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PET imaging of NG2 cells in the adult rat brain
Abstracts / Neuroscience Research 68S (2010) e335–e446
P3-d10 Effects of methylmercury on the differentiation of monkey ES cell-derived neural stem cells Masayoshi Shibata 1 , Nobue Kume 1 , Masahiro Otsu 2 , Takuya Yoshie 1 , Risa Ueda 1 , Hiroyuki Omori 1 , Takashi Nakayama 3 , Yutaka Suzuki 4 , Yasushi Kondo 4 , Nobuo Inoue 1 1
Department of Frontier Health Sci., Tokyo Metropolitan University Grad. School of Human Health Sci., Tokyo 2 Department of Chem., Kyorin University School of Med., Tokyo 3 Department of Biochem, Yokohama City Univ School of Med., Yokohama 4 Regene Med, Adv. Med. Res. Lab., Mitsubishi Tanabe Pharma Co Methylmercury (MeHg), a cause of Minamata disease, has been reported to act as a neurotoxic agent during brain development and to produce fetal Minamata disease, which is characterized by brain malformation and malfunction. We have reported that proliferation of neural stem cells (NSCs), prepared from mouse and monkey embryonic stem (ES) cells by the Neural Stem Sphere (NSS) method, is dose-dependently inhibited by MeHg. In this study, effects of MeHg on differentiation of monkey NSCs were investigated. When the NSCs were induced to differentiate into neurons in the presence of MeHg (from 100 nM to 3 M), the cell number was dose-dependently decreased. In addition, the cells differentiating into neurons and the neurons differentiated from the NSCs were demonstrated to be more susceptive to MeHg than the proliferating NSCs. The effects of MeHg on the cells were supported by gene expression analysis using RT-PCR and immunofluorescent analysis. These results suggest that MeHg inhibits both proliferation and neuronal differentiation of the NSCs and that it consequently causes neurotoxic effects in the development of central nervous system. doi:10.1016/j.neures.2010.07.1590
P3-d11 Significance of remyelination in the functional recovery after transplantation of NSPCs to SCI Akimasa Yasuda 1,2 , Osahiko Tsuji 1 , Kanehiro Fujiyoshi 1 , Shinsuke Shibata 2 , Yuichiro Takahashi 1,2 , Satoshi Nori 1,2 , Yoshiomi Kobayashi 1,2 , Yoshiaki Toyama 1 , Masaya Nakamura 1 , Hideyuki Okano 2 1
Department Orthopaedic Surgery, Keio University School of Medicine 2 Keio University School of Medicine, Department of pysiology
Purpose: Previous studies demonstrated that delayed transplantation of neural stem/progenitor cells (NS/PCs) into the injured spinal cord promoted functional recovery in rodents and non-human primates. However the mechanism of functional recovery has remained unclear. There are several possible explanations for this functional improvement as follows: (1) synapse formation by graft-derived neurons, (2) re-myelination by graftderived oligodendrocytes, (3) trophic effects. In the present study we focused on re-myelination by grafted NS/PCs-derived oligodendroctyes to elucidate the mechanism of functional recovery after NS/PCs transplantation. Methods: NS/PCs were obtained from striata of E14.5 myelin-deficient shiverer mice embryo (shi-NS/PCs) and wild-type mice (wt-NS/PCs), and in vitro differentiation assay was performed. shi-NSPC or wild-type mouse derived NS/PCs (wt-NS/PCs) were grafted into the injured spinal cord of wild-type mice 9 days after injury and motor function was evaluated by BBB and BMS scoring scales for 6 weeks. Immunohistological analysis was also performed. Results: shi-NS/PCs differentiated into Tuj1+ neurons, GFAP+ astrocytes, and CNPase+ oligodendrocytes, but not into MBP+ matured oligodendrocytes in vitro. Consistently, shi-NS/PCs differentiated into Hu+ neurons, GFAP+ astrocytes, and APC+ oligodendrocytes but not into MBP+ matured oligodendrocytes in vivo. Transplantation of shi-NS/PCs was not able to promote locomotor function significantly though whereas wt-NS/PCs showed significant promotion in locomotor functional recovery in vivo. Conclusion: Re-myelination is likely to be involved in the locomotor functional recovery after NS/PCs-transplantation to SCI. doi:10.1016/j.neures.2010.07.1591
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P3-d12 PET imaging of NG2 cells in the adult rat brain Yasuhisa Tamura 1 , Akiko Tachibana 2 , Kaori Okuyama 1 , Emi Hayashinaka 3 , Yasuhiro Wada 3 , Kazuhiro Takahashi 2 , Yosky Kataoka 1 1 Cellular Function Imaging Lab., RIKEN Center for Molecular Imaging Science, Kobe 2 Molecular Imaging Integration Unit, RIKEN Center for Molecular Imaging Science, Kobe 3 Molecular Probe Dynamics Lab., RIKEN Center for Molecular Imaging Science, Kobe
NG2 cells are ubiquitously distributed throughout the gray and white matter in the brain. NG2 cells undergo cell division and can generate oligodendrocytes and astrocytes as well as neurons even in the adult brain. It is known that NG2 cells become rapidly activated with their morphological changes including hypertrophy of the cell body and processes, and proliferate in response to several brain insults including traumatic injury, excitotoxic lesions and viral infections. Thus, establishment of in vivo imaging technique of NG2 cells could help us evaluate the extent of brain injury and repair processes. In this study, we generated a transgenic rat strain which preferentially overexpressed human estrogen receptor␣ ligand binding domain (hERL) in the NG2 cells (NG2-hERL Tg rat), and performed PET imaging of the cells in the brain using 16␣-[18 F]fluoro-17-estradiol (FES), a PET tracer for hERL. doi:10.1016/j.neures.2010.07.1592
P3-d13 Analysis of the mechanism that defines the stagedependent function of neocortical progenitor cells Mayumi Okamoto 1 , Takaki Miyata 1 , Fumio Matsuzaki 2 , Ayano Kawaguchi 1 1 Department of Anatomy and Cell Biol., Nagoya University Grad. School of Med 2 Lab. for Cell Asymmetry, CDB, RIKEN
In mammalian cerebral cortex, neural progenitor cells change their cytogenetic behavior dynamically as development proceeds: While progenitors in early embryonic days mainly generate two progenitor cells in a symmetric manner, those in the middle stage give rise to two different types of daughters in an asymmetric manner (i.e., a stem-like daughter cell and a daughter cell to soon proceed to differentiation). To understand how this stage-dependent cytogenesis is achieved, we sought to identify genes that are expressed by cortical progenitor cells in a temporally regulated manner. We first compared the gene expression profiles of progenitor cells isolated from mouse cortex at E11, E14 and E16 using the single-cell cDNA amplification method followed by microarray analysis. Single-cell gene expression profiles could classify the neocortical progenitor cells into the apical (stem-like) progenitors and the basal (non-stem-like) progenitors at all developmental stages examined. Next, the unsupervised principal component analysis (PCA) revealed that the expression pattern of E11 apical progenitors is distinct from that of E14 apical progenitors (e.g. the expression level of Notch signaling related genes was different), suggesting that apical progenitors may change their property between E11 and E14. We identified several genes whose expression increases or decreases during this E11-E14 period. To ask whether these genes are involved in the stage-dependent change of cytogenesis pattern (from symmetric or purely proliferative to asymmetric or more differentiating), we are now doing “heterochronic” in utero erectroporation experiments. For example, a gene that is normally expressed strongly by E14 apical progenitors but not by E11 apical progenitors is artificially expressed in the cortical wall at E10-E11. doi:10.1016/j.neures.2010.07.1593
P3-d14 Developmental Analysis of spinal V0 neurons in zebrafish Chie Satou , Yukiko Kimura, Shinichi Higashijima Okazaki Institute for Integrative Bioscience Neuronal identity in the spinal cord is primarily determined by the expression of distinct classes of transcriptional factors to divide the progenitor cells into discrete dorsoventral domains. Progenitor cells of different domains generate different classes of neurons. Furthermore, recent studies showed that progenitor cells within a domain also produce multiple types of neurons. For example, p0 progenitor cells, which are defined by the expression of dbx1, are known to generate commissural V0 neurons but both excitatory and inhibitory neurons. However, the mechanisms how multiple types of neurons are generated from one domain are not fully understood. Toward understanding the mechanism, we performed a developmental analysis of
P3-d10 Effects of methylmercury on the differentiation of monkey ES cell-derived neural stem cells Masayoshi Shibata 1 , Nobue Kume 1 , Masahiro Otsu 2 , Takuya Yoshie 1 , Risa Ueda 1 , Hiroyuki Omori 1 , Takashi Nakayama 3 , Yutaka Suzuki 4 , Yasushi Kondo 4 , Nobuo Inoue 1 1
Department of Frontier Health Sci., Tokyo Metropolitan University Grad. School of Human Health Sci., Tokyo 2 Department of Chem., Kyorin University School of Med., Tokyo 3 Department of Biochem, Yokohama City Univ School of Med., Yokohama 4 Regene Med, Adv. Med. Res. Lab., Mitsubishi Tanabe Pharma Co Methylmercury (MeHg), a cause of Minamata disease, has been reported to act as a neurotoxic agent during brain development and to produce fetal Minamata disease, which is characterized by brain malformation and malfunction. We have reported that proliferation of neural stem cells (NSCs), prepared from mouse and monkey embryonic stem (ES) cells by the Neural Stem Sphere (NSS) method, is dose-dependently inhibited by MeHg. In this study, effects of MeHg on differentiation of monkey NSCs were investigated. When the NSCs were induced to differentiate into neurons in the presence of MeHg (from 100 nM to 3 M), the cell number was dose-dependently decreased. In addition, the cells differentiating into neurons and the neurons differentiated from the NSCs were demonstrated to be more susceptive to MeHg than the proliferating NSCs. The effects of MeHg on the cells were supported by gene expression analysis using RT-PCR and immunofluorescent analysis. These results suggest that MeHg inhibits both proliferation and neuronal differentiation of the NSCs and that it consequently causes neurotoxic effects in the development of central nervous system. doi:10.1016/j.neures.2010.07.1590
P3-d11 Significance of remyelination in the functional recovery after transplantation of NSPCs to SCI Akimasa Yasuda 1,2 , Osahiko Tsuji 1 , Kanehiro Fujiyoshi 1 , Shinsuke Shibata 2 , Yuichiro Takahashi 1,2 , Satoshi Nori 1,2 , Yoshiomi Kobayashi 1,2 , Yoshiaki Toyama 1 , Masaya Nakamura 1 , Hideyuki Okano 2 1
Department Orthopaedic Surgery, Keio University School of Medicine 2 Keio University School of Medicine, Department of pysiology
Purpose: Previous studies demonstrated that delayed transplantation of neural stem/progenitor cells (NS/PCs) into the injured spinal cord promoted functional recovery in rodents and non-human primates. However the mechanism of functional recovery has remained unclear. There are several possible explanations for this functional improvement as follows: (1) synapse formation by graft-derived neurons, (2) re-myelination by graftderived oligodendrocytes, (3) trophic effects. In the present study we focused on re-myelination by grafted NS/PCs-derived oligodendroctyes to elucidate the mechanism of functional recovery after NS/PCs transplantation. Methods: NS/PCs were obtained from striata of E14.5 myelin-deficient shiverer mice embryo (shi-NS/PCs) and wild-type mice (wt-NS/PCs), and in vitro differentiation assay was performed. shi-NSPC or wild-type mouse derived NS/PCs (wt-NS/PCs) were grafted into the injured spinal cord of wild-type mice 9 days after injury and motor function was evaluated by BBB and BMS scoring scales for 6 weeks. Immunohistological analysis was also performed. Results: shi-NS/PCs differentiated into Tuj1+ neurons, GFAP+ astrocytes, and CNPase+ oligodendrocytes, but not into MBP+ matured oligodendrocytes in vitro. Consistently, shi-NS/PCs differentiated into Hu+ neurons, GFAP+ astrocytes, and APC+ oligodendrocytes but not into MBP+ matured oligodendrocytes in vivo. Transplantation of shi-NS/PCs was not able to promote locomotor function significantly though whereas wt-NS/PCs showed significant promotion in locomotor functional recovery in vivo. Conclusion: Re-myelination is likely to be involved in the locomotor functional recovery after NS/PCs-transplantation to SCI. doi:10.1016/j.neures.2010.07.1591
e359
P3-d12 PET imaging of NG2 cells in the adult rat brain Yasuhisa Tamura 1 , Akiko Tachibana 2 , Kaori Okuyama 1 , Emi Hayashinaka 3 , Yasuhiro Wada 3 , Kazuhiro Takahashi 2 , Yosky Kataoka 1 1 Cellular Function Imaging Lab., RIKEN Center for Molecular Imaging Science, Kobe 2 Molecular Imaging Integration Unit, RIKEN Center for Molecular Imaging Science, Kobe 3 Molecular Probe Dynamics Lab., RIKEN Center for Molecular Imaging Science, Kobe
NG2 cells are ubiquitously distributed throughout the gray and white matter in the brain. NG2 cells undergo cell division and can generate oligodendrocytes and astrocytes as well as neurons even in the adult brain. It is known that NG2 cells become rapidly activated with their morphological changes including hypertrophy of the cell body and processes, and proliferate in response to several brain insults including traumatic injury, excitotoxic lesions and viral infections. Thus, establishment of in vivo imaging technique of NG2 cells could help us evaluate the extent of brain injury and repair processes. In this study, we generated a transgenic rat strain which preferentially overexpressed human estrogen receptor␣ ligand binding domain (hERL) in the NG2 cells (NG2-hERL Tg rat), and performed PET imaging of the cells in the brain using 16␣-[18 F]fluoro-17-estradiol (FES), a PET tracer for hERL. doi:10.1016/j.neures.2010.07.1592
P3-d13 Analysis of the mechanism that defines the stagedependent function of neocortical progenitor cells Mayumi Okamoto 1 , Takaki Miyata 1 , Fumio Matsuzaki 2 , Ayano Kawaguchi 1 1 Department of Anatomy and Cell Biol., Nagoya University Grad. School of Med 2 Lab. for Cell Asymmetry, CDB, RIKEN
In mammalian cerebral cortex, neural progenitor cells change their cytogenetic behavior dynamically as development proceeds: While progenitors in early embryonic days mainly generate two progenitor cells in a symmetric manner, those in the middle stage give rise to two different types of daughters in an asymmetric manner (i.e., a stem-like daughter cell and a daughter cell to soon proceed to differentiation). To understand how this stage-dependent cytogenesis is achieved, we sought to identify genes that are expressed by cortical progenitor cells in a temporally regulated manner. We first compared the gene expression profiles of progenitor cells isolated from mouse cortex at E11, E14 and E16 using the single-cell cDNA amplification method followed by microarray analysis. Single-cell gene expression profiles could classify the neocortical progenitor cells into the apical (stem-like) progenitors and the basal (non-stem-like) progenitors at all developmental stages examined. Next, the unsupervised principal component analysis (PCA) revealed that the expression pattern of E11 apical progenitors is distinct from that of E14 apical progenitors (e.g. the expression level of Notch signaling related genes was different), suggesting that apical progenitors may change their property between E11 and E14. We identified several genes whose expression increases or decreases during this E11-E14 period. To ask whether these genes are involved in the stage-dependent change of cytogenesis pattern (from symmetric or purely proliferative to asymmetric or more differentiating), we are now doing “heterochronic” in utero erectroporation experiments. For example, a gene that is normally expressed strongly by E14 apical progenitors but not by E11 apical progenitors is artificially expressed in the cortical wall at E10-E11. doi:10.1016/j.neures.2010.07.1593
P3-d14 Developmental Analysis of spinal V0 neurons in zebrafish Chie Satou , Yukiko Kimura, Shinichi Higashijima Okazaki Institute for Integrative Bioscience Neuronal identity in the spinal cord is primarily determined by the expression of distinct classes of transcriptional factors to divide the progenitor cells into discrete dorsoventral domains. Progenitor cells of different domains generate different classes of neurons. Furthermore, recent studies showed that progenitor cells within a domain also produce multiple types of neurons. For example, p0 progenitor cells, which are defined by the expression of dbx1, are known to generate commissural V0 neurons but both excitatory and inhibitory neurons. However, the mechanisms how multiple types of neurons are generated from one domain are not fully understood. Toward understanding the mechanism, we performed a developmental analysis of