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Inductions of neural stem cells and neurons from mouse induced pluripotent stem cells
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Abstracts / Neuroscience Research 68S (2010) e335–e446
highly expressed at microvessels of the CVOs in adult CNS and suggests that microvessels of the CVOs may possess the immature feature to reconstruct microvascular structures. Cells may be able to undergo not only neurogenesis and gliogenesis, but also angiogenesis in adult CVOs. doi:10.1016/j.neures.2010.07.1585
P3-d07 Roles of Runx1 in proliferation and neuronal differentiation of progenitor cell subpopulation in the mouse dorsal root ganglion Kouji Senzaki , Azusa Kobayashi, Masaaki Yoshikawa, Shigeru Ozaki, Satoru Takahashi, Takashi Shiga Graduate School of Comprehensive Human Sciences, Univ. of Tsukuba
P3-d05 Inductions of neural stem cells and neurons from mouse induced pluripotent stem cells Anna Sato , Tomoyuki Kanamatsu, Banri Yamanoha Environ. Eng. for Symbio. major, Grad. Sch. of Eng., Soka University Induced pluripotent stem (iPS) cells hold great promise for safety evaluation and drug efficacy evaluation to chemical substances which is environmental chemicals and medical drugs. Neurogenesis of central nerve system is a complex phenomenon and it occur sequential activation of the genes related with the neural development in the early developmental stage. The use of iPS cells as a source of neurons for fate of their types will require the development of simple and reliable cell differentiation protocol. The neural cell differentiation have been developed for the sequential differentiation of ES cells into neural stem cells and neurons (Nakayama and Inoue, 2003). We have performed that mouse iPS cells (Takahashi and Yamanaka, 2006) were cultured on mouse embryonic fibroblast (MEF) in medium with differentiation inhibitory factor, leukemia inhibitory factor (LIF). It was compared in two type media, DMEM containing with KSR and FBS, under the undifferentiated conditions. In both media, iPS cells are proliferated and formed colonies, whereas colonies in morphology are appeared different type, termed dome, flat and sheet types. The colonies are picked up and transferred to medium containing with ACM (astrocyte conditions medium) and FGF-2. They are differentiated as neural stem spheres with floating culture. The colonies are assessed to yield highly efficient differentiation into neural stem cells (NSCs) with RT-PCR (primers: Oct3/4 and nestin) and immunocytochemistry (antibody: nestin). A high quality of the differentiation in colony would be found in the flat type of colonies than others. The spheres were transferred to a matrigel-coated dish to yield neurons. The neural stem cells on a matrigel are differentiated to neurons with some different morphology in differentiation media without FGF-2 by adhesion culture. We have analyzed isotype of their neurons by Immunocytochemistry. Reference
T. Nakayama and N. Inoue., Div.Neuro.,15, 2003 K. Takahashi and S. Yamanaka, Cell, 126, 2006
doi:10.1016/j.neures.2010.07.1586
P3-d06 Induced pluripotent stem cells transplanted in mouse ischemic brain Hiromi Kawai , Toru Yamashita, Yasuyuki Ohta, Kentaro Deguchi, Shoko Deguchi, Xuemei Zhang, Yoshio Ikeda, Tohru Matsuura, Koji Abe Department of Neurology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Okayama University Stroke is a major neurologic disorder. Induced pluripotent stem (iPS) cells can be produced frombasically any part of patients, with high reproduction ability and pluripotency to differentiate into various types of cells, suggesting that iPS cells can provide a hopeful therapy for celltransplantation. However, transplantation of iPS cells into ischemic brain has not been reported. In this study, we showed that the iPS cells fate in a mouse model of transient middle cerebralartery occlusion (MCAO). Undifferentiated iPS cells (5 × 105 ) were transplanted into ipsilateral striatum and cortex at 24 h after 30 min of transient MCAO. Behavioral and histologic analyses were performed at 28 day after the cell transplantation. To our surprise, the transplanted iPS cells expanded and formed much larger tumors in mice postischemic brain than in sham-operated brain. The clinical recovery of the MCAO + iPS group was delayed as compared with the MCAO + PBS(phosphate-buffered saline) group. iPS cells formed tridermal teratoma, but could supply a great number of Dcx-positive neuroblasts and a few mature neurons in the ischemic lesion. iPS cells have a promising potential to provide neural cells after ischemic brain injury, if tumorigenesis is properly controlled. doi:10.1016/j.neures.2010.07.1587
Transcription factor Runx1 controls the cell type specification of peptidergic and nonpeptidergic nociceptive dorsal root ganglion (DRG) neurons by repressing the expression of neurotrophin receptor TrkA and calcitonin generelated peptide and activating the expression of a receptor for Glial cell line-derived neurotrophic factor Ret during late embryonic and early postnatal periods (Chen et al., 2006; Kramer et al., 2006; Yoshikawa et al., 2007). Because Runx1 is expressed in DRG cells from the initial stage of DRG development, we studied the roles of Runx1 in the proliferation and differentiation of DRG cells using Runx1-deficient (Runx1−/− ::Tg) mice. We found that the number of TrkA-expressing (TrkA+ ) DRG neurons was decreased at embryonic day (E) 12.5 in spite of the fact that the number of TrkA+ DRG neurons was increased at E17.5 in Runx1−/− ::Tg mice. The number of DRG neurons which express neuronal markers NeuN, Islet1 and Hu was also reduced in Runx1−/− ::Tg mice at E12.5. The cell cycle analysis using BrdU/IDU revealed that the number of DRG cells in S-phase and G2/M-phase was increased in E12.5 Runx1−/− ::Tg mice, while the length of S-phase was not changed between Runx1+/+ ::Tg and Runx1−/− ::Tg mice, suggesting that Runx1 negatively controls the cell proliferation of DRG progenitor cell subpopulation in early embryonic periods. Hes1 is a negative regulator of neuronal differentiation (Ishibashi et al., 1995; Tomita et al., 1996). We found that the number of Hes1+ DRG neurons was increased in Runx1−/− ::Tg mice at E12.5. In summary, the present study suggests that Runx1 represses the cell proliferation and activates the neuronal differentiation of DRG progenitor cell subpopulation through the repression of Hes1 expression in early embryonic period. doi:10.1016/j.neures.2010.07.1588
P3-d08 Functional differentiation of exogenous neural stem cells into excitatory and inhibitory neurons in adult mice cortex Mitsunori D. Arai , Shoji Komai Graduate School of Biological Sciences, Division of Structural Cellular Biology, NAIST Many researchers have studied about neural stem cells (NSCs) and its transplantation into several brain regions to treat neurological disorders, such as Parkinson’s disease, ischemia, and spinal cord injuries, caused by damage or loss of neurons and glial cells. These therapies indeed have been helped to recovery of motor functions such as dyskinesia. The recovery have been shown by testing roterod or BBB scale and some behavioral tests. However, little is known about contribution of transplantation to recover motor functions and how neural activities form the appropriate local neural circuits with transplanted cells correctly processing the information with endogenous neurons. It is essential for not only genetic factor but also experience after born to develop correct neural circuits. Therefore we examined whether transplanted NSCs in the intact somatosensory cortex could be involved in functional neural circuits morphologically and physiologically manipulated by using of genetic factors and neural activities. Firstly we investigated whether Neurogenin2 and Mash-1 can differentiate transplanted NSCs either into excitatory or inhibitory neurons, which are thought to regulate differentiation of excitatory and inhibitory neuron respectively in cortical development. Moreover we examined how influence the differentiation of transplanted cells given sensory inputs in diversity ways. In this report we analyzed histologically what kind of cell types NSCs differentatied after gene manipulations and environmental enrichment. This approach will allow us to reveal the importance of the experience or neuronal activity for building up the appropriate neuronal circuit in the cortex. Additionally this method could be helpful to care the patients suffered from neurological disorders. doi:10.1016/j.neures.2010.07.1589
Abstracts / Neuroscience Research 68S (2010) e335–e446
highly expressed at microvessels of the CVOs in adult CNS and suggests that microvessels of the CVOs may possess the immature feature to reconstruct microvascular structures. Cells may be able to undergo not only neurogenesis and gliogenesis, but also angiogenesis in adult CVOs. doi:10.1016/j.neures.2010.07.1585
P3-d07 Roles of Runx1 in proliferation and neuronal differentiation of progenitor cell subpopulation in the mouse dorsal root ganglion Kouji Senzaki , Azusa Kobayashi, Masaaki Yoshikawa, Shigeru Ozaki, Satoru Takahashi, Takashi Shiga Graduate School of Comprehensive Human Sciences, Univ. of Tsukuba
P3-d05 Inductions of neural stem cells and neurons from mouse induced pluripotent stem cells Anna Sato , Tomoyuki Kanamatsu, Banri Yamanoha Environ. Eng. for Symbio. major, Grad. Sch. of Eng., Soka University Induced pluripotent stem (iPS) cells hold great promise for safety evaluation and drug efficacy evaluation to chemical substances which is environmental chemicals and medical drugs. Neurogenesis of central nerve system is a complex phenomenon and it occur sequential activation of the genes related with the neural development in the early developmental stage. The use of iPS cells as a source of neurons for fate of their types will require the development of simple and reliable cell differentiation protocol. The neural cell differentiation have been developed for the sequential differentiation of ES cells into neural stem cells and neurons (Nakayama and Inoue, 2003). We have performed that mouse iPS cells (Takahashi and Yamanaka, 2006) were cultured on mouse embryonic fibroblast (MEF) in medium with differentiation inhibitory factor, leukemia inhibitory factor (LIF). It was compared in two type media, DMEM containing with KSR and FBS, under the undifferentiated conditions. In both media, iPS cells are proliferated and formed colonies, whereas colonies in morphology are appeared different type, termed dome, flat and sheet types. The colonies are picked up and transferred to medium containing with ACM (astrocyte conditions medium) and FGF-2. They are differentiated as neural stem spheres with floating culture. The colonies are assessed to yield highly efficient differentiation into neural stem cells (NSCs) with RT-PCR (primers: Oct3/4 and nestin) and immunocytochemistry (antibody: nestin). A high quality of the differentiation in colony would be found in the flat type of colonies than others. The spheres were transferred to a matrigel-coated dish to yield neurons. The neural stem cells on a matrigel are differentiated to neurons with some different morphology in differentiation media without FGF-2 by adhesion culture. We have analyzed isotype of their neurons by Immunocytochemistry. Reference
T. Nakayama and N. Inoue., Div.Neuro.,15, 2003 K. Takahashi and S. Yamanaka, Cell, 126, 2006
doi:10.1016/j.neures.2010.07.1586
P3-d06 Induced pluripotent stem cells transplanted in mouse ischemic brain Hiromi Kawai , Toru Yamashita, Yasuyuki Ohta, Kentaro Deguchi, Shoko Deguchi, Xuemei Zhang, Yoshio Ikeda, Tohru Matsuura, Koji Abe Department of Neurology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Okayama University Stroke is a major neurologic disorder. Induced pluripotent stem (iPS) cells can be produced frombasically any part of patients, with high reproduction ability and pluripotency to differentiate into various types of cells, suggesting that iPS cells can provide a hopeful therapy for celltransplantation. However, transplantation of iPS cells into ischemic brain has not been reported. In this study, we showed that the iPS cells fate in a mouse model of transient middle cerebralartery occlusion (MCAO). Undifferentiated iPS cells (5 × 105 ) were transplanted into ipsilateral striatum and cortex at 24 h after 30 min of transient MCAO. Behavioral and histologic analyses were performed at 28 day after the cell transplantation. To our surprise, the transplanted iPS cells expanded and formed much larger tumors in mice postischemic brain than in sham-operated brain. The clinical recovery of the MCAO + iPS group was delayed as compared with the MCAO + PBS(phosphate-buffered saline) group. iPS cells formed tridermal teratoma, but could supply a great number of Dcx-positive neuroblasts and a few mature neurons in the ischemic lesion. iPS cells have a promising potential to provide neural cells after ischemic brain injury, if tumorigenesis is properly controlled. doi:10.1016/j.neures.2010.07.1587
Transcription factor Runx1 controls the cell type specification of peptidergic and nonpeptidergic nociceptive dorsal root ganglion (DRG) neurons by repressing the expression of neurotrophin receptor TrkA and calcitonin generelated peptide and activating the expression of a receptor for Glial cell line-derived neurotrophic factor Ret during late embryonic and early postnatal periods (Chen et al., 2006; Kramer et al., 2006; Yoshikawa et al., 2007). Because Runx1 is expressed in DRG cells from the initial stage of DRG development, we studied the roles of Runx1 in the proliferation and differentiation of DRG cells using Runx1-deficient (Runx1−/− ::Tg) mice. We found that the number of TrkA-expressing (TrkA+ ) DRG neurons was decreased at embryonic day (E) 12.5 in spite of the fact that the number of TrkA+ DRG neurons was increased at E17.5 in Runx1−/− ::Tg mice. The number of DRG neurons which express neuronal markers NeuN, Islet1 and Hu was also reduced in Runx1−/− ::Tg mice at E12.5. The cell cycle analysis using BrdU/IDU revealed that the number of DRG cells in S-phase and G2/M-phase was increased in E12.5 Runx1−/− ::Tg mice, while the length of S-phase was not changed between Runx1+/+ ::Tg and Runx1−/− ::Tg mice, suggesting that Runx1 negatively controls the cell proliferation of DRG progenitor cell subpopulation in early embryonic periods. Hes1 is a negative regulator of neuronal differentiation (Ishibashi et al., 1995; Tomita et al., 1996). We found that the number of Hes1+ DRG neurons was increased in Runx1−/− ::Tg mice at E12.5. In summary, the present study suggests that Runx1 represses the cell proliferation and activates the neuronal differentiation of DRG progenitor cell subpopulation through the repression of Hes1 expression in early embryonic period. doi:10.1016/j.neures.2010.07.1588
P3-d08 Functional differentiation of exogenous neural stem cells into excitatory and inhibitory neurons in adult mice cortex Mitsunori D. Arai , Shoji Komai Graduate School of Biological Sciences, Division of Structural Cellular Biology, NAIST Many researchers have studied about neural stem cells (NSCs) and its transplantation into several brain regions to treat neurological disorders, such as Parkinson’s disease, ischemia, and spinal cord injuries, caused by damage or loss of neurons and glial cells. These therapies indeed have been helped to recovery of motor functions such as dyskinesia. The recovery have been shown by testing roterod or BBB scale and some behavioral tests. However, little is known about contribution of transplantation to recover motor functions and how neural activities form the appropriate local neural circuits with transplanted cells correctly processing the information with endogenous neurons. It is essential for not only genetic factor but also experience after born to develop correct neural circuits. Therefore we examined whether transplanted NSCs in the intact somatosensory cortex could be involved in functional neural circuits morphologically and physiologically manipulated by using of genetic factors and neural activities. Firstly we investigated whether Neurogenin2 and Mash-1 can differentiate transplanted NSCs either into excitatory or inhibitory neurons, which are thought to regulate differentiation of excitatory and inhibitory neuron respectively in cortical development. Moreover we examined how influence the differentiation of transplanted cells given sensory inputs in diversity ways. In this report we analyzed histologically what kind of cell types NSCs differentatied after gene manipulations and environmental enrichment. This approach will allow us to reveal the importance of the experience or neuronal activity for building up the appropriate neuronal circuit in the cortex. Additionally this method could be helpful to care the patients suffered from neurological disorders. doi:10.1016/j.neures.2010.07.1589