- Email: [email protected]
Identification of novel regulatory genes of human post-embryonic hematopoiesis
Poster Presentations/ Experimental Hematology 41 (2013) S23–S75
P1032 - QUANTIFICATION OF STEM CELL / NICHE INTERACTIONS BY COUPLING IN VIVO IMAGING AND IN SILICO SIMULATION Ingo Roeder1, Axel Krinner1, Nico Scherf1, Marc Scott2, Narges Rashidi2, Tilo Pompe3, and Cristina Lo Celso2 1 Institute for Medical Informatics and Biometrie, TU Dresden, Medical School, Dresden, Germany; 2Department of Life Sciences, Imperial College, London, UK; 3 Instiute of Biochemistry, University Leipzig, Leipzig, Germany For decades the bone marrow has been recognized as the home of hematopoietic stem cells (HSCs). Their regulation has been studied extensively, in particular regarding single molecules and pathways, but a comprehensive and dynamic picture is still elusive. This holds especially true for spatial organization. Recent advances in imaging techniques now allow acquisition of data on position and migration of HSCs in vivo. However, those techniques are limited by a relatively small observation volume and time span due to experimental limitations. Here, we complement in vivo imaging of HSCs in a mouse model by a mathematical model of spatio-temporal HSC dynamics and exploit its potential to identify basic principles of stem cell / niche organization. The computer model allows us to test a number of general mechanisms of HSC / niche interaction independent of molecular details. Specifically, we investigate various modes of cell migration and niche geometries with respect to their influence on HSC organization. The in silico model demonstrates that spatial distribution of HSCs crucially depends on different mechanisms, such as motility of cells, attraction forces of HSCs towards niche sites, but potentially also repulsion of HSCs from already occupied niches. The simulation results are compared to in vivo experiments, which demonstrate that the distances of transplanted hematopoietic cells to osteoblasts and bone surface varies between different cell types. Interestingly, simulated distance distributions of various hematopoietic cell types to osteoblasts turned out to be most similar to experimental data for random motion and not for an explicit niche-mediated attraction.
P1033 - DECLINED PRESENTATION CD34 FAMILY PROTEINS ARE KEY REGULATORS OF HEMATOPOIETIC CELL MIGRATION AND VASCULAR PERMEABILITY Michael Hughes1, Erin DeBruin1, Bernard Lo1, Matthew Gold1, Julie Nielsen3, Poh Tan1, Marie-Renee Blanchet2, and Kelly McNagny1 1 The Biomedical Research Centre, University of British Columbia, Vancouver, British Columbia, Canada; 2Department of Medicine, Universite Laval, Quebec City, Quebec, Canada; 3Trev and Joyce Deeley Research Centre, BC Cancer Agency, Victoria, British Columbia, Canada CD34 is a widely used marker of hematopoietic progenitors and vascular endothelia yet, its function remains obscure. It is the founding member of a family of three proteins (CD34, podocalyxin and endoglycan) with overlapping expression patterns on progenitors and endothelia, but with distinct expression patterns on other tissues. For example, CD34 is uniquely expressed by mast cells, eosinophils and dendritic cell precursors, and we find that mice lacking CD34 are remarkably resistant to a variety of mucosal inflammatory diseases including allergic asthma, hypersensitivity pneumonitis and colitis. This resistance reflects defective inflammatory cell trafficking due to enhanced adhesion and impaired chemotaxis, which is reversed through ectopic expression of a human CD34 transgene. Podocalyxin, in contrast, is uniquely expressed by a variety of embryonic tissues and inactivation of the Podxl gene leads to perinatal lethality due to a failure of tissues to undergo appropriate morphogenesis. Furthermore, selective inactivation of Podxl in adult endothelial cells results in increased vascular leakage and development of an emphysema-like phenotype due to poor vessel patency in the lung. This suggests that podocalyxin plays an important function in maintaining homeostasis in the adult vasculature. Finally, these proteins also play key roles in hematopoietic precursor homing; podocalyxin, in particular, facilitates CXCR4-dependent chemotaxis, likely by stabilizing polarized signaling through CXCR4. Intriguingly, despite the impaired homing of Podxl-/progenitors, they exhibit a competitive advantage in engraftment of transplanted mice that is highly reminiscent of CXCR4-/- progenitors. In summary, our data suggest that CD34-type proteins play critical roles in a variety of normal developmental processes, hematopoiesis and immunity. Mechanistically, they act as key regulators of adhesion, chemotaxis, cell morphogenesis and, potentially, stem cell expansion.
S31
P1034 - IDENTIFICATION OF NOVEL REGULATORY GENES OF HUMAN POST-EMBRYONIC HEMATOPOIESIS Shayda Hemmati1, Friederike Herbst1, Claudia Ball1, Anna Paruzynski1, Christoph Klein2, Maximilian Witzel2, Kaan Boztug3, Manfred Schmidt1, Christof von Kalle1, and Hanno Glimm1 1 Translational Oncology, National Center for Tumor Diseases (NCT) and German Cancer Research Center (DKFZ), Heidelberg, Germany; 2Pediatrics, Dr. von Hauner Children’s Hospital, Ludwig-Maximilians-Universit€at Munich, Munich, Germany; 3 Pediatrics and Adolescent Medicine, Medical University of Vienna, Vienna, Austria Hematopoietic stem cell (HSC) fate is controlled by intrinsic and extrinsic factors regulating proliferation, self-renewal, apoptosis or differentiation thereby adapting blood cell production to the need of the organism. We and others have shown that genetic manipulation of hematopoiesis using retroviral vectors (RV) can trigger clonal expansion and malignancy by the integration of the therapeutic vector into specific gene regions. In a total of 16 human gene therapy trials we detected genetic loci which are highly overrepresented, indicating for a selective advantage of clones carrying these common vector integration sites (CIS) in vivo. We therefore hypothesize that CIS significantly enriched in genetically modified blood cells point to a regulatory function of nearby genes in post-embryonic hematopoiesis. To identify potential stem cell regulatory genes we systematically analyzed the insertion site (IS) repertoire of gene corrected blood cells within a cohort of 10 gene therapy patients with Wiskott-Aldrich-Syndrome. Using highly sensitive linear amplification mediated PCR combined with high-throughput 454 sequencing a total of 12.887 unique IS in the vicinity of 3.268 genes were identified. Next, we selected all genes with at least 10 different IS within a 200 kb window around the gene (n5588). To enrich for genes with increased probability of transcriptional activation we then chose those genes with at least 10 IS within a 50kb window around the transcriptional start site (n5424). After stringent exclusion of all genes located within gene clusters 32 candidate genes were identified. To evaluate the hematopoietic activity of gene corrected cell clones with CIS we monitored their contribution to blood formation post transplantation. IS close to candidate genes were detectable 15 to 93 times in a total of 102 analyzed patient samples during a time period of 4 years, demonstrating long term activity of HSC clones carrying IS near candidates . Interestingly, 12 of 32 identified genes have not been linked to hematopoiesis so far. Of these, 4 genes are known proto-oncogenes and 3 are highly expressed in HSC. The highest ranked genes EVI1, CCND2 and LMO2 are known as hematopoietic regulators and key factors in malignant transformation, thereby validating our candidate selection strategy. These data demonstrate that RV IS datasets derived from gene therapy patients can be used to identify regulatory genes of hematopoiesis. Systematic identification of novel regulatory genes in meta datasets derived from a larger number of gene therapy studies and subsequent validation in vitro and in vivo will allow new insights into the biology of post-embryonic hematopoiesis.
P1032 - QUANTIFICATION OF STEM CELL / NICHE INTERACTIONS BY COUPLING IN VIVO IMAGING AND IN SILICO SIMULATION Ingo Roeder1, Axel Krinner1, Nico Scherf1, Marc Scott2, Narges Rashidi2, Tilo Pompe3, and Cristina Lo Celso2 1 Institute for Medical Informatics and Biometrie, TU Dresden, Medical School, Dresden, Germany; 2Department of Life Sciences, Imperial College, London, UK; 3 Instiute of Biochemistry, University Leipzig, Leipzig, Germany For decades the bone marrow has been recognized as the home of hematopoietic stem cells (HSCs). Their regulation has been studied extensively, in particular regarding single molecules and pathways, but a comprehensive and dynamic picture is still elusive. This holds especially true for spatial organization. Recent advances in imaging techniques now allow acquisition of data on position and migration of HSCs in vivo. However, those techniques are limited by a relatively small observation volume and time span due to experimental limitations. Here, we complement in vivo imaging of HSCs in a mouse model by a mathematical model of spatio-temporal HSC dynamics and exploit its potential to identify basic principles of stem cell / niche organization. The computer model allows us to test a number of general mechanisms of HSC / niche interaction independent of molecular details. Specifically, we investigate various modes of cell migration and niche geometries with respect to their influence on HSC organization. The in silico model demonstrates that spatial distribution of HSCs crucially depends on different mechanisms, such as motility of cells, attraction forces of HSCs towards niche sites, but potentially also repulsion of HSCs from already occupied niches. The simulation results are compared to in vivo experiments, which demonstrate that the distances of transplanted hematopoietic cells to osteoblasts and bone surface varies between different cell types. Interestingly, simulated distance distributions of various hematopoietic cell types to osteoblasts turned out to be most similar to experimental data for random motion and not for an explicit niche-mediated attraction.
P1033 - DECLINED PRESENTATION CD34 FAMILY PROTEINS ARE KEY REGULATORS OF HEMATOPOIETIC CELL MIGRATION AND VASCULAR PERMEABILITY Michael Hughes1, Erin DeBruin1, Bernard Lo1, Matthew Gold1, Julie Nielsen3, Poh Tan1, Marie-Renee Blanchet2, and Kelly McNagny1 1 The Biomedical Research Centre, University of British Columbia, Vancouver, British Columbia, Canada; 2Department of Medicine, Universite Laval, Quebec City, Quebec, Canada; 3Trev and Joyce Deeley Research Centre, BC Cancer Agency, Victoria, British Columbia, Canada CD34 is a widely used marker of hematopoietic progenitors and vascular endothelia yet, its function remains obscure. It is the founding member of a family of three proteins (CD34, podocalyxin and endoglycan) with overlapping expression patterns on progenitors and endothelia, but with distinct expression patterns on other tissues. For example, CD34 is uniquely expressed by mast cells, eosinophils and dendritic cell precursors, and we find that mice lacking CD34 are remarkably resistant to a variety of mucosal inflammatory diseases including allergic asthma, hypersensitivity pneumonitis and colitis. This resistance reflects defective inflammatory cell trafficking due to enhanced adhesion and impaired chemotaxis, which is reversed through ectopic expression of a human CD34 transgene. Podocalyxin, in contrast, is uniquely expressed by a variety of embryonic tissues and inactivation of the Podxl gene leads to perinatal lethality due to a failure of tissues to undergo appropriate morphogenesis. Furthermore, selective inactivation of Podxl in adult endothelial cells results in increased vascular leakage and development of an emphysema-like phenotype due to poor vessel patency in the lung. This suggests that podocalyxin plays an important function in maintaining homeostasis in the adult vasculature. Finally, these proteins also play key roles in hematopoietic precursor homing; podocalyxin, in particular, facilitates CXCR4-dependent chemotaxis, likely by stabilizing polarized signaling through CXCR4. Intriguingly, despite the impaired homing of Podxl-/progenitors, they exhibit a competitive advantage in engraftment of transplanted mice that is highly reminiscent of CXCR4-/- progenitors. In summary, our data suggest that CD34-type proteins play critical roles in a variety of normal developmental processes, hematopoiesis and immunity. Mechanistically, they act as key regulators of adhesion, chemotaxis, cell morphogenesis and, potentially, stem cell expansion.
S31
P1034 - IDENTIFICATION OF NOVEL REGULATORY GENES OF HUMAN POST-EMBRYONIC HEMATOPOIESIS Shayda Hemmati1, Friederike Herbst1, Claudia Ball1, Anna Paruzynski1, Christoph Klein2, Maximilian Witzel2, Kaan Boztug3, Manfred Schmidt1, Christof von Kalle1, and Hanno Glimm1 1 Translational Oncology, National Center for Tumor Diseases (NCT) and German Cancer Research Center (DKFZ), Heidelberg, Germany; 2Pediatrics, Dr. von Hauner Children’s Hospital, Ludwig-Maximilians-Universit€at Munich, Munich, Germany; 3 Pediatrics and Adolescent Medicine, Medical University of Vienna, Vienna, Austria Hematopoietic stem cell (HSC) fate is controlled by intrinsic and extrinsic factors regulating proliferation, self-renewal, apoptosis or differentiation thereby adapting blood cell production to the need of the organism. We and others have shown that genetic manipulation of hematopoiesis using retroviral vectors (RV) can trigger clonal expansion and malignancy by the integration of the therapeutic vector into specific gene regions. In a total of 16 human gene therapy trials we detected genetic loci which are highly overrepresented, indicating for a selective advantage of clones carrying these common vector integration sites (CIS) in vivo. We therefore hypothesize that CIS significantly enriched in genetically modified blood cells point to a regulatory function of nearby genes in post-embryonic hematopoiesis. To identify potential stem cell regulatory genes we systematically analyzed the insertion site (IS) repertoire of gene corrected blood cells within a cohort of 10 gene therapy patients with Wiskott-Aldrich-Syndrome. Using highly sensitive linear amplification mediated PCR combined with high-throughput 454 sequencing a total of 12.887 unique IS in the vicinity of 3.268 genes were identified. Next, we selected all genes with at least 10 different IS within a 200 kb window around the gene (n5588). To enrich for genes with increased probability of transcriptional activation we then chose those genes with at least 10 IS within a 50kb window around the transcriptional start site (n5424). After stringent exclusion of all genes located within gene clusters 32 candidate genes were identified. To evaluate the hematopoietic activity of gene corrected cell clones with CIS we monitored their contribution to blood formation post transplantation. IS close to candidate genes were detectable 15 to 93 times in a total of 102 analyzed patient samples during a time period of 4 years, demonstrating long term activity of HSC clones carrying IS near candidates . Interestingly, 12 of 32 identified genes have not been linked to hematopoiesis so far. Of these, 4 genes are known proto-oncogenes and 3 are highly expressed in HSC. The highest ranked genes EVI1, CCND2 and LMO2 are known as hematopoietic regulators and key factors in malignant transformation, thereby validating our candidate selection strategy. These data demonstrate that RV IS datasets derived from gene therapy patients can be used to identify regulatory genes of hematopoiesis. Systematic identification of novel regulatory genes in meta datasets derived from a larger number of gene therapy studies and subsequent validation in vitro and in vivo will allow new insights into the biology of post-embryonic hematopoiesis.