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Molecular mechanism of sox2 expression in neural stem cells
Abstracts / Neuroscience Research 58S (2007) S1–S244
S19
S3A-A6 Molecular mechanism underlying maintenance of fear
S3A-B3 Regulatory mechanisms of neural differentiation from
memory
pluripotent cells
Kaoru Inokuchi 1,2 1 Memory Formation Research Group, Mitsubishi Kagaku Institute of Life Sciences, MITILS, Tokyo, Japan; 2 CREST, JST, Kawaguchi, Japan
Yoshiki Sasai Organogenesis and Neurogenesis Group, RIKEN CDB, Kobe, Japan
Memory process consists of at least three distinct phases, acquisition, maintenance, and retrieval. To address functional role activin plays in memory formation process, we generated activin and follistatin (that antagonizes activin function) transgenic mice (BIA/tTA and BIF/tTA, respectively) in which the transgene expression was tightly regulated by DOX in a forebrain-specific manner (Tet OFF system). Transgene expression was turned OFF or ON within 3d by (±) DOX. Contextual fear conditioning with these mice revealed that activin function is required during maintenance phase of fear memory for one week-retention. Furthermore, activin overexpression during maintenance phase enhanced long-term memory. Thus, according to the activin level in the forebrain during maintenance phase, fear memory that was once acquired tightly could be erased or further consolidated. Research fund: CREST, JST
S3A-B1 Epigenetic mechanism regulating fate specification and plasticity of neural cells Kinichi Nakashima 1 , Eriko Takatsuka 1 , Toru Yamashita 2,3 , Jenny Hsieh 4 , Fred H. Gage 5 , Masakazu Namihira 1 , Hideyuki Okano 2 , Kazunobu Sawamoto 3,6 , Jun Kohyama 1 1 Laboratory of Molecular Neuroscience, Graduate School of Biological Science, NAIST, Nara, Japan; 2 Department of Physiology, Keio University School of Medicine, Tokyo, Japan; 3 Bridgestone Laboratory of Developmental and Regenerative Neurobiology, Keio University School of Medicine, Tokyo, Japan; 4 Department of Molecular Biology, Cecil H. Ida Green Center of Reproductive and Biological Science, UT Southwestern Medical Center, Dallas, USA; 5 Laboratory of Genetics, Salk Institute, La Jolla, USA; 6 Nagoya City University Graduate School of Medical Science, Nagoya, Japan Methyl-CpG binding protein transcriptional repressors (MBDs) are expressed predominantly in neurons in the central nervous system. We report here that oligodendrocytes, which are devoid of MBDs, can transdifferentiate into astrocytes after injury in response to extracellular stimuli activating the JAK/STAT pathway, suggesting that differentiation plasticity in neural cells is regulated by cell-intrinsic epigenetic mechanisms in collaboration with ambient cell-extrinsic cues. These findings imply that demyelination after injury is due in part to oligodendrocyte to astrocyte transdifferentiation.
S3A-B2 Signals regulating the differentiation vs. maintenance of neural progenitor cells Takehiko Sunabori, Hideyuki Okano Department of Physiology, Keio University School of Medicine, Tokyo, Japan Signals regulating the maintenance and commitment of the neural progenitor cell have been intensively studied. However, many of the evidence are based on in vitro studies and also little is known about the relationship between those well-learned signaling cascades. Here, we would like to discuss the more detailed timing of the mammalian neural progenitor cell to determine its cell fate by verifying the activation of each signal especially in vivo. Research fund: SORST
We previously reported an efficient method that induces selective neural differentiation from mouse ES cells using coculture with PA6 stromal cells. This method, named SDIA, can be used to generate a wide variety of neural tissues including midbrain dopaminergic neurons and motor neurons. To apply a similar principle to human application, we recently established a culture method of hES cells that induces neural differentiation using human amniotic membrane matrix (AMED). On the other hand, SDIA (or AMED)-treated ES cells rarely differentiate into telencephalic cells. We demonstrate directed differentiation of telencephalic precursors from mES cells using serum-free suspension culture (SFEB). Treatment with Wnt and Nodal antagonists during the first five days of SFEB culture causes selective neural differentiation in ES cells. SFEB induces efficient generation (35%) of Bf1+ telencephalic cells from both mouse and huma ES cells. Thus, floating aggregates of ES cells in default of caudalizing signals generate naive telencephalic precursors that acquire subregional identities by responding to extracellular patterning signals.
S3A-B4 Molecular mechanism of sox2 expression in neural stem cells Toru Kondo Laboratory for Cell Lineage Modulation, RIKEN Center for Developmental Biology, Kobe, Japan Oligodendrocyte precursor cells (OPC) can behave as multipotent neural stem cells (NSC) when the cells are cultured in a specific condition. We have shown that a number of NSC markers are activated when OPC revert to NSC-like cells (NSLC). Among them, we focused Sox2, which is an essential transcription factor in NSC, and found that Sox2 is involved in the proliferation and maintenance of NSLC. Moreover we found that a SWI/SNF chromatin-remodeling complex is recruited to the sox2 enhancer and histone H3 around the enhancer are converted to the active form in the reversion. Because the SWI/SNF complex does not recognize any specific DNA sequences, it is speculated that an unknown DNA-binding factor in NSC binds and recruits the SWI/SNF complex to the enhancer. Recently, we have identified a candidate factor that recognizes the sox2 enhancer and associates with Brm, which is one of essential factors in SWI/SNF complex, in NSC. Thus, these findings suggest that the OPC reversion depends on the epigenetic modification and that OPC might be an alternative source of NSC for CNS therapy. Research fund: RIKEN internal fund
S3A-B5 Generation of high quality iPS cells Kazutoshi Takahashi 1 , Keisuke Okita 1 , Masato Nakagawa 1 , Takashi Aoi 1 , Tomoko Ichisaka 1,2 , Shinya Yamanaka 1,2 1 Department of Stem Cell Biology, Institute for Frontier Medical Sciences, Kyoto University, Japan; 2 CREST, JST, Japan Human ES cells faces difficulties regarding use of human embryos and rejection following implantation. One way to circumvent these issues is to generate pluripotent cells directly from somatic cells. We have previously shown that pluripotent stem cells can be induced from mouse fibroblasts by four factors (Oct3/4, Sox2, c-Myc and Klf4), and by selection for Fbx15 expression. These iPS (induced pluripotent stem) cells showed morphology and proliferation, and teratoma formation similar to ES cells. These data demonstrated that pluripotent cells can be generated from fibroblast culture with a few defined factors. However, microarray analyses revealed significant difference in gene expression between ES cells and iPS cells. Furthermore, iPS cells failed to produce adult or germ-line chimeras when transplanted into blastocysts. We are now trying to improve the quality of iPS cells by using other selection markers or other cell sources. Research fund: CREST, JST, Japan.
S19
S3A-A6 Molecular mechanism underlying maintenance of fear
S3A-B3 Regulatory mechanisms of neural differentiation from
memory
pluripotent cells
Kaoru Inokuchi 1,2 1 Memory Formation Research Group, Mitsubishi Kagaku Institute of Life Sciences, MITILS, Tokyo, Japan; 2 CREST, JST, Kawaguchi, Japan
Yoshiki Sasai Organogenesis and Neurogenesis Group, RIKEN CDB, Kobe, Japan
Memory process consists of at least three distinct phases, acquisition, maintenance, and retrieval. To address functional role activin plays in memory formation process, we generated activin and follistatin (that antagonizes activin function) transgenic mice (BIA/tTA and BIF/tTA, respectively) in which the transgene expression was tightly regulated by DOX in a forebrain-specific manner (Tet OFF system). Transgene expression was turned OFF or ON within 3d by (±) DOX. Contextual fear conditioning with these mice revealed that activin function is required during maintenance phase of fear memory for one week-retention. Furthermore, activin overexpression during maintenance phase enhanced long-term memory. Thus, according to the activin level in the forebrain during maintenance phase, fear memory that was once acquired tightly could be erased or further consolidated. Research fund: CREST, JST
S3A-B1 Epigenetic mechanism regulating fate specification and plasticity of neural cells Kinichi Nakashima 1 , Eriko Takatsuka 1 , Toru Yamashita 2,3 , Jenny Hsieh 4 , Fred H. Gage 5 , Masakazu Namihira 1 , Hideyuki Okano 2 , Kazunobu Sawamoto 3,6 , Jun Kohyama 1 1 Laboratory of Molecular Neuroscience, Graduate School of Biological Science, NAIST, Nara, Japan; 2 Department of Physiology, Keio University School of Medicine, Tokyo, Japan; 3 Bridgestone Laboratory of Developmental and Regenerative Neurobiology, Keio University School of Medicine, Tokyo, Japan; 4 Department of Molecular Biology, Cecil H. Ida Green Center of Reproductive and Biological Science, UT Southwestern Medical Center, Dallas, USA; 5 Laboratory of Genetics, Salk Institute, La Jolla, USA; 6 Nagoya City University Graduate School of Medical Science, Nagoya, Japan Methyl-CpG binding protein transcriptional repressors (MBDs) are expressed predominantly in neurons in the central nervous system. We report here that oligodendrocytes, which are devoid of MBDs, can transdifferentiate into astrocytes after injury in response to extracellular stimuli activating the JAK/STAT pathway, suggesting that differentiation plasticity in neural cells is regulated by cell-intrinsic epigenetic mechanisms in collaboration with ambient cell-extrinsic cues. These findings imply that demyelination after injury is due in part to oligodendrocyte to astrocyte transdifferentiation.
S3A-B2 Signals regulating the differentiation vs. maintenance of neural progenitor cells Takehiko Sunabori, Hideyuki Okano Department of Physiology, Keio University School of Medicine, Tokyo, Japan Signals regulating the maintenance and commitment of the neural progenitor cell have been intensively studied. However, many of the evidence are based on in vitro studies and also little is known about the relationship between those well-learned signaling cascades. Here, we would like to discuss the more detailed timing of the mammalian neural progenitor cell to determine its cell fate by verifying the activation of each signal especially in vivo. Research fund: SORST
We previously reported an efficient method that induces selective neural differentiation from mouse ES cells using coculture with PA6 stromal cells. This method, named SDIA, can be used to generate a wide variety of neural tissues including midbrain dopaminergic neurons and motor neurons. To apply a similar principle to human application, we recently established a culture method of hES cells that induces neural differentiation using human amniotic membrane matrix (AMED). On the other hand, SDIA (or AMED)-treated ES cells rarely differentiate into telencephalic cells. We demonstrate directed differentiation of telencephalic precursors from mES cells using serum-free suspension culture (SFEB). Treatment with Wnt and Nodal antagonists during the first five days of SFEB culture causes selective neural differentiation in ES cells. SFEB induces efficient generation (35%) of Bf1+ telencephalic cells from both mouse and huma ES cells. Thus, floating aggregates of ES cells in default of caudalizing signals generate naive telencephalic precursors that acquire subregional identities by responding to extracellular patterning signals.
S3A-B4 Molecular mechanism of sox2 expression in neural stem cells Toru Kondo Laboratory for Cell Lineage Modulation, RIKEN Center for Developmental Biology, Kobe, Japan Oligodendrocyte precursor cells (OPC) can behave as multipotent neural stem cells (NSC) when the cells are cultured in a specific condition. We have shown that a number of NSC markers are activated when OPC revert to NSC-like cells (NSLC). Among them, we focused Sox2, which is an essential transcription factor in NSC, and found that Sox2 is involved in the proliferation and maintenance of NSLC. Moreover we found that a SWI/SNF chromatin-remodeling complex is recruited to the sox2 enhancer and histone H3 around the enhancer are converted to the active form in the reversion. Because the SWI/SNF complex does not recognize any specific DNA sequences, it is speculated that an unknown DNA-binding factor in NSC binds and recruits the SWI/SNF complex to the enhancer. Recently, we have identified a candidate factor that recognizes the sox2 enhancer and associates with Brm, which is one of essential factors in SWI/SNF complex, in NSC. Thus, these findings suggest that the OPC reversion depends on the epigenetic modification and that OPC might be an alternative source of NSC for CNS therapy. Research fund: RIKEN internal fund
S3A-B5 Generation of high quality iPS cells Kazutoshi Takahashi 1 , Keisuke Okita 1 , Masato Nakagawa 1 , Takashi Aoi 1 , Tomoko Ichisaka 1,2 , Shinya Yamanaka 1,2 1 Department of Stem Cell Biology, Institute for Frontier Medical Sciences, Kyoto University, Japan; 2 CREST, JST, Japan Human ES cells faces difficulties regarding use of human embryos and rejection following implantation. One way to circumvent these issues is to generate pluripotent cells directly from somatic cells. We have previously shown that pluripotent stem cells can be induced from mouse fibroblasts by four factors (Oct3/4, Sox2, c-Myc and Klf4), and by selection for Fbx15 expression. These iPS (induced pluripotent stem) cells showed morphology and proliferation, and teratoma formation similar to ES cells. These data demonstrated that pluripotent cells can be generated from fibroblast culture with a few defined factors. However, microarray analyses revealed significant difference in gene expression between ES cells and iPS cells. Furthermore, iPS cells failed to produce adult or germ-line chimeras when transplanted into blastocysts. We are now trying to improve the quality of iPS cells by using other selection markers or other cell sources. Research fund: CREST, JST, Japan.