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Two definitive adult-type HSC subsets coexistent in mouse
Poster Presentations/ Experimental Hematology 41 (2013) S23–S75
P1086 - TWO DEFINITIVE ADULT-TYPE HSC SUBSETS COEXISTENT IN MOUSE Mihaela Crisan1, Chris Vink1, Tomoko Yamada-Inagawa1, Alex Neagu1, Caterina Purini1, Karine Bollerot1, Sofia Karkanpouna2, Reinir van der Linden1, Alvin Chan1, and Elaine Dzierzak1 1 Cell Biology, Erasmus MC Stem Cell Institute, Rotterdam, Netherlands; 2Molecular and Cell Biology, Centre of Biomedical genetics, Leiden, Netherlands Adult-type hematopoietic stem cells (HSCs) emerge in the aorta-gonad-mesonephros (AGM) region in the mid-gestation mouse embryo. Thereafter, HSCs migrate to the fetal liver (FL) and at birth, colonize the adult bone marrow. The molecular signaling cascades controlling HSCs in the AGM region is not yet known. Exogenous Hedgehog (Hh) added to AGM explants prior to HSC generation induces AGM HSCs and BMP pathway inhibition abolishes AGM HSC activity. In the FL, the BMP signaling pathway is not required for hematopoiesis. A role for the MAPK signaling pathway has been demonstrated in the adult hematopoiesis, but whether this pathway also controls AGM HSCs is not known. We hypothesize that Hh and BMP control the induction/expansion of AGM HSCs and cooperate with the MAPK pathway to control their maintenance and differentiation. To test whether HSCs are directly activated by BMP, we used transgenic BMP-response element (BRE)-gfp mice, in which GFP expression is an indicator of BMP pathway activation. When BRE-gfp E11 AGM cells were sorted for GFP expression and injected into adult irradiated mice, all long term repopulating HSCs were found in the BMP activated (GFP+) fraction. We next asked whether HSCs in the FL are activated by BMP. Surprisingly, we found high level, multilineage, self-renewing HSCs in both GFP+ and GFP- fractions. This suggests that subsets of HSCs shift from an exclusively BMP-activated state, to a state of non-activation. This shift may be involved in the expansion of HSCs in the FL. Since HSCs expand in explant cultures of E11 AGMs, we examined whether after explant, AGM HSCs remain BMP-activated. Interestingly, after explant, both GFP+ and GFP- fractions contained multilineage, self-renewing HSCs. In such explants when Hh or MAPK pathways are inhibited, only the non-BMP-activated HSC activity is abolished. The Hh inhibition effect on AGM HSCs could be rescued by exogeneous VEGF addition, but VEGF could not rescue the HSC defects caused by MAPK inhibition. These results suggest that VEGF acts downstream of Hh and further activates the MAPK pathway to control AGM HSCs. In conclusion, our data provide clear evidence of an interactive molecular cascade involving the BMP, Hh and MAPK signaling pathways in the control of AGM HSC development.
S45
P1087 - TWO ADULT-TYPE HSC SUBSETS COEXISTENT IN MOUSE Mihaela Crisan1, Chris Vink1, Tomoko Yamada-Inagawa1, Alex Neagu1, Caterina Purini1, Sofia Karkanpouna2, Reinir van der Linden1, and Elaine Dzierzak1 1 Cell Biology, Erasmus MC Stem Cell Institute, Rotterdam, Netherlands; 2Dept. of Molecular and Cell Biology, Centre of Biomedical Genetics, Leiden, Netherlands Adult-type hematopoietic stem cells (HSCs) emerge in the aorta-gonad-mesonephros (AGM) region in the mid-gestation mouse embryo. Thereafter, HSCs migrate to the fetal liver (FL) and at birth, colonize the adult bone marrow. The molecular signaling cascades controlling HSCs in the AGM region is not yet known. Exogenous Hedgehog (Hh) added to AGM explants prior to HSC generation induces AGM HSCs and BMP pathway inhibition abolishes AGM HSC activity. In the FL, the BMP signaling pathway is not required for hematopoiesis. A role for the MAPK signaling pathway has been demonstrated in the adult hematopoiesis, but whether this pathway also controls AGM HSCs is not known. We hypothesize that Hh and BMP control the induction/expansion of AGM HSCs and cooperate with the MAPK pathway to control their maintenance and differentiation. To test whether HSCs are directly activated by BMP, we used transgenic BMP-response element (BRE)-gfp mice, in which GFP expression is an indicator of BMP pathway activation. When BRE-gfp E11 AGM cells were sorted for GFP expression and injected into adult irradiated mice, all long term repopulating HSCs were found in the BMP activated (GFP+) fraction. We next asked whether HSCs in the FL are activated by BMP. Surprisingly, we found high level, multilineage, self renewing HSCs in both GFP+ and GFP- fractions. This suggests that subsets of HSCs shift from an exclusively BMP-activated state, to a state of non-activation. This shift may be involved in the expansion of HSCs in the FL. Since HSCs expand in explant cultures of E11 AGMs, we examined whether after explant, AGM HSCs remain BMP-activated. Interestingly, after explant, both GFP+ and GFP- fractions contained multilineage, self-renewing HSCs. In such explants when Hh or MAPK pathways are inhibited, only the non-BMP-activated HSC activity is abolished. The Hh inhibition effect on AGM HSCs could be rescued by exogeneous VEGF addition, but VEGF could not rescue the HSC defects caused by MAPK inhibition. These results suggest that VEGF acts downstream of Hh and further activates the MAPK pathway to control AGM HSCs. In conclusion, our data provide clear evidence of an interactive molecular cascade involving the BMP, Hh and MAPK signaling pathways in the control of AGM HSC development. P1088 - THE CYTOKINE-INDUCED MICRORNA193B MODULATES CKIT EXPRESSION AND STAT5 SIGNALING Nadine Haetscher1,2, Yonatan Feuermann3, Susanne Wingert1,2, Frederic Thalheimer1,2, Christian Weiser1, Andreas Kowarsch4, Fabian Theis4, Timm Schroeder5, Michael Rieger1,2, and Lothar Hennighausen3 1 Haematology Oncology, J.W. Goethe University Clinics Frankfurt, Frankfurt (Main), Hessen, Germany; 2Stem Cell Biology and Stem Cell Therapy, GeorgSpeyer-Haus, Frankfurt, Germany; 3Laboratory of Genetics and Physiology, NIDDK, National Institute of Health, Bethesda, Maryland, USA; 4Institute of Bioinformatics, Helmholtz Zentrum M€unchen, Neuherberg, Germany; 5Stem Cell Dynamics Research Unit, Helmholtz Zentrum M€unchen, Neuherberg, Germany Normal hematopoiesis requires the right composition of microRNAs (miRNA) controlled in a developmental stage and cell type-dependent fashion. MiRNAs are small, non-coding RNAs with the ability to simultaneously target multiple mRNAs for the orchestration of complex gene expression networks in development, regeneration and disease. In a screening for STAT5A/B -induced miRNAs in highly purified murine LT-HSCs, we found miR193b to be upregulated by the cytokine Thrombopoietin. Constitutive activation of STAT5A/B enhances HSC self-renewal and thereby can contribute to myeloproliferative disease. Videomicroscopy-based single cell tracking revealed that miR193b expression in HSCs leads to increased cell death and slowed proliferation in vitro, and a loss of functional HSCs in vivo, both caused by a targeted downregulation of ckit surface expression. To determine the physiological function of miR193b in hematopoiesis, we generated a knock-out mouse model. In miR193b-/mice, peripheral white blood cell numbers of all lineages are increased, hematopoietic progenitors show accelerated proliferation capacity, and aged animals gain an enlarged HSC pool. During the stress response to 5-FU treatment, miR193b-/- mice show an altered cytokine profile and an upregulated platelet production. The potential molecular mechanism explaining these observations is the increased basal activation of STAT5A/ B, which we observed in miR193b-/- bone marrow and spleen cells. RNA sequencing of defined stem and progenitor cells before and after cytokine stimulation will shed light on the regulatory network governed by miR193b. The role of miR193b in contributing to feedback regulation of cytokine responsiveness and STAT5 signaling warrants further investigation and may reveal new therapeutic strategies for hematologic diseases.
P1086 - TWO DEFINITIVE ADULT-TYPE HSC SUBSETS COEXISTENT IN MOUSE Mihaela Crisan1, Chris Vink1, Tomoko Yamada-Inagawa1, Alex Neagu1, Caterina Purini1, Karine Bollerot1, Sofia Karkanpouna2, Reinir van der Linden1, Alvin Chan1, and Elaine Dzierzak1 1 Cell Biology, Erasmus MC Stem Cell Institute, Rotterdam, Netherlands; 2Molecular and Cell Biology, Centre of Biomedical genetics, Leiden, Netherlands Adult-type hematopoietic stem cells (HSCs) emerge in the aorta-gonad-mesonephros (AGM) region in the mid-gestation mouse embryo. Thereafter, HSCs migrate to the fetal liver (FL) and at birth, colonize the adult bone marrow. The molecular signaling cascades controlling HSCs in the AGM region is not yet known. Exogenous Hedgehog (Hh) added to AGM explants prior to HSC generation induces AGM HSCs and BMP pathway inhibition abolishes AGM HSC activity. In the FL, the BMP signaling pathway is not required for hematopoiesis. A role for the MAPK signaling pathway has been demonstrated in the adult hematopoiesis, but whether this pathway also controls AGM HSCs is not known. We hypothesize that Hh and BMP control the induction/expansion of AGM HSCs and cooperate with the MAPK pathway to control their maintenance and differentiation. To test whether HSCs are directly activated by BMP, we used transgenic BMP-response element (BRE)-gfp mice, in which GFP expression is an indicator of BMP pathway activation. When BRE-gfp E11 AGM cells were sorted for GFP expression and injected into adult irradiated mice, all long term repopulating HSCs were found in the BMP activated (GFP+) fraction. We next asked whether HSCs in the FL are activated by BMP. Surprisingly, we found high level, multilineage, self-renewing HSCs in both GFP+ and GFP- fractions. This suggests that subsets of HSCs shift from an exclusively BMP-activated state, to a state of non-activation. This shift may be involved in the expansion of HSCs in the FL. Since HSCs expand in explant cultures of E11 AGMs, we examined whether after explant, AGM HSCs remain BMP-activated. Interestingly, after explant, both GFP+ and GFP- fractions contained multilineage, self-renewing HSCs. In such explants when Hh or MAPK pathways are inhibited, only the non-BMP-activated HSC activity is abolished. The Hh inhibition effect on AGM HSCs could be rescued by exogeneous VEGF addition, but VEGF could not rescue the HSC defects caused by MAPK inhibition. These results suggest that VEGF acts downstream of Hh and further activates the MAPK pathway to control AGM HSCs. In conclusion, our data provide clear evidence of an interactive molecular cascade involving the BMP, Hh and MAPK signaling pathways in the control of AGM HSC development.
S45
P1087 - TWO ADULT-TYPE HSC SUBSETS COEXISTENT IN MOUSE Mihaela Crisan1, Chris Vink1, Tomoko Yamada-Inagawa1, Alex Neagu1, Caterina Purini1, Sofia Karkanpouna2, Reinir van der Linden1, and Elaine Dzierzak1 1 Cell Biology, Erasmus MC Stem Cell Institute, Rotterdam, Netherlands; 2Dept. of Molecular and Cell Biology, Centre of Biomedical Genetics, Leiden, Netherlands Adult-type hematopoietic stem cells (HSCs) emerge in the aorta-gonad-mesonephros (AGM) region in the mid-gestation mouse embryo. Thereafter, HSCs migrate to the fetal liver (FL) and at birth, colonize the adult bone marrow. The molecular signaling cascades controlling HSCs in the AGM region is not yet known. Exogenous Hedgehog (Hh) added to AGM explants prior to HSC generation induces AGM HSCs and BMP pathway inhibition abolishes AGM HSC activity. In the FL, the BMP signaling pathway is not required for hematopoiesis. A role for the MAPK signaling pathway has been demonstrated in the adult hematopoiesis, but whether this pathway also controls AGM HSCs is not known. We hypothesize that Hh and BMP control the induction/expansion of AGM HSCs and cooperate with the MAPK pathway to control their maintenance and differentiation. To test whether HSCs are directly activated by BMP, we used transgenic BMP-response element (BRE)-gfp mice, in which GFP expression is an indicator of BMP pathway activation. When BRE-gfp E11 AGM cells were sorted for GFP expression and injected into adult irradiated mice, all long term repopulating HSCs were found in the BMP activated (GFP+) fraction. We next asked whether HSCs in the FL are activated by BMP. Surprisingly, we found high level, multilineage, self renewing HSCs in both GFP+ and GFP- fractions. This suggests that subsets of HSCs shift from an exclusively BMP-activated state, to a state of non-activation. This shift may be involved in the expansion of HSCs in the FL. Since HSCs expand in explant cultures of E11 AGMs, we examined whether after explant, AGM HSCs remain BMP-activated. Interestingly, after explant, both GFP+ and GFP- fractions contained multilineage, self-renewing HSCs. In such explants when Hh or MAPK pathways are inhibited, only the non-BMP-activated HSC activity is abolished. The Hh inhibition effect on AGM HSCs could be rescued by exogeneous VEGF addition, but VEGF could not rescue the HSC defects caused by MAPK inhibition. These results suggest that VEGF acts downstream of Hh and further activates the MAPK pathway to control AGM HSCs. In conclusion, our data provide clear evidence of an interactive molecular cascade involving the BMP, Hh and MAPK signaling pathways in the control of AGM HSC development. P1088 - THE CYTOKINE-INDUCED MICRORNA193B MODULATES CKIT EXPRESSION AND STAT5 SIGNALING Nadine Haetscher1,2, Yonatan Feuermann3, Susanne Wingert1,2, Frederic Thalheimer1,2, Christian Weiser1, Andreas Kowarsch4, Fabian Theis4, Timm Schroeder5, Michael Rieger1,2, and Lothar Hennighausen3 1 Haematology Oncology, J.W. Goethe University Clinics Frankfurt, Frankfurt (Main), Hessen, Germany; 2Stem Cell Biology and Stem Cell Therapy, GeorgSpeyer-Haus, Frankfurt, Germany; 3Laboratory of Genetics and Physiology, NIDDK, National Institute of Health, Bethesda, Maryland, USA; 4Institute of Bioinformatics, Helmholtz Zentrum M€unchen, Neuherberg, Germany; 5Stem Cell Dynamics Research Unit, Helmholtz Zentrum M€unchen, Neuherberg, Germany Normal hematopoiesis requires the right composition of microRNAs (miRNA) controlled in a developmental stage and cell type-dependent fashion. MiRNAs are small, non-coding RNAs with the ability to simultaneously target multiple mRNAs for the orchestration of complex gene expression networks in development, regeneration and disease. In a screening for STAT5A/B -induced miRNAs in highly purified murine LT-HSCs, we found miR193b to be upregulated by the cytokine Thrombopoietin. Constitutive activation of STAT5A/B enhances HSC self-renewal and thereby can contribute to myeloproliferative disease. Videomicroscopy-based single cell tracking revealed that miR193b expression in HSCs leads to increased cell death and slowed proliferation in vitro, and a loss of functional HSCs in vivo, both caused by a targeted downregulation of ckit surface expression. To determine the physiological function of miR193b in hematopoiesis, we generated a knock-out mouse model. In miR193b-/mice, peripheral white blood cell numbers of all lineages are increased, hematopoietic progenitors show accelerated proliferation capacity, and aged animals gain an enlarged HSC pool. During the stress response to 5-FU treatment, miR193b-/- mice show an altered cytokine profile and an upregulated platelet production. The potential molecular mechanism explaining these observations is the increased basal activation of STAT5A/ B, which we observed in miR193b-/- bone marrow and spleen cells. RNA sequencing of defined stem and progenitor cells before and after cytokine stimulation will shed light on the regulatory network governed by miR193b. The role of miR193b in contributing to feedback regulation of cytokine responsiveness and STAT5 signaling warrants further investigation and may reveal new therapeutic strategies for hematologic diseases.