一般ポスター

一般ポスターエピジェネティクス・リプログラミング

7月8日（土）　12:50-13:50　ポスター会場① 3P④-1 iSCからの神経幹細胞様細胞の誘導 Induction of neural stem cell-like cells from iSCs 湊　雄介¹, 土居　亜紀子², 大谷　佐知³, 前田　誠司¹, 松山　知弘⁴, 中込　隆之², 八木　秀司¹ 1. 兵庫医科大学　解剖・細胞生物, 2. 兵庫医科大学　先端医研, 3. 兵庫医科大学　薬, 4. 兵庫医科大学　先進脳治療 Yusuke Minato¹, Akiko Doi², Sachi Kuwahara-Otani³, Seishi Maeda¹, Tomohiro Matsuyama⁴, Takayuki Nakagomi², Hideshi Yagi¹ 1. Sch. of Med., Dept. of Anat. and Cell Biol., Hyogo Med. Univ., Hyogo, Japan, 2. Inst. for Adv. Med. Sci., Hyogo Med. Univ., 3. Sch. of Pharm., Hyogo Med. Univ., 4. Dept. of Therap. Prog. in Brain Dis., Hyogo Med. Univ. We have found ischemia-induced multipotent stem cells (iSCs) appeared in the infarct area of the post-stroke brain. Since iSCs can differentiate into various types of neuronal cells including neurons, they have clinical application potential for cerebral infarction. However, iSCs rapidly lose their proliferation and differentiation potential through repeated passaging. In this report, we attempted to overcome these limitations.High density culture of iSCs was able to maintain the growth of iSCs that was stopped usually within one month. In the presence of LDN193189, the expression of neural stem cell markers was induced in iSCs, whereas the expression of pericytic and mesenchymal markers was decreased. In addition, iSCs grown in the presence of LDN193189 differentiated into Map2 and Tau-positive neurons when they were cultured in neural induction medium. Furthermore, these differentiated neurons had an electrophysiological activity. Astrocytes could also be induced from these cells.These results indicate that high density culture and LDN193189 overcome the limitation of iSCs. Further, LDN193189 induces iSCs to become neural stem cell-like cells. We developed an iSC culture method that preserves proliferative and neuronal differentiation potential.		7月8日（土）　12:50-13:50　ポスター会場① 3P④-2 筋萎縮性側索硬化症患者由来のiPS細胞を用いた運動神経細胞への分化 Disease modeling using iPS cells derived motor neuron from Amyotrophic lateral sclerosis patient. 山崎　晃, 清水　由梨香, 伊藤　達男川崎医科大学　衛生学 Akira Yamasaki, Yurika Shimizu, Tatsuo Ito Department of Hygiene, Kawasaki Medical School, Kurashiki, Japan Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by selective and progressive degeneration and loss of upper and lower motor neurons. The pathological mechanism is unknown, and some familial ALS cases have mutations in genes encoding RNA-binding proteins. Although motor neurons from ALS patients show the abnormal formation of stress granules, the RNA involved in the stress granules is unknown.In the present study, we tried to differentiate motor neurons from ALS-derived iPS cells to investigate the long non-coding RNAs whose expression is altered in ALS. The iPS cells were maintained feeder-free using iMatrix 511. Next, differentiation into neural progenitor cells was induced by activating the Wnt/RA pathway involved in anterior-posterior patterning with CHIR99021 and Retinoic acid and simultaneously activating the Shh pathway involved in dorsal-ventral patterning with purmophamine. The cells were then cultured in a neuronal medium and differentiated, matured, and maintained into motor neurons. To assess differentiation efficiency, we constructed a fluorescent reporter strain of olig2, a marker of motor neuronal progenitor cells. As a result, motor neurons were obtained from iPS cells derived from both healthy ALS patients. We are currently investigating RNAs that are altered in their expression in the motor neurons of ALS patients by RNAseq.

7月8日（土）　12:50-13:50　ポスター会場① 3P④-3 Development of cell-based regenerative medicine in neurological diseases and spinal cord injury 池野　正史, 笹倉　寛之, 武内　恒成愛知医科大学　医　細胞生物 Masashi Ikeno, Hiroyuki Sasakura, Kosei Takeuchi Dept. of Medical Cell Biol., Sch. of Med., Aichi Medical University, Aichi, Japan Stem/progenitor cells are useful in regenerative medicine and cell therapy. Spinal cord injury (SCI) is a common neurological disease that results in loss of sensory function and mobility. Regenerative medicine and cell therapy are well studied for a potential therapeutic strategy for treatment of SCI. However, recovery mechanism from SCI and tumorigenicity of human induced pluripotent stem cell derived-stem/progenitor cells is in the process of research and development. Separately, application of readily available hMSC or stem cells from human exfoliated deciduous teeth (SHED) is making progress for regenerative medicine and cell therapy. Limited life span and donor-dependent variation of hMSC and SHED present hurdles to reproducible experiments and applications. Therefore, our major aim is establishment of primary cell lines that retain properties for cell therapy. Limited replication capacity of primary cells was overcome by expression of hTERT. We demonstrated that transfection of hTERT was sufficient to extend the life span of hMSC and SHED without significantly perturbing their phenotype or biological behavior. The availability of functionally identified cells will be helpful for development of regenerative medicine and cell therapy. To validate recovery from SCI, we are now undergoing transplantation experiment to mouse SCI model using hTERT-expanded hMSC or SHED.		7月8日（土）　12:50-13:50　ポスター会場① 3P④-4 ヒトiPS細胞由来ミクログリア前駆細胞と大脳皮質神経または脳オルガノイドの共培養系におけるアミロイドβストレス負荷の解析 Amyloid-beta stress in a co-culture of human iPS cell-derived cortical neurons and organoids with microglial progenitors 原田　考輝¹, 西村　周泰^1,2, 岩崎　良太¹, 安藤　ももな¹, 山田　志歩¹, 高田　和幸¹ 1. 京都薬科大学　統合薬科学系, 2. 同志社大学大学院　脳科学研究科 Koki Harada¹, Kaneyasu Nishimura^1,2, Ryota Iwasaki¹, Momona Ando¹, Shiho Yamada¹, Kazuyuki Takata¹ 1. Div. Integ. Pharm. Sci., Kyoto Pharm. Univ., Kyoto, Japan, 2. Grad. Sch. Brain Sci., Doshisha Univ., Kyoto, Japan Alzheimer's disease (AD) induces amyloid-β (Aβ) accumulation and neurodegeneration in the brain; Aβ plaques are surrounded by brain macrophages called microglia, and a unique microglial subpopulation, disease-associated microglia (DAM), appears in the AD brain. Cell culture systems that can precisely reproduce the pathological microenvironment of the AD brain may contribute to elucidating the roles of microglia including DAM in AD. In this study, we established a co-culture system of cortical neurons or organoids with microglial progenitor cells (hiMacs), all derived from human induced pluripotent stem (hiPS) cells, and analyzed their response to Aβ stress. 26-O-acyl isoAβ, highly cytotoxic due to stable oligomer formation, was treated as Aβ stress. Although Aβ induced marked neurodegeneration in cortical neurons and organoids, adding hiMacs suppressed it. Aβ cytotoxicity was not detected in hiMacs, rather than hiMacs phagocytosed Aβ and showed DAM-like transitions. Furthermore, in cortical organoids, jagged Aβ plaques were formed with hot spot-like structures but are more round and smoother surfaces near hiMacs. Thus, hiMacs showed neuroprotective effects might be phagocytosis of Aβ and morphological modification of Aβ plaques. The co-culture model of cortical neurons and organoids with hiMacs is helpful for elucidating microglial function in AD pathology.

7月8日（土）　12:50-13:50　ポスター会場① 3P④-5 プラダー・ウィリー症候群患者由来iPS細胞と視床下部オルガノイドのエピゲノム編集 Epigenome editing of Prader-Willi syndrome patient-derived induced pluripotent stem cells and hypothalamic organoids 根本　晶沙, 今泉　研人, 奥野　博庸, 岡野　栄之慶應義塾大学医学部 Akisa Nemoto, Kent Imaizumi, Hironobu Okuno, Hideyuki Okano Keio University School of Medicine Prader-Willi Syndrome (PWS) is a genomic imprinting disorder caused by the loss of function of paternally expressed genes in the chromosome 15q11-13 region, and is closely related to hypothalamic dysfunction. However, little is known about the pathomechanism of PWS and effective treatment has not yet been developed. PWS patients lack paternal expression of 15q11-13 locus but possess an epigenetically silent set of these genes on the maternal allele. Thus, the activation of this silent maternal allele can serve as a therapeutic target for PWS. Here, we controlled the epigenetic DNA methylation status of the silent maternal genes in PWS patient-derived induced pluripotent stem cells (iPSCs) by CRISPR/dCas9-TET1-mediated epigenome editing. These epigenome-edited iPSCs restored the expression of the imprinting genes even after repeated passages. We then differentiate these epigenome-edited PWS-iPSCs into hypothalamic organoids and confirmed the retained activation of the imprinting gene expression. Furthermore, RNA-seq analysis suggested that the transcriptomic change in PWS was partially rescued by this epigenome editing. This study highlights the therapeutic potential of epigenome editing for treating PWS.