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Megakaryocyte expansion by the fetal liver microenvironment
Poster Presentations / Experimental Hematology 42 (2014) S23–S68
P1008 - PHENOTYPIC SCREENING IDENTIFIES PROSTACYCLIN AGONISTS AS DIFFERENTIATION AGENTS IN THE TREATMENT OF ACUTE MYELOID LEUKEMIA David Sykes1, Youmna Kfoury1, Andres Blanco3, and David Scadden1,2 1 Massachusetts General Hospital, Boston, Massachusetts, USA; 2Harvard Stem Cell Institute, Boston, Massachusetts, USA; 3Children’s Hospital, Boston, Massachusetts, USA One success story in AML treatment has been the discovery of drugs that trigger the differentiation of leukemic blasts in the subset of patients with acute promyelocytic leukemia. Differentiation therapy is not available for the remaining 90% of AML patients. To identify small molecules that trigger differentiation, we developed a novel model of AML, with a built-in GFP reporter of differentiation. In this model, primary bone marrow cells from a lysozyme-GFP knock-in mouse were arrested in differentiation by an ER-HoxA9 fusion protein. The cells grow indefinitely in the presence of SCF and estradiol, but upon withdrawal of estradiol (inactivation of HoxA9), the cells undergo terminal myeloid differentiation, becoming brightly GFP(+). This model formed the basis of a screen of 350,000 small molecules using the flow-cytometric detection of GFP as a marker of myeloid differentiation. Our screen identified treprostinil, a prostacyclin agonist, as a pro-differentiation agent; prostacyclin agonists have not previously been reported as active in myelopoiesis. The importance of prostacyclin signaling in AML is supported by a number of observations. (1) AML expression databases demonstrate that the prostacyclin receptor (PTGIR) is present on a subset of blasts, making this an attractive therapeutic target. (2) Murine cell lines arrested in differentiation by HoxA9, E2a/Pbx1 and MLL/AF9 upregulate CD11b in response to treprostinil suggesting that stimulation through PTGIR acts via a general mechanism rather than in a HoxA9-specific manner. (3) A subset of primary human AML samples upregulate CD11b when treated with treprostinil ex vivo. (4) Mice bearing a HoxA9-driven secondary leukemia were treated with treprostinil or PBS for 10-days. Those mice treated with treprostinil showed a marked reduction in spleen size and a significant reduction in marrow leukemic burden. Leukemic cells isolated from these mice showed a similar pattern of expression changes as those cells treated in vitro. Prostacyclin agonists may represent a new form of differentiation therapy in the treatment of AML.
P1009 - MULTILINEAGE RECONSTITUTION FROM SINGLE HEMATOPOIETIC PROGENITORS IN VIVO Scott Boyer, Stephanie Smith-Berdan, Anna Beaudin, and Camilla Forsberg UC Santa Cruz, Capitola, California, USA Single-cell assays are essential for accurate assessment of the lineage potential of stem and progenitor cells. Single hematopoietic stem cell (HSC) transplantation has demonstrated that a single HSC has the ability to reconstitute all hematopoietic lineages for the long term, indicating that lineage commitment occurs in downstream progenitor cells. Where the first lineage commitment event occurs during hematopoietic differentiation and which lineage potential(s) are lost first is currently unclear. Here, we tested the production of all major mature cell types by comprehensive and quantitative analysis of mature cell production from HSCs and multiple hematopoietic progenitor populations upon transplantation. Strikingly, our results revealed that Flk2-expressing multipotent progenitors (MPPs) generate w1,000 times as many red blood cells and w100 times as many platelets as any white blood cell type upon transplantation, similar to ratios produced by HSCs. These results show that MPPs, as a population, can reconstitute all hematopoietic lineages, but overwhelmingly appear to propagate erythroid and megakaryocytic lineages. To test whether MPPs are multipotent at the single cell level or, alternatively, consist of a heterogeneous mixture of lineage-committed cells, we implemented two in vivo approaches capable of evaluating multilineage readout from single progenitor cells: single-progenitor transplants and analysis of single-progenitor derived CFU-S. Remarkably, single MPPs were able to reconstitute erythroid, megakaryocytic, myeloid, and lymphoid lineages in both assays. These results demonstrate that MPPs contribute substantially to all hematopoietic lineages and that a significant fraction of MPPs have multilineage potential at the single-cell level. Given that both HSCs and MPPs are capable of multilineage reconstitution at the single cell level, we conclude that lineage commitment occurs after the upregulation of Flk2 expression and the subsequent generation of MPPs.
S25
P1011 - MEGAKARYOCYTE EXPANSION BY THE FETAL LIVER MICROENVIRONMENT Fanny Siad, Catherine Strassel, Francois Lanza, Christian Gachet, and Nathalie Brouard UMR-S949 INSERM, Strasbourg, France Blood platelets are produced by megakaryocytes (MK) that derive from hematopoietic stem cells (HSC) through a process termed megakaryopoiesis. While a large range of studies are focusing on the specific microenvironment regulating the maintenance and proliferation of HSC (the niche), little is known about of the microenvironment regulating megakaryopoiesis, in particular the cellular elements involved. The fetal liver is the site of a major expansion of hematopoietic cells. Hence we hypothesized that the fetal liver niche could be the source of specific determinants capable of supporting a large expansion of megakaryocytes and their progenitors. The present study focuses on defining the cellular components of the fetal liver that promote the production of MK from HSC. We first determined, using a combination of cell surface markers, the cell populations present in the mouse fetal liver at day 13.5. We then identified cell populations that can produce adherent layers that will be referred to as stromal cells. We tested in serum free conditions, with minimal addition of cytokines their capacity to support the production of MK from purified bone marrow HSC (SLAM phenotype). In the co-cultures with stromal layers derived from CD45-TER119-CD31-CD51+VCAM-1+PDGFR-cells (V+P-), we observed the production of erythroid progenitors and a large number of megakaryocytes identified by the expression of CD41 and CD42c. In contrast, very few megakaryocytes were produced in co-culture with other fetal liver derived stromal layers. While the proportion of MK produced in absence of stromal layers was similar, their number was dramatically increased (up to 1000 fold) in the co-culture with the V+P-derived stromal layers. The megakaryocytes produced were then sub-cultured in conditions allowing full maturation and efficiently produced proplatelet extensions. We propose that the V+P-stromal cell population represents an essential component of the fetal microenvironment supporting the engagement toward the megakaryocytic lineage and, more importantly, the expansion of MK progenitors. Further characterization of these stromal cells and the factors they produce may guide the development of new methods for the in vitro production of platelets.
P1012 - DPP4 REGULATES IN VITRO AND IN VIVO HEMATOPOIESIS MEDIATED BY MYELOSUPPRESSIVE CHEMOKINES Hal Broxmeyer, Scott Cooper, Timothy Campbell, Heather O’Leary, and Giao Hangoc Department of Microbiology/Immunology, Indiana University School of Medicine, Indianapolis, Indiana, USA We reported that Dipeptidylpeptidase 4 (DPP4) truncates GM-CSF, G-CSF, IL-3, EPO and CXCL12 (Broxmeyer et al, 2012, Nature Med 18:1786). Truncated (TR)-CSFs are decreased in hematopoietic progenitor cell (HPC) stimulating activity, and TRCXCL12/SDF-1 is inactive as an HPC chemotactic, survival or expansion factor. Moreover, TR-molecules block positively acting effects of their own full length (FL)-molecule. Many chemokines have DPP4 TR sites. To determine a role for DPP4 truncation on negatively acting cytokines, we assessed in vitro effects of FL, TR, and FL plus TR myelosuppressive CCL3/MIP-1a, CCL2/MCP-1, CXCL4/PF4, CXCL5/ENA-74, CXCL6/GCP-2, GXCL8/IL-8, CXCL9/MIG, and CXL10/IP-10, and non-myelosuppressive CCL4/MIP-1b and CCL5/RANTES on multi-cytokine stimulated HPC. Myelosuppressive XCL1/Lymphotoxin, without a DPP4 TR site, was also assessed. Myelosupressive chemokines with, but not without, a DPP4-TR site were 100-1000 fold enhanced in suppressor potency by pretreating target BM cells with Diprotin A, a DPP4 inhibitor, or by assaying activity on dpp4 -/- marrow. Non myelosuppressive chemokines remained non suppressive. DPP4-treatment of chemokines with myelosuppressive activity produced non-myelosuppressive molecules that blocked suppression by their FL-chemokine, likely through receptor interference. In vitro effects were reproduced in vivo after IV injection of FL, TR, and FL plus TR CCL3, CXCL8, and CXCL9. CCL3 is myelosuppressive for immature subsets of HPC responsive to stimulation by multiple cytokines, but CCL3 and non-myelosuppressive CCL4 enhance single cytokine (GM-CSF or M-CSF) stimulated colony formation in vitro by more mature HPC (Broxmeyer et al, 1989, JEM 170:1583; 1990, Blood 76:1110). We now show that DPP4-TR CCL3 and CCL4 lose their enhancing activity for GM-CSF and M-CSF stimulated HPC in vitro and in vivo, and TR-CCL3 and -CCL4 each block enhancement by FL CCL3 and CCL4. This highlights intricate in vitro and in vivo influences of DPP4 that should be considered when evaluating regulatory cytokine/chemokine effects and means to modulate hematopoiesis for biological and clinical insight.
P1008 - PHENOTYPIC SCREENING IDENTIFIES PROSTACYCLIN AGONISTS AS DIFFERENTIATION AGENTS IN THE TREATMENT OF ACUTE MYELOID LEUKEMIA David Sykes1, Youmna Kfoury1, Andres Blanco3, and David Scadden1,2 1 Massachusetts General Hospital, Boston, Massachusetts, USA; 2Harvard Stem Cell Institute, Boston, Massachusetts, USA; 3Children’s Hospital, Boston, Massachusetts, USA One success story in AML treatment has been the discovery of drugs that trigger the differentiation of leukemic blasts in the subset of patients with acute promyelocytic leukemia. Differentiation therapy is not available for the remaining 90% of AML patients. To identify small molecules that trigger differentiation, we developed a novel model of AML, with a built-in GFP reporter of differentiation. In this model, primary bone marrow cells from a lysozyme-GFP knock-in mouse were arrested in differentiation by an ER-HoxA9 fusion protein. The cells grow indefinitely in the presence of SCF and estradiol, but upon withdrawal of estradiol (inactivation of HoxA9), the cells undergo terminal myeloid differentiation, becoming brightly GFP(+). This model formed the basis of a screen of 350,000 small molecules using the flow-cytometric detection of GFP as a marker of myeloid differentiation. Our screen identified treprostinil, a prostacyclin agonist, as a pro-differentiation agent; prostacyclin agonists have not previously been reported as active in myelopoiesis. The importance of prostacyclin signaling in AML is supported by a number of observations. (1) AML expression databases demonstrate that the prostacyclin receptor (PTGIR) is present on a subset of blasts, making this an attractive therapeutic target. (2) Murine cell lines arrested in differentiation by HoxA9, E2a/Pbx1 and MLL/AF9 upregulate CD11b in response to treprostinil suggesting that stimulation through PTGIR acts via a general mechanism rather than in a HoxA9-specific manner. (3) A subset of primary human AML samples upregulate CD11b when treated with treprostinil ex vivo. (4) Mice bearing a HoxA9-driven secondary leukemia were treated with treprostinil or PBS for 10-days. Those mice treated with treprostinil showed a marked reduction in spleen size and a significant reduction in marrow leukemic burden. Leukemic cells isolated from these mice showed a similar pattern of expression changes as those cells treated in vitro. Prostacyclin agonists may represent a new form of differentiation therapy in the treatment of AML.
P1009 - MULTILINEAGE RECONSTITUTION FROM SINGLE HEMATOPOIETIC PROGENITORS IN VIVO Scott Boyer, Stephanie Smith-Berdan, Anna Beaudin, and Camilla Forsberg UC Santa Cruz, Capitola, California, USA Single-cell assays are essential for accurate assessment of the lineage potential of stem and progenitor cells. Single hematopoietic stem cell (HSC) transplantation has demonstrated that a single HSC has the ability to reconstitute all hematopoietic lineages for the long term, indicating that lineage commitment occurs in downstream progenitor cells. Where the first lineage commitment event occurs during hematopoietic differentiation and which lineage potential(s) are lost first is currently unclear. Here, we tested the production of all major mature cell types by comprehensive and quantitative analysis of mature cell production from HSCs and multiple hematopoietic progenitor populations upon transplantation. Strikingly, our results revealed that Flk2-expressing multipotent progenitors (MPPs) generate w1,000 times as many red blood cells and w100 times as many platelets as any white blood cell type upon transplantation, similar to ratios produced by HSCs. These results show that MPPs, as a population, can reconstitute all hematopoietic lineages, but overwhelmingly appear to propagate erythroid and megakaryocytic lineages. To test whether MPPs are multipotent at the single cell level or, alternatively, consist of a heterogeneous mixture of lineage-committed cells, we implemented two in vivo approaches capable of evaluating multilineage readout from single progenitor cells: single-progenitor transplants and analysis of single-progenitor derived CFU-S. Remarkably, single MPPs were able to reconstitute erythroid, megakaryocytic, myeloid, and lymphoid lineages in both assays. These results demonstrate that MPPs contribute substantially to all hematopoietic lineages and that a significant fraction of MPPs have multilineage potential at the single-cell level. Given that both HSCs and MPPs are capable of multilineage reconstitution at the single cell level, we conclude that lineage commitment occurs after the upregulation of Flk2 expression and the subsequent generation of MPPs.
S25
P1011 - MEGAKARYOCYTE EXPANSION BY THE FETAL LIVER MICROENVIRONMENT Fanny Siad, Catherine Strassel, Francois Lanza, Christian Gachet, and Nathalie Brouard UMR-S949 INSERM, Strasbourg, France Blood platelets are produced by megakaryocytes (MK) that derive from hematopoietic stem cells (HSC) through a process termed megakaryopoiesis. While a large range of studies are focusing on the specific microenvironment regulating the maintenance and proliferation of HSC (the niche), little is known about of the microenvironment regulating megakaryopoiesis, in particular the cellular elements involved. The fetal liver is the site of a major expansion of hematopoietic cells. Hence we hypothesized that the fetal liver niche could be the source of specific determinants capable of supporting a large expansion of megakaryocytes and their progenitors. The present study focuses on defining the cellular components of the fetal liver that promote the production of MK from HSC. We first determined, using a combination of cell surface markers, the cell populations present in the mouse fetal liver at day 13.5. We then identified cell populations that can produce adherent layers that will be referred to as stromal cells. We tested in serum free conditions, with minimal addition of cytokines their capacity to support the production of MK from purified bone marrow HSC (SLAM phenotype). In the co-cultures with stromal layers derived from CD45-TER119-CD31-CD51+VCAM-1+PDGFR-cells (V+P-), we observed the production of erythroid progenitors and a large number of megakaryocytes identified by the expression of CD41 and CD42c. In contrast, very few megakaryocytes were produced in co-culture with other fetal liver derived stromal layers. While the proportion of MK produced in absence of stromal layers was similar, their number was dramatically increased (up to 1000 fold) in the co-culture with the V+P-derived stromal layers. The megakaryocytes produced were then sub-cultured in conditions allowing full maturation and efficiently produced proplatelet extensions. We propose that the V+P-stromal cell population represents an essential component of the fetal microenvironment supporting the engagement toward the megakaryocytic lineage and, more importantly, the expansion of MK progenitors. Further characterization of these stromal cells and the factors they produce may guide the development of new methods for the in vitro production of platelets.
P1012 - DPP4 REGULATES IN VITRO AND IN VIVO HEMATOPOIESIS MEDIATED BY MYELOSUPPRESSIVE CHEMOKINES Hal Broxmeyer, Scott Cooper, Timothy Campbell, Heather O’Leary, and Giao Hangoc Department of Microbiology/Immunology, Indiana University School of Medicine, Indianapolis, Indiana, USA We reported that Dipeptidylpeptidase 4 (DPP4) truncates GM-CSF, G-CSF, IL-3, EPO and CXCL12 (Broxmeyer et al, 2012, Nature Med 18:1786). Truncated (TR)-CSFs are decreased in hematopoietic progenitor cell (HPC) stimulating activity, and TRCXCL12/SDF-1 is inactive as an HPC chemotactic, survival or expansion factor. Moreover, TR-molecules block positively acting effects of their own full length (FL)-molecule. Many chemokines have DPP4 TR sites. To determine a role for DPP4 truncation on negatively acting cytokines, we assessed in vitro effects of FL, TR, and FL plus TR myelosuppressive CCL3/MIP-1a, CCL2/MCP-1, CXCL4/PF4, CXCL5/ENA-74, CXCL6/GCP-2, GXCL8/IL-8, CXCL9/MIG, and CXL10/IP-10, and non-myelosuppressive CCL4/MIP-1b and CCL5/RANTES on multi-cytokine stimulated HPC. Myelosuppressive XCL1/Lymphotoxin, without a DPP4 TR site, was also assessed. Myelosupressive chemokines with, but not without, a DPP4-TR site were 100-1000 fold enhanced in suppressor potency by pretreating target BM cells with Diprotin A, a DPP4 inhibitor, or by assaying activity on dpp4 -/- marrow. Non myelosuppressive chemokines remained non suppressive. DPP4-treatment of chemokines with myelosuppressive activity produced non-myelosuppressive molecules that blocked suppression by their FL-chemokine, likely through receptor interference. In vitro effects were reproduced in vivo after IV injection of FL, TR, and FL plus TR CCL3, CXCL8, and CXCL9. CCL3 is myelosuppressive for immature subsets of HPC responsive to stimulation by multiple cytokines, but CCL3 and non-myelosuppressive CCL4 enhance single cytokine (GM-CSF or M-CSF) stimulated colony formation in vitro by more mature HPC (Broxmeyer et al, 1989, JEM 170:1583; 1990, Blood 76:1110). We now show that DPP4-TR CCL3 and CCL4 lose their enhancing activity for GM-CSF and M-CSF stimulated HPC in vitro and in vivo, and TR-CCL3 and -CCL4 each block enhancement by FL CCL3 and CCL4. This highlights intricate in vitro and in vivo influences of DPP4 that should be considered when evaluating regulatory cytokine/chemokine effects and means to modulate hematopoiesis for biological and clinical insight.