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Assessment of candidate microRNAs to supress leukemic cell function using an inducible lentiviral vector system
S92
Poster Presentations/Experimental Hematology 43 (2015) S51–S106
3164 - PLURIPOTENT STEM CELL MODELS OF RETICULAR DYSGENESIS AS A TOOL FOR ELUCIDATING THE POTENTIAL ROLE OF INTRACELLULAR BIOENERGETIC SYSTEMS ON CONTROLLING THE FATE OF HEMATOPOIETIC PROGENITORS Norikazu Saiki1,2, Koichi Oshima1, Akiyoshi Hirayama3, Tomoyoshi Soga3, Masaru Tomita3, Tatsutoshi Nakahata1, and Megumu K. Saito1 1 Center for iPS cell Research and Application, Kyoto Univ., Kyoto, Japan; 2Graduate school of medicine, Kyoto Univ., Kyoto, Japan; 3Institute for Advanced Biosciences, Keio Univ., Tsuruoka, Japan
3166 - DEVELOPMENT OF HUMAN HEMATO-LYMPHOID SYSTEM IN HUMAN CYTOKINE KNOCK-IN RAG2-/-IL2RG-/- MICE ENGRAFTED WITH PERIPHERAL BLOOD MOBILIZED CD34+ CELLS Yasuyuki Saito1,2, Jana M. Ellegast2, Daniel Kull3, Mathias Heikenw€alder3, Richard A. Flavell4, and Markus G. Manz2 1 Kobe University Graduate School of Medicine, Kobe, Hyogo, Japan; 2University Hospital Zurich, Zurich, Zurich, Switzerland; 3Technische Universit€at M€unchen and Helmholtz Zentrum M€unchen, Munich, Germany; 4Yale University, New Haven, Connecticut, USA
Metabolic plasticity of cytosolic to mitochondrial oxidative phosphorylation (OXPHOS) plays a critical role in differentiation process. However, the contribution of metabolic communication among subcellular components to the fate of progenitor cells remains unclear. Adenylate kinase 2(AK2) is an adenylate phosphotransferase through the reaction ATP + AMP 4 2ADP localized in the mitochondrial intermembrane space. Although AK2 mutations in human can cause a severe combined immuno deficiency with neutropenia, named reticular dysgenesis (RD), underlying mechanisms have not yet been elucidated. To address relationship between mitochondrial phosphotransfer and differentiation disorder in RD, we established induced pluripotent stem cells (iPSCs) from two RD patients. We differentiated RD-iPSCs into hematopoietic cells to evaluate whether the in vivo phenotype of RD is recapitulated and to examine which progenitors were responsible for the hematopoietic defect. The differentiation of mature neutrophils and T-lymphocytes and the output of CD34+KDR+ hemoangiogenic progenitors (HPCs) from RD-iPSCs ware profoundly impaired compared to AK2-supplemented RD-iPSCs. Next, using highthroughput metabolomics, we found that RD-iPSCs have latent alteration of metabolic network caused by AK2 deficiency. For detailed description of metabolic flux distribution of energy metabolism, we performed 13C-labeled substance tracer analysis. The distribution of isotopomer appeared to be unbalanced in AK2(-) clones, which indicated that AK2 mediates the intracellular metabolic communication. Moreover, we also applied metabolome analysis to differentiated cells containing HPCs. We extracted TCA cycle intermediate malate as featured metabolite which contributed to distinguish AK2(+) and AK2(-) clones. Perturbation of intracellular bioenergetics caused by differentiation may cause inappropriate optimization of AK2(-) metabolic state during transition into OXPHOS.
Transplantation of human CD34+ hematopoietic stem and progenitor cells (HSPCs) into severe immunocompromised newborn mice allows the development of a human hemato-lymphoid system (HHLS) in vivo. While fetal liver- or cord blood- derived CD34+ cells lead to high levels of engraftment, only low engraftment levels could be achieved after adult donor-derived CD34+ cell transplantation in the existing humanized mice models. We recently generated novel mouse strains in which human versions of cytokines (M-CSF and TPO with or without IL-3/GM-CSF) are knocked into Rag2-/-Il2rg-/- strains (MISTRG or MSTRG, respectively). In addition, human Sirpa is expressed as transgene in both strains. To evaluate human adult CD34+ cell engraftment in these mice, G-CSF mobilized CD34+ cells obtained from healthy donors were injected intrahepatically into sub-lethally irradiated newborn MISTRG or MSTRG mice, or into NOD/scid/Il2rg-/- (NSG) or Rag2-/-Il2rg-/-hSIRPATg (RGS) mice as controls. Both frequency as well as absolute number of human CD45+ cells 10-16 wks after injection were significantly higher in the bone marrow (BM) of MISTRG/MSTRG mice than that of NSG/RGS mice. We also observed substantial engraftment of human hematopoietic stem and progenitor cells in the BM of MISTRG/MSTRG mice. In addition, MISTRG/MSTRG mice supported higher level of human thymocyte development in the thymus. Besides lymphoid organs, development of human cells, in particular myeloid lineage cells, was significantly promoted in the peripheral tissues of MISTRG/MSTRG mice compared to those of NSG/RGS mice. Taken together, our humanized mice model supports adult donor-derived HSC engraftment and development of HHLS in primary lymphoid and non-lymphoid tissues. Our model thus has the potential for personalized studies of healthy hematopoiesis as well as hemato-immune system diseases derived from adult individuals.
3165 - ASSESSMENT OF CANDIDATE MICRORNAS TO SUPRESS LEUKEMIC CELL FUNCTION USING AN INDUCIBLE LENTIVIRAL VECTOR SYSTEM Jens Ruschmann1, Tobias Maetzig1, Kathrin Krowiorz2, Florian Kuchenbauer2, Oleh Petriv3, Carl Hansen3, and Keith Humphries1 1 BC Cancer Agency, Vancouver, British Columbia, Canada; 2Department of Internal Medicine III, University Hospital of Ulm, Ulm, Germany; 3Centre for High Throughput Biology, University of BC, Vancouver, British Columbia, Canada
3167 - MAINTENANCE OF HEMATOPOIETIC STEM AND PROGENITOR CELL PHENOTYPE OF INTRA-AORTIC CELL CLUSTERS IN THE AGM REGION THROUGH THE SOX17-NOTCH1-HES1 AXIS Kiyoka Saito, Ikuo Nobuhisa, Maha Anani, Kaho Harada, Satomi Takahashi, and Tetsuya Taga Tokyo medical and dental university, Tokyo, Japan
Using a high-throughput microfluidic real-time quantitative PCR platform, we developed miRNA-based expression signatures from enriched malignant subpopulations purified from over 50 patients with AML. From 25 miRNAs with significant differential expression between more mature leukemic cell populations (bulk, myeloid enriched and/or CD34-) and more primitive stem/progenitor leukemic populations (CD34+ and/or CD34+CD38-), we identified miRNAs including miR-150 that were upregulated in the more mature leukemic cell populations for further studies. To test whether overexpression of such miRNAs would induce differentiation and suppress leukemogenic activity in a murine leukemia model, we established a lentiviral system for the doxycycline inducible expression of miRNAs and assessed the impact of miR-150 expression in HoxA9/Meis1 (HA9M) leukemia model using a variety of in vitro and in vivo readouts. In vitro, there was a 50 fold increase of mature miR-150 expression upon addition of doxycycline within 72h that was associated with an increase in the differentiation markers CD11b and Gr1, a O10-fold growth disadvantage and a 60% reduction of c-kit+ cells relative to un-induced HA9M cells. After establishing HA9M leukemia in vivo for 21 days, miRNA expression was induced by switching mice to doxycycline containing chow, and peripheral blood was analyzed weekly. HA9M cells expressing miR-150 revealed a strong disadvantage compared to un-induced cells, resulting in an almost complete loss of miR-150 expressing cells after 2 weeks of miRNA induction and prolonged survival. Our results demonstrate that leads from patient based gene expression profiling coupled to an inducible lentiviral vector system have provided preliminary evidence of the potent ability of selective miRNA expression to overcome the differentiation block in a murine leukemia model that may significantly suppress leukemogenic activity.
The aorta-gonad-mesonephros (AGM) region, from which definitive hematopoiesis firstly arises in midgestation mouse embryo, has intra-aortic cell clusters (IACCs) containing hematopoietic stem cells and hematopoietic progenitor cells. In the previous study, we reported that a transcription factor Sox17 is expressed in the IACCs and that overexpression of Sox17 in CD45lowc-Kithigh cells comprising IACCs maintained the formation of such cell clusters and the multipotency in vitro during several passages. We and other groups showed that Notch1 is expressed in the IACCs, and Sox17 induces the Notch1 promoter activity. In the present study, we investigated a role of Notch1 in the IACCs and its detailed relationship with Sox17. The expression level of Notch1 was high in Sox17-transduced cells and the binding of Sox17 to the Notch1 promoter was observed by chromatin immunoprecipitation assay. We then examined whether Notch1 has a function to maintain the undifferentiated state of the IACCs. Cells transduced with the cleaved Notch1 intracellular domain (NICD, the active form) maintained the multipotent colony-forming activity in semisolid media during at least three passages. Moreover, cells stimulated by Jagged 1, which is known as the ligand of Notch, had a capacity to form multilineage colonies. In contrast, Jagged 1 stimulation of CD45-c-Kit-CD31+ endothelial cells, which are known to contain the hemogenic endothelium, inhibited the production of CD45+ hematopoietic cells. We also examined the function of Hes1 and Hes5, which are induced by the binding of NICD to Hes1 and Hes5 promoters, in the IACCs. Hes1-transduced cells have a capacity to form multilineage colonies and express marker genes for the undifferentiation state. In contrast, Hes5-transduced cells have a capacity to form erythroid colonies and express erythroid marker genes. These results suggest that Notch1 has an ability to maintain an undifferentiated state of the IACCs and Hes1 and Hes5 play different roles in the AGM hematopoiesis.
Poster Presentations/Experimental Hematology 43 (2015) S51–S106
3164 - PLURIPOTENT STEM CELL MODELS OF RETICULAR DYSGENESIS AS A TOOL FOR ELUCIDATING THE POTENTIAL ROLE OF INTRACELLULAR BIOENERGETIC SYSTEMS ON CONTROLLING THE FATE OF HEMATOPOIETIC PROGENITORS Norikazu Saiki1,2, Koichi Oshima1, Akiyoshi Hirayama3, Tomoyoshi Soga3, Masaru Tomita3, Tatsutoshi Nakahata1, and Megumu K. Saito1 1 Center for iPS cell Research and Application, Kyoto Univ., Kyoto, Japan; 2Graduate school of medicine, Kyoto Univ., Kyoto, Japan; 3Institute for Advanced Biosciences, Keio Univ., Tsuruoka, Japan
3166 - DEVELOPMENT OF HUMAN HEMATO-LYMPHOID SYSTEM IN HUMAN CYTOKINE KNOCK-IN RAG2-/-IL2RG-/- MICE ENGRAFTED WITH PERIPHERAL BLOOD MOBILIZED CD34+ CELLS Yasuyuki Saito1,2, Jana M. Ellegast2, Daniel Kull3, Mathias Heikenw€alder3, Richard A. Flavell4, and Markus G. Manz2 1 Kobe University Graduate School of Medicine, Kobe, Hyogo, Japan; 2University Hospital Zurich, Zurich, Zurich, Switzerland; 3Technische Universit€at M€unchen and Helmholtz Zentrum M€unchen, Munich, Germany; 4Yale University, New Haven, Connecticut, USA
Metabolic plasticity of cytosolic to mitochondrial oxidative phosphorylation (OXPHOS) plays a critical role in differentiation process. However, the contribution of metabolic communication among subcellular components to the fate of progenitor cells remains unclear. Adenylate kinase 2(AK2) is an adenylate phosphotransferase through the reaction ATP + AMP 4 2ADP localized in the mitochondrial intermembrane space. Although AK2 mutations in human can cause a severe combined immuno deficiency with neutropenia, named reticular dysgenesis (RD), underlying mechanisms have not yet been elucidated. To address relationship between mitochondrial phosphotransfer and differentiation disorder in RD, we established induced pluripotent stem cells (iPSCs) from two RD patients. We differentiated RD-iPSCs into hematopoietic cells to evaluate whether the in vivo phenotype of RD is recapitulated and to examine which progenitors were responsible for the hematopoietic defect. The differentiation of mature neutrophils and T-lymphocytes and the output of CD34+KDR+ hemoangiogenic progenitors (HPCs) from RD-iPSCs ware profoundly impaired compared to AK2-supplemented RD-iPSCs. Next, using highthroughput metabolomics, we found that RD-iPSCs have latent alteration of metabolic network caused by AK2 deficiency. For detailed description of metabolic flux distribution of energy metabolism, we performed 13C-labeled substance tracer analysis. The distribution of isotopomer appeared to be unbalanced in AK2(-) clones, which indicated that AK2 mediates the intracellular metabolic communication. Moreover, we also applied metabolome analysis to differentiated cells containing HPCs. We extracted TCA cycle intermediate malate as featured metabolite which contributed to distinguish AK2(+) and AK2(-) clones. Perturbation of intracellular bioenergetics caused by differentiation may cause inappropriate optimization of AK2(-) metabolic state during transition into OXPHOS.
Transplantation of human CD34+ hematopoietic stem and progenitor cells (HSPCs) into severe immunocompromised newborn mice allows the development of a human hemato-lymphoid system (HHLS) in vivo. While fetal liver- or cord blood- derived CD34+ cells lead to high levels of engraftment, only low engraftment levels could be achieved after adult donor-derived CD34+ cell transplantation in the existing humanized mice models. We recently generated novel mouse strains in which human versions of cytokines (M-CSF and TPO with or without IL-3/GM-CSF) are knocked into Rag2-/-Il2rg-/- strains (MISTRG or MSTRG, respectively). In addition, human Sirpa is expressed as transgene in both strains. To evaluate human adult CD34+ cell engraftment in these mice, G-CSF mobilized CD34+ cells obtained from healthy donors were injected intrahepatically into sub-lethally irradiated newborn MISTRG or MSTRG mice, or into NOD/scid/Il2rg-/- (NSG) or Rag2-/-Il2rg-/-hSIRPATg (RGS) mice as controls. Both frequency as well as absolute number of human CD45+ cells 10-16 wks after injection were significantly higher in the bone marrow (BM) of MISTRG/MSTRG mice than that of NSG/RGS mice. We also observed substantial engraftment of human hematopoietic stem and progenitor cells in the BM of MISTRG/MSTRG mice. In addition, MISTRG/MSTRG mice supported higher level of human thymocyte development in the thymus. Besides lymphoid organs, development of human cells, in particular myeloid lineage cells, was significantly promoted in the peripheral tissues of MISTRG/MSTRG mice compared to those of NSG/RGS mice. Taken together, our humanized mice model supports adult donor-derived HSC engraftment and development of HHLS in primary lymphoid and non-lymphoid tissues. Our model thus has the potential for personalized studies of healthy hematopoiesis as well as hemato-immune system diseases derived from adult individuals.
3165 - ASSESSMENT OF CANDIDATE MICRORNAS TO SUPRESS LEUKEMIC CELL FUNCTION USING AN INDUCIBLE LENTIVIRAL VECTOR SYSTEM Jens Ruschmann1, Tobias Maetzig1, Kathrin Krowiorz2, Florian Kuchenbauer2, Oleh Petriv3, Carl Hansen3, and Keith Humphries1 1 BC Cancer Agency, Vancouver, British Columbia, Canada; 2Department of Internal Medicine III, University Hospital of Ulm, Ulm, Germany; 3Centre for High Throughput Biology, University of BC, Vancouver, British Columbia, Canada
3167 - MAINTENANCE OF HEMATOPOIETIC STEM AND PROGENITOR CELL PHENOTYPE OF INTRA-AORTIC CELL CLUSTERS IN THE AGM REGION THROUGH THE SOX17-NOTCH1-HES1 AXIS Kiyoka Saito, Ikuo Nobuhisa, Maha Anani, Kaho Harada, Satomi Takahashi, and Tetsuya Taga Tokyo medical and dental university, Tokyo, Japan
Using a high-throughput microfluidic real-time quantitative PCR platform, we developed miRNA-based expression signatures from enriched malignant subpopulations purified from over 50 patients with AML. From 25 miRNAs with significant differential expression between more mature leukemic cell populations (bulk, myeloid enriched and/or CD34-) and more primitive stem/progenitor leukemic populations (CD34+ and/or CD34+CD38-), we identified miRNAs including miR-150 that were upregulated in the more mature leukemic cell populations for further studies. To test whether overexpression of such miRNAs would induce differentiation and suppress leukemogenic activity in a murine leukemia model, we established a lentiviral system for the doxycycline inducible expression of miRNAs and assessed the impact of miR-150 expression in HoxA9/Meis1 (HA9M) leukemia model using a variety of in vitro and in vivo readouts. In vitro, there was a 50 fold increase of mature miR-150 expression upon addition of doxycycline within 72h that was associated with an increase in the differentiation markers CD11b and Gr1, a O10-fold growth disadvantage and a 60% reduction of c-kit+ cells relative to un-induced HA9M cells. After establishing HA9M leukemia in vivo for 21 days, miRNA expression was induced by switching mice to doxycycline containing chow, and peripheral blood was analyzed weekly. HA9M cells expressing miR-150 revealed a strong disadvantage compared to un-induced cells, resulting in an almost complete loss of miR-150 expressing cells after 2 weeks of miRNA induction and prolonged survival. Our results demonstrate that leads from patient based gene expression profiling coupled to an inducible lentiviral vector system have provided preliminary evidence of the potent ability of selective miRNA expression to overcome the differentiation block in a murine leukemia model that may significantly suppress leukemogenic activity.
The aorta-gonad-mesonephros (AGM) region, from which definitive hematopoiesis firstly arises in midgestation mouse embryo, has intra-aortic cell clusters (IACCs) containing hematopoietic stem cells and hematopoietic progenitor cells. In the previous study, we reported that a transcription factor Sox17 is expressed in the IACCs and that overexpression of Sox17 in CD45lowc-Kithigh cells comprising IACCs maintained the formation of such cell clusters and the multipotency in vitro during several passages. We and other groups showed that Notch1 is expressed in the IACCs, and Sox17 induces the Notch1 promoter activity. In the present study, we investigated a role of Notch1 in the IACCs and its detailed relationship with Sox17. The expression level of Notch1 was high in Sox17-transduced cells and the binding of Sox17 to the Notch1 promoter was observed by chromatin immunoprecipitation assay. We then examined whether Notch1 has a function to maintain the undifferentiated state of the IACCs. Cells transduced with the cleaved Notch1 intracellular domain (NICD, the active form) maintained the multipotent colony-forming activity in semisolid media during at least three passages. Moreover, cells stimulated by Jagged 1, which is known as the ligand of Notch, had a capacity to form multilineage colonies. In contrast, Jagged 1 stimulation of CD45-c-Kit-CD31+ endothelial cells, which are known to contain the hemogenic endothelium, inhibited the production of CD45+ hematopoietic cells. We also examined the function of Hes1 and Hes5, which are induced by the binding of NICD to Hes1 and Hes5 promoters, in the IACCs. Hes1-transduced cells have a capacity to form multilineage colonies and express marker genes for the undifferentiation state. In contrast, Hes5-transduced cells have a capacity to form erythroid colonies and express erythroid marker genes. These results suggest that Notch1 has an ability to maintain an undifferentiated state of the IACCs and Hes1 and Hes5 play different roles in the AGM hematopoiesis.