- Email: [email protected]
P71. Stem cells and gene expression during hemichordate head regeneration
S40
Abstract / Differentiation 80 (2010) S17–S63
Stem cells are tightly linked to their niche or microenvironment, which regulates their behaviors. The germline stem cell (GSC) niche in Drosophila and Caenorhabditis elegans containing easily identifiable GSCs has been extensively characterized on the molecular and genetic levels, revealing the importance of multiple signaling pathways and cellular processes. However, the mammalian GSC niche is less defined because unequivocal identification of GSCs has also not been achieved. In the mouse testis, a subset of primitive spermatogonia termed ‘‘undifferentiated spermatogonia’’ or ‘‘Aundiff’’ includes stem cells that constitute an as-yet-unidentified subpopulation. The Aundiff population are located on the basal membrane within the seminiferous tubules, and are biased toward vascular network in the interstitial tissue. Differentiating spermatogonia left these regions and dispersed throughout the basal compartment of the seminiferous tubules. According to these findings the mammalian GSC niche is likely to be located around the vascular-associated regions. However, no specialized niche substructure and their cellular components within the seminiferous tubules have not been identified. To explore the detailed substructute and cellular components of the GSC niche, we initiated a comprehensive identification of the genes expressed around the vascular-associated regions within the seminiferous tubules. First, we isolated distinct cell-type populations between vasculature-associated regions and the other tubule bounding regions by Laser Capture Microdissection (LCM). Using these samples, we analyzed their gene expression differences by microarray. Until now, we verified 18 genes preferentially expressed around the vascular-associated regions by in situ hybridization. Within this subset, 2 and 16 genes were expressed in germ-line and somatic cells, respectively. We anticipate that characterization of spatio-temporal expression patterns corresponding to putative stem cells will lead to greater understanding of stem cell–niche interactions. doi: 10.1016/j.diff.2010.09.074
P69 Towards CNS injury regeneration: Derivation of oligodendrocyte precursor cells from bone marrow stromal cells
Y.P. Tsui a,b, Y.S. Chan b, D.K.Y. Shum a
suggesting maturation into myelinating oligodendrocytes. Our findings indicate BMSCs as a possible source of OPCs for autologous transplantation. The results also support the possibility of directed differentiation BMSCs along specific neural/glial lineages. doi: 10.1016/j.diff.2010.09.075
P70 Collective migration of keratinocytes induced by EGF requires their proliferative capacity
D. Nanba a,b, F. Toki a,b, Y. Barrandon b a
Ehime University, Toon, Japan ´de´rale de Lausanne (EPFL), Lausanne, Ecole Polytechnique Fe Switzerland E-mail address: [email protected] (D. Nanba)
b
Activation of EGFR signaling induces collective migration of keratinocytes during wound healing and tumor invasion. However, collective behaviors of keratinocytes by EGFR activation with different growth capacity have not been elucidated. Here, we show coupling of proliferative and EGF-induced migratory capacities of cultured human epidermal keratinocytes. EGF immediately induced collective migration and contraction of keratinocytes with significant and limited proliferative capacities, respectively. These collective behaviors were caused through the phosphorylation of myosin regulatory light chain and subsequent actomyosin interaction. PI3K/Rac1 activity directed the actin filament organization and modes of EGF response in keratinocytes. Furthermore, Rac1 activity was also essential for reproductive capacity of keratinocytes. This study links proliferative and EGF-induced migratory capacities of keratinocytes through PI3K/ Rac1 signaling and suggests that collective migration of keratinocytes activated by EGFR signaling with higher reproductive and tumor-initiating capacities contributes considerably to re-epithelialization, and tumor invasion and metastasis. doi: 10.1016/j.diff.2010.09.076
a
Department of Biochemistry, The University of Hong Kong, Hong Kong, China b Department of Physiology, The University of Hong Kong, Hong Kong, China E-mail address: [email protected] (Y.P. Tsui)
CNS injury induces demyelination and severe loss of function. We hypothesize that transplantation of adult bone marrowderived oligodendrocyte precursor cells (OPCs) serves roles in neuroprotection and myelin repair. We attempted therefore to direct differentiation of bone marrow stromal cells (BMSCs, adult rats) along the oligodendroglial lineage in vitro. Haematopoietic cells were removed upon subcultures of the BMSCs. The BMSCs were then expanded as non-adherent spheres until they expressed markers of neural/glial progenitors (BM-NGPs). These BM-NGPs were then maintained in adherent culture supplemented with b-heregulin (b-Her), PDGF-AA, bFGF and forskolin. In the course of 2–3 weeks, Z95% of the BM-NGPs expressed OPC markers—NG2 and PDGFRa. These differentiated cells could be maintained in culture for up to 3 months with no decline in OPC marker expression. To test for myelinating function of these BMSC-derived OPCs (BM-OPCs), the cells were cocultured with dorsal root ganglion neurons. In 2 weeks, the BM-OPCs extended myelin basic protein-immunopositive processes along neurites,
P71 Stem cells and gene expression during hemichordate head regeneration
Tom Humphreys University of Hawaii, Manoa, Honolulu, Hawaii, HI 96822, USA E-mail address: [email protected] Hemichordate acorn worms grow a new head on the anterior wound surface when the body is transected. In Ptychodera flava, a blastema is evident by 3 days and a new full-sized, functional head differentiates on the cut stump by 12 days. The hemichordate nervous system provides the most ancestral extant example of the dorsal hollow brain, formed by neurulation, characteristic of chordates and humans. Only in hemichordates does such a nervous system regenerate significantly. Animals throughout the phylogenetic scale share similar developmental gene regulatory networks, and our work, along with that of others, supports the conclusion that the same gene regulatory networks occur in hemichordate and chordate neural differentiation. Examination of the regeneration process by whole mount
Abstract / Differentiation 80 (2010) S17–S63
in situ hybridization (WISH) of sequences from Yamanaka pluripotency factors or ‘‘stem cell’’ genes, as defined by genes inducing pluripotency in mammalian somatic cells, and developmental organizer genes reveals a cascade of gene expression associated with the steps of regeneration. A subset of the stem cell genes produce the first WISH signals before a blastema is formed. The second set of stem cell genes begins to generate signal as the blastema is established and this signal persists in the blastema as organizer and early development gene signals appear as differentiation proceeds. These results suggest that activation of cells with regenerative capability involves the early expression of stem cell genes in the cells that will form the blastema. Signal from some stem cell genes continues as the expression of typical embryonic gene regulatory networks are activated for the differentiation of the tissues. In planarians, regeneration is based on mobilization of stem cells residing in the tissue of the animal to form a regeneration blastema. During regeneration of an amphibian limb, a blastema is also formed, apparently by dedifferentiation of tissue cells in the cut limb to stem cells which accumulate to establish the blastema. The nature of the cells and the tissue origin of the cells that participate in hemichordate blastema formation and head regeneration are being investigated. Supported by the PBRC Biomedical Fund UHF#12-209-04. doi: 10.1016/j.diff.2010.09.077
P72 Mesenchymal progenitors distinct from muscle satellite cells contribute to ectopic fat cell formation in skeletal muscle
A. Uezumi a, S. Fukada b, H. Yamada a, S. Takeda c, K. Tsuchida a a
Fujita Health University, Toyoake, Japan Osaka University, Osaka, Japan c National Institute of Neuroscience, NCNP, Tokyo, Japan E-mail address: [email protected] (A. Uezumi) b
Adult skeletal muscle possesses a remarkable regenerative ability. This depends on satellite cells that function as adult muscle stem cells. Despite such a high regenerative capacity, ectopic fat cells emerge in skeletal muscle in several pathological conditions. Since occurrence of ectopic fat is usually associated with loss or extreme atrophy of muscle, the idea that dysregulation of fate-switch between muscle and adipose lineages in satellite cells may underlie this pathological change has been emerged. However, the contribution of satellite cells to in vivo fat formation has not been demonstrated. Here, we prospectively identified PDGFRa + mesenchymal progenitors, distinct from satellite cells, in the mouse muscle interstitium. We show that, of the muscle-derived cell populations, only PDGFRa + cells exhibit efficient adipogenic differentiation both in vitro and in vivo. Reciprocal transplantations between regenerating and degenerating muscles, and co-culture experiments reveal that adipogenesis of PDGFRa + cells is strongly inhibited by the presence of satellite cell-derived myofibers. PDGFRa + mesenchymal progenitors can be isolated from human muscle, and adipogenic potential is highly enriched in this population. Taken together, our results suggest that PDGFRa + mesenchymal progenitors are the major contributor to ectopic fat cell formation in skeletal muscle, and emphasize that cellular interaction between muscle cells and PDGFRa + mesenchymal progenitors, not the fate decision of satellite cells, has a considerable impact on muscle homeostasis. doi: 10.1016/j.diff.2010.09.078
S41
P73 Regulation of myeloid leukemia by the cell fate determinant Musashi
Takahiro Ito a, Hyog Young Kwon a, Bryan Zimdahl a, Kendra L. Congdon a, Jordan Blum a, William E. Lento a, Chen Zhao a, Anand Lagoo a, Gareth Gerrard b, Letizia Foroni b, John Goldman b, Harriet Goh c, Soo-Hyun Kim c, Dong-Wook Kim c, Charles Chuah d, Vivian G. Oehler e, Jerald P. Radich e, Craig T. Jordan f, Tannishtha Reya a a
Duke University Medical Center, Durham, USA Imperial College London, London, UK c The Catholic University of Korea, Seoul, Republic of Korea d Duke-NUS Graduate Medical School, Singapore e Fred Hutchinson Cancer Research Center, Seattle, USA f University of Rochester School of Medicine, Rochester, USA E-mail address: [email protected] (T. Reya) b
Chronic myelogenous leukemia (CML) can progress from an indolent chronic phase to an aggressive blast crisis phase but the molecular basis of this transition remains poorly understood. Here we have used mouse models of CML to show that disease progression is regulated by the Musashi-Numb signaling axis. Specifically we find that chronic phase is marked by high and blast crisis phase by low levels of Numb expression, and that ectopic expression of Numb promotes differentiation and impairs advanced phase disease in vivo. As a possible explanation for the decreased levels of Numb in blast crisis phase, we show that NUP98-HOXA9, an oncogene associated with blast crisis CML, can trigger expression of Musashi 2 (Msi2) an RNA binding protein which in turn represses Numb. Importantly, loss of Msi2 significantly impairs the development and propagation of blast crisis CML in vitro and in vivo. Finally, we show that Msi2 expression is not only highly upregulated during human CML progression but is also an early indicator of poorer prognosis. These data show that the Musashi-Numb pathway can control the differentiation of CML cells, and raise the possibility that targeting this pathway may provide a new strategy for therapy of aggressive leukemias. doi: 10.1016/j.diff.2010.09.079
P74 Age-related changes in prospectively isolated muscle satellite cells
M. Ikemoto-Uezumi a, A. Uezumi b, K. Tsuchida b, S. Fukada c, N. Hashimoto a a
National Center for Geriatrics and Gerontology, Aichi, Japan Fujita Health University, Aichi, Japan c Osaka University, Osaka, Japan E-mail address: [email protected] (M. Ikemoto-Uezumi) b
Sarcopenia is the loss of skeletal muscle mass and strength with age. The causes of sarcopenia are probably manifold and remain to be completely elucidated. One of the possible causes could be the decline of stem cell function. Satellite cell is muscle-specific stem cell and is responsible for the postnatal muscle maintenance, growth and regeneration. We showed that muscle regenerative potential of aged mice (24–30 months old) remarkably declined compared with that of young mice (2–3 months old) after injection with cardiotoxin (CTX). To study age-related changes of satellite cells, we prospectively
Abstract / Differentiation 80 (2010) S17–S63
Stem cells are tightly linked to their niche or microenvironment, which regulates their behaviors. The germline stem cell (GSC) niche in Drosophila and Caenorhabditis elegans containing easily identifiable GSCs has been extensively characterized on the molecular and genetic levels, revealing the importance of multiple signaling pathways and cellular processes. However, the mammalian GSC niche is less defined because unequivocal identification of GSCs has also not been achieved. In the mouse testis, a subset of primitive spermatogonia termed ‘‘undifferentiated spermatogonia’’ or ‘‘Aundiff’’ includes stem cells that constitute an as-yet-unidentified subpopulation. The Aundiff population are located on the basal membrane within the seminiferous tubules, and are biased toward vascular network in the interstitial tissue. Differentiating spermatogonia left these regions and dispersed throughout the basal compartment of the seminiferous tubules. According to these findings the mammalian GSC niche is likely to be located around the vascular-associated regions. However, no specialized niche substructure and their cellular components within the seminiferous tubules have not been identified. To explore the detailed substructute and cellular components of the GSC niche, we initiated a comprehensive identification of the genes expressed around the vascular-associated regions within the seminiferous tubules. First, we isolated distinct cell-type populations between vasculature-associated regions and the other tubule bounding regions by Laser Capture Microdissection (LCM). Using these samples, we analyzed their gene expression differences by microarray. Until now, we verified 18 genes preferentially expressed around the vascular-associated regions by in situ hybridization. Within this subset, 2 and 16 genes were expressed in germ-line and somatic cells, respectively. We anticipate that characterization of spatio-temporal expression patterns corresponding to putative stem cells will lead to greater understanding of stem cell–niche interactions. doi: 10.1016/j.diff.2010.09.074
P69 Towards CNS injury regeneration: Derivation of oligodendrocyte precursor cells from bone marrow stromal cells
Y.P. Tsui a,b, Y.S. Chan b, D.K.Y. Shum a
suggesting maturation into myelinating oligodendrocytes. Our findings indicate BMSCs as a possible source of OPCs for autologous transplantation. The results also support the possibility of directed differentiation BMSCs along specific neural/glial lineages. doi: 10.1016/j.diff.2010.09.075
P70 Collective migration of keratinocytes induced by EGF requires their proliferative capacity
D. Nanba a,b, F. Toki a,b, Y. Barrandon b a
Ehime University, Toon, Japan ´de´rale de Lausanne (EPFL), Lausanne, Ecole Polytechnique Fe Switzerland E-mail address: [email protected] (D. Nanba)
b
Activation of EGFR signaling induces collective migration of keratinocytes during wound healing and tumor invasion. However, collective behaviors of keratinocytes by EGFR activation with different growth capacity have not been elucidated. Here, we show coupling of proliferative and EGF-induced migratory capacities of cultured human epidermal keratinocytes. EGF immediately induced collective migration and contraction of keratinocytes with significant and limited proliferative capacities, respectively. These collective behaviors were caused through the phosphorylation of myosin regulatory light chain and subsequent actomyosin interaction. PI3K/Rac1 activity directed the actin filament organization and modes of EGF response in keratinocytes. Furthermore, Rac1 activity was also essential for reproductive capacity of keratinocytes. This study links proliferative and EGF-induced migratory capacities of keratinocytes through PI3K/ Rac1 signaling and suggests that collective migration of keratinocytes activated by EGFR signaling with higher reproductive and tumor-initiating capacities contributes considerably to re-epithelialization, and tumor invasion and metastasis. doi: 10.1016/j.diff.2010.09.076
a
Department of Biochemistry, The University of Hong Kong, Hong Kong, China b Department of Physiology, The University of Hong Kong, Hong Kong, China E-mail address: [email protected] (Y.P. Tsui)
CNS injury induces demyelination and severe loss of function. We hypothesize that transplantation of adult bone marrowderived oligodendrocyte precursor cells (OPCs) serves roles in neuroprotection and myelin repair. We attempted therefore to direct differentiation of bone marrow stromal cells (BMSCs, adult rats) along the oligodendroglial lineage in vitro. Haematopoietic cells were removed upon subcultures of the BMSCs. The BMSCs were then expanded as non-adherent spheres until they expressed markers of neural/glial progenitors (BM-NGPs). These BM-NGPs were then maintained in adherent culture supplemented with b-heregulin (b-Her), PDGF-AA, bFGF and forskolin. In the course of 2–3 weeks, Z95% of the BM-NGPs expressed OPC markers—NG2 and PDGFRa. These differentiated cells could be maintained in culture for up to 3 months with no decline in OPC marker expression. To test for myelinating function of these BMSC-derived OPCs (BM-OPCs), the cells were cocultured with dorsal root ganglion neurons. In 2 weeks, the BM-OPCs extended myelin basic protein-immunopositive processes along neurites,
P71 Stem cells and gene expression during hemichordate head regeneration
Tom Humphreys University of Hawaii, Manoa, Honolulu, Hawaii, HI 96822, USA E-mail address: [email protected] Hemichordate acorn worms grow a new head on the anterior wound surface when the body is transected. In Ptychodera flava, a blastema is evident by 3 days and a new full-sized, functional head differentiates on the cut stump by 12 days. The hemichordate nervous system provides the most ancestral extant example of the dorsal hollow brain, formed by neurulation, characteristic of chordates and humans. Only in hemichordates does such a nervous system regenerate significantly. Animals throughout the phylogenetic scale share similar developmental gene regulatory networks, and our work, along with that of others, supports the conclusion that the same gene regulatory networks occur in hemichordate and chordate neural differentiation. Examination of the regeneration process by whole mount
Abstract / Differentiation 80 (2010) S17–S63
in situ hybridization (WISH) of sequences from Yamanaka pluripotency factors or ‘‘stem cell’’ genes, as defined by genes inducing pluripotency in mammalian somatic cells, and developmental organizer genes reveals a cascade of gene expression associated with the steps of regeneration. A subset of the stem cell genes produce the first WISH signals before a blastema is formed. The second set of stem cell genes begins to generate signal as the blastema is established and this signal persists in the blastema as organizer and early development gene signals appear as differentiation proceeds. These results suggest that activation of cells with regenerative capability involves the early expression of stem cell genes in the cells that will form the blastema. Signal from some stem cell genes continues as the expression of typical embryonic gene regulatory networks are activated for the differentiation of the tissues. In planarians, regeneration is based on mobilization of stem cells residing in the tissue of the animal to form a regeneration blastema. During regeneration of an amphibian limb, a blastema is also formed, apparently by dedifferentiation of tissue cells in the cut limb to stem cells which accumulate to establish the blastema. The nature of the cells and the tissue origin of the cells that participate in hemichordate blastema formation and head regeneration are being investigated. Supported by the PBRC Biomedical Fund UHF#12-209-04. doi: 10.1016/j.diff.2010.09.077
P72 Mesenchymal progenitors distinct from muscle satellite cells contribute to ectopic fat cell formation in skeletal muscle
A. Uezumi a, S. Fukada b, H. Yamada a, S. Takeda c, K. Tsuchida a a
Fujita Health University, Toyoake, Japan Osaka University, Osaka, Japan c National Institute of Neuroscience, NCNP, Tokyo, Japan E-mail address: [email protected] (A. Uezumi) b
Adult skeletal muscle possesses a remarkable regenerative ability. This depends on satellite cells that function as adult muscle stem cells. Despite such a high regenerative capacity, ectopic fat cells emerge in skeletal muscle in several pathological conditions. Since occurrence of ectopic fat is usually associated with loss or extreme atrophy of muscle, the idea that dysregulation of fate-switch between muscle and adipose lineages in satellite cells may underlie this pathological change has been emerged. However, the contribution of satellite cells to in vivo fat formation has not been demonstrated. Here, we prospectively identified PDGFRa + mesenchymal progenitors, distinct from satellite cells, in the mouse muscle interstitium. We show that, of the muscle-derived cell populations, only PDGFRa + cells exhibit efficient adipogenic differentiation both in vitro and in vivo. Reciprocal transplantations between regenerating and degenerating muscles, and co-culture experiments reveal that adipogenesis of PDGFRa + cells is strongly inhibited by the presence of satellite cell-derived myofibers. PDGFRa + mesenchymal progenitors can be isolated from human muscle, and adipogenic potential is highly enriched in this population. Taken together, our results suggest that PDGFRa + mesenchymal progenitors are the major contributor to ectopic fat cell formation in skeletal muscle, and emphasize that cellular interaction between muscle cells and PDGFRa + mesenchymal progenitors, not the fate decision of satellite cells, has a considerable impact on muscle homeostasis. doi: 10.1016/j.diff.2010.09.078
S41
P73 Regulation of myeloid leukemia by the cell fate determinant Musashi
Takahiro Ito a, Hyog Young Kwon a, Bryan Zimdahl a, Kendra L. Congdon a, Jordan Blum a, William E. Lento a, Chen Zhao a, Anand Lagoo a, Gareth Gerrard b, Letizia Foroni b, John Goldman b, Harriet Goh c, Soo-Hyun Kim c, Dong-Wook Kim c, Charles Chuah d, Vivian G. Oehler e, Jerald P. Radich e, Craig T. Jordan f, Tannishtha Reya a a
Duke University Medical Center, Durham, USA Imperial College London, London, UK c The Catholic University of Korea, Seoul, Republic of Korea d Duke-NUS Graduate Medical School, Singapore e Fred Hutchinson Cancer Research Center, Seattle, USA f University of Rochester School of Medicine, Rochester, USA E-mail address: [email protected] (T. Reya) b
Chronic myelogenous leukemia (CML) can progress from an indolent chronic phase to an aggressive blast crisis phase but the molecular basis of this transition remains poorly understood. Here we have used mouse models of CML to show that disease progression is regulated by the Musashi-Numb signaling axis. Specifically we find that chronic phase is marked by high and blast crisis phase by low levels of Numb expression, and that ectopic expression of Numb promotes differentiation and impairs advanced phase disease in vivo. As a possible explanation for the decreased levels of Numb in blast crisis phase, we show that NUP98-HOXA9, an oncogene associated with blast crisis CML, can trigger expression of Musashi 2 (Msi2) an RNA binding protein which in turn represses Numb. Importantly, loss of Msi2 significantly impairs the development and propagation of blast crisis CML in vitro and in vivo. Finally, we show that Msi2 expression is not only highly upregulated during human CML progression but is also an early indicator of poorer prognosis. These data show that the Musashi-Numb pathway can control the differentiation of CML cells, and raise the possibility that targeting this pathway may provide a new strategy for therapy of aggressive leukemias. doi: 10.1016/j.diff.2010.09.079
P74 Age-related changes in prospectively isolated muscle satellite cells
M. Ikemoto-Uezumi a, A. Uezumi b, K. Tsuchida b, S. Fukada c, N. Hashimoto a a
National Center for Geriatrics and Gerontology, Aichi, Japan Fujita Health University, Aichi, Japan c Osaka University, Osaka, Japan E-mail address: [email protected] (M. Ikemoto-Uezumi) b
Sarcopenia is the loss of skeletal muscle mass and strength with age. The causes of sarcopenia are probably manifold and remain to be completely elucidated. One of the possible causes could be the decline of stem cell function. Satellite cell is muscle-specific stem cell and is responsible for the postnatal muscle maintenance, growth and regeneration. We showed that muscle regenerative potential of aged mice (24–30 months old) remarkably declined compared with that of young mice (2–3 months old) after injection with cardiotoxin (CTX). To study age-related changes of satellite cells, we prospectively