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SIRT1 deacetylase is essential for hematopoietic stem cell activity
Poster Presentations/ Experimental Hematology 41 (2013) S23–S75
P1047 - MOLECULAR MECHANISM OF HEMATOPOIETIC LINEAGE CHOICE INSTRUCTION Max Endele1 and Timm Schroeder2,1 1 Stem Cell Dynamics, Helmholtz Center Munich, Neuherberg, Germany; 2 Department of Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland Stem and progenitor cell fate decisions are regulated by the timed integration of extracellular signals and intracellular molecular states. Through continuous single cell observations we could recently provide evidence for the long disputed instructive effect of the hematopoietic cytokines M- and G-CSF on granulocyte-macrophage progenitor (GMP) lineage choice. M-CSF activates a multitude of signalling pathways which can differentially affect different cell fates. However, which of these pathways are involved in mediating lineage choice instruction is yet unknown. Combining M-CSF receptor loss of function studies with novel bioimaging technologies allowing long-term quantification of single GMP behaviour, we are investigating the molecular mechanism orchestrating M-CSF mediated lineage choice instruction.
P1048 - RETROVIRAL INSERTIONAL MUTAGENESIS AS A PLATFORM TO STUDY PROTO-ONCOGENES AND THEIR COLLABORATING PARTNERS Teng Cheong Ha, Maike Stahlhut, Olga Kustikova, Axel Schambach, and Christopher Baum Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany Retroviral insertional mutagenesis (RIM) is a well known adverse effect in gene therapy trials which can lead to leukemia or preleukemic conditions. However, it can also serve as a powerful tool to study cancer and stem cell biology by studying genes adjacent to insertion sites. This study is to demonstrate the capacity of RIM to study proto-oncogenes in stem cell regulation and their potential collaborating partners in leukemogenesis. By recording retroviral insertion sites from hematopoietic dominant clones in murine bone marrow transplantation (BMT) model, our group has developed the Insertional Dominance Database (IDDb). Bcl2l1, an apoptosis regulator, is one of the top listed common insertion sites in the IDDb, with multiple roles in normal and malignant hematopoiesis. Here, we focus on the effect of Bcl2l1 overexpression in Lin- Sca1+ Ckit+ cells (LSK) using murine BMT model and perform insertional analysis to identify potential collaborating partners of Bcl2l1 in causing malignancy. 18 weeks after BMT, expression of eGFP was detected at high percentage in the blood, bone marrow (BM) and spleen of the Bcl2l1 group but limited in control group. Spleen enlargement was observed in the Bcl2l1 group without further sign of malignancy. In addition a higher LSK number was observed in the Bcl2l1 transplanted animals. Progeny output per transplanted LSK showed no difference between the reconstitution ability of eGFP and Bcl2l1 LSK in short term (6 weeks), but clear advantage of Bcl2l1 input cells in long term (12 weeks). With all the animals exhibiting normal phenotype up to 18 weeks, homeostatic regulations must have occurred between transgene expressing and nonexpressing LSK in balancing their progeny output. Preliminary clonality studies supported by insertional analysis showed oligo- or polyclonal contribution to the blood, BM and spleen of recipients. In conclusion, overexpression of Bcl2l1 supports long term hematopoiesis without establishing a strong bias for clonal selection. Either a longer latency is needed for clonal outgrowth, or multiple collaborating partners contribute to a common pathway are present in these samples. Sequencing the insertion sites and identifying adjacent genes will reveal further details in this regard.
S35
P1049 - SIRT1 DEACETYLASE IS ESSENTIAL FOR HEMATOPOIETIC STEM CELL ACTIVITY Pauline Rimmele1, Carolina Bigarella1, Valentina d’Escamard1, Brigitte Izac1, David Sinclair2, and Saghi Ghaffari1 1 Developmental and regenerative biology, Mount Sinai School of Medicine, New York, New York, USA; 2Genetics, Harvard Medical School, Boston, Massachusetts, USA SIRT1 NAD-dependent sirtuin deacetylase has critical functions in cellular metabolism, cancer and aging. SIRT1 has recently been implicated in the pathogenesis of chronic myeloid leukemia (CML). As inhibition of SIRT1 appears to be an effective treatment for CML and several groups using germline deleted SIRT1 mice found SIRT1 to be dispensable for normal adult hematopoietic stem cell (HSC), SIRT1 inhibitors are proposed to be used in the treatment of CML. We have shown using an adult-tamoxifen inducible SIRT1 knockout mouse model circumventing the potential developmental adaptation of germ-line deleted SIRT1 mice (only 10% of mice reach adulthood), that loss of SIRT1 compromises severely the CD48-CD150+LSK long-term repopulating HSC frequency and function. Importantly, loss of SIRT1 in young adult mice leads to anemia, significant increase in peripheral blood neutrophils, monocytes and eosinophils and significant decrease overtime of lymphocytes. Interestingly, these abnormalities are associated with a specific and significant increase in granulocyte-monocyte progenitors (GMP), a known hallmark of aging in both mouse and human, and a significant decline overtime in common lymphoid progenitors (CLP) altogether suggesting that SIRT1-deficient HSC recapitulates prematurely key features of aging HSC and hematopoiesis. In agreement with an aging phenotype, we show an increase in reactive oxygen species (ROS) and an accumulation of DNA damage overtime in SIRT1-/- HSC. Importantly, we find that SIRT1 expression is decreased in aged HSC (16 months old) and identify Foxo3 transcription factor that is essential for the HSC maintenance, as a key target of SIRT1 in HSC. Our findings contrast with previously published data obtained from germline deleted SIRT1 mice, and suggest a critical function for SIRT1-Foxo3 in the control of HSC aging.
P1050 - A NOVEL HIGH-CONTENT SCREEN LEADS TO THE IDENTIFICATION OF PROMISING NEW COMPOUNDS FOR HEMATOPOIETIC STEM AND PROGENITOR CELL EXPANSION Veronika Gann1,2, Andreas Radek1, Silke Schult1, Jan Drewes1, and Veit Bergendahl1 1 Miltenyi Biotec GmbH, Bergisch Gladbach, Germany; 2Johannes Gutenberg University, Mainz, Germany Despite significant advances in the hematopoietic research field in the last two decades, hematopoietic stem cell (HSC) transplantation still suffers by the limitation to expand these cells ex vivo. A high-content screen allows a systematic search for new compounds and may uncover new mechanisms that mediate expansion, survival or other effects on HSCs. The main challenge in the development of cell-based screens is the establishment of robust read-outs using suitable formats. In order to minimize the false-positive and even more important the true-negative rate, we combined cell-count, phenotype and functional analysis in the screening protocol. Therefore, we integrated a flow cytometer in a liquid handling system. With this system we were able to establish an automated multiplexed high-content analysis enabling enumeration and detection of surface proteins of interest (e.g. CD34, CD133, CD45RA, CD90, CD38). For the functional analysis the colony forming cell (CFC) standard assay was translated into a screening suitable 96-well format. In this regard, we also invented a new analysis algorithm for the enumeration of colonies. We found a clear correlation of CD34 and CFC counts proving the robustness of both assays. To address our purpose to find new conditions for HSC expansion we prepared a cytokine library counting 103 cytokines. Additionally to our own cytokine library we got the commercially available chemical libraries from Merck (Stem Select and Inhibitor Select) and Micro Source (Spectrum Collection). Finally, we screened in total 3195 compounds based on CD34 peripheral blood derived cells and identified several promising compounds for HSC expansion which we are currently validating. This unique combination of enumeration, phenotyping and functional assay performance already in the screen ensures the identification of highly qualified compounds. Additionally, the risk to lose active compounds is mitigated by obtaining true-negative results. Furthermore, the proposed high-content screening is not only suitable for the analysis of HSC expansion, but will also help to address many further questions.
P1047 - MOLECULAR MECHANISM OF HEMATOPOIETIC LINEAGE CHOICE INSTRUCTION Max Endele1 and Timm Schroeder2,1 1 Stem Cell Dynamics, Helmholtz Center Munich, Neuherberg, Germany; 2 Department of Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland Stem and progenitor cell fate decisions are regulated by the timed integration of extracellular signals and intracellular molecular states. Through continuous single cell observations we could recently provide evidence for the long disputed instructive effect of the hematopoietic cytokines M- and G-CSF on granulocyte-macrophage progenitor (GMP) lineage choice. M-CSF activates a multitude of signalling pathways which can differentially affect different cell fates. However, which of these pathways are involved in mediating lineage choice instruction is yet unknown. Combining M-CSF receptor loss of function studies with novel bioimaging technologies allowing long-term quantification of single GMP behaviour, we are investigating the molecular mechanism orchestrating M-CSF mediated lineage choice instruction.
P1048 - RETROVIRAL INSERTIONAL MUTAGENESIS AS A PLATFORM TO STUDY PROTO-ONCOGENES AND THEIR COLLABORATING PARTNERS Teng Cheong Ha, Maike Stahlhut, Olga Kustikova, Axel Schambach, and Christopher Baum Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany Retroviral insertional mutagenesis (RIM) is a well known adverse effect in gene therapy trials which can lead to leukemia or preleukemic conditions. However, it can also serve as a powerful tool to study cancer and stem cell biology by studying genes adjacent to insertion sites. This study is to demonstrate the capacity of RIM to study proto-oncogenes in stem cell regulation and their potential collaborating partners in leukemogenesis. By recording retroviral insertion sites from hematopoietic dominant clones in murine bone marrow transplantation (BMT) model, our group has developed the Insertional Dominance Database (IDDb). Bcl2l1, an apoptosis regulator, is one of the top listed common insertion sites in the IDDb, with multiple roles in normal and malignant hematopoiesis. Here, we focus on the effect of Bcl2l1 overexpression in Lin- Sca1+ Ckit+ cells (LSK) using murine BMT model and perform insertional analysis to identify potential collaborating partners of Bcl2l1 in causing malignancy. 18 weeks after BMT, expression of eGFP was detected at high percentage in the blood, bone marrow (BM) and spleen of the Bcl2l1 group but limited in control group. Spleen enlargement was observed in the Bcl2l1 group without further sign of malignancy. In addition a higher LSK number was observed in the Bcl2l1 transplanted animals. Progeny output per transplanted LSK showed no difference between the reconstitution ability of eGFP and Bcl2l1 LSK in short term (6 weeks), but clear advantage of Bcl2l1 input cells in long term (12 weeks). With all the animals exhibiting normal phenotype up to 18 weeks, homeostatic regulations must have occurred between transgene expressing and nonexpressing LSK in balancing their progeny output. Preliminary clonality studies supported by insertional analysis showed oligo- or polyclonal contribution to the blood, BM and spleen of recipients. In conclusion, overexpression of Bcl2l1 supports long term hematopoiesis without establishing a strong bias for clonal selection. Either a longer latency is needed for clonal outgrowth, or multiple collaborating partners contribute to a common pathway are present in these samples. Sequencing the insertion sites and identifying adjacent genes will reveal further details in this regard.
S35
P1049 - SIRT1 DEACETYLASE IS ESSENTIAL FOR HEMATOPOIETIC STEM CELL ACTIVITY Pauline Rimmele1, Carolina Bigarella1, Valentina d’Escamard1, Brigitte Izac1, David Sinclair2, and Saghi Ghaffari1 1 Developmental and regenerative biology, Mount Sinai School of Medicine, New York, New York, USA; 2Genetics, Harvard Medical School, Boston, Massachusetts, USA SIRT1 NAD-dependent sirtuin deacetylase has critical functions in cellular metabolism, cancer and aging. SIRT1 has recently been implicated in the pathogenesis of chronic myeloid leukemia (CML). As inhibition of SIRT1 appears to be an effective treatment for CML and several groups using germline deleted SIRT1 mice found SIRT1 to be dispensable for normal adult hematopoietic stem cell (HSC), SIRT1 inhibitors are proposed to be used in the treatment of CML. We have shown using an adult-tamoxifen inducible SIRT1 knockout mouse model circumventing the potential developmental adaptation of germ-line deleted SIRT1 mice (only 10% of mice reach adulthood), that loss of SIRT1 compromises severely the CD48-CD150+LSK long-term repopulating HSC frequency and function. Importantly, loss of SIRT1 in young adult mice leads to anemia, significant increase in peripheral blood neutrophils, monocytes and eosinophils and significant decrease overtime of lymphocytes. Interestingly, these abnormalities are associated with a specific and significant increase in granulocyte-monocyte progenitors (GMP), a known hallmark of aging in both mouse and human, and a significant decline overtime in common lymphoid progenitors (CLP) altogether suggesting that SIRT1-deficient HSC recapitulates prematurely key features of aging HSC and hematopoiesis. In agreement with an aging phenotype, we show an increase in reactive oxygen species (ROS) and an accumulation of DNA damage overtime in SIRT1-/- HSC. Importantly, we find that SIRT1 expression is decreased in aged HSC (16 months old) and identify Foxo3 transcription factor that is essential for the HSC maintenance, as a key target of SIRT1 in HSC. Our findings contrast with previously published data obtained from germline deleted SIRT1 mice, and suggest a critical function for SIRT1-Foxo3 in the control of HSC aging.
P1050 - A NOVEL HIGH-CONTENT SCREEN LEADS TO THE IDENTIFICATION OF PROMISING NEW COMPOUNDS FOR HEMATOPOIETIC STEM AND PROGENITOR CELL EXPANSION Veronika Gann1,2, Andreas Radek1, Silke Schult1, Jan Drewes1, and Veit Bergendahl1 1 Miltenyi Biotec GmbH, Bergisch Gladbach, Germany; 2Johannes Gutenberg University, Mainz, Germany Despite significant advances in the hematopoietic research field in the last two decades, hematopoietic stem cell (HSC) transplantation still suffers by the limitation to expand these cells ex vivo. A high-content screen allows a systematic search for new compounds and may uncover new mechanisms that mediate expansion, survival or other effects on HSCs. The main challenge in the development of cell-based screens is the establishment of robust read-outs using suitable formats. In order to minimize the false-positive and even more important the true-negative rate, we combined cell-count, phenotype and functional analysis in the screening protocol. Therefore, we integrated a flow cytometer in a liquid handling system. With this system we were able to establish an automated multiplexed high-content analysis enabling enumeration and detection of surface proteins of interest (e.g. CD34, CD133, CD45RA, CD90, CD38). For the functional analysis the colony forming cell (CFC) standard assay was translated into a screening suitable 96-well format. In this regard, we also invented a new analysis algorithm for the enumeration of colonies. We found a clear correlation of CD34 and CFC counts proving the robustness of both assays. To address our purpose to find new conditions for HSC expansion we prepared a cytokine library counting 103 cytokines. Additionally to our own cytokine library we got the commercially available chemical libraries from Merck (Stem Select and Inhibitor Select) and Micro Source (Spectrum Collection). Finally, we screened in total 3195 compounds based on CD34 peripheral blood derived cells and identified several promising compounds for HSC expansion which we are currently validating. This unique combination of enumeration, phenotyping and functional assay performance already in the screen ensures the identification of highly qualified compounds. Additionally, the risk to lose active compounds is mitigated by obtaining true-negative results. Furthermore, the proposed high-content screening is not only suitable for the analysis of HSC expansion, but will also help to address many further questions.