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A stromal organiser cell that directs neonatal spleen regeneration
Poster Presentations/Experimental Hematology 43 (2015) S51–S106
3185 - A NEW GENE TRANSFER SYSTEM FOR HEMATOPOIETIC STEM CELLS UNDER PHYSIOLOGICAL CONDITION Yoko Tajima, Satoshi Yamazaki, and Hiromitsu Nakauchi Division of Stem Cell Therapy, The institute of medical science, the University of Tokyo, Tokyo, Japan To date, most studies on hematopoietic stem cells (HSCs) have been conducted by the transplantation of donor-derived HSCs into lethally irradiated recipient mice together with competitor cells. Indeed, much progress has been made in characterizing the cell types that repopulate in peripheral blood, and a number of genes involved were identified. However, the developmental process of HSCs in steady state under the unstressed physiological condition remains largely unknown. To address this issue, we undertook to establish a transplantation-independent assay system . In the current study, we focused on the RCAS/TVA system with a cell type specific marking strategy. RCAS is a retroviral vector derived from avian leukosis virus (ALV) and recognizes its specific receptor TVA, expressed only on avian cells. The advantage of this system is that any cloned gene can be introduced into TVA expressing mammalian cells using RCAS virus. Moreover it is also possible to conduct not only lineage tracing under normal physiological conditions, but also gain-offunction and loss-of-function assays in vivo. We have generated some TVA knock-in mouse lines, anti-TVA monoclonal antibody and ALV pseudotyped lentivirus vector. Using these items, we are now trying to establish an efficient gene transfer system for integrating the gene of interest into HSCs. Finally, these strategies will lead to basic understanding of gene function under normal hematopoiesis without transplantation.
3186 - DECLINED PRESENTATION P38a–ACTIVATED PURINE METABOLISM PLAYS A CRUCIAL ROLE IN PROLIFERATION OF TRANSPLANTED HEMATOPOIETIC STEM CELLS Keiyo Takubo National Center for Global Health and Medicine, Tokyo, Japan Hematopoietic stem cells (HSCs) are maintained in a quiescent state through the activation of specific metabolic pathways including glycolysis. However, how stress hematopoiesis including bone marrow transplantation (BMT) induces metabolic alteration in HSCs is still unclear. Here we report an unexpected role of the dominant isoform of p38MAPK family, p38a, in the metabolic homeostasis of proliferating HSCs. p38MAPK is reported as a major inducer of replicative senescence in various somatic cells. Contrary to our expectation, loss of p38a in HSCs did not confer a senescence-free phenotype but showed defective transplantation capacity. In addition, 5-FU treatment in p38a-deficient mice exhibited defective recovery of hematopoiesis. These observations suggested a requirement of p38a in proper maintenance of HSCs during stress hematopoiesis. To identify a downstream event in p38a-deficient HSCs after stress, we evaluated phosphorylation status of p38MAPK by intracellular FACS. p38MAPK was dynamically phosphorylated on day 1 after BMT, but the phosphorylation returned to normal on day 7. In parallel with this, p38a deficiency delayed cell cycle progression immediately after transplantation. In addition, p38a deficiency altered the amount of amino acid and purine-related metabolites. The expression levels of purine synthesizing enzymes were reduced in p38a-deficient HSCs after BMT. Thus p38a modulates cell cycle progression of HSCs during stress through the tuning of purine metabolism.
S97
3187 - A NEW STRATEGY FOR MANIPULATING EXPRESSION AND ACTIVITY OF GEMININ COULD MAKE IT POSSIBLE TO REGULATE CELL FATES OF HSCS Yoshihiro Takihara1, Yoshinori Ohno1, Kyoko Suzuki-Takedachi1, Toshiaki Kurogi1, Mimoko Santo1, Kazuhito Naka1, and Shin’ichiro Yasunaga1,2 1 RIRBM, Hiroshima University, Hiroshima, Japan; 2Department of Biochemistry, Faculty of Medicine, Fukuoka University., Fukuoka, Japan Hematopoietic stem cell (HSC) transplantation therapy has provided an epochal clinical outcome. There developed has, however, been no practically available technology for expanding HSCs ex vivo, which has hampered the further advance and application of HSCs for immunotherapy and gene therapy. Since hematopoietic cytokines and niche molecules have been shown not to be enough for the expansion, we have focused on the cell intrinsic factors. By utilizing a biochemical as well as genetic technology, we previously demonstrated that Polycomb-group complex 1 and Hoxb4/Hoxa9 regulate HSCs through the ubiquitin proteasome system-mediated direct regulation of Geminin protein, and shRNA-mediated knockdown of Geminin and visualization of Geminin in Geminin-EYFP knock-in mice further supported our hypothesis that Geminin acts as a key regulator for determining cell fate of HSCs, i.e., cellular quiescence, self-renewal and cellular differentiation. Geminin negatively regulates DNA replication and chromatin remodeling through the direct interaction with Cdt1 and Brahma/Brg1, respectively. In this study we generated a recombinant Geminin protein fused with a membrane translocating motif (MTM) of FGF4, which was designated as cell-penetrating (CP-) Geminin. We have demonstrated that Geminin expression in NIH 3T3 cells was altered by CP-Geminin. Biologically, CP-Geminin transduction controlled the cell cycle, i.e., CP-Geminin efficiently suppressed G0/G1 to S-phase transition to keep cellular quiescence. Next, we further showed that Geminin with a mutation in each of the functional domains acted in a dominant-negative fashion against endogenous Geminin. By using the Geminin derivatives we currently aim at regulating activity as well as expression of Geminin to perform detailed verification of our hypothesis described above and further to generate a new strategy for manipulating the HSC activity.
3188 - A STROMAL ORGANISER CELL THAT DIRECTS NEONATAL SPLEEN REGENERATION Jon Tan1,2 and Takeshi Watanabe3,2 1 The Australian National University, Acton, Australian Capital Territory, Australia; 2 Kyoto University, Sakyo-ku, Kyoto, Japan; 3The Tazuke Kofukai Medical Research Institute/Kitano Hospital, Kita-ku, Osaka, Japan Development of lymphoid tissue is determined by interactions between stromal lymphoid tissue organiser (LTo) and hematopoietic lymphoid tissue inducer cells. A failure for LTo to receive appropriate lymphotoxin engagement during embryogenesis leads to a complete cessation of lymph node and Peyer’s patch development. This identifies LTo as a key stromal population for lymphoid tissue organogenesis. However, little is known about the equivalent stromal cells that induce spleen development. In order to identify cell populations that specifically drive spleen organogenesis, a model for spleen tissue regeneration involving cell aggregate graft construction and transplantation was developed. Dissociation of neonatal murine spleen stromal tissue into a single cell suspension, followed by aggregation over collagen sheets and transplantation under the kidney capsule of adult mouse recipients, led to the induction of spleen tissue regeneration in a highly reproducible manner. By manipulating the cell input of graft aggregates, a spleen stromal organiser cell-type critical for spleen tissue formation was identified. We now demonstrate that spleen regeneration from neonatal tissue is governed by a lineage of CD45-MAdCAM-1+CD31+CD201+ stromal cells, distinct from previously reported mesenchymal organisers directing lymph node development.
3185 - A NEW GENE TRANSFER SYSTEM FOR HEMATOPOIETIC STEM CELLS UNDER PHYSIOLOGICAL CONDITION Yoko Tajima, Satoshi Yamazaki, and Hiromitsu Nakauchi Division of Stem Cell Therapy, The institute of medical science, the University of Tokyo, Tokyo, Japan To date, most studies on hematopoietic stem cells (HSCs) have been conducted by the transplantation of donor-derived HSCs into lethally irradiated recipient mice together with competitor cells. Indeed, much progress has been made in characterizing the cell types that repopulate in peripheral blood, and a number of genes involved were identified. However, the developmental process of HSCs in steady state under the unstressed physiological condition remains largely unknown. To address this issue, we undertook to establish a transplantation-independent assay system . In the current study, we focused on the RCAS/TVA system with a cell type specific marking strategy. RCAS is a retroviral vector derived from avian leukosis virus (ALV) and recognizes its specific receptor TVA, expressed only on avian cells. The advantage of this system is that any cloned gene can be introduced into TVA expressing mammalian cells using RCAS virus. Moreover it is also possible to conduct not only lineage tracing under normal physiological conditions, but also gain-offunction and loss-of-function assays in vivo. We have generated some TVA knock-in mouse lines, anti-TVA monoclonal antibody and ALV pseudotyped lentivirus vector. Using these items, we are now trying to establish an efficient gene transfer system for integrating the gene of interest into HSCs. Finally, these strategies will lead to basic understanding of gene function under normal hematopoiesis without transplantation.
3186 - DECLINED PRESENTATION P38a–ACTIVATED PURINE METABOLISM PLAYS A CRUCIAL ROLE IN PROLIFERATION OF TRANSPLANTED HEMATOPOIETIC STEM CELLS Keiyo Takubo National Center for Global Health and Medicine, Tokyo, Japan Hematopoietic stem cells (HSCs) are maintained in a quiescent state through the activation of specific metabolic pathways including glycolysis. However, how stress hematopoiesis including bone marrow transplantation (BMT) induces metabolic alteration in HSCs is still unclear. Here we report an unexpected role of the dominant isoform of p38MAPK family, p38a, in the metabolic homeostasis of proliferating HSCs. p38MAPK is reported as a major inducer of replicative senescence in various somatic cells. Contrary to our expectation, loss of p38a in HSCs did not confer a senescence-free phenotype but showed defective transplantation capacity. In addition, 5-FU treatment in p38a-deficient mice exhibited defective recovery of hematopoiesis. These observations suggested a requirement of p38a in proper maintenance of HSCs during stress hematopoiesis. To identify a downstream event in p38a-deficient HSCs after stress, we evaluated phosphorylation status of p38MAPK by intracellular FACS. p38MAPK was dynamically phosphorylated on day 1 after BMT, but the phosphorylation returned to normal on day 7. In parallel with this, p38a deficiency delayed cell cycle progression immediately after transplantation. In addition, p38a deficiency altered the amount of amino acid and purine-related metabolites. The expression levels of purine synthesizing enzymes were reduced in p38a-deficient HSCs after BMT. Thus p38a modulates cell cycle progression of HSCs during stress through the tuning of purine metabolism.
S97
3187 - A NEW STRATEGY FOR MANIPULATING EXPRESSION AND ACTIVITY OF GEMININ COULD MAKE IT POSSIBLE TO REGULATE CELL FATES OF HSCS Yoshihiro Takihara1, Yoshinori Ohno1, Kyoko Suzuki-Takedachi1, Toshiaki Kurogi1, Mimoko Santo1, Kazuhito Naka1, and Shin’ichiro Yasunaga1,2 1 RIRBM, Hiroshima University, Hiroshima, Japan; 2Department of Biochemistry, Faculty of Medicine, Fukuoka University., Fukuoka, Japan Hematopoietic stem cell (HSC) transplantation therapy has provided an epochal clinical outcome. There developed has, however, been no practically available technology for expanding HSCs ex vivo, which has hampered the further advance and application of HSCs for immunotherapy and gene therapy. Since hematopoietic cytokines and niche molecules have been shown not to be enough for the expansion, we have focused on the cell intrinsic factors. By utilizing a biochemical as well as genetic technology, we previously demonstrated that Polycomb-group complex 1 and Hoxb4/Hoxa9 regulate HSCs through the ubiquitin proteasome system-mediated direct regulation of Geminin protein, and shRNA-mediated knockdown of Geminin and visualization of Geminin in Geminin-EYFP knock-in mice further supported our hypothesis that Geminin acts as a key regulator for determining cell fate of HSCs, i.e., cellular quiescence, self-renewal and cellular differentiation. Geminin negatively regulates DNA replication and chromatin remodeling through the direct interaction with Cdt1 and Brahma/Brg1, respectively. In this study we generated a recombinant Geminin protein fused with a membrane translocating motif (MTM) of FGF4, which was designated as cell-penetrating (CP-) Geminin. We have demonstrated that Geminin expression in NIH 3T3 cells was altered by CP-Geminin. Biologically, CP-Geminin transduction controlled the cell cycle, i.e., CP-Geminin efficiently suppressed G0/G1 to S-phase transition to keep cellular quiescence. Next, we further showed that Geminin with a mutation in each of the functional domains acted in a dominant-negative fashion against endogenous Geminin. By using the Geminin derivatives we currently aim at regulating activity as well as expression of Geminin to perform detailed verification of our hypothesis described above and further to generate a new strategy for manipulating the HSC activity.
3188 - A STROMAL ORGANISER CELL THAT DIRECTS NEONATAL SPLEEN REGENERATION Jon Tan1,2 and Takeshi Watanabe3,2 1 The Australian National University, Acton, Australian Capital Territory, Australia; 2 Kyoto University, Sakyo-ku, Kyoto, Japan; 3The Tazuke Kofukai Medical Research Institute/Kitano Hospital, Kita-ku, Osaka, Japan Development of lymphoid tissue is determined by interactions between stromal lymphoid tissue organiser (LTo) and hematopoietic lymphoid tissue inducer cells. A failure for LTo to receive appropriate lymphotoxin engagement during embryogenesis leads to a complete cessation of lymph node and Peyer’s patch development. This identifies LTo as a key stromal population for lymphoid tissue organogenesis. However, little is known about the equivalent stromal cells that induce spleen development. In order to identify cell populations that specifically drive spleen organogenesis, a model for spleen tissue regeneration involving cell aggregate graft construction and transplantation was developed. Dissociation of neonatal murine spleen stromal tissue into a single cell suspension, followed by aggregation over collagen sheets and transplantation under the kidney capsule of adult mouse recipients, led to the induction of spleen tissue regeneration in a highly reproducible manner. By manipulating the cell input of graft aggregates, a spleen stromal organiser cell-type critical for spleen tissue formation was identified. We now demonstrate that spleen regeneration from neonatal tissue is governed by a lineage of CD45-MAdCAM-1+CD31+CD201+ stromal cells, distinct from previously reported mesenchymal organisers directing lymph node development.