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Human definitive hematopoiesis is regulated by CDX4 gene expression in early HPSC-derived mesoderm
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Poster Presentations/ Experimental Hematology 44 (2016) S56–S110
3138 - TYPE I INTERFERONS DRIVE HEMATOPOIETIC PROGENITOR CELL DEPLETION VIA NECROPTOSIS IN A MODEL OF SEVERE RICKETTSIAL INFECTION Julianne N.P. Smith1, Amanda McCabe2, and Katherine MacNamara1 1 Albany Medical College, Albany, USA; 2Harvard Medical School, Boston, USA Type I interferons (IFNa/b) modulate immune responses to most pathogens and regulate hematopoietic stem and progenitor (HSC/HSPC) proliferation and death upon noninfectious immune-stimulation in vivo. The impact of type I IFNs on HSPC regulation during infection has not yet been investigated, however. Human monocytic ehrlichiosis (HME) is a tick-borne disease characterized by severe cytopenias, thus we used a murine model of severe ehrlichial infection to investigate the impact of infection-induced type I IFNs on HSPC function. We recently reported that the tick-borne pathogen Ixodes ovatus ehrlichia (IOE) drives pathology in an IFNa/b-dependent manner. At the peak of IFNa/b induction, myeloid progenitors and HSCs were reduced in wild-type (WT) BM, but were significantly protected and even expanded in mice lacking the IFNa/b receptor (IFNaR1). Ifnar1-/- HSCs derived from infected mice engrafted recipient BM and provided multilineage repopulation, while HSCs derived from WT mice were only capable of transient, myeloid lineage-restricted repopulation. IFNa/b also limited infection-induced extramedullary hematopoiesis in the spleen. To determine if IFNa/b impairs hematopoiesis via reduced HSPC proliferation and/or increased cell death, we examined WT and Ifnar1-/- HSPCs in IOE-infected mixed BM chimeras. Although IFNa/b neither directly depleted quiescent HSCs nor impacted the magnitude of HSPC cycle entry, IFNa/b directly promoted caspase-independent HSPC death. Progenitors lacking IFNaR1 were protected from depletion, showed enhanced caspase 8 activation, and preferentially underwent apoptosis during infection. During infection, HSPCs exposed to IFNa/b showed cytoplasmic RIP3 accumulation and significantly greater RIP3-RIP1 colocalization when compared to HSPCs from Ifnar1-/- mice. Moreover, administration of the RIP1 inhibitor Necrostatin-1s to IOE-infected WT mice preserved BM cellularity and resulted in HSPC expansion, similar to the phenotype of Ifnar1-/- mice. Our data identify a novel mechanism by which type I IFNs directly impair hematopoiesis, via sensitization of HSPCs to pro-inflammatory necroptotic death, during shock-like rickettsial infection.
3139 - PROXIMITY-BASED ANALYSIS OF THE BONE MARROW NICHE IDENTIFIES NOVEL EXTRINSIC REGULATORS OF HEMATOPOIETIC REGENERATION Lev Silberstein1, Kevin Goncalves2, Peter Kharchenko3, Raphael Turcotte4, Youmna Kfoury1, Francois Mercier1, Nicolas Severe1, Ninib Baryawno1, Charles Lin3, and David Scadden5 1 Massachusetts General Hospital, Boston, USA; 2Tufts University, Medford, USA; 3Harvard University, Boston, USA; 4HHMI, Chevy Chase, USA; 5Center for Regenerative Medicine, North Miami, USA Sluggish hematopoietic recovery post-transplant or following chemotherapy is a major cause of morbidity and mortality. We undertook a study of the bone marrow niche to identify novel extrinsic factors which may influence hematopoietic stem and progenitor cell (HSPC) behavior. We transplanted fluorescently labeled HSPC into neonatal recipients with GFP-labeled osteolineage cells (OLC), an established cellular component of the niche, extracted individual OLCs which were located close (proximal OLC) and further away (distal OLC) from transplanted HSPC and compared their transcriptional profile by single cell RNA-Seq, thus defining a proximal OLC signature. Using this signature, we selected three secreted factors with previously unknown HSPC regulatory function which were preferentially expressed in proximal OLCs- cytokine Interleukin 18, cell adhesion molecule Embigin and secreted RNAse Angiogenin – for testing their effect on HSPC in vivo. We found that all three molecules acted as regulators of quiescence affecting distinct HSPC subsets: IL18 predominantly regulated short-term progenitors, while Embigin and Angiogenin also controlled long-term HSC, as we confirmed in knock-out models (IL18 and Angiogenin) or using in vivo antibody treatment (Embigin). Testing each molecule in a therapeutically relevant setting revealed their potential clinical applications. Absence of IL18 significantly accelerated short-term multi-lineage post-transplant reconstitution and recovery post-chemotherapy, such as 5-fluorouracil (5FU), and improved post-transplant animal survival. Neutralization of Embigin also resulted in increased post-transplant HSPC proliferation and faster
post-5U recovery. Somewhat counter-intuitively, ex-vivo treatment of mouse or human HSPC with Angiogenin, while promoting quiescence, endowed these cells with a markedly enhanced repopulation capacity following transplantation which persisted into secondary transplants. Further mechanistic studies revealed that this effect of Angiogenin was mediated through tiRNA (also known as tRNA-halves) which are produced upon enzymatic cleavage of tRNA by this molecule. Thus, our study of HSPC niche regulatory function revealed the complexity of nichederived quiescence-controlling pathways and identified novel molecular tools to enhance hematopoietic regeneration.
3140 - HUMAN DEFINITIVE HEMATOPOIESIS IS REGULATED BY CDX4 GENE EXPRESSION IN EARLY HPSC-DERIVED MESODERM Christopher Sturgeon, Carissa Dege, Qihao Ren, and Mark Valentine 1 Washington University School of Medicine, St. Louis, USA The generation of hematopoietic stem cells (HSCs) from human pluripotent stem cells (hPSCs) is a major goal for regenerative medicine, as it will provide an unlimited source of these cells for transplantation, and, a unique platform for the study of both normal and disease hematopoietic processes. To reproducibly achieve this goal in all hPSC lines, we must first fully understand hematopoietic ontogeny. We have recently developed a novel method to obtain pure KDR+CD235a+ primitive or pure KDR+CD235a- definitive progenitors from hPSCs through stage-specific manipulation of Wnt signaling. We performed whole-transcriptome gene expression analyses on these isolated populations and identified strong CDX and HOX gene enrichment within KDR+CD235a- definitive hematopoietic mesoderm. CDX1 and CDX2 were expressed in broad mesendoderm, while CDX4 was expressed later exclusively within definitive hematopoietic mesoderm. To test whether CDX4 expression regulates definitive hematopoietic specification, we generated an inducible CDX4 expression hPSC line, and a tandem shRNA CDX4 knockdown hPSC line, using the ‘‘safe-harbor’’ AAVS1 locus. Interestingly, while knockdown of CDX4 had no effect on the human primitive hematopoietic specification, it strongly decreased definitive CD34+CD73-CD184- hemogenic endothelial specification. Conversely, CDX4 overexpression during mesoderm specification repressed primitive hematopoietic commitment by O90%. Critically, CDX4 overexpression solely during mesoderm specification conferred 10-fold greater definitive hematopoietic potential within CD34+CD73-CD184- hemogenic endothelium, giving rise to definitive erythroid-myeloid-lymphoid progenitors. These findings provide novel insight into the genetic regulation of human definitive hematopoiesis and identify CDX4 as one of the first differentially expressed transcription factors in hematopoietic development. This understanding will enable the identification of additional signal pathways required for efficient definitive hematopoietic specification from hPSCs.
Poster Presentations/ Experimental Hematology 44 (2016) S56–S110
3138 - TYPE I INTERFERONS DRIVE HEMATOPOIETIC PROGENITOR CELL DEPLETION VIA NECROPTOSIS IN A MODEL OF SEVERE RICKETTSIAL INFECTION Julianne N.P. Smith1, Amanda McCabe2, and Katherine MacNamara1 1 Albany Medical College, Albany, USA; 2Harvard Medical School, Boston, USA Type I interferons (IFNa/b) modulate immune responses to most pathogens and regulate hematopoietic stem and progenitor (HSC/HSPC) proliferation and death upon noninfectious immune-stimulation in vivo. The impact of type I IFNs on HSPC regulation during infection has not yet been investigated, however. Human monocytic ehrlichiosis (HME) is a tick-borne disease characterized by severe cytopenias, thus we used a murine model of severe ehrlichial infection to investigate the impact of infection-induced type I IFNs on HSPC function. We recently reported that the tick-borne pathogen Ixodes ovatus ehrlichia (IOE) drives pathology in an IFNa/b-dependent manner. At the peak of IFNa/b induction, myeloid progenitors and HSCs were reduced in wild-type (WT) BM, but were significantly protected and even expanded in mice lacking the IFNa/b receptor (IFNaR1). Ifnar1-/- HSCs derived from infected mice engrafted recipient BM and provided multilineage repopulation, while HSCs derived from WT mice were only capable of transient, myeloid lineage-restricted repopulation. IFNa/b also limited infection-induced extramedullary hematopoiesis in the spleen. To determine if IFNa/b impairs hematopoiesis via reduced HSPC proliferation and/or increased cell death, we examined WT and Ifnar1-/- HSPCs in IOE-infected mixed BM chimeras. Although IFNa/b neither directly depleted quiescent HSCs nor impacted the magnitude of HSPC cycle entry, IFNa/b directly promoted caspase-independent HSPC death. Progenitors lacking IFNaR1 were protected from depletion, showed enhanced caspase 8 activation, and preferentially underwent apoptosis during infection. During infection, HSPCs exposed to IFNa/b showed cytoplasmic RIP3 accumulation and significantly greater RIP3-RIP1 colocalization when compared to HSPCs from Ifnar1-/- mice. Moreover, administration of the RIP1 inhibitor Necrostatin-1s to IOE-infected WT mice preserved BM cellularity and resulted in HSPC expansion, similar to the phenotype of Ifnar1-/- mice. Our data identify a novel mechanism by which type I IFNs directly impair hematopoiesis, via sensitization of HSPCs to pro-inflammatory necroptotic death, during shock-like rickettsial infection.
3139 - PROXIMITY-BASED ANALYSIS OF THE BONE MARROW NICHE IDENTIFIES NOVEL EXTRINSIC REGULATORS OF HEMATOPOIETIC REGENERATION Lev Silberstein1, Kevin Goncalves2, Peter Kharchenko3, Raphael Turcotte4, Youmna Kfoury1, Francois Mercier1, Nicolas Severe1, Ninib Baryawno1, Charles Lin3, and David Scadden5 1 Massachusetts General Hospital, Boston, USA; 2Tufts University, Medford, USA; 3Harvard University, Boston, USA; 4HHMI, Chevy Chase, USA; 5Center for Regenerative Medicine, North Miami, USA Sluggish hematopoietic recovery post-transplant or following chemotherapy is a major cause of morbidity and mortality. We undertook a study of the bone marrow niche to identify novel extrinsic factors which may influence hematopoietic stem and progenitor cell (HSPC) behavior. We transplanted fluorescently labeled HSPC into neonatal recipients with GFP-labeled osteolineage cells (OLC), an established cellular component of the niche, extracted individual OLCs which were located close (proximal OLC) and further away (distal OLC) from transplanted HSPC and compared their transcriptional profile by single cell RNA-Seq, thus defining a proximal OLC signature. Using this signature, we selected three secreted factors with previously unknown HSPC regulatory function which were preferentially expressed in proximal OLCs- cytokine Interleukin 18, cell adhesion molecule Embigin and secreted RNAse Angiogenin – for testing their effect on HSPC in vivo. We found that all three molecules acted as regulators of quiescence affecting distinct HSPC subsets: IL18 predominantly regulated short-term progenitors, while Embigin and Angiogenin also controlled long-term HSC, as we confirmed in knock-out models (IL18 and Angiogenin) or using in vivo antibody treatment (Embigin). Testing each molecule in a therapeutically relevant setting revealed their potential clinical applications. Absence of IL18 significantly accelerated short-term multi-lineage post-transplant reconstitution and recovery post-chemotherapy, such as 5-fluorouracil (5FU), and improved post-transplant animal survival. Neutralization of Embigin also resulted in increased post-transplant HSPC proliferation and faster
post-5U recovery. Somewhat counter-intuitively, ex-vivo treatment of mouse or human HSPC with Angiogenin, while promoting quiescence, endowed these cells with a markedly enhanced repopulation capacity following transplantation which persisted into secondary transplants. Further mechanistic studies revealed that this effect of Angiogenin was mediated through tiRNA (also known as tRNA-halves) which are produced upon enzymatic cleavage of tRNA by this molecule. Thus, our study of HSPC niche regulatory function revealed the complexity of nichederived quiescence-controlling pathways and identified novel molecular tools to enhance hematopoietic regeneration.
3140 - HUMAN DEFINITIVE HEMATOPOIESIS IS REGULATED BY CDX4 GENE EXPRESSION IN EARLY HPSC-DERIVED MESODERM Christopher Sturgeon, Carissa Dege, Qihao Ren, and Mark Valentine 1 Washington University School of Medicine, St. Louis, USA The generation of hematopoietic stem cells (HSCs) from human pluripotent stem cells (hPSCs) is a major goal for regenerative medicine, as it will provide an unlimited source of these cells for transplantation, and, a unique platform for the study of both normal and disease hematopoietic processes. To reproducibly achieve this goal in all hPSC lines, we must first fully understand hematopoietic ontogeny. We have recently developed a novel method to obtain pure KDR+CD235a+ primitive or pure KDR+CD235a- definitive progenitors from hPSCs through stage-specific manipulation of Wnt signaling. We performed whole-transcriptome gene expression analyses on these isolated populations and identified strong CDX and HOX gene enrichment within KDR+CD235a- definitive hematopoietic mesoderm. CDX1 and CDX2 were expressed in broad mesendoderm, while CDX4 was expressed later exclusively within definitive hematopoietic mesoderm. To test whether CDX4 expression regulates definitive hematopoietic specification, we generated an inducible CDX4 expression hPSC line, and a tandem shRNA CDX4 knockdown hPSC line, using the ‘‘safe-harbor’’ AAVS1 locus. Interestingly, while knockdown of CDX4 had no effect on the human primitive hematopoietic specification, it strongly decreased definitive CD34+CD73-CD184- hemogenic endothelial specification. Conversely, CDX4 overexpression during mesoderm specification repressed primitive hematopoietic commitment by O90%. Critically, CDX4 overexpression solely during mesoderm specification conferred 10-fold greater definitive hematopoietic potential within CD34+CD73-CD184- hemogenic endothelium, giving rise to definitive erythroid-myeloid-lymphoid progenitors. These findings provide novel insight into the genetic regulation of human definitive hematopoiesis and identify CDX4 as one of the first differentially expressed transcription factors in hematopoietic development. This understanding will enable the identification of additional signal pathways required for efficient definitive hematopoietic specification from hPSCs.