Inflammasome-Mediated Regulation of Hematopoiesis in the Vertebrate Embryo

Abstract Hematopoietic stem and progenitor cells (HSPCs) arise in the embryonic dorsal aorta (DA) through a Runx1-dependent process of endothelial-to-hematopoietic transition (EHT). Subsequently, HSPCs colonize secondary niches to proliferate, differentiate and maintain lifelong hematopoiesis. We previously reported that elevated glucose metabolism and sterile inflammatory signaling each stimulate zebrafish HSPC production from hemogenic endothelium. Consistent with the hypothesis that transient metabolic activation induces an inflammatory response to influence HSPC formation, we found that glucose stimulation from 12-36 hours post fertilization (hpf) increased expression of pro-inflammatory cytokines and receptors by quantitative PCR. In particular, morpholino-mediated knockdown of il1b blunted the inductive effects of glucose metabolism on runx1 expression in the DA and numbers of CD41+ HSPCs in the caudal hematopoietic tissue (CHT). In contrast, overexpression of mature il1b increased HSPC numbers as assessed by flow cytometry and runx1/cmyb in situ hybridization. As IL-1β is cleaved and activated by the inflammasome, which is a well-known sensor of metabolic stimuli, we treated embryos with validated inflammasome activators, such as nigericin, and found that they also increased runx1/cmyb expression and Flk1+cMyb+ HSPCs. Inflammasome stimulation required NF-κB activity, and upregulated canonical IL-1β targets, including IL-6 and IL-8, which have been previously shown by our laboratory to play significant roles in embryonic HSPC formation. Importantly, loss of the inflammasome adapter pycard or effector caspa reduced HSPC numbers, and prevented expansion of the HSPC pool in response to glucose stimulation. In further support of a role for sterile inflammatory activity in regulating HSPC number downstream of metabolic activation, we found that inflammasome-mediated IL-1β action bypassed the suppressive effect of antioxidant treatment on runx1/cmyb expression in the DA. Conversely, inflammasome inhibition partially blocked effects of metabolism-associated HIF1α activation on runx1/cmyb expression, indicating that the inflammasome acts downstream of reactive-oxygen-species (ROS) generation and HIF1α activation to promote HSPC production and/or expansion from hemogenic endothelium. Inflammasome components are highly expressed in myeloid cells; targeted knockdown and cell ablation studies indicate that macrophages are necessary to mediate the effects of inflammasome stimulation on HSPC numbers in the CHT at 72hpf. Interestingly, prolonged inflammasome stimulation also expands myeloid and lymphoid progenitors, as cmyb, rag1 and mpo expression were each elevated at 120hpf; increased numbers of Rag2+ and Mpo+ cells were confirmed by flow cytometry. To determine whether the effects of inflammasome activation were conserved in higher vertebrates, we induced inflammasome activation during differentiation of human induced pluripotent stem cells (iPSC) into hematopoietic progenitors. In this system, nigericin treatment of iPSC-derived hemogenic endothelial cells yielded an increased frequency of functional colony-forming units by day 7 of EHT culture. Taken together, inflammasome-mediated IL-1β action appears to serve as an integrator of metabolic activity, downstream of ROS/ HIF1α, to promote HSPC formation and development of myeloid and lymphoid lineages in vivo and in vitro. These studies identify the inflammasome as a promising target to promote human HSPC production from iPSCs for therapeutic purposes. Disclosures No relevant conflicts of interest to declare.

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Development of Hras-Induced Zebrafish Leukemia Models

Blood ◽

10.1182/blood.v126.23.2459.2459 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2459-2459

Author(s):

Elisa Alghisi ◽

Martina Konantz ◽

Marina Mione ◽

Claudia Lengerke

Keyword(s):

Flow Cytometry ◽

Conflicts Of Interest ◽

Experimental Models ◽

Pcr Analysis ◽

Human Leukemia ◽

Hematopoietic Tissue ◽

Hematopoietic Stem ◽

Primitive Hematopoiesis ◽

Transgenic Lines ◽

Early Lethality

Abstract Background: Over the last years, the zebrafish has emerged as a versatile novel experimental model for hematopoietic studies. The major genetic pathways have proven conserved between fish and mammalian hematopoiesis. Several oncogenes involved in human leukemia have been successfully overexpressed in a transient fashion in zebrafish embryos. However, despite first encouraging results these experimental models often failed to fully recapitulate human myeloid malignancy, perhaps due to early lethality caused by off-target expression or lack of secondary events necessary for full malignant transformation. Material and Methods: Here we took advantage of the Gal4/UAS binary system and of existing transgenic lines to overexpress the human oncogenic HRAS gene in zebrafish hematopoietic cells under control of specific promoters (fli.1, pu.1, runx.1). HRAS-transgenic lines were generated and fish followed by microscopy from early development until, if possible, sexual maturity. Hematopoietic cell development was studied at embryonic (fli.1, pu.1), larval (pu.1, runx1) and adult stages (runx1) by in situ hybridization and real-time PCR analysis of hematopoietic gene expression, flow cytometry, immunohistochemistry and/or blood smear morphological assessment. Results: All HRAS transgenic lines showed hematopoietic abnormalities. However, different phenotypes were observed depending on the promoter driving the oncogene expression. HRAS induction via the early hematopoietic promoter fli.1 affected primitive hematopoiesis inducing a myelo-erythroid proliferation characterized by the expansion of the caudal hematopoietic tissue, enhanced expression of myelo-erythroid genes and delayed erythrocyte maturation (Alghisi et al. 2013). Surprisingly, no obvious effects were noted on the emerging hematopoietic stem cells (HSCs) in the aorta-gonado-mesonephros (AGM) region and studies at later stages were hampered by early lethality of the fish due to vascular defects and cardiac edema. The lethality at early stages was also observed using the myeloid promoter pu.1 that induced the expansion of primitive myeloid cells along with severe developmental defects. In contrast, HRAS expression driven by runx1, a known HSC marker, did not affect primitive hematopoiesis and allowed studies at later developmental stages. Supporting the results obtained with the fli.1 promoter, no alteration was noted in the AGM of runx1-HRAS induced fish. However, at 1 month post-fertilization, flow cytometry analyses revealed a prominent cellular expansion of the hematopoietic stem/progenitor cells in the kidney marrow, the zebrafish definitive hematopoietic compartment. Kidney marrow cytospin preparation and flow cytometry analysis confirmed high numbers of undifferentiated cells indicating that HRAS -overexpression in this model induced stem/progenitor cell proliferation. Interestingly, first analyses suggested that the numbers of differentiated cells might be reduced in these fish, implying that HRAS-induced stem/progenitors have impaired differentiation capacity. Outlook and conclusion: We are currently further investigating the effects of runx1-driven HRAS on the hematopoietic compartment and generate tools to explore potential cooperation of HRAS with other oncogenes during leukemogenesis. Successfully established zebrafish leukemia models shall be eventually used for identification of therapeutically active compounds in small molecule screens. Disclosures No relevant conflicts of interest to declare.

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CongenitalSTAT3mutation Mediates Primary Persistent Polymorphic Inflammatory Activation and T Cell Leukemogenesis

Blood ◽

10.1182/blood-2019-132138 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1054-1054 ◽

Cited By ~ 1

Author(s):

Hongxing Liu

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

T Cell ◽

Lymph Nodes ◽

Conflicts Of Interest ◽

Sh2 Domain ◽

Janus Kinase ◽

Gingival Hyperplasia ◽

Hematopoietic Stem ◽

T Cell Leukemogenesis

Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathways play a pivotal role in inflammation and immunity, among which, JAK/STAT3 pathway is the most potent and leads the crosstalk of immunity and oncogenesis. Somatic STAT3 activatingmutations have been found in about 40% of T cell large granular lymphocytic leukemia (T-LGLL) patients, most of which are located in exon 21 which encodes Src homology 2 (SH2) domain leading to the increased activity of aberrant STAT3 protein and the upregulation of its transcriptional targets. While germline STAT3activatingmutations represent a newly defined entity of immune dysregulations named infantile-onset multisystem autoimmune disease-1 (ADMIO1, #MIM 615952). Both the two diseases are rare and poorly understood. Here, we report a pedigree including a proband, a six-year-old girl, primarily manifesting as thrombocytopenia and lymphadenopathy and her father diagnosed as T-LGLL with pure red cell aplastic anemia without autoimmune disorders preceding or during his disease course. Morphology of the bone marrow smears of the proband indicated normal hyperplasia without evident dyspepsia or increased blast cells. However, the vacuoles in monocytes and the density and size of granules in neutrophils increased, and megaloblast transformation was observed in some neutrophils. (Fig. 1A, 1B) Biopsy of an enlarged lymph node showed the reactive follicular hyperplasia. (Fig. 1C) Whole exon sequencing and pedigree analysis of the family revealed the germline STAT3 c.833G>A/p.R278Hmutation harbored by the proband which originated de novo from her father who additionally carried a germline TAL1G62Rmutation and somatically accumulated an FLT3-ITD mutation. (Fig. 2) Through single-cell RNA sequencing, we also found the increase of circulating CD8+ T cells and the decrease of NK cells of the proband. (Fig. 3) The STAT3 target genes were generally overactivated, and the expression of cytokines decreased in transcription level. In the genes participating in JAK/STATs pathways, the expression of JAK3, STAT1, and STAT3was up-regulated significantly. (data not shown) Immunophenotype of the proband by flow cytometry confirmed change in immunocyte compartments, (Fig. 4) but the serum cytokine concentrations measured by flow cytometry yielded controversial results, that most of cytokines were moderately elevated, and IL-1β, IL-5, TNF-α, and IFN-γ were of the most evident. (data not shown) During the treatment and follow-up, Cyclosporin A (CsA) was efficient in maintaining her circulating platelets in the range of 166×109/L to 302×109/L, but the enlarged lymph nodes and hepatosplenomegaly had no response. Eleven months later, CsA was replaced by tacrolimusfor the severe gingival hyperplasia, which has efficiently stabilized her platelets count and normalized the enlarged lymph nodes, liver, and spleen. On the contrary, in the three and a half years' span of illness, the father was refractory to CsA and methotrexate (MTX), moreover, lethal bone marrow suppression was induced by one course of fludarabine. For the high level of HLA-I and HLA-II antibodies in the circulation, plantlets transfusions were only efficient after plasmapheresis. The father eventually died from pulmonary and gastrointestinal infection due to the failure of maternal HLA-haploidentical hematopoietic stem cell transplantation (HSCT). We comprehensively elaborated the immunophenotype of the proband and thoroughly elucidated the genetic alternations of the father which led to the T cell leukemogenesis, which brought new insight on these two rare diseases and highlighted a more scrupulous therapeutic strategy in T-LGLL with congenital mutations. Figure 1 Disclosures No relevant conflicts of interest to declare.

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Metabolic Regulation of Hematopoietic Stem Cells During Stress

Blood ◽

10.1182/blood.v120.21.sci-42.sci-42 ◽

2012 ◽

Vol 120 (21) ◽

pp. SCI-42-SCI-42

Author(s):

Toshio Suda

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Metabolic Regulation ◽

Conflicts Of Interest ◽

Hypoxia Inducible Factor ◽

Ros Generation ◽

Hematopoietic Stem

Abstract Abstract SCI-42 Tissue homeostasis over the life of an organism relies on both self-renewal and multipotent differentiation of stem cells. Hematopoietic stem cells (HSCs) are sustained in a specific microenvironment known as the stem cell niche. Adult HSCs are kept quiescent during the cell cycle in the endosteal niche of the bone marrow. Normal HSCs maintain intracellular hypoxia, stabilize the hypoxia-inducible factor-1a (HIF-1a) protein, and generate ATP by anaerobic metabolism. In HIF-1a deficiency, HSCs became metabolically aerobic, lost cell cycle quiescence, and finally became exhausted. An increased dose of HIF-1a protein in VHL-mutated HSCs and their progenitors induced cell cycle quiescence and accumulation of HSCs in the bone marrow (BM), which were not transplantable. This metabolic balance promotes HSC maintenance by limiting the production of reactive oxygen species (ROS), but leaves HSCs susceptible to changes in redox status (1). We have performed the metabolomic analysis in HSCs. Upregulation of pyruvate dehydrogenase kinases enhanced the glycolytic pathway, cell cycle quiescence, and stem cell capacity. Thus, HSCs directly utilize the hypoxic microenvironment to maintain their slow cell cycle by HIF-1a-dependent metabolism. Downregulation of mitochondrial metabolism might be reasonable, since it reduces ROS generation. On the other hand, at the time of BM transplantation, HSCs activate oxidative phosphorylation to acquire more ATP for proliferation. Autophagy also energizes HSCs by providing amino acids during transplantation. ATG (autophagy-related) 7 is essential for transplantation and metabolic homeostasis. The relationship between mitochondrial heat shock protein, mortalin, and metabolism in HSCs will also be discussed. Disclosures: No relevant conflicts of interest to declare.

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Disease-Relevant Expression Of TRAF6 In Mouse HSC Results In An MDS-Like Disease

Blood ◽

10.1182/blood.v122.21.101.101 ◽

2013 ◽

Vol 122 (21) ◽

pp. 101-101

Author(s):

Jing Fang ◽

Xiaona Liu ◽

Brenden Barker ◽

Lyndsey Bolanos ◽

Yue Wei ◽

...

Keyword(s):

Gene Expression ◽

Conflicts Of Interest ◽

Early Stage ◽

Bone Marrow Failure ◽

Gene Signature ◽

Sublethal Dose ◽

Hematopoietic Tissue ◽

Hematopoietic Stem ◽

Disease Evolution ◽

Cd34 Cells

Abstract Overexpression of immune-related genes is widely reported in Myelodysplastic Syndrome (MDS), and chronic immune stimulation increases the risk for developing MDS. We find that TNF receptor associated factor 6 (TRAF6), an innate immune protein, is overexpressed approximately 2-fold in CD34+ cells from 40% of MDS patients, and may explain immune pathway activation in the MDS-initiating hematopoietic stem/progenitor cell (HSPC). In support of these observations and our hypothesis that TRAF6 is important in the pathophysiology of MDS, a gene expression analysis revealed that TRAF6 controls an MDS gene signature in human cells. We, and others, have previously shown that retroviral overexpression of TRAF6 in mouse HSPC results in MDS and Acute Myeloid Leukemia (AML). However, interpretations of these findings are hampered by supra-physiological levels of TRAF6 (>10-fold overexpression) and the stress associated with HSPC transduction/transplantation. To investigate the consequences of TRAF6 overexpression to MDS, we generated a transgenic mouse model overexpressing TRAF6 from a hematopoietic-specific Vav promoter. Expression of TRAF6 in HSPC was approximately 2-fold higher as compared to endogenous TRAF6 and in line with MDS patient CD34+ cells. By 15 months of age, half of Vav-TRAF6 mice succumbed to a hematologic disease resembling MDS associated with bone marrow failure (BMF). In contrast to the retroviral overexpression approach, Vav-TRAF6 mice did not develop AML. Examination of sick mice revealed stage-specific disease evolution. Initially, all Vav-TRAF6 mice exhibit an inversion of myeloid/lymphoid proportions. For Vav-TRAF6 mice that develop a fatal disease, they present with a hypocellular marrow, dysplasic myeloid cells, and neutropenia. A subset of mice also display anemia with nucleated red blood cells, poikilocytosis, and extramedullular erythropoiesis. In support of a BMF phenotype, HSPC from Vav-TRAF6 mice form fewer colonies in methylcellulose. To investigate the consequences of an acute exposure to pathogen, early-stage Vav-TRAF6 mice were treated with a single sublethal dose of lipopolysaccharide (LPS). Unlike wild-type (WT) mice, Vav-TRAF6 mice developed a rapid and reversible anemia, suggesting environmental factors can influence the severity of the disease. To gain insight into the mechanism contributing to BMF, gene expression profiling was performed in WT and Vav-TRAF6 HSPC. One of the enriched pathways consisted of AKT activation and FOXO downregulation. Consistent with the microarray analysis, AKT is constitutively phosphorylated at Thr308 in hematopoietic tissue from Vav-TRAF6 mice. SOD2, a transcriptional target of FoxO3a that is suppressed by activated AKT, is decreased in Vav-TRAF6 HSPC. Given that AKT/FOXO regulate reactive oxygen species (ROS) in cells, we investigated ROS levels in HPSC from Vav-TRAF6 and WT mice. Intracellular ROS is significantly elevated in BM cells from Vav-TRAF6 mice, and restored to normal levels when AKT was inhibited. In conclusion, we propose the potential role of TRAF6 in the development of MDS-associated BMF, partly due to constitutive activation of AKT and subsequent ROS elevation in HSPC cells. Disclosures: No relevant conflicts of interest to declare.

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Sf3b1 Regulation of Jak/Stat Signaling Is Essential for Hematopoietic Stem Cell Formation

Blood ◽

10.1182/blood-2018-99-118158 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1268-1268

Author(s):

Teresa V. Bowman ◽

Rosannah C. Cameron ◽

Kathryn S Potts ◽

Mia McKinstry ◽

Varun Gupta ◽

...

Keyword(s):

Rna Splicing ◽

Conflicts Of Interest ◽

Active Form ◽

Janus Kinase ◽

Splicing Factor ◽

Loss Of Function ◽

Wild Type ◽

Hemogenic Endothelium ◽

Hematopoietic Stem ◽

Stat Signaling

Abstract Hematopoietic stem cells (HSCs) maintain the hematopoietic system throughout the lifetime of an organism. During embryonic development, HSCs emerge through an endothelial-to-hematopoietic transition (EHT) from specialized hemogenic endothelial (HE) cells in the dorsal aorta. HSC fate specification depends on gene expression, which is the culmination of coordinated transcription, RNA splicing, and translation. Although transcriptional regulation of HSC fate choice is well studied, the regulatory role of RNA splicing in this process is poorly understood. Using zebrafish loss-of-function mutants for the spliceosomal component splicing factor 3b, subunit 1 (sf3b1), we identified that impaired splicing hindered HSC production. Surprisingly, we found that this constitutive splicing factor selectively regulates the fate of hemogenic endothelium while leaving the identity of closely-related non-hemogenic endothelium unperturbed. To identify Sf3b1-regulated transcripts important in EHT, we performed RNA-sequencing on purified kdrl:gfp+ endothelial cells from sf3b1 mutant and wild-type siblings at 24 hpf. Approximately 900 genes were mis-spliced, 144 of which were differentially expressed. Ingenuity Pathway Analysis identified Janus Kinase (Jak)/Signaling Transducer and Activator of Transcription (Stat) signaling, in particular Stat3, as one of the top perturbed pathways in the mis-spliced gene set. Stat3 is a transcription factor activated in response to several cytokine and inflammatory signals. To determine if altered splicing of stat3 was critical for HSC formation, we injected antisense splice-blocking morpholinos (MO) targeting the Sf3b1-sensitive stat3 exon19 into wild-type and sf3b1 heterozygous embryos, which normally generate equivalent levels of HSCs. We observed an impairment of HSC production in stat3 morpholino-injected sf3b1 heterozygotes, but not wild-type siblings, indicating a synthetic lethal interaction between sf3b1 and stat3. We also found that overexpression of a constitutively active form of Stat3 significantly suppressed the HSC defects in sf3b1 homozygous mutants. Together, these data indicate that Sf3b1-mediated splicing regulation of the Jak/Stat pathway is critical for HSC emergence. Disclosures No relevant conflicts of interest to declare.

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Clinical Utility of Flow Cytometry Immunophenotyping in Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2019-127099 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5191-5191

Author(s):

Gustavo Henrique de Medeiros Oliveira ◽

Lenilton Silva DA Silva Júnior ◽

Antonia Eduarda Martins Oliveira Elói Silva ◽

João Pedro Andrade Lima ◽

Victor lima Soares ◽

...

Keyword(s):

Flow Cytometry ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Clinical Laboratory ◽

Conflicts Of Interest ◽

Demographic Data ◽

Hematopoietic Tissue ◽

Diagnostic Challenge ◽

Acute Leukemias ◽

Acute Myeloid

INTRUDUCTION: Acute myeloid leukemia (AML) is a heterogeneous group of clonal disease of hematopoietic tissue, characterized by proliferation of abnormal myeloid progenitor cells, suppressing the normal hematopoietic activity and constituting a great diagnostic challenge. With the advent of immunophenotyping by flow cytometry, the diagnosis of these neoplasms became more faithful, facilitating the treatment and follow-up of the patients. METHODS: In this study a flow cytometric immunophenotyping study was performed in bone marrow aspirate and / or peripheral blood samples from 38 patients with AML, using a panel of monoclonal antibodies specific for acute leukemias, also investigating clinical, laboratory and demographic data of these patients. RESULTS AND DISCUSSION: Of the 38 subjects studied, 23 were male and 15 females. In relation to the age, we found a higher number of cases in adult patients. According to clinical data, splenomegaly and hepatomegaly were present in most cases. The immunophenotyping demonstrated a characteristic profile of AML with expression of CD13 and CD33 in all cases and CD34 and CD117 in most cases. The CD14 was reactive in monocytic leukemia (M4 and M5-AML). Were observed negativity for lymphoid antigens CD19, CD10 and CD3 and positivity to CD7, CD56 and CD2, present in 10, 5 and one cases respectively. Regarding cytomorphology, there was a direct correlation with FAB classification, with the prevalence of type myeloid-monocytic (AML-M4). CONCLUSION: These data demonstrate the importance of immunophenotyping in the differential diagnosis of AML and monitoring of these neoplasms. Disclosures No relevant conflicts of interest to declare.

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Blood Flow Is Required for the Release of Hematopoietic Stem- and Progenitor Cells From Hemogenic Endothelium in the Placenta.

Blood ◽

10.1182/blood.v114.22.698.698 ◽

2009 ◽

Vol 114 (22) ◽

pp. 698-698

Author(s):

Katrin E Rhodes ◽

Ben Van Handel ◽

Michele Wang ◽

Yanling Wang ◽

Akanksha Chhabra ◽

...

Keyword(s):

Endothelial Cells ◽

Blood Flow ◽

Progenitor Cells ◽

Conflicts Of Interest ◽

Yolk Sac ◽

Positive Staining ◽

Hemogenic Endothelium ◽

Hematopoietic Stem ◽

Notch1 Signaling ◽

Stem And Progenitor Cells

Abstract Abstract 698 Hematopoietic stem cells (HSCs) are required for continuous blood cell production throughout life. HSCs emerge only within a short developmental time window during embryogenesis. Mounting evidence posits that HSCs arise directly from hemogenic endothelial cells during midgestation within the large arteries of the conceptus, which include the dorsal aorta, the umbilical and vitelline arteries and the chorioallantoic vessels of the placenta. However, the microenvironmental signals that mediate this temporally regulated process remain unclear. Here we examine, by using Ncx1−/− embryos that lack heartbeat and circulation, how blood flow imparts instructive cues that ensure proper HSC development. Immunostaining revealed that CD41+ hematopoietic cells, although present, were markedly decreased in Ncx1-/-placentas as compared to wild-type controls. Furthermore, mutant placentas evidenced large clusters of round CD31+ cells protruding into the lumens of the chorioallantoic vessels. Based on these data, we hypothesized that lack of blood flow may impede the generation of hematopoietic stem and progenitor cells (HS/PCs) and that the endothelial clusters represent hemogenic intermediates. FACS analysis and colony forming assays confirmed a dramatic reduction in the number of clonogenic progenitors in the placenta and the embryo proper of Ncx mutants, while the yolk sac was unaffected. However, HS/PC generation in the placenta and embryo could be rescued by culturing explants on OP9 stroma before plating in colony forming assays, verifying intact hematopoietic potential. To determine if the rescue observed was due to expansion of existing progenitors or generation of new HS/PCs, we sorted CD41medckit+hematopoietic progenitors and CD31+CD41− endothelial cells from hematopoietic tissues and co-cultured them on stroma. These experiments demonstrated that endothelial cells from placenta, embryo proper and yolk sac can generate HS/PCs following stroma stimulation, confirming the presence of hemogenic endothelium in these organs. Immunostaining of Ncx−/− placentas revealed that although the development of the arterio-venous vascular network was impaired, Notch1 signaling, required for both arterial specification and HSC development, was robust in cells of the endothelial clusters. Furthermore, positive staining for Runx1 and c-myb indicated that cells in the clusters had activated the hematopoietic program. Interestingly, electron microscopy demonstrated that cells in the clusters were tethered to each other via adherens junctions, a characteristic of endothelial cells. In addition, they also maintained high levels of Flk1, expressed VEGF and were actively proliferating, consistent with exposure to extended hypoxia. These data suggest that although cells in the clusters have initiated hematopoietic commitment, they are unable to down-regulate their endothelial identity and complete hematopoietic emergence, resulting in the formation of clusters of hemogenic intermediates. These results imply that cues imparted via circulation are required to complete the commitment to a hematopoietic fate from hemogenic endothelium. Data from co-culture experiments suggest that prolonged Notch1 signaling impairs hematopoietic emergence from hemogenic endothelial cells, and may account for the HSC emergence defect in the absence of blood flow. Overall, these data suggest that blood flow and circulating primitive red blood cells are critical components of the dynamic microenvironment necessary to both relieve the hypoxia required for the specification and proliferation of hemogenic endothelium and provide important mechanical and/or molecular signals required by HSCs to fully commit to the hematopoietic fate and complete emergence. Disclosures: No relevant conflicts of interest to declare.

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Latent Cardiogenic Potential in Endocardium and Hemogenic Endothelium Revealed in the Absence of Scl/tal1

Blood ◽

10.1182/blood.v118.21.2362.2362 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2362-2362

Author(s):

Amelie Montel-Hagen ◽

Ben Van Handel ◽

Roberto Ferrari ◽

Rajkumar Sasidharan ◽

Tonis Org ◽

...

Keyword(s):

Endothelial Cells ◽

Progenitor Cells ◽

Conflicts Of Interest ◽

Lineage Tracing ◽

Bhlh Transcription Factor ◽

Wild Type ◽

Transcriptional Program ◽

Hemogenic Endothelium ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells

Abstract Abstract 2362 The endothelium in embryonic and extraembryonic hematopoietic tissues has the capacity to generate hematopoietic stem and progenitor cells (HS/PC). However, it is unknown how this unique endothelium is specified. Microarray analysis of endothelial cells from hematopoietic tissues of embryos deficient for the bHLH transcription factor Scl/tal1 revealed that Scl establishes a robust hematopoietic transcriptional program in the endothelium. Surprisingly, lack of Scl also induced an unexpected fate switching of the prospective hemogenic endothelium to the cardiac lineage. Scl deficient embryos displayed a dramatic upregulation of cardiac transcription factors and structural proteins within the yolk sac vasculature, resulting in the generation of spontaneously beating cardiomyocytes. Ectopic cardiac potential in Scl deficient embryos arose from endothelial-derived CD31+Pdgfrα+ cardiogenic progenitor cells (CPCs), which were present in all sites of HS/PC generation. Analysis of Runx1-deficient embryos revealed, that although Runx1 acts downstream of Scl during the emergence of definitive HS/PCs, it is not required for the suppression of the cardiac fate in the endothelium. The only wild type tissue that contained CD31+Pdgfrα+ putative CPCs was the heart, and this population was greatly expanded in Scl deficient embryos. Strikingly, endocardium in Scl−/− hearts also activated a robust cardiomyogenic transcriptional program and generated Troponin T+ cardiomyocytes both in vivo and in vitro. Although CD31+Pdgfrα+ CPCs from wild type hearts did not generate readily beating cells in culture, they produced cells expressing endothelial, smooth muscle and cardiomyocyte specific genes, implying multipotentiality of this novel CPC population. Furthermore, CD31+Pdgfrα+ CPCs were greatly reduced in Isl1−/− hearts, which fail to generate functional, multipotential CPCs. Lineage tracing using VE-cadherin Cre Rosa-YFP mouse strain demonstrated that, in addition to generating HS/PCs in hematopoietic tissues, endothelial cells are also the cell of origin for CD31+Pdgfrα+ CPCs in the heart. Together, these data suggest a broader role for embryonic endothelium as a potential source of tissue-specific stem and progenitor cells and implicate Scl/tal1 as an important regulator of endothelial fate choice. Disclosures: No relevant conflicts of interest to declare.

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Erythropoietin Treatment Enhanced Cell Surface CXCR4 Expression On Neonatal CD34-Positive Cells

Blood ◽

10.1182/blood.v120.21.4724.4724 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4724-4724

Author(s):

Norioki Ohno ◽

Seiichi Hayakawa ◽

Shuhei Karakawa ◽

Masao Kobayashi

Keyword(s):

Stem Cells ◽

Flow Cytometry ◽

Cell Surface ◽

Mononuclear Cells ◽

Conflicts Of Interest ◽

Cxcr4 Expression ◽

Stem Cell Marker ◽

Cell Marker ◽

Low Oxygen ◽

Hematopoietic Stem

Abstract Abstract 4724 Erythropoietin (EPO), which is used to treat anemic immature neonates, is thought to be effective for treating neonatal hypoxic encephalopathy by stimulating nervous system cells or vascular stem cells. On stem cells, CXCR4 is an important chemokine receptor for cell migration and proliferation. Previously, we reported that increased expression of CXCR4 on cord blood (CB)-derived CD34-positive cells, caused by low oxygen tension, enhanced cell homing activity and engraftment (Ohno et al., ASH. 2010). In human endothelial stem cells, CXCR4 expression is controlled by hypoxia-inducible factor-1α (HIF-1α), and EPO expression is enhanced by hypoxia via HIF-1α. In this study, we examined the effect of EPO treatment on CXCR4 expression on premature neonatal endothelial stem cells and cultured CB-derived CD34-positive cells. First, we examined the cell surface markers CD34, CD133, and CXCR4 in preterm infants diagnosed with anemia and treated with EPO. The 10 enrolled neonates were first administered EPO when they were over 32 weeks old and were not given oxygen therapy. Blood samples were collected twice before and once 24 h after EPO administration. Mononuclear cells were isolated from peripheral blood and stained for the hematopoietic stem cell marker CD34, vascular endothelial progenitor cell marker CD133, and surface CXCR4. The cells were analyzed by flow cytometry. No change in the proportions of CD34- and CD133-positive cells were found after the treatment (CD34 positive: 0.59%→0.57%; CD133 positive: 0.52%→0.49%). The proportion of cell surface CXCR4 on the CD133-positive cells did not change after EPO administration, whereas the proportion of cell surface CXCR4 on the CD34-positive cells increased significantly after EPO was administered (average: 7.6%→11.0%, p < 0.01). In particular, slightly CD34-positive cells showed enhanced cell surface CXCR4. Next, we examined CXCR4 enhancement in EPO-treated CB-derived CD34-positive enriched samples. Samples were divided into three aliquots and cultured. The first aliquot was incubated for 24 h in RPMI-1640 medium alone as a control; the second, for 24 h in RPMI-1640 medium with EPO (10 U/ml); and the third, for 24 h in RPMI-1640 medium with EPO (10 U/ml) containing the specific HIF-1 antagonist rapamycin. Flow cytometry revealed significantly increased surface CXCR4 expression on CD34-positive cells after incubation with EPO compared with the control expression (average: 51.7% vs. 30.3%, p< 0.05). This enhancement was inhibited completely by the addition of rapamycin. Intracellular HIF-1 was enhanced significantly in the EPO-treated cells compared with the control expression on flow cytometry. The enhanced CXCR4 expression on CD34-positive cells reflects an amplification of the chemotactic and homing abilities of stem cells. The CXCR4 enhancement caused by EPO may increase tissue migration of CD34-positive cells, although this is probably a result of an active HIF-1 pathway. Disclosures: No relevant conflicts of interest to declare.

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Do Leukemic Cells and Mesenchymal Stem Cells (MSCs) From AML Patients Share The Same Chromosomal Defect? A Cytogenetics, FISH and aCGH/Snpa Study

Blood ◽

10.1182/blood.v122.21.2602.2602 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2602-2602

Author(s):

Paolo Bernasconi ◽

Irene Dambruoso ◽

Manuel Gotti ◽

Marina Boni ◽

Rita Zappatore ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Conflicts Of Interest ◽

Gradient Centrifugation ◽

Leukemic Cells ◽

Hematopoietic Tissue ◽

Trisomy 8 ◽

Commercial Fish ◽

Monosomy 7 ◽

Microarray Scanner

Abstract Recent evidence suggests that leukemia is not solely a cancer autonomous process, but rather a disease in which the bone marrow microenvironment, the niche, plays a crucial role too (Raaijmakers, 2011). MSCs are key component of the niche. Thus, several studies have tested whether these cells from haematological patients contain chromosomal defects identical or different from those present in leukemic cells. Based on these findings the principal aim of the present study was to evaluate whether leukemic and MSC from six AML patients shared the same cytogenetic defects after examination with three different technologies, conventional cytogenetics (CC), FISH and aCGH/SNPa. At the onset of the disease and after informed consent all the six patients were submitted to bone marrow (BM) aspiration. BM cells were submitted to CC and FISH analyses. In addition, MSC were isolated from BM cell suspension (10-15 ml) as previously described. Briefly, mononucleated cells were isolated from BM by density gradient centrifugation using Lympholyte®-H and seeded in 75 cm2 cell culture flasks at a cell density of 106 cells/cm2. Cells were cultured at 37°C, 5% CO2 in MEM-alpha medium containing 1% Penicillin/Streptomycin, 1% L-Glutamine and 10% fetal bovine serum. After 48-h adhesion, non-adherent cells were removed and culture medium replaced (Achille et al, 2011). Growth medium was changed every three days. MSCs were examined after the first passage and their phenotype was evaluated by flow cytometry. Cells were detached from culture using Tripsin-EDTA, washed twice with PBS and stained for ten minutes with the following fluorochrome-conjugated antibodies: anti-CD90-FITC, anti-CD105-PE, anti-CD14-FITC, anti-CD73-PE, anti-CD34-FITC, anti-CD80-PE, anti-CD133-APC, anti-CD31-PE and anti-CD45-APC-Alexa750. Stained cells were acquired with a Beckman Coulter Navios instrument and data analyzed with Kalooza software. The commercial FISH probes used were LSI D7S486/CEP7, LSI AMLETO from Abbot Molecular Inc. (Chicago, Il, USA) and ON c-Myc/SE8, SE10(D10Z1) from Kreatech (Amsterdam, NL). These probes were applied according to manufactures guidelines and cut-off values determined by applying a one-sided 95% confidence interval using a binomial distribution. aCGH/SNPa was carried out with the SureScan Microarray Scanner G4900DA (Agilent Technologies Inc. Santa Clara, CA). CC revealed a monosomy 7 in two patients, a del(7)(q31) in one, a trisomy 8 and a trisomy 10 in one patient each, a t(8;21)(q24,q22) translocation in the last patient. All these defects were confirmed by FISH. In order to establish whether leukemic cells and MSCs shared these same abnormalities, MSCs cultures were tested with FISH. MSC purity assessed by flow-cytometry was 50-87%. FISH revealed a normal pattern in all the cultures examined. In contrast, aCGH/SNPa revealed neither gains/losses nor LOH in four patients, a trisomy 5 in one and the LOH of a 3.8 Mb sized region located on 13q31.1 in one patient. This study, the first one that applied aCGH/SNPa to investigate the MSC chromosomal pattern, suggests that i) MSCs from chromosomally abnormal AML patients may show a normal FISH pattern, but may be either normal or contain chromosomal aberrations different from those present in leukemic cells on aCGH/SNPa analysis; ii) these defects are uncommonly seen in AML; iii) MSCs defects may flag that the leukemogenic event targets not only the hematopoietic tissue but also the stromal cell compartment, i.e. the niche; iii) aCGH/SNPa provides an in-depth view of MSC chromosomal pattern allowing the identification of potential clonal markers. Disclosures: No relevant conflicts of interest to declare.

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