ○近藤孝之1,2, 高橋良輔2, 井上治久1,3 ○Takayuki Kondo1,2, Ryosuke Takahashi2, Haruhisa Inoue1,3 京都大学iPS細胞研究所1, 京都大学大学院医学研究科　臨床神経学2 Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto1, Department of Neurology, Graduate School of Medicine, Kyoto University, Kyoto2, CREST, Japan Science and Technology Agency, Tokyo, Japan3 [Background] Astrocytes, one subtype of glial cells, are the most abundant cells in the vertebrate central nervous system. Similarly to neurons, astrocyte is also important key to understand neural function and disorders. However, access to human astrocyte for scientific research is limited and astrocytes research have been depend on tumor cell lines or model animals. Recent establishment of human induced pluripotent (hiPSCs) technology enabled us to utilize variety of differentiated cells as research materials, including neural cells. Here we establish a method to differentiate hiPSCs into astrocytes and evaluate hiPSCs-derived astrocytes. [Method] We started to differentiate hiPSCs by blocking SMAD signaling and modify serum-free floating culture method to obtain neural progenitor cells efficiently. After neuronal differentiation, we enriched astroglial population, exploiting the difference of cell adhesion ability, every month up to more than nine months. [Results] We could observe 1) glial marker expression, 2) glutamate transport ability and 3) signal transduction between neighboring astrocytes. [Conclusion] We established astrocyte differentiation method from hiPSCs, which can be beneficial to understand astrocyte physiological functions and neuronal disorders.	P1-2-34 感知系脳室周囲器官のダイナミックな血管構築変化とアストロサイトバリア：血中情報感知システムにおける役割 Dynamic vascular remodeling and astrocytic barrier play a role to sense blood-derived information in the sensory circumventricular organs ○森田晶子1,2, 鵜飼真璃2, 奥田洋明1, 辰巳晃子1, 和中明生1, 宮田清司2 ○Shoko Morita1,2, Shinri Ukai2, Hiroaki Okuda1, Kouko Tatsumi1, Akio Wanaka1, Seiji Miyata2 奈良県立医科大学・医・第2解剖学1, 京都工芸繊維大学・応用生物学部門2 Department of Anatomy and Neuroscience, Nara Medical University, Kashihara, Nara1, Department of Applied Biology, Kyoto Institute of Technology, Kyoto2 In the sensory circumventricular organs (CVOs), including the organum vasculosum of the lamina terminalis (OVLT), subfornical organ (SFO), and area postrema (AP), the vasculature lacks the complete blood-brain barrier (BBB) and allows parenchyma cells to sense a wide variety of blood-derived substances and convey their information into other brain regions for the control of autonomic and inflammatory reactions and/or behaviors. However, it remains unclear fundamental mechanisms how the sensory CVOs are distinguished from other brain regions that contain complete BBB vasculature. Here, we found that continuous angiogenesis occur together with continuous gliogenesis and neurogenesis in the OVLT, SFO, and AP. A major angiogenic factor vascular endothelial growth factor (VEGF) was highly expressed at neurons and astrocytes and the proliferation of endothelial cells was significantly attenuated by an anti-angiogenic inhibitor SU11248 possibly via VEGF receptor 2. The immunohistochemistry of tight junction proteins and electron microscopic observation revealed the presence of intercellular tight junction at dense astrocytic network that surrounded fenestrated vasculature and absence of vascular tight junction. The tracer experiment revealed that blood-derived low-molecular-mass (LMM) molecules, Dextran 3,000 and fluorescein isothiocyanate (FITC), accessed to parenchyma, but scarcely diffused beyond dense astrocytic network and contacted with neuronal somata. The attenuation of endothelial cell proliferation by a mitotic inhibitor cytosine arabinoside significantly decreased parenchymal access of blood-derived FITC, whereas the disruption of astrocytic network by an astroglial toxin alpha-amino-adipic acid prominently increased parenchymal access of FITC. Our present data indicate that continuous angiogenesis possibly contribute to fenestrated vasculature and dense astrocytic network acts as a barrier against blood-derived LMM molecules instead of the BBB.
P1-2-35 視細胞変性後Müllerグリアに起こるDNA損傷と細胞死について DNA damage-induced cell death of Müller glia after photoreceptor injury ○野村かおり1, 齋藤文典1, 根岸春樹1, 早川亨1, 藤枝弘樹1 ○Kaori Nomura1, Fuminori Saito1, Haruki Negishi1, Tooru Hayakawa1, Hiroki Fujieda1 東京女子医科大学　解剖学教室1 Department of Anatomy, Tokyo Women's Medical University, Tokyo, JAPAN1 Müller glia are the principal glial cell type in the retina and have a range of functions to support retinal neurons, such as transmitter recycling and regulation of ion and water homeostasis. Recent evidence has further indicated that Müller glia have the potential to regenerate neurons in certain pathological conditions. In the fish retina, damage stimulates Müller glia to proliferate and regenerate retinal neurons to restore the structure and function of the retina. Unlike fish, the regenerative response of Müller glia is extremely limited in mammals. To understand the mechanism that limits the regenerative capacity of Müller glia in mammals, we examined the response of Müller glia to photoreceptor damage induced by methylnitrosourea (MNU) in two rodent species, mice and rats. In mice, virtually no Müller glia reentered the cell cycle after photoreceptor damage while, in rats, many Müller glia incorporated BrdU and expressed a variety of cell cycle markers such as Ki67 and pH3. Cell counting revealed a significant increase in the number of Müller glia by day 4 after MNU treatment, indicating that many, if not all, Müller glia completed a full mitotic division. However, cell cycle reentry of Müller glia was accompanied by DNA damage response with H2AX phosphorylation and upregulation of p53 and p21. There was a significant decline in the number of Müller glia between day 4 and day 7, during which apoptotic cell death of Müller glia was observed by TUNEL assay. These findings suggest that unscheduled cell cycle reentry of Müller glia causes DNA damage followed by cell cycle arrest and cell death, which may be one of the mechanisms that limit retinal regeneration in mammals.	P1-2-36 無血清下でのマウスiPS細胞からのオリゴデンドロサイト分化誘導 Oligodendrocytes differentiation from mouse induced pluripotent stem (iPS) cells under serum free condition ○三角吉代1, 上田佳朋1, 西垣瑠里子1, 石田章真1, 鄭且均1, 飛田秀樹1 ○Sachiyo Misumi1, Yoshitomo Ueda1, Ruriko Nishigaki1, Akimasa Ishida1, Cha-Gyun Jung1, Hideki Hida1 名古屋市立大学大学院　医学研究科　脳神経生理1 Dept of Neurophysiol & Brain Sci, Nagoya City Univ Grad Sch Med Sci, Nagoya, Japan1 Developing diffuse white matter injury (DWMI) caused by hypoxia is associated with permanent neurodevelopmental disabilities in preterm infants. As selective loss of oligodendrocytes progenitor cells (OPC) is reported in some case of DWMI, OPC transplantation seems to be a hopeful treatment. We are challenging OPC transplantation using mouse induced pluripotent stem (iPS) cells. To obtain OPC lineage cells suitable for transplantation, we try to induce iPS cell-derived OPC effectively and characterized OPC lineage cells. Firstly, to investigate whether serum-free condition induces iPS cells into embryonic bodies (EB), we estimated gene expression of three germ layers. In serum-free groups, significantly high level of FGF5 and nestin (ectoderm gene makers) mRNA expression was found compared with serum-containing group. Following to selection and expansion of neural stem cells, cells were differentiated into glial progenitors with FGF2 and EGF. These cells expanded and formed small cluster. Most cells in the cluster were A2B5 positive glial progenitors. Some cells expressed NG2 and olig2, while few were nestin positive neural stem cells. Cells were then induced into OPC with PDGF. After one week, cells showed OPC morphology with multipolar process. At this stage 47.4% of total cells were PDGFRα positive OPC. NG2 and olig2 double positive cells and A2B5 and NG2 double positive cells were also detected. Some cells were GFAP positive astrocytes without olig2. After terminal differentiation under T3 and CNTF, cells expressed an immature oligodendrocyte marker O4 (24.3 % of total cells) and mature oligodendrocyte makers MBP. Inward rectifying potassium (IKir) current was found as property of mature oligodendrocytes. Data suggest that functional oligodendrocytes are effectively induced from mouse iPS cells under serum-free condition.
P1-2-37 発達早期段階におけるヒトiPS細胞由来神経細胞の免疫細胞化学的解析 Immunocytochemical analysis of human iPSC-derived neurons at early developmental stages ○大原由貴1, 山崎博幸1, 白尾智明1 ○Yuki Ohara1, Hiroyuki Yamazaki1, Tomoaki Shirao1 群馬大学大学院　医学系研究科　神経薬理学1 Department of Neurobiology and Behavior, Gunma University Graduate School of Medicine, Maebashi, Japan1 Recent advances in human induced pluripotent stem cells (hiPSCs) offer new possibilities for biomedical research and clinical applications. Differentiated neurons from hiPSCs are expected to be a tool for developing a new method of treatment for various neurological diseases. However, the detail process of neuronal development from hiPSCs has not yet been known. In this study, we analyzed neuronal development of hiPSC-derived neurons particularly focusing on their early developmental stages. We cultured iCell Neuron (Cellular Dynamics International) and compared their development with that of the primary cultured neurons derived from rat hippocampus. In 2 days in vitro (DIV) culture of rat neurons we observed three different stages, which were stages 1, 2 and 3 in developmental classification proposed by Dotti. Most developed stage 3 neurons had several short neurites with one long neurite, which is destined for an axon. In contrast, we could observe only stage 1 and 2 neurons at 5 DIV as well as 1 DIV in iCell neuron culture. We could not observe stage 3 iCell neurons which had one longer neurite with several neuritis of similar length. This suggests that the human neurons develop at a slow speed compared with rat neurons. We further double labeled these cells for drebrin and F-actin, and found that the localization patterns of F-actin and drebrin in growth cones of iCell neurons were similar to those of rat neurons. However, iCell neurons showed large dot-like pattern with F-actin and drebrin signals in soma. These drebrin-actin structures were not observed in rat neurons. In addition, strong drebrin immunostaining was observed in some regions where F-actin signal was weak. Additionally, the size of cell body of iCell neurons is smaller than that of rat neurons at stage 2. These data suggest that the development of iCell neurons shows qualitatively difference in addition with the quantitative difference at developmental speed.	P1-2-38 神経前駆細胞のダイナミクスが大脳皮質原基脳室帯の維持に及ぼす機能 Physiological meanings of the dynamics of progenitor cells for the maintenance of ventricular zone ○篠田友靖1, 長坂新1, 三浦岳2, 月田早智子3, 藤森俊彦4, 宮田卓樹1 ○Tomoyasu Shinoda1, Arata Nagasaka1, Takashi Miura2, Sachiko Tsukita3, Toshihiko Fujimori4, Takaki Miyata1 名古屋大院・医・細胞生物1, 京大院・医・形態形成機構2, 大阪大院・生命機能3, 基生研・初期発生4 Dept Anatomy and Cell Biol, Nagoya Univ, Nagoya1, Dept Anatomy and Dev Biol, Kyoto Univ, Kyoto2, Lab Bioscience, Grad Sch Frontier Bioscience and Grad Sch Med, Osaka Univ, Osaka3, Div Embryology, NIBB, Okazaki4 In the pseudostratified neuroepithelium (NE) or ventricular zone (VZ) of developing mammalian cerebral cortex, each neural progenitor cell shows nuclear migration along the apical-basal axis correlated with cell cycle, i.e., M-phase at the apical surface and S-phase in the basal region (called interkinetic nuclear migration, INM). How INM of each progenitor cell is three-dimensionally coordinated to maintain the whole structure of the NE/VZ , however, still remains unclear. To investigate the possible community-level regulatory mechanisms, we initiated large-scale monitoring of all NE/VZ cells. ZO-1-EGFP, H2B-mCherry or Lyn-Venus-transgenic mice enabled us to visualize all the cellular elements in a given tangential optical slice. We found: (1) While an M-phase cell approach the apical surface and then divide, the neighboring endofeet also moved following the M-phase cell's enlargement and cytokinesis. (2) The initial basal-directed movement of daughter cells generated at the apical surface seemed to be influenced by neighboring M-phase cells. (3) The apical process neighboring an M-phase cell showed a highly stretched structure. Our computational modeling also suggested that rapid basal-directed movement of newborn G1 cells at the periventricular area is indispensable for the continuous INM of the progenitor cells in VZ. These findings suggest that the basal-directed migration of G1 cells adjacent to the apical surface, possibly generated by surrounding apical process or M-phase cells, is important for the coordinated INM and the maintenance of pseudostratificated structure of VZ.
P1-2-39 ES細胞由来-視床下部培養系におけるMCHニューロンの発生 Generation of melanin-concentrating hormone neurons in ES cell-derived hypothalamic culture ○小谷侑1, 長崎弘1,2, 須賀英隆2,3, 綿谷崇史3, 金子葉子1, 中島昭1, 大磯ユタカ2, 笹井芳樹3, 太田明1 ○Yu Kodani1, Hiroshi Nagasaki1,2, Hidetaka Suga2,3, Takafumi Wataya3, Yoko S Kaneko1, Akira Nakashima1, Yutaka Oiso2, Yoshiki Sasai3, Akira Ota1 藤田保健衛生大・医・生理I1, 名古屋大院・医・糖尿病内分泌2, 理研・発生・再生研3 Dept Physiol, Fujita Health Univ Sch Med, Toyoake, Japan1, Dept Endocrinol & Diabetes, Nagoya Univ Grad Sch Med, Nagoya, Japan2, RIKEN CDB3 In vitro organogenesis of the hypothalamic tissue from mouse embryonic stem (ES) cells has been accomplished by Wataya et al (2008, PNAS). The ES cell-derived hypothalamic culture (ES-hypo) is reported to generate peptidergic neurons producing vasopressin, neuropeptide Y, or agouti-related protein. However, it remains unclear in many aspects: whether ES-hypo shares a variety of peptidergic neurons with the native hypothalamus; also whether those peptidergic neurons are functional in ES-hypo. The present study focused on the generation of melanin-concentrating hormone (MCH) neurons and their morphological and histological profiles in ES-hypo. MCH is a hypothalamic neuropeptide involved in the regulation of feeding behavior and energy metabolism. ES-hypo was prepared as embryoid body-like cell aggregates according to the original method and subjected to quantitative real time PCR or immunohistochemistry. MCH mRNA was detectable after 3 weeks of the induction and exponentially increased thereafter. Consistently, a significant number of MCH-immunoreactive cell bodies and fibers were found in the aggregates after 4 weeks of the induction. These MCH neurons showed histological characters common to native ones in vivo: co-expression of MCH with GABA or cocaine- and amphetamine-regulated transcript (CART), and reciprocal synaptic connections with tyrosine hydroxylase-positive (i.e., dopaminergic) or orexin/hypocretin-positive neurons. In the dissociation culture of ES-hypo, MCH neurons exhibited various morphologies identical to those found in the developing stages. In summary, we have identified MCH neurons co-exist with other peptidergic neurons in ES-hypo, and also they share many characters with native MCH neurons. It is suggested that ES-hypo provides a new experimental system to investigate the development and neurophysiology of the hypothalamic MCH system.	P1-2-40 小脳顆粒細胞前駆体の分化制御におけるProx1の役割 Prox1 regulate the transition from proliferative state to mature state of cerebellar granule cell precursors ○宮下聡1,2, 瀬戸祐介1, 川口義弥3, 宗田孝之2, 星野幹雄1 ○Satoshi Miyashita1,2, Yusuke Seto1, Yoshiya Kawaguchi3, Takayuki Souta2, Mikio Hoshino1 国立精神・神経医療研究センター　神経研究所　病態生化学研究部1, 早稲田大学先進理工学部2, 京都大学iPS細胞研究所3 Dept Biochem & Cell Biol, NCNP,Tokyo1, Sch. of Adv Sci and Eng Waeda Univ, Tokyo, Japan2, Dept of Clinic Appli, Center for iPS Cell Res and Appli, Kyoto University, Kyoto, Japan.3 Cerebellar granule cells are excitatory neurons that comprise a majority of cerebellar cells. During development, granule cell precursors (GCPs) leave the rhombic rip, the origin of cerebellar excitatory neurons, migrate along the pial surface of the cerebellum and then form the external granule cell layer (EGL) that consists of two sublayers, outer EGL (oEGL) and inner EGL (iEGL). GCPs proliferate in the oEGL, and then exit from the cell cycle to migrate into iEGL. After staying in iEGL for a few days, GCPs leave iEGL for internal granule cell layer (IGL) to become mature granule cells. Although these processes for differentiation and maturation of granule cells are well described, the underlying molecular mechanisims are poorly understood. A failure in these processes causes medulloblastoma, common brain tumor found in childhood, convincing the importance to understand the underlying machinery. We found that GCPs express a homeobox transcriptional factor, Prox1 (Prospero related homeobox 1), at the timing when GCPs migrate from oEGL to iEGL. Prox1 is a homolog of Drosophila transcriptional factor Prospero (Pros), which is known to regulate cell cycle and maturation of Drosophila cells in the neuronal lineage. We observed that Prox1 introduction into oEGL cells by in vivo electroporation induced GCPs to exit from the cell cycle and to express early neuron markers. Conversely, cell cycle exit and maturation of GCPs are delayed or disorganized in the cerebellum of Prox1 conditional KO mice. Furthermore, by analyzing several related genes for Prox1, we found molecular pathways that regulate the transition from proliferative state to mature state of GCPs. Our findings may contribute to understanding the molecular machinery for differentiation and maturation of cerebellar granule cells as well as the pathology of medulloblastoma.
P1-2-41 BMPシグナルは胎生期海馬に存在するGFAPを発現する神経幹細胞の産生に重要である BMP signal are crucial for production of GFAP-expressing NSCs in the developing hippocampus ○柏木太一1, 塩田清二2, 石龍徳1 ○Taichi Kashiwagi1, Seiji Shioda2, Tatsunori Seki1 東京医科大学　組織・神経解剖学1, 昭和大学　医学部　第一解剖学2 Dept Histol and Neuroanat, Tokyo Med Univ, Tokyo1, Dept Anat, Showa Univ Sch of Med, Tokyo2 In the hippocampus, neural stem cells (NSCs) are presented even in adulthood, and the granule cells continue to be generated throughout life. Therefore, the hippocampal NSCs presumably possess the special mechanisms to maintain stemness for long-term. Unlike general embryonic NSCs, adult hippocampal NSCs are known to express glial fibrillary acidic protein (GFAP). However, it remains unclear the embryonic origin of adult hippocampal NSCs. Our previous analysis using the transgenic mice expressing GFP under GFAP promoter (GFAP-GFP mice) reveals that GFP+ cells are confined to in the dentate primordium, suggesting that they are candidates for the source of the adult NSCs. Here we studied the mechanisms to generate GFAP-expressing cells in hippocampal primodium. First, hippocampal cells were isolated from GFAP-GFP mouse embryos and were cultured in the medium with FGF2. We found cultured cells expressing GFP and NSC markers. Further GFAP-expressing cells were able to form neurospheres, suggesting that they contain NSCs. Next, we focused on bone morphogenetic proteins (BMPs) as a possible factor to induce the generation of GFP+ NSCs, since BMPs are strongly expressed by the hippocampal primodium compared to the neocortex where few GFP+ cells exist. When neocortical cells from GFAP-GFP mouse embryos were cultured with BMP4, GFP expression was induced. GFP + cells induced by BMP4 also express nestin, that is a NSC marker, and could form neurospheres. Furthermore, those cells generate Prox1+ cells that form granular cell layer in the dentate gyrus (DG). These results suggest that BMP signal endows NSCs with a character of dentate ones and contributes to the generation of granular cells in the DG.	P1-2-42 興奮性の大脳皮質神経細胞は誕生時においてRbファミリー依存的に細胞分裂能を失う Cortical excitatory neurons become protected from cell division during neurogenesis in an Rb family-dependent manner ○押川未央1, 岡田桂1, 味岡逸樹1 ○Mio Oshikawa1, Kei Okada1, Itsuki Ajioka1 東京医科歯科大学 脳統合機能研究センター 分子生物学分野1 Tokyo Medical and Dental University, Center for Brain Integration Research, Tokyo, Japan1 During development, cell cycle exit is tightly coordinated with the initiation of differentiation. In the central nervous system (CNS), once the daughter cells initiate neuronal differentiation, they become post-mitotic cells and are believed to be protected from cell division. The tumor suppressor retinoblastoma protein (Rb) is characterized as a key regulator of cell proliferation, differentiation, and death in a context-specific manner. The inactivation of Rb pathway in post-mitotic neurons triggers the cell cycle re-entry, followed by cell death associated with the progression of neuronal degeneration. Nevertheless, some evidence shows that differentiating neurons can proliferate and form tumors when Rb is inactivated in their progenitors. Beyond the context specificities of Rb functions, the complicated compensations and redundancies in Rb and its related family members (p107 and p130) are likely to mask the functions of the Rb family. In this study, to elucidate the Rb family roles in cortical excitatory neurons and their progenitors, all the Rb family members were inactivated in differentiating neurons and progenitors using a neuron-specific pMAP2 promoter and a ubiquitous pCAG promoter, respectively, with the in utero electroporation method. We found that pMAP2-induced Rb-TKO differentiating neurons undergo S-phase, but not cell division. Whereas pCAG-induced Rb-TKO cells initiate differentiation without undergoing cell cycle exit, proliferate, and form tumors. These results suggest that newly born cortical neurons from progenitors become protected from cell division in an Rb family-dependent manner.
P1-2-43 腸管神経系の新たな細胞起源の同定 Schwann cell precursor-like cells from the mesentery are a cellular origin of enteric neurons ○上坂敏弘1, 榎本秀樹1 ○Toshihiro Uesaka1, Hideki Enomoto1 理化学研究所　発生・再生科学総合研究センター　神経・発生研究室1 Lab NDR, CDB, RIKEN, Kobe, Japan1 The enteric nervous system (ENS) is the largest and most complex division of the peripheral nervous system. Current consensus holds that the enteric neurons are derived from either vagal or sacral neural crest (NC) cells. Here, we report a third cellular origin of enteric neurons. Genetic labeling of NC-derived cells associated with the extrinsic nerves projecting to the gut revealed that a subset of Schwann cell precursor-like cells (SCPLCs) takes a neuronal fate and contributes up to 20% of neurons in the ENS depending on the gut regions. SCPLCs display higher neuronal differentiation capacity in a mouse model for Hirschsprung disease (intestinal aganglionosis) than a normal mouse, suggesting plasticity and a compensatory effect of SCPLC-derived neurogenesis in pathological conditions. Identification of SCPLCs as a novel cellular origin of the ENS provides deeper insights into understanding of development, developmental disorders and regeneration of the ENS.

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