- Email: [email protected]
P49. Cell reprogramming factors of neural crest cells
Abstract / Differentiation 80 (2010) S17–S63
NPR-A knockdown also resulted in a marked downregulation of phosphorylated Akt, which is essential for ES cell self-renewal. Furthermore, NPR-A knockdown induced accumulation of ES cells in the G1-phase of the cell cycle. Interestingly, we found that ANP was expressed in self-renewing ES cells, whereas its level was reduced after ES cell differentiation by the withdrawal of leukemia inhibitory factor. Treatment of ES cells with exogenous ANP upregulated the expression of Oct4 and Nanog, and this upregulation depended on NPR-A signaling, because it was completely reversed by pretreatment with NPR-A antagonist or cGMP-dependent protein kinase inhibitor. Also, ANP increased the expression level of phosphorylated Akt. These findings provide a novel role for NPR-A in the maintenance of self-renewal and pluripotency of ES cells. doi: 10.1016/j.diff.2010.09.053
P48 Formation of blood vessels is controlled by neural progenitor cells in the central nervous system
Teruaki Takahashi, Yuta Takase, Ryosuke Tadokoro, Yoshiko Takahashi Nara Institute of Science and Technology (NAIST), Nara, Japan E-mail address: [email protected] (T. Takahashi)
Vascular networks of blood vessels are stereotypically patterned in early developing embryos. It remains unknown how the vascular patterns are established in three-dimensional tissues including peripheral organs and the central nervous system (CNS). We study the mechanisms underlying the patterning of blood vessels in CNS using early spinal cord as a model. In chicken E4 embryos, endothelial cells of somitic origin penetrate into the spinal cord through the ventral edge. Subsequently, forming vessels extend dorsally along determined paths. We found that these paths coincide with the interface between the layer of progenitor cells (medial) and the layer of differentiated neurons and glial cells (lateral). Since the blood vessels do not invade the progenitor layer, we reasoned that the progenitor layer elicits inhibitory influence on the blood vessel formation. To test this, we experimentally promoted neural/glial differentiation in the progenitor territory. The small GTPase RhoA is known to maintain progenitor cells, thus delaying neurogenesis. When the progenitor layer was electroporated with dominant-negative RhoA (DNRhoA), the cells underwent precocious neural differentiation. Remarkably, in the electroporated region, an ectopic formation of blood vessels was observed. These observations support the idea that the blood vessel patterning in CNS is regulated by local environment, where neural progenitor cells appear to prevent the vessel formation. We will discuss relationships between vascular patterning and neural differentiation. doi: 10.1016/j.diff.2010.09.054
P49 Cell reprogramming factors of neural crest cells
K. Watanabe, Y. Takase, Y. Takahashi Nara Institute of Science and Technology, Nara, Japan E-mail address: [email protected] (K. Watanabe)
S33
The iPS-technology has opened a previously unexplored way to study the mechanisms of cell differentiation. Thus, cells that have already differentiated or committed to a certain cell type can potentially be reprogrammed to either a less differentiated state or a different type of cells. Such cell reprogramming technology has so far been applied to an in vitro situation. Toward the goal of achieving cell reprogramming directly in vivo, we have focused on neural crest cells (NCCs), multipotent precursors of peripheral nervous system. NCCs are highly amenable for in vivo gene manipulation using in ovo electroporation technique, particularly in chicken embryos. In addition, the entire cell lineage derived from NCCs has been described, and many transcriptional factors are available that regulate the development of NCCs and their derivatives. Combining these advantages of NCCs with recently established technologies such as tet-on inducible expression (Watanabe et al., 2007. Dev. Biol.) and transposon-mediated stable integration of electroporated genes (Sato et al., 2007. Dev. Biol.), NCCs have become an excellent model to achieve the in vivo cell reprogramming. We have found that Sox9, Sox 10, and canonical Wnt signal are powerful candidates with which NCCs and their derivatives can be reprogrammed directly in vivo. doi: 10.1016/j.diff.2010.09.055
P50 Role of glioma produced CM1 in the brain tumor tropism of human neural stem cells
J. Jeon a, S. Cho b, K. Cho c, Y. Lee b, M. Lee a a
Medical Science Research Center, School of Medicine, Ajou University, Suwon, Republic of Korea b Division of Molecular Life Science, Hanyang University, Ansan, Republic of Korea c Department of Neurosurgery, School of Medicine, Ajou University, Suwon, Republic of Korea E-mail address: [email protected] (J. Jeon)
Stem cells from various lineages have well known for its migration tendency toward glioma and become attractive vehicles to deliver therapeutic genes to brain tumors. However, which factors and mechanisms work in these functions is not yet known. To identify those factors and mechanisms, we analyzed the brain tumor-specific gene expression profile using microarray analysis. We choose 14 candidate genes, reported to play roles in tumor metastasis, angiogenesis, and cell motility to further experiments. Chemoattract molecule 1 (CM1) is one of them, and highly expressed in malignancy tumors. Real time polymerase chain reaction and Immuno-histochemistry assay results support that. To check possibility of NSC motility reinforcement by CM1, in vitro boyden chamber assay was performed. CM1 dramatically induced the migration of human neural stem cells (NSC) two times as VEGF, previously known chemoattract molecule, did. Interestingly, when CM1 over-expressing cells were transplanted in rat brain, it attracted NSCs, which were transplanted in the same or contra-lateral hemisphere. These migrations were mediated by FAK downstream signaling pathway controlling actin and tubulin dynamics. We prepared cytosine deaminase gene encoding NSC (CD-NSC) line by retrovirus transduction. We explored their therapeutic potential on brain tumor model in the presence of prodrug 5-fluorocytosine (5-FC). CD-NSCs injected to animal that transplanted CM1 over expressing cells mixed with C6 rat glioma cell followed by 5-FC administration for 2 weeks. In more CM1 over-expressing cells were transplanted to the animal, tumor volume was significantly decreased compare less transplanted it. Additionally, many stem cells showed dramatic
NPR-A knockdown also resulted in a marked downregulation of phosphorylated Akt, which is essential for ES cell self-renewal. Furthermore, NPR-A knockdown induced accumulation of ES cells in the G1-phase of the cell cycle. Interestingly, we found that ANP was expressed in self-renewing ES cells, whereas its level was reduced after ES cell differentiation by the withdrawal of leukemia inhibitory factor. Treatment of ES cells with exogenous ANP upregulated the expression of Oct4 and Nanog, and this upregulation depended on NPR-A signaling, because it was completely reversed by pretreatment with NPR-A antagonist or cGMP-dependent protein kinase inhibitor. Also, ANP increased the expression level of phosphorylated Akt. These findings provide a novel role for NPR-A in the maintenance of self-renewal and pluripotency of ES cells. doi: 10.1016/j.diff.2010.09.053
P48 Formation of blood vessels is controlled by neural progenitor cells in the central nervous system
Teruaki Takahashi, Yuta Takase, Ryosuke Tadokoro, Yoshiko Takahashi Nara Institute of Science and Technology (NAIST), Nara, Japan E-mail address: [email protected] (T. Takahashi)
Vascular networks of blood vessels are stereotypically patterned in early developing embryos. It remains unknown how the vascular patterns are established in three-dimensional tissues including peripheral organs and the central nervous system (CNS). We study the mechanisms underlying the patterning of blood vessels in CNS using early spinal cord as a model. In chicken E4 embryos, endothelial cells of somitic origin penetrate into the spinal cord through the ventral edge. Subsequently, forming vessels extend dorsally along determined paths. We found that these paths coincide with the interface between the layer of progenitor cells (medial) and the layer of differentiated neurons and glial cells (lateral). Since the blood vessels do not invade the progenitor layer, we reasoned that the progenitor layer elicits inhibitory influence on the blood vessel formation. To test this, we experimentally promoted neural/glial differentiation in the progenitor territory. The small GTPase RhoA is known to maintain progenitor cells, thus delaying neurogenesis. When the progenitor layer was electroporated with dominant-negative RhoA (DNRhoA), the cells underwent precocious neural differentiation. Remarkably, in the electroporated region, an ectopic formation of blood vessels was observed. These observations support the idea that the blood vessel patterning in CNS is regulated by local environment, where neural progenitor cells appear to prevent the vessel formation. We will discuss relationships between vascular patterning and neural differentiation. doi: 10.1016/j.diff.2010.09.054
P49 Cell reprogramming factors of neural crest cells
K. Watanabe, Y. Takase, Y. Takahashi Nara Institute of Science and Technology, Nara, Japan E-mail address: [email protected] (K. Watanabe)
S33
The iPS-technology has opened a previously unexplored way to study the mechanisms of cell differentiation. Thus, cells that have already differentiated or committed to a certain cell type can potentially be reprogrammed to either a less differentiated state or a different type of cells. Such cell reprogramming technology has so far been applied to an in vitro situation. Toward the goal of achieving cell reprogramming directly in vivo, we have focused on neural crest cells (NCCs), multipotent precursors of peripheral nervous system. NCCs are highly amenable for in vivo gene manipulation using in ovo electroporation technique, particularly in chicken embryos. In addition, the entire cell lineage derived from NCCs has been described, and many transcriptional factors are available that regulate the development of NCCs and their derivatives. Combining these advantages of NCCs with recently established technologies such as tet-on inducible expression (Watanabe et al., 2007. Dev. Biol.) and transposon-mediated stable integration of electroporated genes (Sato et al., 2007. Dev. Biol.), NCCs have become an excellent model to achieve the in vivo cell reprogramming. We have found that Sox9, Sox 10, and canonical Wnt signal are powerful candidates with which NCCs and their derivatives can be reprogrammed directly in vivo. doi: 10.1016/j.diff.2010.09.055
P50 Role of glioma produced CM1 in the brain tumor tropism of human neural stem cells
J. Jeon a, S. Cho b, K. Cho c, Y. Lee b, M. Lee a a
Medical Science Research Center, School of Medicine, Ajou University, Suwon, Republic of Korea b Division of Molecular Life Science, Hanyang University, Ansan, Republic of Korea c Department of Neurosurgery, School of Medicine, Ajou University, Suwon, Republic of Korea E-mail address: [email protected] (J. Jeon)
Stem cells from various lineages have well known for its migration tendency toward glioma and become attractive vehicles to deliver therapeutic genes to brain tumors. However, which factors and mechanisms work in these functions is not yet known. To identify those factors and mechanisms, we analyzed the brain tumor-specific gene expression profile using microarray analysis. We choose 14 candidate genes, reported to play roles in tumor metastasis, angiogenesis, and cell motility to further experiments. Chemoattract molecule 1 (CM1) is one of them, and highly expressed in malignancy tumors. Real time polymerase chain reaction and Immuno-histochemistry assay results support that. To check possibility of NSC motility reinforcement by CM1, in vitro boyden chamber assay was performed. CM1 dramatically induced the migration of human neural stem cells (NSC) two times as VEGF, previously known chemoattract molecule, did. Interestingly, when CM1 over-expressing cells were transplanted in rat brain, it attracted NSCs, which were transplanted in the same or contra-lateral hemisphere. These migrations were mediated by FAK downstream signaling pathway controlling actin and tubulin dynamics. We prepared cytosine deaminase gene encoding NSC (CD-NSC) line by retrovirus transduction. We explored their therapeutic potential on brain tumor model in the presence of prodrug 5-fluorocytosine (5-FC). CD-NSCs injected to animal that transplanted CM1 over expressing cells mixed with C6 rat glioma cell followed by 5-FC administration for 2 weeks. In more CM1 over-expressing cells were transplanted to the animal, tumor volume was significantly decreased compare less transplanted it. Additionally, many stem cells showed dramatic