- Email: [email protected]
P50. Role of glioma produced CM1 in the brain tumor tropism of human neural stem 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
S34
Abstract / Differentiation 80 (2010) S17–S63
migratory capacity in much more CM1 over-expressing cell transplanted groups. These results suggest that malignant tumor derived CM1, induce FAK downstream signal pathway, could play an important role in NSC tropism for glioma cells for tumor growth inhibition gene deliver and may provide valuable information for establishment of a new stem cell base cancer therapeutic strategy. doi: 10.1016/j.diff.2010.09.056
P52 The role of heparan sulfate proteoglycans in Drosophila germline stem cell niche
Yoshiki Hayashi a, Satoru Kobayashi b, Hiroshi Nakato b a
National Institute for Basic Biology, Aichi, Japan University of Minnesota, Minnesota, USA E-mail address: [email protected] (Y. Hayashi) b
P51 Bistable cell fate specification as a result of stochastic fluctuations and collective spatial cell behaviour
Pa ldi Andra s, Stockholm Daniel, Edom-Vovard Fre´de´rique, Coutant Sophie, Sanatine Peggy, Yamagata Yoshiaki, Corre Guillaume, Laurent Le Guillou, Neildez-Nguyen Thi My Anh GENETHON, INSERM U951, Ecole Pratique des Hautes Etudes, Evry F91002, France E-mail address: [email protected] (P. Andra s)
Cell differentiation is traditionally seen as a sequence of genetically predetermined gene expression changes. This view has been challenged recently by the discovery of important stochastic fluctuations in gene expression and by the identification of the role these fluctuations may play in the cell fate decision. In particularly, it has been proposed that fully multipotent cells fluctuate slowly between states with varying likelihoods of differentiation and this spontaneous heterogeneity or ‘‘noise’’ is the central driving force behind multipotency. It has been proposed that the different phenotypic states correspond to attractor states in the ‘‘epigenetic landscape’’ defined by the network of genes. In our study we used myoblasts that express the differentiation marker CD56 considered as a sign of definitive fate commitment to the muscle pathway. We show experimentally that these cells fluctuate between two phenotypes. Using spatial distribution analysis of the cells in growing population, computer simulations of these observations and experimental test of the new predictions made by the model we show that the fluctuations follow a bistable dynamics driven by a microenvironment and phenotype dependent noise. We show that fluctuation between phenotypes is not a distinguishing feature of multipotent cells. Committed tissue cells (myoblasts, used in our study) also may fluctuate between different phenotypes. We suggest that phenotypic fluctuations are a general feature of any non-terminally differentiated cell, thus, reversion to a previous differentiation state is a normal event. The cellular microenvironment contributes actively to the fluctuations by increasing the noise level in cells with a given phenotype and decreasing it in others. Importantly, the microenvironment is created by the cells themselves as a consequence of their motility that creates random cell interactions. In this way each cell contributes to put together its own microenvironment that in turn stimulates it to fluctuate between the phenotypes until the most appropriate phenotypic state with low noise is found. This means that the attractor state is not cell intrinsic; rather it is specified by the joint action of the cell-intrinsic gene network and the network of cell-cell interactions. Both networks are subject to stochastic fluctuations. doi: 10.1016/j.diff.2010.09.057
Stem cell posses the remarkable characteristics to produce its daughter cell that retain a stem-cell identity and the other that differentiates. Stem cells reside in dedicated cellular microenvironments termed stem-cell niches. These niches dictate a stem-cell identity, maintain the stem cell population, and coordinate proper homeostatic production of differentiated cells. Drosophila germ line stem cells (GSCs) are one of the ideal models for stem cell study in vivo. In apical tips of both male and female gonads, GSCs associate with specially differentiated somatic gonadal cells, the niche cells. These niche cells generate niches for GSCs by secreting signaling molecules, such as TGF-beta ligand, Dpp and JAK/STAT ligand Upd, which are essential for GSC maintenance. Although these facts indicate that region of niche is defined by the spatial distribution of these ligands, molecular mechanisms that regulate ligand distribution within the niche is not characterized. Heparan sulfate proteoglycans (HSPGs) are one kind of glycoprotein which are expressed on the cell surface and/or in the extracellular matrix. Recent in vivo studies have shown that HSPGs play critical roles in regulating signaling during development by a variety of mechanisms including regulation of extracellular ligand distribution. Here we show that Drosophila HSPGs are essential components of GSC niche. Drosophila two glypians, a subfamily of HSPGs, dally and dally-like were expressed in niche cells of female and male gonads, respectively and were required for GSC maintenance. In GSCs of these glypican mutants, signaling which is essential for GSC maintenance were impaired. Conversely, when dally was ectopically expressed in ovaries, we observed expansion of GSC niche. These results showed that these glypicans define niche region by regulating range of niche signal activity, presumably by controlling ligand distribution. HSPGs are evolutionally conserved molecules, thus our results imply HSPGs are general niche components that define niche region molecularly. doi: 10.1016/j.diff.2010.09.058
P53 Towards a functional characterization of Rx genes in teleost retinal stem cells
S. Kirchmaier, J. Wittbrodt University of Heidelberg, Institute of Zoology, Heidelberg, Germany E-mail address: [email protected] (S. Kirchmaier)
Retinal homeobox (Rx) genes are master regulators of retinal development in all vertebrate species. They are implicated in the differentiation of photoreceptor cells (Arendt et al., 2004) and also in the regulation of cell movements during optic cup formation (Rembold et al., 2006). Interestingly, the loss of Rx gene expression is also associated with a reduction of proliferation in the developing
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
S34
Abstract / Differentiation 80 (2010) S17–S63
migratory capacity in much more CM1 over-expressing cell transplanted groups. These results suggest that malignant tumor derived CM1, induce FAK downstream signal pathway, could play an important role in NSC tropism for glioma cells for tumor growth inhibition gene deliver and may provide valuable information for establishment of a new stem cell base cancer therapeutic strategy. doi: 10.1016/j.diff.2010.09.056
P52 The role of heparan sulfate proteoglycans in Drosophila germline stem cell niche
Yoshiki Hayashi a, Satoru Kobayashi b, Hiroshi Nakato b a
National Institute for Basic Biology, Aichi, Japan University of Minnesota, Minnesota, USA E-mail address: [email protected] (Y. Hayashi) b
P51 Bistable cell fate specification as a result of stochastic fluctuations and collective spatial cell behaviour
Pa ldi Andra s, Stockholm Daniel, Edom-Vovard Fre´de´rique, Coutant Sophie, Sanatine Peggy, Yamagata Yoshiaki, Corre Guillaume, Laurent Le Guillou, Neildez-Nguyen Thi My Anh GENETHON, INSERM U951, Ecole Pratique des Hautes Etudes, Evry F91002, France E-mail address: [email protected] (P. Andra s)
Cell differentiation is traditionally seen as a sequence of genetically predetermined gene expression changes. This view has been challenged recently by the discovery of important stochastic fluctuations in gene expression and by the identification of the role these fluctuations may play in the cell fate decision. In particularly, it has been proposed that fully multipotent cells fluctuate slowly between states with varying likelihoods of differentiation and this spontaneous heterogeneity or ‘‘noise’’ is the central driving force behind multipotency. It has been proposed that the different phenotypic states correspond to attractor states in the ‘‘epigenetic landscape’’ defined by the network of genes. In our study we used myoblasts that express the differentiation marker CD56 considered as a sign of definitive fate commitment to the muscle pathway. We show experimentally that these cells fluctuate between two phenotypes. Using spatial distribution analysis of the cells in growing population, computer simulations of these observations and experimental test of the new predictions made by the model we show that the fluctuations follow a bistable dynamics driven by a microenvironment and phenotype dependent noise. We show that fluctuation between phenotypes is not a distinguishing feature of multipotent cells. Committed tissue cells (myoblasts, used in our study) also may fluctuate between different phenotypes. We suggest that phenotypic fluctuations are a general feature of any non-terminally differentiated cell, thus, reversion to a previous differentiation state is a normal event. The cellular microenvironment contributes actively to the fluctuations by increasing the noise level in cells with a given phenotype and decreasing it in others. Importantly, the microenvironment is created by the cells themselves as a consequence of their motility that creates random cell interactions. In this way each cell contributes to put together its own microenvironment that in turn stimulates it to fluctuate between the phenotypes until the most appropriate phenotypic state with low noise is found. This means that the attractor state is not cell intrinsic; rather it is specified by the joint action of the cell-intrinsic gene network and the network of cell-cell interactions. Both networks are subject to stochastic fluctuations. doi: 10.1016/j.diff.2010.09.057
Stem cell posses the remarkable characteristics to produce its daughter cell that retain a stem-cell identity and the other that differentiates. Stem cells reside in dedicated cellular microenvironments termed stem-cell niches. These niches dictate a stem-cell identity, maintain the stem cell population, and coordinate proper homeostatic production of differentiated cells. Drosophila germ line stem cells (GSCs) are one of the ideal models for stem cell study in vivo. In apical tips of both male and female gonads, GSCs associate with specially differentiated somatic gonadal cells, the niche cells. These niche cells generate niches for GSCs by secreting signaling molecules, such as TGF-beta ligand, Dpp and JAK/STAT ligand Upd, which are essential for GSC maintenance. Although these facts indicate that region of niche is defined by the spatial distribution of these ligands, molecular mechanisms that regulate ligand distribution within the niche is not characterized. Heparan sulfate proteoglycans (HSPGs) are one kind of glycoprotein which are expressed on the cell surface and/or in the extracellular matrix. Recent in vivo studies have shown that HSPGs play critical roles in regulating signaling during development by a variety of mechanisms including regulation of extracellular ligand distribution. Here we show that Drosophila HSPGs are essential components of GSC niche. Drosophila two glypians, a subfamily of HSPGs, dally and dally-like were expressed in niche cells of female and male gonads, respectively and were required for GSC maintenance. In GSCs of these glypican mutants, signaling which is essential for GSC maintenance were impaired. Conversely, when dally was ectopically expressed in ovaries, we observed expansion of GSC niche. These results showed that these glypicans define niche region by regulating range of niche signal activity, presumably by controlling ligand distribution. HSPGs are evolutionally conserved molecules, thus our results imply HSPGs are general niche components that define niche region molecularly. doi: 10.1016/j.diff.2010.09.058
P53 Towards a functional characterization of Rx genes in teleost retinal stem cells
S. Kirchmaier, J. Wittbrodt University of Heidelberg, Institute of Zoology, Heidelberg, Germany E-mail address: [email protected] (S. Kirchmaier)
Retinal homeobox (Rx) genes are master regulators of retinal development in all vertebrate species. They are implicated in the differentiation of photoreceptor cells (Arendt et al., 2004) and also in the regulation of cell movements during optic cup formation (Rembold et al., 2006). Interestingly, the loss of Rx gene expression is also associated with a reduction of proliferation in the developing