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P3-1-61 マウスES細胞由来ニューロン／神経前駆細胞に対する細胞外直流電流負荷の作用 Effects of extracellular direct current electrical stimulation on neuron / neural progenitor cells derived from mouse ES cells ○小林麻美1, 川端佑果1, 蜷川菜々1, 鳥橋茂子1 ○Mami Kobayashi1, Yuka Kawabata1, Nana Ninagawa1, Shigeko Torihashi1 名古屋大学大学院医学系研究科リハビリテーション療法学専攻1 Department of Health Sciences, Nagoya University Graduate School of Medicine,Nagoya1 In the development of the embryonic nervous system and the regeneration of adult brain following injury, neural cells and their progenitors proliferate and migrate toward sites where they differentiate into specific neuron types and/or glia. Any guidance cue besides growth factors is necessary to lead neural cells to the differentiating specific sites to make and synaptic contacts. Previous studies have indicated that a direct current electrical field (DC EF) occurs in frog and chick embryos during neurogenesis. Disruption of the DC EF causes failure in the formation of the intact nervous system. Therefore, DC EF is essential for neurogenesis. DC EF also guides neuronal stem/progenitor cell migration and induces outgrowth of neuritis in vitro. Therefore, in cell transplantation for the recovery of the nervous system from injury, application of DC EF seems to be useful. In the present study, we examined the effect of DC EF on neuron/ neural progenitor cells derived from ES cells in vitro to evaluate clinical usage. Undifferentiated ES cells were induced to differentiate into neuronal cells by retinoic acid. After that PSA-NCAM+cells were collected by Magnetic Cell Sorting and were plated on poly-L-lysine/laminin coated dishes.After 1day or 3days incubation, 0.2mA of DC EF orpulse DC was applied for 3 hours a day.Immunohistochemistry and RT-PCR were used todetect Islet1 or Hb9 expression. We also analyzedintracellular Ca2+ influx using fluo-4. As a result,The DC EF slightly increased Islet1-possitive cells,which is a maker for motor neurons, comparing to the control (no-EF). It indicates that DC EF on neural progenitor cells promotes differentiation into motor neurons. RT-PCR dates showed DC EFenhanced both HB9 and Islet1 expressions in DC EF groups. Both two expressions in DC EF for 2days were higher than those for 4days. Intracellular Ca2+levels increased after electrical stimulation. Further studies are necessary to clarify the mechanism under DC EF.	P3-1-62 Withdrawn
P3-1-63 リーリン強制発現による異所性灰白質のマウスモデル Mouse model of subcortical heterotopia caused by ectopic expression of Reelin ○石井一裕1, 久保健一郎1, 仲嶋一範1 ○Kazuhiro Ishii1, Ken-ichiro Kubo1, Kazunori Nakajima1 慶應義塾大学医学部　解剖学1 Dep. of Anatomy, Keio Univ. Sch. of Med., Tokyo, Japan1 Cortical malformation is associated with several pathological conditions in human, including developmental disorder, mental retardation and epilepsy. Most of these cortical malformations result from impairment of neuronal migration and laminar formation in the developing cortex. The Reelin is one of the most well-known mechanisms involved in the laminar formation and neuronal migration in the developing cortex. Reelin, an extracellular matrix protein, binds to cell-surface receptors, ApoER2 and VLDLR, and induces tyrosine phosphorylation of Dab1. In the reelin-deficient mutant mouse, reeler, the cortical lamination is largely inverted. Therefore, Reelin is required for correct neuronal lamination during brain development. We recently reported that ectopically expressed Reelin induces neuronal aggregation in the developing mouse cortex, which mimics the marginal zone and cortical plate in vivo. However, it remains unclear that the neuronal connection between the aggregate and other cortical areas and whether any abnormal behavior is exhibited by the postnatal mouse with ectopic aggregates. In this study, we investigated the effects of the ectopic Reelin expression in postnatal stages. We found that the subcortical heterotopia persisted at least until postnatal day 28. The heterotopias mainly consisted of excitatory cortical neurons that expressed superficial layer markers. Moreover, the efferent and afferent projections of heterotopia were investigated. Collectively, the present study provides a new model of subcortical heterotopia produced by in utero gene transfer into the developing mouse cortex. Further analysis of our newly generated mouse model would be helpful to elucidate the pathophysiology of cortical developmental disorders.

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