ポスター発表 (Poster Sessions)

幹細胞、ニューロンとグリアの分化 Stem Cells, Neuronal and Glial Production/Differentiation
P2-1-65 n-6/n-3多価不飽和脂肪酸摂取比の乱れは大脳皮質形成を妨げる Nutritional high ratio of n-6 to n-3 polyunsaturated fatty acids disturbs corticogenesis ○酒寄信幸^1,2, 大隅典子¹ ○Nobuyuki Sakayori^1,2, Noriko Osumi¹ 東北大院・医・発生発達¹, 日本学術振興会特別研究員² Div. Dev. Neurosci., Grad. Sch. Med., Tohoku Univ., Miyagi¹, JSPS Res. Fellow² Polyunsaturated fatty acids (PUFAs) are important nutrients because they compose biological structures and produce biologically active substances. Especially, n-6 and n-3 PUFAs are the dominant PUFAs in the brain and are involved in brain functions. It is known that not only the amount but also the ratio of these PUFAs are important for various biological functions due to their mutual competition for the metabolism, transport, and incorporation into cell membrane. In the modern society, intake of n-6 PUFAs has dramatically increased mainly due to the intake of seed oils. Actually, the ratio of n-6/n-3 PUFAs in the diet has steadily increased to 10-25 in industrialized countries, although appropriate n-6/n-3 PUFA ratio in nutrition is considered to be1-4. Here, we investigated effects of n-6 excess/n-3 deficient diet on embryonic corticogenesis. We fed control or n-6 excess/n-3 deficient diet to pregnant mice and measured mean thickness of the cortical primordium on embryonic day 14.5. The mice that were fed with n-6 excess/n-3 deficient diet showed decrease in the mean thickness of the cortical primordium. We also found decrease in the thickness of the cortical plate (composed of neurons) but not in that of the ventricular zone (neural stem cells, NSCs) or the subventricular zone (basal progenitors, BPs). We further observed that proliferation of NSCs and BPs was decreased with no significant difference in the cell death. These results demonstrate that maternal intake of n-6 excess/n-3 deficient diet during pregnancy alters embryonic cortical neurogenesis. This study could be a warning for consuming n-6 excess/n-3 deficient diet that is popular in modern industrialized countries.	P2-1-66 大脳皮質梗塞後の視床における内因性神経幹細胞の発現 Generation of endogenous neural stem cells in the ipsilateral thalamus following cortical infarction ○柴田啓貴^1,2, 百田義弘^1,2, 中込隆之¹, 中野亜紀子¹, 河原麻衣子¹, 中田雅代^1,2, 盧山^1,3, 小谷順一郎², 松山知弘¹ ○Hiroki Shibata^1,2, Yoshihiro Momota^1,2, Takayuki Nakagomi¹, Akiko Nakano-Doi¹, Maiko Kawahara¹, Masayo Nakata^1,2, Shan Lu^1,3, Junichiro Kotani², Tomohiro Matsuyama¹ 兵庫医科大学先端医学研究所神経再生部門¹, 大阪歯科大学歯科麻酔学講座², 兵庫医科大学内科学総合診療科³ Institute for Advanced Medical Sciences, Hyogo College of Medicine, Hyogo, Japan¹, Department of Anesthesiology, Osaka Dental University, Osaka, Japan², Department of Internal Medicine, Hyogo College of Medicine, Hyogo, Japan³ Rationale: We have reported the generation of neural stem/progenitor cells (NSPCs) using a reproducible model of cortical infarction in mice. We found that the focal cortical infarction induced secondary damage of the ipsilateral ventroposterior nucleus (VPN) of the thalamus. Objective: In this study, we examined whether neurogenesis would occur even at the region undergoing such secondary degeneration. Methods: All procedures were performed under auspices of an approved protocol from the Animal Care and Use Committee. Focal cerebral ischemia was produced by occluding the left middle cerebral artery (MCA) of adult SCID mice. Animals were perfusion-fixed 14 or 28 days after MCA occlusion, and frozen brain sections were subjected to immunohistochemistry for Nestin, PDGFR-β, MAP2, GFAP and Iba-1. In other groups, total RNA was extracted from the region of VPN, and RT-PCR was carried out to confirm the expression of nestin. Cells separated from same region were incubated in medium promoting formation of neurosphere-like cell.clusters. Results: All post-stroke mice showed secondary neuronal damage selectively at the ipsilateral VPN where loss of MAP2-staining with expression of GFAP-positive astrocytes and Iba-1-positive microglial cells were shown. Nestin-positive cells were obtained from the ipsilateral thalamus, and RT-PCR confirmed its expression. No nestin expression was seen in the contralateral thalamus. Conclusion: These results demonstrate that neural stem/progenitor cells were generated in the secondary degenerated region following cerebral infarction, suggesting that neurogenesis in the region affected indirectly by ischemia is involved in the repair of post-stroke brain.
P2-1-67 軽度の虚血は大脳皮質における神経再生を促進する Mild ischemic insult promotes neurogenesis in the cerebral cortex ○中田雅代^1,2, 百田義弘^1,2, 中込隆之¹, 中野亜希子¹, 河原麻衣子¹小谷順一郎², 松山知弘¹ ○Masayo Nakata^1,2, Yoshihiro Momota^1,2, Takayuki Nakagomi¹, Akiko Nakano¹, Maiko Kawahara¹, Shan Lu¹, Junichiro Kotani², Tomohiro Matsuyama¹ 兵庫医科大学　先端医学研究所　神経再生部門¹, 大阪歯科大学　歯科麻酔学講座² Institute for Advanced Medical Sciences, Hyogo College of Medicine, Hyogo¹, Department of Anesthesiology, Osaka Dental University, Osaka² Introduction: We recently have demonstrated that ischemia-induced neural stem/progenitor cells (iNSPCs) are generated in the post-infarct cerebral cortex (Stem Cells Dev, 2011). We also have shown the presence of nestin-positive cells close to the blood vessels following non-lethal ischemia, suggesting that iNSPCs can be generated by mild ischemic insult. In the present study, we attributed to produce the neurospheres from the cerebral cortex following transient ischemia, which is non-lethal to cortical neurons, by using a highly reproducible murine model of cortical ischemia in the territory of middle cerebral artery (MCA).Materials and methods: Left MCA of male C.B-17-SCID mice was exposed, and transient ischemia for 15 min was produced using a 7.0 nylon monofilament. The ischemic cortex including pia mater was removed 3 days after the ischemia, and dissociated by passage through 23- and 27-gage needles. Cell suspensions obtained were incubated in medium promoting formation of neurosphere-like cell clusters. In another experiment animals were perfusion-fixed 3 days after the ischemia, and frozen brain sections were subjected to immunohistochemistry for nestin, PDGFR-ß, MAP2 and GFAP. Results: Loss of MAP2-staining was not apparent in the ischemic cortex where GFAP-positive astrocytes were proliferated. Nestin-immunoreactivity was expressed both in GFAP-positive astrocytes and PDGFR-ß-positive pericytes, the latter was associated with the endothelial cells. Following incubation in culture medium, a few neurosphere-like cell clusters were obtained, although the number is less than that of clusters obtained from the infarct cortex. Conclusion: These results suggest that neurogenesis is promoted even after non-lethal ischemic insult, though the precise source of the stem cells has to be determined.	P2-1-68 神経細胞の分化における統合失調症リスク因子miR-137の役割 The role of miR-137 in neuronal differentiation of neuroblastoma cells ○梅田知美¹, 橋本亮太^2,3, 山森英長^1,3, 安田由華³, 大井一高³, 藤本美智子³, 伊藤彰¹, 武田雅俊³ ○Satomi Umeda-Yano¹, Ryota Hashimoto^2,3, Hidenaga Yamamori^1,3, Yuka Yasuda³, Kazutaka Ohi³, Michiko Fujimoto³, Akira Ito¹, Masatoshi Takeda³ 大阪大学大学院医学系研究科分子精神神経学（大日本住友製薬）寄附講座¹, 大阪大学大学院連合小児発達学研究科附属子どものこころの分子統御機構研究センター², 大阪大学大学院医学系研究科精神医学教室³ Department of Molecular Neuropsychiatry, Osaka University Graduate School of Medicine, Osaka¹, Molecular Research Center for Children's Mental Development, United Graduate School of Child Development, Osaka², Department of Psychiatry, Osaka University Graduate School of Medicine, Osaka³ MicroRNAs (miRNAs) are a class of small noncoding RNAs that regulate gene expression at the posttranscriptional level. It has been reported that miRNAs have been implicated in several steps of neuronal maturation including dendritic and axonal growth, spine development, and synaptogenesis. A recently completed genome-wide association study (GWAS) showed that single-nucleotide polymorphisms (SNPs) in the MIR137 gene, which encodes one of the brain-enriched miRNAs (miR-137), is highly associated with schizophrenia. In addition, the GWAS showed that not only MIR137 but also putative target genes of miR-137 (CSMD1, C10orf26, CACNA1C and TCF4) have significant associations with schizophrenia. Further, genes which have been reported to be associated with schizophrenia are enriched in predicted miR-137 target genes. In this study, we demonstrate that miR-137 was significantly upregulated during differentiation induced by all-trans-retinoic acid (RA) and brain-derived neurotrophic factor (BDNF) in human neuroblastoma SH-SY5Y cells, suggesting that miR-137 has an important role in regulating neuronal differentiation. Now, we are investigating the role of miR-137 and its target genes during neuronal differentiation.
P2-1-69 単一細胞遺伝子プロファイリングによって明らかになった大脳発生過程における神経前駆細胞の発生時期依存的な変化 Temporal change in cortical progenitor cells as revealed by single cell gene expression profiles ○川口綾乃¹, 岡本麻友美¹, 宮田卓樹¹, 松崎文雄² ○Ayano Kawaguchi¹, Mayumi Okamoto¹, Takaki Miyata¹, Fumio Matsuzaki² 名古屋大学大学院　医学系研究科　細胞生物学分野¹, 理研CDB、非対称細胞分裂研究グループ² Dept of Anatomy and Cell Biology, Grad Sch of Med, Nagoya Univ.¹, Lab for Cell Asymmetry, CDB, RIKEN, Kobe, Japan² During mammalian cerebral development, apical progenitor cells (APs) generate daughter cells which undergo different fate in a defined temporal sequence. To examine how APs change their gene expression during neurogenic period, we generated genome-wide transcriptome profiles of single progenitor cells from mouse cerebrum at different stages, and identified a set of temporal genes in APs and basal progenitors (BPs). APs gradually changed their gene profiles along development, and this change seemed to occur simultaneously in APs within small region. Most of temporal change of gene expression in APs was not inherited to BPs directly. In AP population, cell-to-cell variation of expression levels of Notch signaling related genes was varied during development. Furthermore, profiles of single APs that had been transfected NotchIC with/without cdk inhibitor p18 showed that neither Notch activation nor cell cycle arrest stop temporal change of gene expression in APs. These findings suggest that cell cycle progression does not necessary for progressive restriction in fate potential of APs.	P2-1-70 グルタミントランスポーターslc38a1による胚性腫瘍細胞株P19細胞の神経細胞分化促進 Promotion by the glutamine transporter slc38a1 of neuronal differentiation in pluripotent P19 cells ○國保博史¹, 宝田剛志¹, 福森良¹, 米田幸雄¹ ○Hiroshi Kokubo¹, Takeshi Takarada¹, Ryo Fukumori¹, Yukio Yoneda¹ 金沢大院・薬・薬物学¹ Dept Pharmacal, Univ of Kanazawa, Ishikawa, Japan¹ We have previously demonstrated the functional expression in newborn rat neocortical astrocytes of glutamine transporter (GlnT=slc38a1) believed to predominate in neurons over astroglia in the brain. In order to evaluate the possible role of this transporter in neurogenesis, in this study, we attempted to establish stable transfectants of slc38a1 in mouse embryonal carcinoma P19 cells endowed to proliferate for self-renewal and differentiate into progeny cells such as neurons and astroglia. The full-length coding region of rat slc38a1 was inserted into a vector for gene transfection along with selection by G418, followed by culture with all-trans retinoic acid under floating conditions and subsequent dispersion for spontaneous differentiation under adherent conditions. Stable overexpression of slc38a1 led to marked increases in the size of round spheres formed during the culture for 4 days and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide reduction, with concomitant promotion of subsequent differentiation into cells immunoreactive for a neuronal marker protein. In these stable slc38a1 transfectants before differentiation, drastic upregulation was seen for mRNA expression of several proneural genes with a basic helix-loop-helix domain such as NeuroD1. Taken together, slc38a1 would promote both proliferation and neuronal differentiation through a mechanism relevant to upregulation of particular proneural genes in undifferentiated P19 cells.
P2-1-71 発生過程における腸管神経系のグリア細胞系譜に発現する遺伝子群の同定 Identification of genes expressed in glial cell lineages in developing enteric nervous system ○岩崎光泰^1,2, 榎本秀樹^1,2 ○Mitsuhiro Iwasaki^1,2, Hideki Enomoto^1,2 理研CDB　神経分化・再生¹, 大阪大学　医学系研究科　医学専攻² Lab for NDR, RIKEN CDB, Hyogo¹, Div of Med, Osaka Univ, Osaka² The enteric nervous system (ENS) constitutes the largest division of the autonomic nervous system and controls motility, secretion and blood flow of the gut. Enteric glial cells (EGCs), one of the main cell populations of the ENS, are essential for the control of gastrointestinal functions; studies using genetic ablation in, animal models to destroy EGCs resulted in a fatal haemorrhagic. Although some of the molecules regulating ENS development are known, the mechanisms underlying differentiation of EGCs are still poorly understood. To identify additional molecules critical for enteric glial differentiation, we used microarray and in situ hybridization to compare total gene expression in ENS cells and neuronal lineage cells. We identified 283 genes with at least 1.5-fold higher expression in ENS cells than in neuronal lineage cells. Among those identified, 44 genes had not previously characterized in EGC differentiation and were thus further subjected to in situ hybridization analysis. We confirmed that half of these genes (22 out of 44) were expressed in the ENS at embryonic day 16.5 (E16.5) when a glial marker, GFAP, starts to be expressed. Most genes were expressed in a restricted fashion in a subpopulation of ENS cells. The list of 22 genes contained 8 transcription factors, 6 uncharacterized genes, 3 transmembrane receptor proteins, 3 adhesion molecules and 3 other molecules. This comprehensive profile of ENS gene expression improves our understanding of the mechanisms underlying EGC differentiation and serves as a valuable resource for future developmental and mouse genetic studies.	P2-1-72 Srcシグナリングを介した機械刺激によるES細胞維持機構 Dual inhibition of Src and GSK3 maintains mouse embryonic stem cells, whose differentiation is mechanically regulated by Src signaling ○清水健史^1,2, 上田潤^3,4岩崎勝彦⁵原田一郎⁵, 澤田泰宏² ○Takeshi Shimizu^1,2, Jun Ueda^3,4, Jolene Ho³, Katsuhiko Iwasaki⁵, Lorenz Poellinger³, Ichiro Harada⁵, Yasuhiro Sawada² 生理研、分子神経生理¹, シンガポール大学、メカノ², シンガポール大学、癌³, 大阪大、微研、生体応答⁴, 東工大、生命理工⁵ Natiol Inst Physiol Sci, Div of Neurobiol and Bioinfo¹, Mechanobiol Inst, Univ of Singapore², Cancer Sci Inst, Univ of Singapore³, Cent Genet Anal of Biol Resp, Osaka Univ⁴, Dept Biomol Eng, Tokyo Inst of Tech⁵ Recent studies reveal that the mechanical environment influences the behavior and function of various types of cells, including stem cells. However, signaling pathways involved in the mechanical regulation of stem cell properties remain largely unknown. Using polyacrylamide gels with varying Young's moduli as substrates, we demonstrate that mouse embryonic stem cells (mESCs) are induced to differentiate on substrates with defined elasticity, involving the Src-ShcA-MAP kinase pathway. While the dual inhibition of MAP kinase and GSK3, termed 2i, was reported to sustain the pluripotency of mESCs, we find it to be substrate elasticity-dependent. In contrast, Src inhibition in addition to 2i allows mESCs to retain their pluripotency independent of substrate elasticity. The alternative dual inhibition of Src and GSK3 (alternative 2i) retains the pluripotency and self-renewal of mESCs in vitro and is instrumental in efficiently deriving mESCs from preimplantation mouse embryos. In addition, the transplantation of mESCs, maintained under the alternative 2i condition, to immunodeficient mice leads to the formation of teratomas that differentiates into three germ layers. Furthermore, mESCs established with alternative 2i contributed to chimeric mice production and transmitted to the germline. These results reveal a role for Src-ShcA-MAP kinase signaling in the mechanical regulation of mESC properties and indicate that alternative 2i is a versatile tool for the maintenance of mESCs in serum-free conditions, as well as for the derivation of mESCs.
P2-1-73 神経栄養因子受容体の白血病関連遺伝子MTGR1による発現調節メカニズム MTGR1, myeloid translocation gene related-1, suppresses GFR α 1 expression by downregulation of NeuroM ○本間俊作¹, 島田孝子¹, 八木沼洋行¹ ○Shunsaku Homma¹, Takako Shimada¹, Hiroyuki Yaginuma¹ 福島県立医科大学　神経解剖発生学講座¹ Dept. of Neurobiol. & Anat., Fukushima Med. Univ.¹ Myeloid Translocation Gene Related-1 (MTGR1), a member of the myeloid Translocation Gene (MTG) family of transcriptional corepressors, is transiently expressed in a subset of nascent neurons in the vertebrate spinal cord. We noticed that the expression pattern of MTGR1 was very similar to that of a bHLH transcriptional factor, NeuroM (Math3/ NeuroD4) and that of a specific receptor for glial cell line derived-neurotrophic factor (GDNF family of receptors α 1: GFR α 1), suggesting potential regulatory roles for MTGR1 in the expression of NeuroM and GFR α 1. In ovo electroporation in the chick embryo revealed that the overexpression of NeuroM alone was sufficient to induce ectopic GFR α 1 expression without overt neuronal differentiation, whereas the suppression of NeuroM activity resulted in the specific loss of GFR α 1 expression. Contrary to the inductive role of NeuroM in GFR α 1 expression, MTGR1 overexpression resembled the effect of the suppression of NeuroM activity: the overexpression of MTGR1 repressed GFR α 1 expression. Moreover, NeuroM was also downregulated after the overexpression of MTGR1. Conversely, a fusion protein of MTGR1 with the VP16 activator domain failed to repress NeuroM and GFR α 1 expression. These data indicate that MTGR1 suppresses GFR α 1 expression by its ability to repress NeuroM expression in differentiating neurons. Thus, MTGR1, together with NeuroM, plays important roles in the differentiation of GFR α 1-positive spinal neurons.	P2-1-74 歯髄幹細胞を用いた霊長類の脊髄損傷後の機能回復 Functional recovery following spinal cord injury in primates using dental pulp stem cells ○加納史也^1,2, 山本朗仁¹, 酒井陽¹, 西村幸男², 伊佐正², 上田実¹ ○Fumiya Kano^1,2, Akihito Yamamoto¹, Kiyoshi Sakai¹, Yukio Nishimura², Tadashi Isa², Minoru Ueda¹ 名大院・医・口腔外科¹, 自然科学研究機構生理学研究所² Department of Oral and Maxillofacial Surgery, Nagoya University Graduate School of Medicine, Nagoya¹, Department of Developmental Physiology, National Institute for Physiological Science, Okazaki² Spinal cord injury (SCI) often leads to persistent functional deficits due to severe neuron and glial loss, and to limited axonal regeneration after injury. Currently no effective treatment has been developed. We have reported remarkable neuroregenerative activities of human adult dental pulp stem cells (DPSCs) and stem cells from human exfoliated deciduous teeth (SHED) using rat SCI model. Engrafted into the completely transected rat SC, these tooth-derived stem cells promote marked recovery of hindlimb locomotion through the inhibition of the SCI-induced apoptosis of multiple cell types and the unique regeneration of the transected axons by directly inhibiting multiple axonal growth inhibitors (AGIs), such as chondroitin sulphate proteoglycans (CSPG) and myelin-associated glycoprotein (MAG). However, the therapeutic effects of the engrafted SHED or DPSC for the SCI-recovery in other species, especially in primate, have not been examined. Here we report our study investigating the neuoregenerative activities of tooth-derived stem cells transplanted into the rhesus monkey with spinal hemisection at the cervical level. Monkeys were pre-trained for behavioral test before operation and its neurological recovery was evaluated with Brinkman board task, Tube task, and Slit task. To examine the regeneration of the neural network we labeled the CST by biotinylated dextran amine (BDA). We also examined the neural pathway involved in the functional recovery by electrophysiological experiments. Together with histological data, we will discuss the levels and mechanisms of the functional recovery of the SCI monkeys receiving SHED.
P2-1-75 グリア発生におけるNFIA転写因子の機能解析と下流因子の探索 Analysis of the function and searching for downstream of NFIA transcription factor in gliogenesis ○津山淳¹, 島崎琢也¹, 岡野栄之¹ ○Jun Tsuyama¹, Takuya Shimazaki¹, Hideyuki Okano¹ 慶應大学大学院医学系研究科生理学¹ Department of Physiology, Keio University, Tokyo¹ In the developing CNS, the all types of neural cells are derived from multipotent neural stem cells (NSCs). However, since the differentiation potential of NSCs is spatiotemporally regulated during development, they cannot always generate all the neural cell types. For instance, NSCs in early gestation cannot differentiate into astrocyte because they have no competency to respond gliogenic differentiation signals yet. Thus, neurons come first then glia in developing brain. Despite of extensive analysis of cell fate determination processes of NSCs, the molecular mechanisms of temporal specification of NSCs is poorly understood. Nuclear Factor I-A(NFIA) transcription factor has been shown to be a key regulatory factor for NSCs to start gliogenesis. To confirm the function of NFIA for gliogenesis, we overexpressed NFIA in ES cells(ESCs)-derived neurospheres via lentivirus vectors. Surprisingly, NFIA seemed to promote astrogliogenesis only if its overexpressing cells are localized with a mosaic-like pattern within ESCs-derived neurospheres. Therefore, we established ESCs lines that express NFIA in a doxycyclinedependent manner so-called Tet-off system and co-cultured NSCs derived from the Tet-off ESCs line and wild-type ESCs to form aggregated and mixed (WT/NFIA-mix) neurospheres. NFIAoverexpressing NSCs derived from WT/NFIA-mix spheres showed significant increase of astrocytic differentiation compared to neurospheres composed of only NFIA-overexpressing cells. Interestingly, this action of NFIA was cancelled by γ-secretase inhibitor, suggesting a possible involvement of Notch signaling in this process. Finally, NFIA-overexpressing cells from the WT/NFIA-mix spheres analysed global gene expression patterns by DNA-microarrray. We are currently performing functional screening of candidate genes to reveal the molecular mechanisms underlying NFIA mediated gliogenesis by NSCs in the CNS development.	P2-1-76 TET1が引き出すiPS細胞の神経誘導性 TET1 educes full human pluripotency toward neural fates ○加藤英政¹ ○Hidemasa Kato¹ 埼玉医科大学　ゲノム医学研究センター　発生・分化・再生部門¹ Div Dev Biol, Research Center for Genomic Medicine, Saitama Medical University¹ Mouse ESCs (mESCs) are largely accepted to have genuine self-renewing capacity and pluripotency. In contrast, human ESCs (hESCs) which are derived from the blastocyst but are more akin to the later epiblast-stage embryos show some resistance and biases toward differentiation. Moreover, human iPS cells (hiPSCs) tend to have lower self-renewal capacity when compared to mESCs and even to hESCs. Why all these cell populations differ this much? We here postulated that this could be because hiPSCs have not passed through a blastocyst-stage like other pluripotent stem cells (PSCs). We focused on proteins which expressions are present in blastocysts but not in the ensuing epiblasts. Tet1 was found to be such a protein. Our investigation indicated that Tet1 is involved in harnessing the full differentiation potential of mESCs. Without Tet1, the differentiation of mESCs decelerates which leads to a differentiation resistance. Also in the mouse, Tet1 is only expressed up to the blastocyst stage. As the current method for deriving hiPSCs stands, we believe that hiPSCs would have little chance to ever express TET1, which may explain the differentiation defects of these cells. Therefore, we over-expressed TET1 in hiPSCs and its effects were studied. By comparing wild-type hiPSCs and TET1-overexpressing hiPSCs (TET1-hiPSCs), we found that the self-renewal ability has been improved upon TET1 expression. Next, we tested the pluripotency of the TET1-hiPSCs. When assigned to neuronal differentiation, these cells exhibited markedly higher efficiencies. Judging from the levels of SOX1, we found a 60-times increase in its efficiency when compared to wild-type hiPSC differentiation. We therefore concluded that the presence of TET1 reduces an inherent differentiation resistance of hiPSC, at least for the neural lineage, educing the bona fide pluripotency of these cells.
P2-1-77 Foxg1による大脳皮質上層ニューロンの分化制御機構 Foxg1 directs the specification of upper-layer projection neurons in late neocortical progenitors ○當麻憲一^1,2, 隈元拓馬¹花嶋かりな¹ ○Ken-ichi Toma^1,2, Takuma Kumamoto¹, Bin Chen³, Carina Hanashima¹ 理研CDB 大脳皮質発生研究チーム¹, 神戸大院・理学研・生物・発生生物学² Lab. for. Neocort. Dev., RIKEN CDB, Kobe¹, Dep. of Bio., Grad. Sch. of Sci., Univ. of Kobe, Kobe², MCD biology, UCSC, Santa Cruz, CA³ The mammalian neocortex comprises of diverse glutamatergic neuron populations, which includes the preplate, deep-layer and upper-layer projection neurons. These layer subtypes are generated from common neural progenitor cells in a stereotypical temporal order during development. Previous in vitro and in vivo studies have shown that the specification of deep-layer subtypes during the early corticogenesis period is largely regulated by intrinsic determinants. However, the mechanisms underlying the progression of progenitor cell competence resulting in the switch from deep- to upper-layer projection neuron production has remained largely unknown.Foxg1 is a forkhead transcription factor that plays pleiotropic roles in the proliferation and differentiation of cortical progenitor cells. We previously identified that Foxg1 is a key regulator of temporal competence to switch to the generation of deep-layer projection neurons during the early corticogenesis period. In contrast, the requirement and specific roles of Foxg1 in late cortical progenitor cells have remained elusive. To directly assess the role of Foxg1 in upper-layer neuron development, we utilized a conditional knockout mouse line to inactivate Foxg1 at E14.5, and examined the development of upper-layer projection neurons. These mutant cortices showed that the expression of upper-layer neuron specific genes was lost, and the migration of newly born neurons into the cortical plate was impaired. Using genome-wide transcriptome and chromatin immunoprecipitation assay, we further demonstrate that Foxg1 regulate distinct molecular events within the early and late cortical progenitor cells to control neuronal differentiation. Collectively, these data imply that Foxg1 is a critical coordinator of neurogenesis program to direct layer-specific neuron differentiation throughout mammalian neocortical development.	P2-1-78 不完全にリプログラミングされたヒトiPS細胞は分化誘導に伴うゲノム不安定化を通してグリオーマ様腫瘍を形成する Incompletely Reprogrammed Human iPSCs Form Glioma-like Tumors Through Genomic Instability During Neural Differentiation ○岡田洋平¹, 宮冬樹², 小池正人³, 冨里周太¹, 戸倉智子¹, 石原康晴¹, 下門大祐¹, 服部千夏¹, 兼松大介⁴, 金村米博⁴, 幸田和久¹, 祖父江元⁵, 山中伸弥⁶, 柚崎通介¹, 内山安男³, 池田栄二⁷, 角田達彦², 岡野栄之¹ ○Yohei Okada¹, Fuyuki Miya², Masato Koike³, Shuta Tomisato¹, Tomoko Tokura¹, Yasuharu Ishihara¹, Daisuke Shimojo¹, Chinatsu Hattori¹, Daisuke Kanematsu⁴, Yonehiro Kanemura⁴, Kazuhisa Kohda¹, Gen Sobue⁵⁵, Shinya Yamanaka⁶, Michisuke Yuzaki¹, Yasuo Uchiyama³, Eiji Ikeda⁷, Tatsuhiko Tsunoda², Hideyuki Okano¹ 慶應義塾大・医・生理学¹, 理化学研究所・ゲノム医化学研究センター², 順天堂大院・医・神経機能構造学³, 大阪医療センター・臨床研究センター⁴, 名古屋大院・医・神経内科⁵, 京都大・ iPS細胞研究所⁶, 山口大院・医・病理形態学⁷ Dept Physiol, Keio Univ, Tokyo¹, Center for Genomic Medicine, RIKEN, Yokohama², Dept Cell Biol and Neurosci, Juntendo Univ, Tokyo³, Inst Clinical Research, Osaka National Hospital, Osaka⁴, Dept Neurol, Nagoya Univ, Nagoya⁵, Center for iPS Cell Research and Application (CiRA), Kyoto Univ, Kyoto⁶, Dept Pathol, Yamaguchi Univ, Yamaguchi⁷ Human-induced pluripotent stem cells (hiPSCs) hold great promise for regenerative medicine. Despite the importance in evaluating their quality for future cell therapy of neural disorders, there are no precise analyses for assessing abnormal differentiation and tumorigenicity. Here, we demonstrate that pre-evaluated hiPSC clones, established by retroviruses or integration-free episomal vectors, may exhibit aberrant neural differentiation or glioma-like tumor formation by the transplantation of hiPSC-derived neural stem/progenitor cells (hiPSC-NS/PCs) into NOD/SCID mice. Notably, the tumorigenicities of hiPSC-NS/PCs were closely associated with incomplete reprogramming of hiPSCs, revealed by the expressions of newly identified human embryonic stem cell signature genes or DNA repair genes. This incomplete reprogramming resulted in genomic instability not in an undifferentiated pluripotent state, but during neural differentiation. Moreover, this newly identified cluster of 60 or 17 DNA repair genes, including TP53, FANCI, ATRX, and SUPT16H, may provide a useful "scorecard" for easy and quick evaluation of the incomplete reprogramming and quality of hiPSCs in the undifferentiated state.
P2-1-79 先天性髄鞘形成不全症iPS細胞由来オリゴデンドロサイトを用いた病態解析 Pathological analysis of human iPS derived oligodendrocytes in dysmyelinating neurological disorder ○沼澤佑子^1,2, 岡田洋平^1,5, 芝田晋介¹, 葛巻直子¹, 岸憲幸¹, 赤松和土¹, 天谷雅行³, 小坂仁⁶, 井上健⁷, 高橋和利⁸, 山中伸弥⁸, 小崎健次郎^2,4, 高橋孝雄², 岡野栄之¹ ○Yuko Numasawa^1,2, Yohei Okada^1,5, Shinsuke Shibata¹, Naoko Kuzumaki¹, Noriyuki Kishi¹, Wado Akamatsu¹, Masayuki Amagai³, Hitoshi Osaka⁶, Ken Inoue⁷, Kazutoshi Takahashi⁸, Shinya Yamanaka⁸, Kenjiro Kosaki^2,4^2,4, Takao Takahashi², Hideyuki Okano¹ 慶應義塾大学医学部生理学教室¹, 慶應義塾大学医学部小児科学教室², 慶應義塾大学医学部皮膚科学教室³, 慶應義塾大学医学部臨床遺伝センター⁴, 慶應・咸臨丸プロジェクト⁵, 神奈川こども医療センター神経内科⁶, 国立精神・神経医療研究センター神経研究所⁷, 京都大学iPS細胞研究所⁸ Department of Physiology, Keio University School of Medicine, Tokyo, Japan¹, Department of Pediatrics, Keio University School of Medicine, Tokyo, Japan², Department of Dermatology, Keio University School of Medicine, Tokyo, Japan³, Center for Medical Genetics, Keio University School of Medicine, Tokyo, Japan⁴, Kanrinmaru Project, Keio University School of Medicine, Tokyo, Japan⁵, Kanagawa Childrens Medical Center, Kanagawa, Japan⁶, National Center of Neurology and Psychiatry, Tokyo, Japan⁷, Center for Induced Pluripotent Stem Cell Research, Kyoto University, Kyoto, Japan⁸ Pelizaeus Merzbacher disease (PMD) is an X-linked leukodystrophy caused by mutations of Proteolipid protein 1 (PLP1) gene. Missense mutations of PLP1 gene has been thought to result in the accumulation of mutant PLP1 proteins in rough endoplasmic reticulum(ER). This phenomenon has been shown to induce an unfolded protein response through the transcriptional activations of chaperone genes and genes involved in apoptosis, by the analyses using cell lines transfected with mutant PLP1 genes or mouse models of PMD. However, none of the previous report has shown whether these pathogenic changes could be really observed in the PMD patients' oligodendrocytes. We established patient specific induced pluripotent stem cells (iPSCs) from two PMD patients with missense point mutation, and differentiated them into mature oligodendrocytes. We confirmed mislocalization and accumulation of mutant PLP1 proteins to ER in PMD-iPSC-derived mature oligodendrocytes. In addition, by transmission electron microscope in vitro, the frequency of the myelin formation and the thickness of the myelin sheath were drastically reduced compared to those of control. This is the first report of modeling dysmyelinating neurological disorders by patient-specific iPSCs, which has shown the usefulness of iPSC-derived mature oligodendrocytes for the analysis of pathogenic process of dysmyelinating neurological disorders.

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