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Single-cell analysis of mixed-lineage states leading to a binary cell fate choice
S24
Invited Speakers/ Experimental Hematology 44 (2016) S23–S38
1003 - HEMATOPOIETIC STEM CELL LOSS AND REGENERATION FOLLOWING INFLAMMATORY STRESS Michael Milsom1,2 1 Heidelberg Institute for Stem Cell Technology and Experimental Medicine (HI-STEM), Germany; 2German Cancer Research Center (DKFZ), Germany DNA damage within the hematopoietic stem cell (HSC) compartment is a likely cause of age-associated decline in hematopoietic function and hematologic transformation. Although DNA damage is known to provoke cell fate decisions that would be consistent with HSC attrition, such as apoptosis, senescence and differentiation, the physiologic source of DNA damage within the HSC compartment is not well defined. We have recently demonstrated that chronic inflammation can induce DNA damage within the HSC compartment in mice in vivo by forcing stem cells out of their homeostatic quiescent status and inducing replication stress (Walter et al., 2015, Nature, 520: 549-52). Extended inflammatory challenge led to significant HSC depletion and myeloid differentiation bias, which are consistent with a premature ageing phenotype. In the setting of Fanconi anemia knockout mice, a clinically relevant model of defective DNA repair, chronic inflammation led to HSC exhaustion and highly penetrant severe aplastic anemia, recapitulating the disease progression within Fanconi anemia patients. By performing detailed dose escalation studies using wild type mice, we have been able to observe a progressive depletion of functional HSCs equating to an approximate 50% reduction in HSC frequency per round of challenge. Importantly, this depletion appears to be irreversible, with HSCs numbers failing to recover even up to 20 weeks after cessation of challenge. We were able to observe that aged mice which had been exposed to chronic inflammation in early life developed mild peripheral blood cytopenias and bone marrow aplasia, reminiscent of anemia of ageing. We hypothesize that such a model is consistent with HSC depletion during ageing and propose that infection/inflammatory stress is an important mediator of the aged hematopoietic phenotype that is not normally encountered in standard murine laboratory models of hematopoiesis.
1004 - SINGLE-CELL ANALYSIS OF MIXED-LINEAGE STATES LEADING TO A BINARY CELL FATE CHOICE Andre Olsson1, Meenakshi Venkatasubramanian2, Viren K. Chaudhri1, Bruce J. Aronow2, Nathan Salomonis2, Harinder Singh1, and H. Leighton Grimes1,3 1 Division of Immunobiology and Center for Systems Immunology, Cincinnati Children’s Hospital Medical Center, Cincinnati, USA; 2 Division of Biomedical Informatics, Cincinnati Children’s Hospital Medical Center, Cincinnati, USA; 3Division of Experimental Hematology and Cancer Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, USA Single-cell RNA-Seq has the potential to become a dominant approach in probing diverse and complex developmental compartments. Its unbiased and comprehensive nature could enable developmental ordering of cellular and regulatory gene hierarchies without prior knowledge. To test general utility we performed single-cell RNA-seq of murine hematopoietic progenitors focusing on the myeloid developmental hierarchy. Using novel unsupervised clustering analysis, ICDS, we correctly ordered known hierarchical states as well as revealed rare intermediates. Regulatory state analysis suggested that the transcription factors Gfi1 and Irf8 function antagonistically to control homeostatic neutrophil and macrophage production, respectively. This prediction was validated by complementary genetic and genomic experiments in granulocyte-macrophage progenitors. Using knock-in reporters for Gfi1 and Irf8 and clonogenic analyses coupled with single-cell RNA-seq we distinguished regulatory states of bi-potential progenitors from their lineage specifying or committed progeny. Thus single-cell RNA-Seq is a powerful developmental tool to characterize hierarchical and rare cellular states along with the regulators that control their dynamics.
Invited Speakers/ Experimental Hematology 44 (2016) S23–S38
1003 - HEMATOPOIETIC STEM CELL LOSS AND REGENERATION FOLLOWING INFLAMMATORY STRESS Michael Milsom1,2 1 Heidelberg Institute for Stem Cell Technology and Experimental Medicine (HI-STEM), Germany; 2German Cancer Research Center (DKFZ), Germany DNA damage within the hematopoietic stem cell (HSC) compartment is a likely cause of age-associated decline in hematopoietic function and hematologic transformation. Although DNA damage is known to provoke cell fate decisions that would be consistent with HSC attrition, such as apoptosis, senescence and differentiation, the physiologic source of DNA damage within the HSC compartment is not well defined. We have recently demonstrated that chronic inflammation can induce DNA damage within the HSC compartment in mice in vivo by forcing stem cells out of their homeostatic quiescent status and inducing replication stress (Walter et al., 2015, Nature, 520: 549-52). Extended inflammatory challenge led to significant HSC depletion and myeloid differentiation bias, which are consistent with a premature ageing phenotype. In the setting of Fanconi anemia knockout mice, a clinically relevant model of defective DNA repair, chronic inflammation led to HSC exhaustion and highly penetrant severe aplastic anemia, recapitulating the disease progression within Fanconi anemia patients. By performing detailed dose escalation studies using wild type mice, we have been able to observe a progressive depletion of functional HSCs equating to an approximate 50% reduction in HSC frequency per round of challenge. Importantly, this depletion appears to be irreversible, with HSCs numbers failing to recover even up to 20 weeks after cessation of challenge. We were able to observe that aged mice which had been exposed to chronic inflammation in early life developed mild peripheral blood cytopenias and bone marrow aplasia, reminiscent of anemia of ageing. We hypothesize that such a model is consistent with HSC depletion during ageing and propose that infection/inflammatory stress is an important mediator of the aged hematopoietic phenotype that is not normally encountered in standard murine laboratory models of hematopoiesis.
1004 - SINGLE-CELL ANALYSIS OF MIXED-LINEAGE STATES LEADING TO A BINARY CELL FATE CHOICE Andre Olsson1, Meenakshi Venkatasubramanian2, Viren K. Chaudhri1, Bruce J. Aronow2, Nathan Salomonis2, Harinder Singh1, and H. Leighton Grimes1,3 1 Division of Immunobiology and Center for Systems Immunology, Cincinnati Children’s Hospital Medical Center, Cincinnati, USA; 2 Division of Biomedical Informatics, Cincinnati Children’s Hospital Medical Center, Cincinnati, USA; 3Division of Experimental Hematology and Cancer Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, USA Single-cell RNA-Seq has the potential to become a dominant approach in probing diverse and complex developmental compartments. Its unbiased and comprehensive nature could enable developmental ordering of cellular and regulatory gene hierarchies without prior knowledge. To test general utility we performed single-cell RNA-seq of murine hematopoietic progenitors focusing on the myeloid developmental hierarchy. Using novel unsupervised clustering analysis, ICDS, we correctly ordered known hierarchical states as well as revealed rare intermediates. Regulatory state analysis suggested that the transcription factors Gfi1 and Irf8 function antagonistically to control homeostatic neutrophil and macrophage production, respectively. This prediction was validated by complementary genetic and genomic experiments in granulocyte-macrophage progenitors. Using knock-in reporters for Gfi1 and Irf8 and clonogenic analyses coupled with single-cell RNA-seq we distinguished regulatory states of bi-potential progenitors from their lineage specifying or committed progeny. Thus single-cell RNA-Seq is a powerful developmental tool to characterize hierarchical and rare cellular states along with the regulators that control their dynamics.