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一般演題(ポスター)
iPS cells
3P-47
Methylcobalamin Protects Motor Neuron Loss in Mutated Human SOD1 and ES Cell Mediated in vitro ALS Model
Ito Shunsuke1,Izumi Yukina1,Niidome Tetsuhiro2,3,Ono Yuichi1
1Regenerat. Med. KAN Res. Inst. Inc.,2Concept Creation Strategy, KAN Res. Inst. Inc.,3KAN PCU, Eisai Co., Ltd.

Amyotrophic lateral sclerosis(ALS)is a neurodegenerative disease characterized by loss of upper and lower motor neurons and progressive decline of muscle function resulting in death caused by respiratory defects. The approved drug for the ALS treatment is only riluzole but its effect on ALS is slight and limited. Therefore, more effective ALS drugs are in great expectations. Most ALS cases are sporadic, which are thought to be caused by multiple factors like environmental stress, life style habits, genetic and epigenetic failures, but 5-10% are familial cases. About 20% of familial cases link with mutations in the SOD1 gene. Causal link of the mutation was proven by the transgenic mice expressing ALS-linked mutant of human SOD1(G93A)that die around age 140-150 days with drastic motor dysfunction like ALS. It has been also known that the astrocytes expressing mutant human SOD1(G93A)have killing activity to motor neurons in in vitro co-culture. Here we established the mutant human SOD1-mediated in vitro ALS model using pluripotent stem cell technologies and examined that the effect of methylcobalamin(MBL)treatment in this model. MBL is an activated form of vitamin B12 known as an effective drug for the wide variety of neuronal diseases like palsy and diabetic neuropathy through regulating Akt/mTOR signaling pathway. In the present study, MBL treatment significantly prevented the motor neuron loss induced by mutant human SOD1-expressing astrocytes in a concentration dependent manner in an effective range at 10-100nmol/L. This result suggests that the MBL prevents ALS-like death in the motor neurons and will be a new choice for the treatment of ALS.
3P-48
Induced pluripotent stem cells-derived neurons of the patients with discordant schizophrenia
Toritsuka Michihiro1,Makinodan Manabu1,Yoshino Hiroki1,Hamano-Iwasa Kaori1,Yamamuro Kazuhiko1,Akamatsu Wado2,Okada Yohei2,4,Kimoto Sohei1,Ikawa Daisuke1,Hashimoto Kazumichi1,Fukami Shin-ichi1,Yamashita Yasunori1,Imamura Akira3,Nishihara Koji3,Ozawa Hiroki3,Okazaki Yuji3,Okano Hideyuki2,Kishimoto Toshifumi1
1Department of Psychiatry, Nara Medical University,2Department of Physiology, Keio University School of Medicine,3Department of Neuropsychiatry, Unit of Translation Medicine Nagasaki University Graduate School of Biomedical Sciences,4Department of Neurology, School of Medicine, Aichi Medical University

Schizophrenia is one of the major psychiatric disorders with the onset between late adolescence and early adulthood. Despite its high lifetime prevalence, about 1%, its pathobiology is still unclear. While many kinds of comprehensive researches had revealed a number of aspects of this disorder, there still existed some difficulties because of the methodological limitations such as inaccessibility of the live brain. However, a technology of induced pluripotent stem cells(iPSCs)allows us to investigate living brain cells from patients with neuropsychiatric disorders. Here, we present our studies using iPSCs-deprived neurons of discordant schizophrenia and the healthy controls. Human iPSCs were established via electroporation of episomal plasmid vectors into dermal fibroblasts obtained from skins of 3 subjects;schizophrenia patient, his monozygotic twin sibling without schizophrenia, and the healthy control subject. There was no difference between the subjects in the expression pattern of pluripotent markers of established iPS clones. Neurons were differentiated from iPSCs via neurosphere formation in suspension culture, and subsequent adherent culture on coverslips. There were also no differences in the induction property and the proliferation rate of neural stem cells. Most neurons were considered glutamatergic pyramidal neurons with PAX6 and vGlut1 expression. All of these neurons showed electrophysiologically mature features such as multiple action potentials and spontaneous post synaptic currents recorded with a patch-clump method after 3 months culture in vitro.
3P-49
Human iPS cells-derived neuron ReproNeuro for electrophysiological assay
Inamura Mitsuru1,Kogami Harumi1,Okuda Yuichi1,Yoshida Syunsuke1,Akahira Rina1,Watanabe Tomoaki1,Yamazaki Hiroyuki2,Shirao Tomoaki2
1ReproCELL,2Gunma University Graduate School of Medicine Department of Neurobiology and Behavior All Right Reserved

Human induced pluripotent stem(iPS)cells can proliferate infinitely and differentiate into most cell types in the human body. In addition, production of human iPS cells can be scaled to support large-scale experiments, and human iPS cells are a native cellular source similar to primary cell cultures. And human iPS cells are good source to make primary cell types from them. These features of human iPS cells, and differentiated cells derived from them, are attractive for evaluating pipeline compounds and elucidating pathological conditions in a variety for therapeutic areas.We have developed a comprehensive workflow inclusive of patient specific primary somatic cell isolation, cellular reprogramming, and genetic modification with directed differentiation to the neural lineage. These derived neurons carry Alzheimer’s disease specific mutations. In addition, we have created developed patient-specific neurons that specifically carry the PS1 gene mutation, by using gene-recombination technology starting with healthy cells. By regulating the differentiation conditions for these neurons, the proportion of neuronal subtypes can be controlled, and the resulting neurons can be analyzed functionally and phenotypically such as MEA assays, Ca imaging, and ELISA.ReproNeuroTM has a mixed population of neurons, including dopaminergic, glutamatergic, cholinergic, and GABAergic neurons. ReproNeuroTM is available for Patch clump, Ca imaging, and toxicity assay such as LDH assay. ReproNeuro MQTM is designed for MEA analysis, and these neurons show higher-frequency spikes and better sensitivity to antagonists of glutamate receptors such as AP5 or CNQX.This comprehensive workflow capability enables us to generate customized disease models that target specific neurological disease requirements.
3P-50
Astrocyte-secreted factors promote neurite length of human iPSCs-derived neurons in early developmental stage
Arayama Yuki,Yamazaki Hiroyuki,Ishizuka Yuta,Shirao Tomoaki
Department of Neurobiology and Behavior, Gunma University Graduate School of Medicine

Human induced pluripotent stem cells(hiPSCs)have an enormous potential for biomedical research and clinical applications. Differentiated neurons from hiPSCs are expected to be good tools for developing new methods of treatments for various neurological diseases. However, the detailed processes of neuronal development from hiPSCs have not been shown. In this study, we cultured iCell neuron(Cellular Dynamics International), Repro Glu and Repro Neuro(ReproCELL)under various conditions. We found that neurite outgrowth of hiPSCs-derived neurons is promoted by using culture medium conditioned by astrocytes at 2 days in vitro. In contrast, the number of neurites was not altered by using the medium. These results indicate that astrocytes secrete some factors promotes neurite outgrowth. Therefore, we focused on TSP-1(thrombospondin-1), which is extracellular-matrix glycoprotein secreted by astrocytes. Previous studies showed that TSP-1 promotes synaptogenesis in cultured neurons. We are planning to examine whether TSP-1 promotes neurite outgrowth as well as synaptogenesis.
3P-51
Induction and characterization of synaptic transmission induced synchronized population bursts of the induced pluripotent stem cell-derived neurons
Miyamoto Norimasa,Sawada Kohei
Biopharmaceutical Core Function Unit

Many drugs have been reported to cause seizures. It has been reported that the causes of drug-induced seizures are GABAA antagonism, GABAB agonism, adenosine antagonism, and enhanced excitation through NMDA in the neurons. So far, there is no good in vitro assay system for predicting drug-induced unexpected seizure-risks. Spontaneous neuron activity recordings by multi-electrode array(MEA)system from networks of cultured neurons could be a good risk evaluation system for such drug-induced seizure events[1]. It was reported that long-term electrophysiological activity and pharmacological response of human induced pluripotent stem cell(hiPSC)-derived neurons were accelerated by co-culture with rat astrocytes[2]. In this study, we observed time course generation of population burst spikes from iCell neurons with conditioned medium of mouse primary astrocytes by MEA system. Humoral factor(s)from mouse primary astrocytes was sufficient to generate synchronized population burst spikes in the iPSC-derived neurons. GABA antagonism enhanced the periodic synchronized burst spikes in a dose-dependent manner. P/Q-type and N-type calcium channel blockers eliminated the periodic synchronized burst spikes, suggesting that the burst spikes are mediated by synaptic transmission. We concluded that the observed astrocyte-induced population bursts by MEA system are mediated by synaptic transmission and the periodic synchronized population burst signals could be a good prediction marker of GABAA antagonism.
3P-52
Relevance between the expression of nur family genes and the neurite outgrowth through the histone modification
Yamazoe Ryosuke1,2,Nisihata Yosiki1,2,Tsumura Kazaho1,2,Shimayama Erika1,2,Tomioka Takuma1,2,Maruoka Hiroki2,3,Shimoke Koji1,2
1Grad. Sch. of Sci. and Tech., Kansai Univ,2Dept.Life Sci. and Biotech., Fac. Chem. Mat. Bioengin., Kansai Univ,3Tech. Res. Lab., KURABO

Nerve cells are specialized to transduce an electric signal via the axon, thus it is necessary to construct the neuronal network by the elongation of neurites.
 In this process, specific genes are expressed during neurite formation. Identification and functional analyses of these genes are important in developing a new strategy for regenerative therapy.
 We have previously analyzed that forskolin(FSK), an intracellular cAMP producer, or valproic acid(VPA), a histone deacetylase inhibitor, both are involved in neurite outgrowth. As a result, we have revealed that Nur77 protein induces neurites in PC12 cells. In addition, we have demonstrated that both FSK and VPA induce other genes, which belongs to the Nur nuclear receptor family, along with the nur77 gene, within 4 hours in PC12 cells. FSK was induced nurr1 gene and nor1 gene while VPA alone was induced nurr1 gene only.
In present study, we investigated which genes belonging to the nur family are important in elongation of the neurites in the presence of FSK or VPA. Knock-down experiments showed that siRNA against each of nur family mRNA suppressed neurite outgrowth in response to treatment with FSK or VPA, suggesting that nur77 gene and nurr1 gene are essential for neurite outgrowth in the presence of FSK or VPA. We also found that epigenetic regulation via histone H3 modification was important for the FSK- or VPA-induced neurite outgrowth. These results show that up-regulation of nur77 gene and nurr1 gene are involved in neurite outgrowth induced by FSA or VPA through acetylation of histone H3 at the lysine residue, and suggest that different mechanisms are also involved the nur77 family genes during neurite outgrowth.