scholarly journals Scl and Lyl1 Are Redundant in Erythropoiesis

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1182-1182
Author(s):  
Sung Kai Chiu ◽  
Cedric Tremblay ◽  
Jesslyn Saw ◽  
David J. Curtis
Keyword(s):  
Conflicts Of Interest ◽  
Cell Leukaemia ◽  
Erythroid Progenitors ◽  
Hematopoietic Stem ◽  
Helix Loop Helix ◽  
Mild Anemia ◽  
Adult Mice ◽  
Primitive Erythropoiesis ◽  
Bhlh Transcription Factors

Abstract Stem cell leukaemia (Scl) and Lymphoblastic lymphoma derived sequence 1 (Lyl1) are the only hematopoiesis-specific basic Helix-loop-helix (bHLH) transcription factors. During development, Lyl1 is unable to compensate for Scl; with death of Scl-null embryos at e9.5 due complete absence of primitive hematopoiesis and defective vascular development. In contrast, Lyl1 can compensate for Scl in adult hematopoietic stem cells. To further explore the role of these two bHLH factors during hematopoietic development, we deleted Scl with Cre recombinase under the control of the Epo receptor, which is active in late erythroid progenitors. Surprisingly, embryos lacking Scl in erythroid progenitors (EpoR-Cre SclD/D) were born at the expected Mendelian frequency with only a mild anemia in adult mice. In contrast EpoR-Cre SclD/D mice lacking Lyl1 died at e11.5-12.5 due to erythropoietic collapse (see figure 1). These experiments provide the first evidence for an important role of Lyl1 in erythroid development and suggest that death of Scl-null embryos is due to defects in endothelial development rather than lack of primitive erythropoiesis. Figure 1. (A) Wildtype and (B) Scl/Lyl dko yolk sacs & embryos at e11.5. Benzidine stains of (C) wildtype & (D) Scl/Lyl dko yolk sacs at e11.5. Figure 1. (A) Wildtype and (B) Scl/Lyl dko yolk sacs & embryos at e11.5. / Benzidine stains of (C) wildtype & (D) Scl/Lyl dko yolk sacs at e11.5. Disclosures No relevant conflicts of interest to declare.

scholarly journals Either Scl or Lyl1 Is Required to Maintain Primitive Erythropoiesis

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5057-5057
Author(s):  
Sung Kai Chiu ◽  
Cedric Tremblay ◽  
Jesslyn Saw ◽  
David J. Curtis
Keyword(s):  
Conflicts Of Interest ◽  
Embryonic Stem ◽  
Erythroid Progenitors ◽  
Downstream Target ◽  
Mild Anemia ◽  
Adult Mice ◽  
Conditional Allele ◽  
Primitive Erythropoiesis ◽  
Definitive Erythropoiesis ◽  
Bhlh Transcription Factors

Abstract SCL (TAL1) and LYL1 are the predominant bHLH transcription factors expressed in erythropoiesis. Using a conditional allele of Scl, we have previously demonstrated redundancy of Scl in adult erythropoiesis. Similarly, adult erythropoiesis is maintained in Lyl1-deficient mice. To determine if these factors can compensate for each other, we deleted Scl with Cre recombinase under the control of the Epo receptor, which is active in late erythroid progenitors from embryonic day 8.5. Embryos lacking Scl in erythroid progenitors (EpoR-Cre SclD/D) were born at the expected Mendelian frequency with only a mild anemia in adult mice, indicating Scl was not required for primitive or definitive erythropoiesis. In contrast EpoR-Cre SclD/D mice lacking Lyl1 died at e11.5 due to erythropoietic collapse. Gene expression profiling of yolk sacs prior to the loss of erythrocytes (E9.5) revealed reduced Gata1, Fog1, Klf1 and the primitive b-globins. In contrast, expression of Gata2 and Runx1 were increased despite absence of Scl and Lyl1. Development of embryonic stem cells from these mice will determine if Gata1 is key downstream target of the Scl/Lyl1 complex for primitive erythropoiesis. Disclosures No relevant conflicts of interest to declare.

scholarly journals The role of Selenoprotein N in the differentiation of erythroid progenitors during stress erythropoiesis

2018 ◽  
Author(s):  
Robert F Paulson ◽  
Shaneice K Nettleford ◽  
Chang Liao ◽  
Yuanting Chen ◽  
Siyang Hao ◽  
...  
Keyword(s):  
Terminal Differentiation ◽  
Erythroid Progenitors ◽  
Hematopoietic Stem ◽  
Stress Erythropoiesis ◽  
Sickle Cells ◽  
Mild Anemia ◽  
Constant Rate ◽  
Proliferation And Differentiation ◽  
Human Stress

Low serum Se is independently associated with anemia in elderly population, dialysis patients, sickle-cells patients, and hypothyroidism patients. Previous work from our laboratory showed that dietary Se deficiency in mice showed mild anemia indicating activation of stress- erythropoietic mechanisms. Unlike steady state erythropoiesis that is primarily responsible for homeostasis to produce new erythrocytes at a constant rate, stress erythropoiesis predominates when the bone marrow cannot generate sufficient erythrocytes. During such a process, short-term reconstituting hematopoietic stem cells (CD34+Kit+Sca1+Linneg) migrate to the spleen leading to the proliferation and differentiation of stress-erythroid progenitors (SEPs). These cells lead to stress burst forming unit- erythroid cells (BFU-E) followed by terminal differentiation to erythrocytes. Recent studies demonstrate deficits in selenoproteins block the expansion and development of stress BFU-E with defects in terminal differentiation. Analysis of selenoprotein expression showed that selenoprotein W (SelenoW) was highly expressed in developing SEPs. CRISPR-Cas9 knockout of SelenoW blocked the proliferation of immature SEPs in murine and human stress erythropoiesis cultures demonstrating a central role for SelenoW in stress erythropoiesis. Using the two-culture system to generate SEPs, selenoprotein N (SelenoN) expression increased as the progenitors transition from self-renewing progenitors to form committed erythroid progenitors. SelenoN-/- mice showed significantly slower erythroid recovery following phenylhydrazine (PHZ)-induced acute hemolytic anemia. As in the muscle satellite cells where SelenoN regulates cellular Ca2+ signaling, SelenoN may also regulate Ca2+ signaling in SEPs to modulate pathways of differentiation. In summary, these data suggest that multiple selenoproteins, including SelenoN and SelenoW, coordinately regulate stress erythropoiesis.

Primitive erythropoiesis in theXenopusembryo: the synergistic role of LMO-2, SCL and GATA-binding proteins

Development ◽  
2001 ◽  
Vol 128 (12) ◽  
pp. 2301-2308 ◽  
Author(s):  
Paul E. Mead ◽  
Anne E. Deconinck ◽  
Tara L. Huber ◽  
Stuart H. Orkin ◽  
Leonard I. Zon
Keyword(s):  
Vertebrate Development ◽  
Apparent Paradox ◽  
Hematopoietic Stem ◽  
Blood Island ◽  
Helix Loop Helix ◽  
Blood Formation ◽  
Transcriptional Complex ◽  
Primitive Erythropoiesis

Hematopoietic stem cells are derived from ventral mesoderm during vertebrate development. Gene targeting experiments in the mouse have demonstrated key roles for the basic helix-loop-helix transcription factor SCL and the GATA-binding protein GATA-1 in hematopoiesis. When overexpressed in Xenopus animal cap explants, SCL and GATA-1 are each capable of specifying mesoderm to become blood. Forced expression of either factor in whole embryos, however, does not lead to ectopic blood formation. This apparent paradox between animal cap assays and whole embryo phenotype has led to the hypothesis that additional factors are involved in specifying hematopoietic mesoderm. SCL and GATA-1 interact in a transcriptional complex with the LIM domain protein LMO-2. We have cloned the Xenopus homolog of LMO-2 and show that it is expressed in a similar pattern to SCL during development. LMO-2 can specify hematopoietic mesoderm in animal cap assays. SCL and LMO-2 act synergistically to expand the blood island when overexpressed in whole embryos. Furthermore, co-expression of GATA-1 with SCL and LMO-2 leads to embryos that are ventralized and have blood throughout the dorsal-ventral axis. The synergistic effect of SCL, LMO-2 and GATA-1, taken together with the findings that these factors can form a complex in vitro, suggests that this complex specifies mesoderm to become blood during embryogenesis.

SCL/TAL1 Regulates Erythropoietin Receptor Expression.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1195-1195
Author(s):  
Heather M. Rogers ◽  
Xiaobing Yu ◽  
Constance Tom Noguchi
Keyword(s):  
Erythroid Differentiation ◽  
Erythropoietin Receptor ◽  
Receptor Expression ◽  
Binding Motif ◽  
Erythroid Cells ◽  
Hematopoietic Stem ◽  
Mild Anemia ◽  
Adult Mice ◽  
High Level

Abstract The basic-helix-loop-helix transcription factor SCL/TAL1, is required for erythropoiesis during development, and conditional deletion in adult hematopoiesis results in hematopoietic stem cells with a competitive repopulation disadvantage and defective erythropoiesis in vitro. However, adult mice with a conditional SCL/TAL1 deletion survive with mild anemia, suggesting defective erythroid proliferation and indicating that SCL/TAL1 is important, but not essential in mature red blood cell production. We find that during erythroid differentiation of primary human hematopoietic CD34+ cells, SCL/TAL1 expression peaks at day 8–10 following erythropoietin (EPO) stimulation, concomitant with peak expression of GATA-1 and EKLF. Treatment with SCL/TAL1 antisense oligonucleotides during erythroid differentiation markedly decreases erythroid differentiation as indicated by decreased expression of GATA-1 and both b- and g-globin expression, along with the absence of the characteristic decrease in GATA-2. Microarray analysis of erythroid cells overexpressing SCL/TAL1 indicate increased gene expression for b- and g-globin, and other genes related to erythropoiesis including EPO receptor (EPO-R), and these results are confirmed in stable cell lines with increasing SCL/TAL1 expression. Examination of EPO-R transcription regulation indicates that E-boxes in the 5′ UTR can bind SCL/TAL1 in vitro and, in addition to the GATA-1 binding motif, provide transcription activity in reporter gene assays. These data indicate that in addition to the importance of SCL/TAL1 DNA binding for proliferation of BFU-E and expression of glycophorin A and protein 4.2, SCL/TAL1 is also necessary for high level expression of EPO-R. Reduction in EPO-R expression likely contributes to the anemia associated with the conditional adult deletion of SCL/TAL1 and to the proliferative defect of erythroid cells observed in vitro. Early expression of SCL/TAL1 in hematopoietic cells may activate expression of EPO-R prior to EPO stimulation of erythropoiesis and induction of GATA-1.

Abnormal Distribution of Erythroid Progenitors Populations in Mice Lacking p45 NF-E2.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1988-1988
Author(s):  
Jadwiga Gasiorek ◽  
Gregory Chevillard ◽  
Zaynab Nouhi ◽  
Volker Blank
Keyword(s):  
Bone Marrow ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Conflicts Of Interest ◽  
Globin Genes ◽  
Erythroid Progenitors ◽  
Erythroid Progenitor ◽  
Adult Mice ◽  
Deficient Mice

Abstract Abstract 1988 Poster Board I-1010 The NF-E2 transcription factor is a heterodimer composed of a large hematopoietic-specific subunit called p45 and widely expressed 18 to 20-kDa small Maf subunits. In MEL (mouse erythroleukemia) cells, a model of erythroid differentiatin, the absence of p45 is inhibiting chemically induced differentiation, including induction of globin genes. In vivo, p45 knockout mice were reported to show splenomegaly, severe thrompocytopenia and mild erythroid abnormalities. Most of the mice die shortly after birth due to haemorrhages. The animals that survive display increased bone, especially in bony sites of hematopoiesis. We confirmed that femurs of p45 deficient mice are filled with bone, thus limiting the space for cells. Hence, we observed a decrease in the number of hematopoietic cells in the bone marrow of 3 months old mice. In order to analyze erythroid progenitor populations we performed flow cytometry using the markers Ter119 and CD71. We found that p45 deficient mice have an increased proportion of early erythroid progenitors (proerythroblasts) and a decreased proportion of late stage differentiated red blood cells (orthochromatic erythroblasts and reticulocytes) in the spleen, when compared to wild-type mice. We showed that the liver of p45 knockout adult mice is also becoming a site of red blood cell production. The use of secondary sites, such as the spleen and liver, suggests stress erythropoiesis, likely compensating for the decreased production of red blood cells in bone marrow. In accordance with those observations, we observed about 2 fold increased levels of erythropoietin in the serum of p45 knockout mice.Overall, our data suggest that p45 NF-E2 is required for proper functioning of the erythroid compartment in vivo. Disclosures: No relevant conflicts of interest to declare.

Aberrant Bone Marrow Perivascular Niches are Involved in Multiple Myeloma Progression: Role of Notch–Delta Pathway.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2800-2800
Author(s):  
Sara Lamorte ◽  
Marta Costa ◽  
Giovanni Camussi ◽  
Sergio Dias
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Annexin V ◽  
Pcr Analysis ◽  
Adherent Cells ◽  
Feeder Layer ◽  
Hematopoietic Stem ◽  
Lower Chamber

Abstract Abstract 2800 Poster Board II-776 Bone marrow (BM) angiogenesis is implicated in Multiple Myeloma (MM) progression. In this study, we tested the hypothesis that MM progression occurs when aberrant BM perivascular niches are established. We isolated BM endothelial cells derived from MM patients (MM-BMECs) from BM aspirates using anti-CD31Ab coupled to magnetic beads. FACS analysis showed that of all the cell lines isolated were endothelial: more than 95% expressed Ulex Europaeus Agglutinin-1 and Factor VIII and were negative for monocyte-macrophage (CD14) and plasma cell markers (CD38). To test the hypothesis that in MM patients BM perivascular niches are aberrant we analyzed how MM-BMECs modulate hematopoietic stem cells (HSCs) properties using a BM microvascular endothelial cell line isolated from a healthy donor (BMECs) as control. We co-cultured cord blood cells CD34+ HSCs in the presence of MM-BMECs or BMECs feeder layer and we analyzed the ability of MM-BMECs compared with BMECs to modulate HSCs adhesion, chemotaxis and apoptosis. The results show that MM-BMECs promote CD34+ HSCs adhesion, recruitment and protect them from apoptosis. In detail, we showed that after 24h of co-culture there was a significant increase in the number of adherent HSCs on MM-BMECs than on BMECs: 43±9% versus 25±6%. Moreover, when HSCs were cultured for 48 hours in 1% of serum in the presence of MM-BMECs they were less sensitive to apoptosis (9±11% of Annexin V+ cells) than HSCs cultured in the presence of BMECs (14±1% of Annexin V+ cells) or without a feeder layer, as control (17±3% of Annexin V+ cells). For the migration assay a transwell chamber system, in which the upper and the lower chambers were separated by 5-μm pore-size filter, was used. BMECs, MM-BMECs or nothing was plated in the lower chamber, while HSCs were seeded into the upper chamber. Both chambers were loaded with unsupplemented EBM-2 plus 2% of serum. Cell migration was studied over a 6-8 hours period and evaluated as number of cells migrated into the lower chamber. The results showed a significantly greater migration of HSCs in the presence of MM-BMECs than BMECs: 12±2% versus 5±1% of migrated cells. Taken together, these data showed that MM-BMECs promoted HSCs migration, adhesion and survival. Next we evaluated how MM-BMECs modulate the hemopoiesis recovery after irradiation in a NOD-SCID mouse model. When injected into sub-lethally irradiated (3 Grey) NOD-SCID mice MM-BMECs were detected in the BM integrated within the murine BM vessels and promoted hematopoietic recovery. In detail, MM-BMECs provided signals favoring the commitment towards lymphoid lineage. In fact, 7 days after injection, the BM of mice injected with MM-BMECs showed an increase in the percentage of lymphoblast (2.7%), compared with mice injected with BMECs or PBS, as control (respectively, 1.5% and 1.4%); followed, 14 days after injection, by a significant increase in the percentage of peripheral blood lymphocytes in mice injected with MM-BMECs (75±6%) versus mice injected with BMECS and PBS (respectively 60±0.5% and 47±7%). Since MM is a plasma cells disorder and the Notch-Delta pathway has been shown to play a central role in regulating HSCs properties, including the decisions of HSCs to undergo T- or B-cell differentiation, we investigated the involvement of this pathway in MM-BMECs and HSCs interaction. As determined by FACS and RT-PCR analysis, MM-BMECs, compared to BMECs, over expressed Delta-like Notch ligand 4 (DII4). Thus, we investigated the role of DII4 in the MM-BMECs/BMECs-HSCs adhesion. The first results showed that the expression of DII4 by MM-BMECs is necessary to promote HSCs adhesion. In fact, using a blocking antibody against DII4 (AbαDII4) at 50ug/ml there was an impairment in HSCs adhesion to MM-BMECs (43±9% versus 24±2% of adherent cells without and with AbαDII4 treatment), but not on BMECs (25±6% versus 26±1.4% of adherent cells without and with AbαDII4 treatment). Ongoing experiments are focusing on the role of DII4 in the modulation of HSCs proliferation, protection against apoptosis and in vitro-in vivo B commitment by MM-BMECs. Taken together, all these data suggest that BMECs in MM may function as “aberrant perivascular niches”, modulating HSCs properties. This aberrant phenotype could be due to an alteration of the Notch-Delta pathway in BMECs that favors malignant clonal growth by protecting it from apoptosis, favoring migration, adhesion and providing self-renewing and/or proliferative cues. Disclosures: No relevant conflicts of interest to declare.

Mobilization Studies in Complement-Deficient Mice Reveal That AMD3100 Mobilization Depends On Complement Cascade Activation and Suggest Involvement of “Membrane Attack Complex - MAC” in Egress of Hematopoietic Stem/Progenitor Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 367-367
Author(s):  
Marcin Wysoczynski ◽  
HakMo Lee ◽  
Rui Liu ◽  
Wan Wu ◽  
Janina Ratajczak, ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Conflicts Of Interest ◽  
Membrane Attack Complex ◽  
Classical Pathway ◽  
Compensatory Mechanism ◽  
Complement Cascade ◽  
Cxcr4 Antagonist ◽  
Hematopoietic Stem ◽  
Deficient Mice

Abstract Abstract 367 We reported that complement cascade (CC) becomes activated in bone marrow (BM) during mobilization of hematopoietic stem/progenitor cells (HSPCs) by immunoglobulin (Ig)-dependent pathway and/or by alternative Ig-independent pathway as seen during G-CSF- or Zymosan mobilization, respectively. As a result, several potent bioactive CC anaphylatoxins (C3 and C5 cleavage fragments) are released that regulate egress of HSPCs (Blood 2003;101,3784; Blood 2004;103,2071; Blood 2005;105,40, Leukemia 2009; in press.). This explains why: i) NOD/SCID and RAG-/- animals that do not activate the Ig-dependent CC classical pathway; ii) C2fB-/- and C3-/- mice that do not activate the classical and alternative CC pathways; and iii) C5-/- mice that do not activate the distal pathway of CC are all poor G-CSF- and/or Zymosan mobilizers. In this study, we evaluated the role of CC in mobilization induced by CXCR4 antagonist AMD3100. We noticed that all CC activation-deficient mice mentioned above, except C5-/- mice, mobilize normally in response to AMD3100 administration. Accordingly, the number of mobilized CD34- SKL cells, leucocytes, and CFU-GM clonogeneic progenitors in mutant mice was similar to wt littermates. More important we observed that AMD3100 mobilization of HSPCs was preceded by a massive egress of leucocytes from BM and that AMD3100 was able to stimulate in these cells i) phosphorylation of MAPKp42/44 and ii) secretion of MMP-9. At the same time, ELISA data to detect CC activation revealed that serum levels of CC cleavage fragments, which were low in the initial phase of AMD3100 mobilization during granulocyte egress, become elevated later during HSPC egress. Thus, our data show that despite a fact that G-CSF and AMD3100 mobilize HSPCs by involving different mechanisms, activation of CC is a common phenomenon occurring during mobilization induced by both compounds. This further supports a pivotal role of CC activation in the egress of HSPCs from BM; however, both compounds activate CC differently. While G-CSF administration initiates CC activation at its proximal C1q-C3 level, AMD3100 induces CC activation at the distal C5 level, pointing to a crucial role of C5 cleavage in executing mobilization. To support this, all mice employed in our studies that display defects in activation of proximal stages of CC (NOD/SCID, RAG, C2fB-/-, and C3-/-) are normal AMD3100 mobilizers. However, C5 is cleavage required for mobilization occurs in the plasma of these animals latter on - directly by proteases released from AMD3100-stimulated granulocytes that egress from the BM as a first wave of mobilized cells. This compensatory mechanism cannot occur from obvious reasons in C5-/- mice. We conclude that AMD3100-directed mobilization similarly as G-CSF-induced one depends on activation of CC; however, AMD3100 in contrast to G-CSF activates CC at distal stages – directly by proteases released from mobilized/activated granulocytes. Cleavage of C5 and release of C5a and desArgC5a create a sinusoid-permissive environment in BM for HSPCs egress. This suggests involvement of both C5 cleavage fragments as well as a potential role of downstream elements of CC activation - membrane attack complex - MAC (C5b-C9) in stem cell mobilization. Therefore, some poor AMD3100 patient responders could possess a defect in activation of the distal steps of CC. Disclosures: No relevant conflicts of interest to declare.

Flt3 Kinase Regulates Microvesicle Transfer of miRNA Between AML and Stromal Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1492-1492
Author(s):  
Noah Hornick ◽  
Jianya Huan ◽  
Jeffrey W Tyner ◽  
Peter Kurre
Keyword(s):  
Cell Lines ◽  
Stromal Cells ◽  
Conflicts Of Interest ◽  
Membrane Vesicles ◽  
Mirna Expression Profile ◽  
Cell Phenotype ◽  
Cell Of Origin ◽  
Hematopoietic Stem ◽  
Global Comparison

Abstract Abstract 1492 The presence of an internal tandem duplication in the receptor tyrosine kinase Flt3 (Flt3-ITD) is found in 25–30% of cytogenetically normal AML and confers a worsened prognosis, including an increased likelihood for relapse after hematopoietic stem cell transplantation (HSCT). This tendency toward relapse, combined with the improved capacity of Flt3-ITD+ disease to resist chemotherapy, may imply mechanisms of resistance beyond those present in leukemias lacking this mutation. Microvesicles and exosomes, membrane-bound extracellular vesicles that capture cell-specific protein and RNA, have previously been isolated from the serum of CLL patients (Ghosh et al., Blood 2010 Mar 4;115(9): 1755–64). We recently observed that vesicles are constitutively produced by both AML cell lines and by AML blasts isolated from patients. Using light scatter analysis and transmission electron microscopy, we found that vesicles produced by the HL60 (Flt3-ITD-) and Molm14 (Flt3-ITD+) cell lines, as well as by AML patient cells, predominantly fall within the 30–100nm range, generally considered to specify exosomes. To demonstrate vesicle transfer to neighboring cells, we labeled vesicles produced by the HL-60 cell line with the fluorescent membrane dye PKH-26 and imaged their uptake by stromal cells. Internalization of labeled exosomes was detectable within 15 minutes following exposure, and occurred at approximately 27 particles per cell by 2 hours (n=20). This result indicates rapid uptake of exosomes by non-phagocytic bystander cells, and supports a potential role for the vesicle content in altering the cell phenotype. We next tested for the presence of certain candidate mRNAs in the AML cell lines HEL, HL-60, MOLM-14, and U937, and in vesicles produced by those cell lines. We detected several relevant mRNAs, including nucleophosmin-1 and Flt3-ITD, in the vesicle preparations. During a more global comparison of vesicle and cell-of-origin RNA spectra using bioanalyzer analysis, we found that the RNA in microvesicles from (Flt3-ITD+) MOLM14 cells contained no detectable ribosomal RNA, but a markedly increased proportion of small RNA transcripts, suggesting that miRNA content might be increased. An initial screen of cultured MOLM-14 cells in the presence and absence of a small-molecule Flt3 inhibitor (AC220) and microvesicles isolated from the culture supernatant revealed several candidate miRNAs, including let-7a, miR-99b, and miR-155, whose expression varied with the presence of inhibitor. Our results not only support a role for Flt3 kinase activation in determining the miRNA expression profile, but provide evidence for the kinase-regulated incorporation of miRNA into cell membrane vesicles. The recent description of several AML subtypes, including Flt3-ITD+ disease with unique miRNA profiles, and the role of miRNA as potent regulators of both microenvironmental function and immune responses provides strong motivation to evaluate the role of vesicles in AML therapy evasion. Disclosures: No relevant conflicts of interest to declare.

The bHLH Transcription Factors SCL and LYL1 Awaken Hematopoietic Stem Cells by Repression of p21

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 214-214
Author(s):  
David J. Curtis ◽  
Nhu-Y Nguyen ◽  
Jessica Salmon
Keyword(s):  
Cell Cycle ◽  
Transcription Factors ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Short Term ◽  
Hematopoietic Stem ◽  
Bhlh Factors ◽  
Mild Defect ◽  
Bhlh Transcription Factors

Abstract Abstract 214 The basic helix-loop-helix (bHLH) transcription factors SCL (TAL1) and LYL1 are regulators of adult hematopoietic stem cell (HSC) activity with significant functional redundancy: HSCs lacking SCL (SCLδ/δ) have a mild defect in short-term repopulating activity whilst HSCs lacking LYL1 (LYL1−/−) have normal repopulating activity. In contrast, we have shown previously that HSCs lacking both SCL and LYL1 (DKO) are unable to grow in vitro and have no in vivo repopulating activity. Phenotypic and expression analyses of SCLδ/δ, LYL1−/− and DKO mice were performed to determine how bHLH factors regulate HSC activity. Consistent with the short-term repopulating defects of SCLδ/δ HSC, Lineage negative Sca-1+ c-Kit+ (LSK) bone marrow cells from SCLδ/δ mice had reduced in vitro replating activity associated with increased quiescence – 90% in G0 compared with 70% in normal LSK. Increased quiescence was associated with delayed hematopoietic recovery following treatment of mice with 5-Fluorouracil. Consistent with the increased quiescence, expression of the cell cycle inhibitor, Cdkn1a (p21) was increased three-fold in SCLδ/δ and LYL1−/− LSK. Moreover, p21 levels in LSK isolated from DKO mice were increased 50-fold. To determine the functional relevance of the elevated levels of p21 in DKO HSCs, we generated DKO mice on a p21-deficient (p21−/−) background. Remarkably, loss of p21 rescued in vitro cell growth of DKO progenitors. More importantly, primary and secondary competitive repopulation assays demonstrated multi-lineage repopulating activity of p21−/− DKO HSCs. These results suggest the bHLH factors SCL and LYL1 function as repressors of p21, allowing HSCs to enter cell cycle during stress hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

Non-Canonical NF-Kb Signaling Regulates Hematopoietic Stem Cell Self-Renewal and Microenvironment Interactions

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 859-859 ◽  
Author(s):  
Chen Zhao ◽  
Yan Xiu ◽  
John M Ashton ◽  
Lianping Xing ◽  
Yoshikazu Morita ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Wild Type ◽  
Double Knockout ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Physiological Processes ◽  
Marrow Cells

Abstract Abstract 859 RelB and NF-kB2 are the main effectors of NF-kB non-canonical signaling and play critical roles in many physiological processes. However, their role in hematopoietic stem/progenitor cell (HSPC) maintenance has not been characterized. To investigate this, we generated RelB/NF-kB2 double-knockout (dKO) mice and found that dKO HSPCs have profoundly impaired engraftment and self-renewal activity after transplantation into wild-type recipients. Transplantation of wild-type bone marrow cells into dKO mice to assess the role of the dKO microenvironment showed that wild-type HSPCs cycled more rapidly, were more abundant, and had developmental aberrancies: increased myeloid and decreased lymphoid lineages, similar to dKO HSPCs. Notably, when these wild-type cells were returned to normal hosts, these phenotypic changes were reversed, indicating a potent but transient phenotype conferred by the dKO microenvironment. However, dKO bone marrow stromal cell numbers were reduced, and bone-lining niche cells supported less HSPC expansion than controls. Further, increased dKO HSPC proliferation was associated with impaired expression of niche adhesion molecules by bone-lining cells and increased inflammatory cytokine expression by bone marrow cells. Thus, RelB/NF-kB2 signaling positively and intrinsically regulates HSPC self-renewal and maintains stromal/osteoblastic niches and negatively and extrinsically regulates HSPC expansion and lineage commitment through the marrow microenvironment. Disclosures: No relevant conflicts of interest to declare.

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