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Purification of neural stem cells derived from mouse induced pluripotent stem cells by drug selection
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Abstracts / Neuroscience Research 71S (2011) e108–e415
ation propensity. Therefore, we have to evaluate each hiPS cell line to obtain safe NS/PCs with normal properties to avoid tumorigenesis and inappropriate pathophysiological analysis due to abnormal differentiation. In this study, we derived NS/PCs from hES cells and hiPS cells as neurospheres, and examined their differentiation potentials, functional properties, and gene expression profiles in vitro. We also examined the proportion of residual undifferentiated cells in derived NS/PCs, and the expression of retroviral transgenes which were used for the establishment of hiPS cells, during neural differentiation, to estimate their tumorigenic potentials. Finally, we injected hES, hiPS cell-derived NS/PCs into brains and testes of NOD/SCID mice to evaluate their differentiation properties and tumorigenicities in vivo. As a result of these analyses, we found that one of the hiPS cell lines we used could not differentiate to form neurospheres efficiently, and that NS/PCs derived from some of the hiPS cell lines formed tumors after transplantation, but without teratoma formation. Focusing on these distinct properties of hiPS cell lines, we further analyzed the underlying differences among hiPS cell lines, and evaluated the quality of hiPS cell lines. Research fund: JST-CIRM collaborative program, Keio Kanrinmaru Project. doi:10.1016/j.neures.2011.07.1441
P4-d13 Distribution and differentiation of cephalic neural crest-derived cells in the mouse brain Emiko Yamanishi 1,2 , Masanori Takahashi 1 , Noriko Osumi 1,2 1
Div. of Dev. Neurosci., Grad. Sch. of Med., Tohoku Univ., Sendai, Japan Tohoku Neuroscience Global COE, Basic Translational Research Center for Global Brain Science 2
Neural crest-derived cells (NCDCs) differentiate into various cell types including neurons and glial cells of peripheral nervous system. In the craniofacial region, NCDCs give rise to bones, cartilages, smooth muscles, and pericytes in addition to above mentioned cell types. Curiously, pericytes in the forebrain also originate from NCDCs, although little is known how NCDCs penetrate into the brain and differentiate into pericytes. Furthermore, a previous in vitro study has shown that pericytes derived from the CNS can form spheres that contain stem cells. However, it is unclear whether NCDCs in the brain differentiate into any other cells than pericytes. To address these issues, we examined spatial and temporal distribution patterns of NCDCs in P0-Cre/EGFP mice, in which NCDCs are genetically labeled with EGFP. At E9.5, EGFP+ cells first appeared in the ventral telencephalon through the basement membrane. These cells associated with endothelial cells and migrated toward the dorsal telencephalon in accordance with angiogenesis between E9.5 and E11.5. At E11.5, most EGFP+ cells coexpressed pericyte markers, PDGFR and NG2, whereas about 10% of EGFP+ cells did not express pericyte markers. To further elucidate molecular features of NCDCs that contribute to pericytes in the telencephalon, we analyzed sequential expression patterns of PDGFR and NG2 in EGFP+ cells before the penetration of these cells into the brain. In the craniofacial mesenchyme, expression of PDGFR was uniformly observed in EGFP+ cells from E9.5, although EGFP+ cells did not express PDGFR in the telencephalon until E10.5. Contrastingly, EGFP+/NG2+ cells first emerged in the region adjacent to the ventral neuroepithelium after E10.5 and formed clusters wrapping around blood vessels penetrating into the telencephalon. Taking together, our results suggest that NCDCs differentiate into pericytes after E10.5 and give rise not only to pericytes but also any other cells than pericytes in the telencephalon. doi:10.1016/j.neures.2011.07.1442
P4-d14 Purification of neural stem cells derived from mouse induced pluripotent stem cells by drug selection Masato Maruyama , Yuji Yamashita, Stefan Trifonov, Masahiko Kase, Jun-ichi Shimizu, Tetsuo Sugimoto Department Anat. Brain Sci., Kansai Medical University, Osaka, Japan Reprogramming somatic cells into induced pluripotent stem cells (iPS cells) is an attractive method to produce a new cell source for use in regenerative medicine, disease investigation and drug development. In many fields of iPS research, efficient purification of target cells from differentiated iPS cells is required. One useful strategy is isolation of desired cells from differentiated iPS cells by using drug selection. We managed to purify neural stem cells as a model of target cells, because central nervous system is recognized as a good target for cell therapy. To achieve this, we cloned nestin second intron, known as neural stem cell specific enhancer, and its enhancer activity was determined in nestin positive NE-4C cells by luciferase assay. We confirmed that the 257 bp core sequence of nestin second intron was important for enhancer activity and tandem repeat of this region indicated additive effect. Tandem
enhancer sequence was inserted into the upstream site of 2A-peptide mediated bicistronic expression vector including blasticidin S resistance gene and DsRed. This construct was inserted into the iPS genome mediated by piggyBac transposon system and subclones were established. These cells were differentiated into neural lineage with or without blasticidin S, and we found that addition of blasticidin S purified nestin positive cells. These results suggest that our method is useful to purify the neural stem cells from differentiated iPS cells and could be applicable to obtain other specific cell types. Research Fund: KAKENHI (22790092). doi:10.1016/j.neures.2011.07.1443
P4-d15 Molecular mechanisms of upper-layer neuron specification in mouse neocortex Ken-ichi Toma 1 Hanashima 1
, Yuko Gonda 1 , Ken-ichi Mizutani 2 , Carina
1
Lab. for Neocort. Dev., RIKEN CDB, Kobe 2 Brain Dev. & Aging Res., Doshisha Univ., Kyoto
The mammalian neocortex is comprised of diverse arrays of neurons that are radially organized into six layers and tangentially grouped into areas. Developmentally, cortical layer neurons are sequentially generated from common progenitors within the dorsal telencephalon through multiple rounds of cell division. However, the mechanisms underlying the progression of progenitor competence resulting in a shift from deep- to upper-layer neuron production remain largely elusive. To explore the extent of intrinsic control in upperlayer neuron development, we utilized a Neurog2 inducible Cre-recombinase mouse line to fate-map transient Neurog2-expressing cells. Neurog2 expression peaks in dorsal telencephalic progenitors that are committed to leave the cell cycle, and thus was an ideal candidate to label the cortical lineage as well as temporal cohorts of differentiating neurons. By crossing the Neurog2CreER line with a ROSA26R-EYFP reporter mice, we found that EYFP+ cells labeled upon tamoxifen administration up to E13.5 predominantly gave rise to deep-layer neurons, whereas administration E14.5 onward gave rise to the majority of upper-layer neurons. We further characterized the molecular specificity of each cell population by microarray. These results revealed that upper-layer progenitors are largely distinct from those of deep-layers in molecular expression from the onset of cell cycle exit. To further explore the mechanisms of upper-layer neuron specification, we examined the function of Foxg1, a forkhead transcription factor expressed in the cortical progenitors. By conditionally inactivating Foxg1 at E14.5, we demonstrate that loss of Foxg1 has a critical effect in upper-layer cell development, where these neurons fail to migrate into the cortical plate and expression of specific markers are down-regulated. Together these results imply that the identity of upper-layer neurons is in part determined through early combinatorial gene expression within the progenitors. doi:10.1016/j.neures.2011.07.1444
P4-d16 Characterization of neural stem/progenitor cell properties in the subventricular zone Eunhyuk Chang 1 , Tim Davis 2 , Paul Fairchild 2 , Francis Szele 1 1 2
Dept Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK The Sir William Dunn School of Pathology, University of Oxford, Oxford, UK
The subventricular zone (SVZ) in the adult brain contains a population of stem cells and produces tens of thousands of neurons daily, which migrate actively to the olfactory bulb. However, the mechanisms of SVZ neural stem/progenitor cell (NSPC) maintenance, progenitor cell-fate specification, and differentiation are still not clear. Among these parameters, we are investigating whether Jarid2/Jumonji is necessary for regulating self-renewal and multipotency of the SVZ NSPCs. It has been shown in embryonic stem cells that Jarid2 interacts with polycomb repressive complex 2 (PRC2) to regulate pluripotent gene expression for the balance between self-renewal and differentiation. Jarid2 and PRC2 expression in the SVZ NSPC population were unknown; therefore, we first investigated and confirmed their expression in the SVZ both in vivo and in vitro by RT-PCR and Western blot. Also, we measured the expression level of pluripotent/multipotent stem cell transcription factors (Oct4, Sox2, c-Myc and Klf4) and found only Sox2 and c-Myc were expressed in SVZ tissues and neurospheres. Because it has not been clearly shown how epigenetic regulators modulate SVZ NSPC proliferation, further studies are being carried out to help clarify the role of Jarid2 in self-renewal and multipency. Research Fund: NIH grant in U.S.A. doi:10.1016/j.neures.2011.07.1445
Abstracts / Neuroscience Research 71S (2011) e108–e415
ation propensity. Therefore, we have to evaluate each hiPS cell line to obtain safe NS/PCs with normal properties to avoid tumorigenesis and inappropriate pathophysiological analysis due to abnormal differentiation. In this study, we derived NS/PCs from hES cells and hiPS cells as neurospheres, and examined their differentiation potentials, functional properties, and gene expression profiles in vitro. We also examined the proportion of residual undifferentiated cells in derived NS/PCs, and the expression of retroviral transgenes which were used for the establishment of hiPS cells, during neural differentiation, to estimate their tumorigenic potentials. Finally, we injected hES, hiPS cell-derived NS/PCs into brains and testes of NOD/SCID mice to evaluate their differentiation properties and tumorigenicities in vivo. As a result of these analyses, we found that one of the hiPS cell lines we used could not differentiate to form neurospheres efficiently, and that NS/PCs derived from some of the hiPS cell lines formed tumors after transplantation, but without teratoma formation. Focusing on these distinct properties of hiPS cell lines, we further analyzed the underlying differences among hiPS cell lines, and evaluated the quality of hiPS cell lines. Research fund: JST-CIRM collaborative program, Keio Kanrinmaru Project. doi:10.1016/j.neures.2011.07.1441
P4-d13 Distribution and differentiation of cephalic neural crest-derived cells in the mouse brain Emiko Yamanishi 1,2 , Masanori Takahashi 1 , Noriko Osumi 1,2 1
Div. of Dev. Neurosci., Grad. Sch. of Med., Tohoku Univ., Sendai, Japan Tohoku Neuroscience Global COE, Basic Translational Research Center for Global Brain Science 2
Neural crest-derived cells (NCDCs) differentiate into various cell types including neurons and glial cells of peripheral nervous system. In the craniofacial region, NCDCs give rise to bones, cartilages, smooth muscles, and pericytes in addition to above mentioned cell types. Curiously, pericytes in the forebrain also originate from NCDCs, although little is known how NCDCs penetrate into the brain and differentiate into pericytes. Furthermore, a previous in vitro study has shown that pericytes derived from the CNS can form spheres that contain stem cells. However, it is unclear whether NCDCs in the brain differentiate into any other cells than pericytes. To address these issues, we examined spatial and temporal distribution patterns of NCDCs in P0-Cre/EGFP mice, in which NCDCs are genetically labeled with EGFP. At E9.5, EGFP+ cells first appeared in the ventral telencephalon through the basement membrane. These cells associated with endothelial cells and migrated toward the dorsal telencephalon in accordance with angiogenesis between E9.5 and E11.5. At E11.5, most EGFP+ cells coexpressed pericyte markers, PDGFR and NG2, whereas about 10% of EGFP+ cells did not express pericyte markers. To further elucidate molecular features of NCDCs that contribute to pericytes in the telencephalon, we analyzed sequential expression patterns of PDGFR and NG2 in EGFP+ cells before the penetration of these cells into the brain. In the craniofacial mesenchyme, expression of PDGFR was uniformly observed in EGFP+ cells from E9.5, although EGFP+ cells did not express PDGFR in the telencephalon until E10.5. Contrastingly, EGFP+/NG2+ cells first emerged in the region adjacent to the ventral neuroepithelium after E10.5 and formed clusters wrapping around blood vessels penetrating into the telencephalon. Taking together, our results suggest that NCDCs differentiate into pericytes after E10.5 and give rise not only to pericytes but also any other cells than pericytes in the telencephalon. doi:10.1016/j.neures.2011.07.1442
P4-d14 Purification of neural stem cells derived from mouse induced pluripotent stem cells by drug selection Masato Maruyama , Yuji Yamashita, Stefan Trifonov, Masahiko Kase, Jun-ichi Shimizu, Tetsuo Sugimoto Department Anat. Brain Sci., Kansai Medical University, Osaka, Japan Reprogramming somatic cells into induced pluripotent stem cells (iPS cells) is an attractive method to produce a new cell source for use in regenerative medicine, disease investigation and drug development. In many fields of iPS research, efficient purification of target cells from differentiated iPS cells is required. One useful strategy is isolation of desired cells from differentiated iPS cells by using drug selection. We managed to purify neural stem cells as a model of target cells, because central nervous system is recognized as a good target for cell therapy. To achieve this, we cloned nestin second intron, known as neural stem cell specific enhancer, and its enhancer activity was determined in nestin positive NE-4C cells by luciferase assay. We confirmed that the 257 bp core sequence of nestin second intron was important for enhancer activity and tandem repeat of this region indicated additive effect. Tandem
enhancer sequence was inserted into the upstream site of 2A-peptide mediated bicistronic expression vector including blasticidin S resistance gene and DsRed. This construct was inserted into the iPS genome mediated by piggyBac transposon system and subclones were established. These cells were differentiated into neural lineage with or without blasticidin S, and we found that addition of blasticidin S purified nestin positive cells. These results suggest that our method is useful to purify the neural stem cells from differentiated iPS cells and could be applicable to obtain other specific cell types. Research Fund: KAKENHI (22790092). doi:10.1016/j.neures.2011.07.1443
P4-d15 Molecular mechanisms of upper-layer neuron specification in mouse neocortex Ken-ichi Toma 1 Hanashima 1
, Yuko Gonda 1 , Ken-ichi Mizutani 2 , Carina
1
Lab. for Neocort. Dev., RIKEN CDB, Kobe 2 Brain Dev. & Aging Res., Doshisha Univ., Kyoto
The mammalian neocortex is comprised of diverse arrays of neurons that are radially organized into six layers and tangentially grouped into areas. Developmentally, cortical layer neurons are sequentially generated from common progenitors within the dorsal telencephalon through multiple rounds of cell division. However, the mechanisms underlying the progression of progenitor competence resulting in a shift from deep- to upper-layer neuron production remain largely elusive. To explore the extent of intrinsic control in upperlayer neuron development, we utilized a Neurog2 inducible Cre-recombinase mouse line to fate-map transient Neurog2-expressing cells. Neurog2 expression peaks in dorsal telencephalic progenitors that are committed to leave the cell cycle, and thus was an ideal candidate to label the cortical lineage as well as temporal cohorts of differentiating neurons. By crossing the Neurog2CreER line with a ROSA26R-EYFP reporter mice, we found that EYFP+ cells labeled upon tamoxifen administration up to E13.5 predominantly gave rise to deep-layer neurons, whereas administration E14.5 onward gave rise to the majority of upper-layer neurons. We further characterized the molecular specificity of each cell population by microarray. These results revealed that upper-layer progenitors are largely distinct from those of deep-layers in molecular expression from the onset of cell cycle exit. To further explore the mechanisms of upper-layer neuron specification, we examined the function of Foxg1, a forkhead transcription factor expressed in the cortical progenitors. By conditionally inactivating Foxg1 at E14.5, we demonstrate that loss of Foxg1 has a critical effect in upper-layer cell development, where these neurons fail to migrate into the cortical plate and expression of specific markers are down-regulated. Together these results imply that the identity of upper-layer neurons is in part determined through early combinatorial gene expression within the progenitors. doi:10.1016/j.neures.2011.07.1444
P4-d16 Characterization of neural stem/progenitor cell properties in the subventricular zone Eunhyuk Chang 1 , Tim Davis 2 , Paul Fairchild 2 , Francis Szele 1 1 2
Dept Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK The Sir William Dunn School of Pathology, University of Oxford, Oxford, UK
The subventricular zone (SVZ) in the adult brain contains a population of stem cells and produces tens of thousands of neurons daily, which migrate actively to the olfactory bulb. However, the mechanisms of SVZ neural stem/progenitor cell (NSPC) maintenance, progenitor cell-fate specification, and differentiation are still not clear. Among these parameters, we are investigating whether Jarid2/Jumonji is necessary for regulating self-renewal and multipotency of the SVZ NSPCs. It has been shown in embryonic stem cells that Jarid2 interacts with polycomb repressive complex 2 (PRC2) to regulate pluripotent gene expression for the balance between self-renewal and differentiation. Jarid2 and PRC2 expression in the SVZ NSPC population were unknown; therefore, we first investigated and confirmed their expression in the SVZ both in vivo and in vitro by RT-PCR and Western blot. Also, we measured the expression level of pluripotent/multipotent stem cell transcription factors (Oct4, Sox2, c-Myc and Klf4) and found only Sox2 and c-Myc were expressed in SVZ tissues and neurospheres. Because it has not been clearly shown how epigenetic regulators modulate SVZ NSPC proliferation, further studies are being carried out to help clarify the role of Jarid2 in self-renewal and multipency. Research Fund: NIH grant in U.S.A. doi:10.1016/j.neures.2011.07.1445