scholarly journals Socs2 Is Dispensable for BCR/ABL1-Induced Chronic Myeloid Leukemia-Like Disease in Mice and for Normal Hematopoietic Stem Cell Function,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3743-3743
Author(s):  
Nils Hansen ◽  
Helena Agerstam ◽  
Martin Wahlestedt ◽  
Mats Ehinger ◽  
Petra Johnels ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Myeloid Leukemia ◽  
Cell Function ◽  
Cytokine Signaling ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Cell Counts ◽  
Blood Cell Counts

Abstract Abstract 3743 Background: The P210 BCR/ABL1 fusion protein initiates signaling through several down stream pathways such as STAT5, PI3K, AKT, RAS, and WNT. However, only few down stream mediators have so far been thoroughly studied in vivo in the context of BCR/ABL1-mediated induction of CML-like disorder in mice deficient for the specific genes. Suppressor of cytokine signaling 2 (SOCS2) is known as a feedback inhibitor of cytokine signaling, a negative regulator of the STAT5 pathway, and is markedly upregulated in primary bone marrow cells from patients with chronic myeloid leukemia (CML). However, it has not been clear whether SOCS2 is involved in BCR/ABL1 induced cell transformation or whether it is important for normal hematopoietic stem cell (HSC) function. Methods: To evaluate the stem cell function of Socs2, HSCs from wild type and Socs2 deficient mice were competitively transplanted into lethally irradiated recipients. Blood chimerism and lineage distribution were analyzed by flow cytometry at 4 and 18 weeks after transplantation. To investigate the potential role of Socs2 in CML, c-kit enriched Socs2(−/−) and wild type cells were transduced with a BCR/ABL1 expressing retroviral vector and transplanted into lethally irradiated recipients. The in vivo development of malignant disease was analyzed by peripheral blood cell counts and, upon euthanization, by flow cytometry and histopathology. Socs family gene expression was assessed by Q-RT/PCR and Stat5 phosphorylation by Western blotting on BCR-ABL1 expressing cells. Results: Although Socs2 was previously found to be upregulated in long-term repopulating HSCs by gene expression arrays, Socs2 deficient HSCs were indistinguishable from wild type HSCs when challenged in competitive bone marrow transplantation. Furthermore, when expressing BCR/ABL1, both Socs2 deficient and wild type cells induced a CML-like disease with an average survival of three weeks after transplantation. The leukemic mice suffered from elevated white blood cell counts and splenomegaly. To investigate if other Socs family members were upregulated to compensate for the Socs2 deficiency, we compared the expression of the Socs gene family members in Socs2(−/−) with wild type, leukemic cells. The expression levels of all other Socs genes were similar between Socs2(−/−) and Socs2 wt cells, suggesting that compensatory mechanisms of other Socs genes do not account for the lack of Socs2 function. In addition, the finding that the phosphorylation of Stat5 was unaffected in Socs2(−/−) cells indicates that BCR/ABL1-induced Stat5-phosphorylation is insensitive to Socs2 levels. Conclusions: Collectively, our results clarify that Socs2 is dispensable for normal HSC function and for BCR/ABL1-induced CML-like disease. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Hyperactive Nras and Dose Reduction of Tet2 Collaborate to Dys-Regulate Hematopoietic Stem Cells and Promote Leukemogenesis

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1204-1204
Author(s):  
Xi Jin ◽  
Tingting Qin ◽  
Nathanael G Bailey ◽  
Meiling Zhao ◽  
Kevin B Yang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Double Mutant ◽  
Mutant Mice ◽  
Cytokine Signaling ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Single Mutant ◽  
Tet2 Mutation

Abstract Activating mutations in RAS and somatic loss-of-function mutations in the ten-eleven translocation 2 (TET2) are frequently detected in hematologic malignancies. Global genomic sequencing revealed the co-occurrence of RAS and TET2 mutations in chronic myelomonocytic leukemias (CMMLs) and acute myeloid leukemias (AMLs), suggesting that the two mutations collaborate to induce malignant transformation. However, how the two mutations interact with each other, and the effects of co-existing RAS and TET2 mutations on hematopoietic stem cell (HSC) function and leukemogenesis, remains unknown. In this study, we generated conditional Mx1-Cre+;NrasLSL-G12D/+;Tet2fl/+mice (double mutant) and activated the expression of mutant Nras and Tet2 in hematopoietic tissues with poly(I:C) injections. Double mutant mice had significantly reduced survival compared to mice expressing only NrasG12D/+ or Tet2+/-(single mutants). Hematopathology and flow-cytometry analyses showed that these mice developed accelerated CMML-like phenotypes with higher myeloid cell infiltrations in the bone marrow and spleen as compared to single mutants. However, no cases of AML occurred. Given that CMML is driven by dys-regulated HSC function, we examined stem cell competitiveness, self-renewal and proliferation in double mutant mice at the pre-leukemic stage. The absolute numbers of HSCs in 10-week old double mutant mice were comparable to that observed in wild type (WT) and single mutant mice. However, double mutant HSCsdisplayed significantly enhanced self-renewal potential in colony forming (CFU) replating assays. In vivo competitive serial transplantation assays using either whole bone marrow cells or 15 purified SLAM (CD150+CD48-Lin-Sca1+cKit+) HSCs showed that while single mutant HSCs have increased competitiveness and self-renewal compared to WT HSCs, double mutants have further enhanced HSC competitiveness and self-renewal in primary and secondary transplant recipients. Furthermore, in vivo BrdU incorporation demonstrated that while Nras mutant HSCs had increased proliferation rate, Tet2 mutation significantly reduced the level of HSC proliferation in double mutants. Consistent with this, in vivo H2B-GFP label-retention assays (Liet. al. Nature 2013) in the Col1A1-H2B-GFP;Rosa26-M2-rtTA transgenic mice revealed significantly higher levels of H2B-GFP in Tet2 mutant HSCs, suggesting that Tet2 haploinsufficiency reduced overall HSC cycling. Overall, these findings suggest that hyperactive Nras signaling and Tet2 haploinsufficiency collaborate to enhance HSC competitiveness through distinct functions: N-RasG12D increases HSC self-renewal, proliferation and differentiation, while Tet2 haploinsufficiency reduces HSC proliferation to maintain HSCs in a more quiescent state. Consistent with this, gene expression profiling with RNA sequencing on purified SLAM HSCs indicated thatN-RasG12D and Tet2haploinsufficiencyinduce different yet complementary cellular programs to collaborate in HSC dys-regulation. To fully understand how N-RasG12D and Tet2dose reduction synergistically modulate HSC properties, we examined HSC response to cytokines important for HSC functions. We found that when HSCs were cultured in the presence of low dose stem cell factor (SCF) and thrombopoietin (TPO), only Nras single mutant and Nras/Tet2 double mutant HSCs expanded, but not WT or Tet2 single mutant HSCs. In the presence of TPO and absence of SCF, HSC expansion was only detected in the double mutants. These results suggest that HSCs harboring single mutation of Nras are hypersensitive to cytokine signaling, yet the addition of Tet2 mutation allows for further cytokine independency. Thus, N-RasG12D and Tet2 dose reduction collaborate to promote cytokine signaling. Together, our data demonstrate that hyperactive Nras and Tet2 haploinsufficiency collaborate to alter global HSC gene expression and sensitivity to stem cell cytokines. These events lead to enhanced HSC competitiveness and self-renewal, thus promoting transition toward advanced myeloid malignancy. This model provides a novel platform to delineate how mutations of signaling molecules and epigenetic modifiers collaborate in leukemogenesis, and may identify opportunities for new therapeutic interventions. Disclosures No relevant conflicts of interest to declare.

scholarly journals The histone H2A deubiquitinase Usp16 regulates hematopoiesis and hematopoietic stem cell function

2015 ◽  
Vol 113 (1) ◽  
pp. E51-E60 ◽  
Author(s):  
Yue Gu ◽  
Amanda E. Jones ◽  
Wei Yang ◽  
Shanrun Liu ◽  
Qian Dai ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Ubiquitin Ligase ◽  
Cell Function ◽  
Lymphoid Organ ◽  
Histone H2a ◽  
Hematopoietic Stem

Epigenetic mechanisms play important regulatory roles in hematopoiesis and hematopoietic stem cell (HSC) function. Subunits of polycomb repressive complex 1 (PRC1), the major histone H2A ubiquitin ligase, are critical for both normal and pathological hematopoiesis; however, it is unclear which of the several counteracting H2A deubiquitinases functions along with PRC1 to control H2A ubiquitination (ubH2A) level and regulates hematopoiesis in vivo. Here we investigated the function of Usp16 in mouse hematopoiesis. Conditional deletion of Usp16 in bone marrow resulted in a significant increase of global ubH2A level and lethality. Usp16 deletion did not change HSC number but was associated with a dramatic reduction of mature and progenitor cell populations, revealing a role in governing HSC lineage commitment. ChIP- and RNA-sequencing studies in HSC and progenitor cells revealed that Usp16 bound to many important hematopoietic regulators and that Usp16 deletion altered the expression of genes in transcription/chromosome organization, immune response, hematopoietic/lymphoid organ development, and myeloid/leukocyte differentiation. The altered gene expression was partly rescued by knockdown of PRC1 subunits, suggesting that Usp16 and PRC1 counterbalance each other to regulate cellular ubH2A level and gene expression in the hematopoietic system. We further discovered that knocking down Cdkn1a (p21cip1), a Usp16 target and regulated gene, rescued the altered cell cycle profile and differentiation defect of Usp16-deleted HSCs. Collectively, these studies identified Usp16 as one of the histone H2A deubiquitinases, which coordinates with the H2A ubiquitin ligase PRC1 to regulate hematopoiesis, and revealed cell cycle regulation by Usp16 as key for HSC differentiation.

Loss of FancC Function Results in Decreased Hematopoietic Stem Cell Repopulating Ability

Blood ◽  
1999 ◽  
Vol 94 (1) ◽  
pp. 1-8 ◽  
Author(s):  
Laura S. Haneline ◽  
Troy A. Gobbett ◽  
Rema Ramani ◽  
Madeleine Carreau ◽  
Manuel Buchwald ◽  
...  
Keyword(s):  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Cell Function ◽  
Genetic Disorder ◽  
Test Cell ◽  
Hematopoietic Stem ◽  
Murine Homologue ◽  
Complex Genetic Disorder

Fanconi anemia (FA) is a complex genetic disorder characterized by progressive bone marrow (BM) aplasia, chromosomal instability, and acquisition of malignancies, particularly myeloid leukemia. We used a murine model containing a disruption of the murine homologue ofFANCC (FancC) to evaluate short- and long-term multilineage repopulating ability of FancC −/− cells in vivo. Competitive repopulation assays were conducted where “test”FancC −/− or FancC +/+ BM cells (expressing CD45.2) were cotransplanted with congenic competitor cells (expressing CD45.1) into irradiated mice. In two independent experiments, we determined that FancC −/− BM cells have a profound decrease in short-term, as well as long-term, multilineage repopulating ability. To determine quantitatively the relative production of progeny cells by each test cell population, we calculated test cell contribution to chimerism as compared with 1 × 105 competitor cells. We determined that FancC −/− cells have a 7-fold to 12-fold decrease in repopulating ability compared with FancC +/+cells. These data indicate that loss of FancC function results in reduced in vivo repopulating ability of pluripotential hematopoietic stem cells, which may play a role in the development of the BM failure in FA patients. This model system provides a powerful tool for evaluation of experimental therapeutics on hematopoietic stem cell function.

scholarly journals Cytokine-Mediated Natural Killer Cells Effects Impair Hematopoietic Stem Cell Function

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2641-2641
Author(s):  
Lorena Lobo Figueiredo-Pontes ◽  
Robert S. Welner ◽  
Miroslava Kardosova ◽  
Hong Zhang ◽  
Meritxell Alberich-Jorda ◽  
...  
Keyword(s):  
Stem Cell ◽  
Nk Cells ◽  
Natural Killer ◽  
Cytokine Production ◽  
Cell Function ◽  
Conflicts Of Interest ◽  
Cytokine Signaling ◽  
Dependent Manner ◽  
Hematopoietic Stem ◽  
Nk Cytotoxicity

Abstract Natural killer (NK) cells participate in innate and adaptive immune responses, and upon activation rapidly produce cytokines, chemokines, and growth factors, including IFNγ, TNFα, TGFβ, GM-CSF, MIP1α, MIP1β, IL-10, and others, which can affect the function of other hematopoietic cells. Considering the recent evidences that hematopoietic stem cells (HSCs) respond to cytokine signaling, we hypothesized that NK cell-mediated cytokine production could mediate HSC function. By the use of co-cultures of purified Ly5.1 murine NK cells and congenic Ly5.2 HSCs, we concluded that NK activity affects HSC frequency in vitro as well as hematopoietic reconstitution in vivo. Sorted NK cells (CD3- NK1.1+) and HSCs (Lin-, Sca1+, ckithi, CD48-, CD150+) were co-cultured in the presence or absence of IL2 over an OP9 stromal cells layer for 14 to 28 days. After 14 days, the addition of NK cells to HSC cultures resulted in an approximate 2-fold reduction of lineage negative cells (Lin-) recovered cells, as compared to control HSC cultures, as determined by flow cytometry analysis. Lin- counts were even lower in HSC+NK long-term cultures when compared to HSC only cultures. Ly5.1 HSCs and/or Ly5.2 NK cells were injected into sublethally irradiated Ly5.1/2 chimeric mice in a ratio of 105 NK to 103 HSCs per mouse. The addition of IL2-stimulated NK to injected HSCs reduced engraftment from 15.7% to 1.82% when the 16 weeks bone marrow (BM) chimerism was analyzed. In agreement, donor CD45.1 cells contribution to the LSK and HSC subpopulations was reduced in the HSC+NK transplanted mice. To test whether NK depletion from BM grafts would affect HSC function, we performed limiting dilution transplantation assays where whole BM from Ly5.2 mice was submitted to immunonagnetic NK1.1 or IgG depletion and injected into lethally irradiated Ly5.1 animals. Donor chimerism after 8 and 16 weeks of transplant showed that depleting NK cells improves the engraftment ability of HSC in a cell dose-dependent manner. When 25 x104 BM cells were injected, chimerism increased from 40 to more than 90% in NK depleted group. Of note, HSC frequency was 1 in 1595 in the control and 1 in 95 in the NK depleted group. In order to understand the mechanisms by which NK cells could regulate HSCs, we took advantage of a CCAAT/enhancer-binding protein gamma (C/ebpg) knockout (KO) conditional mouse model generated in our laboratory, considering that C/ebpg had been previously shown to regulate NK cytotoxicity. Using similar culture conditions, HSCs and NK cells isolated from control (CT) or Cebpg KO mice were injected into congenic sublethally irradiated recipients. Results showed that Cebpg-deficient NK cells do not harm HSC engraftment as CT NK cells do. For instance, after 8 weeks, the addition of CT non-stimulated and IL-2-stimulated NK cells to normal transplanted HSCs reduced the engraftment from 40% to 20% and 10%, respectively. In contrast, chimerism was not different when HSCs only or HSCs + stimulated KO NK cells were transplanted. Gene expression and cytokine profiles of deficient and normal NK cells revealed the potential players of this HSC-NK regulation. Of these, interferon gamma (IFNg), was lower produced by the C/ebpg deficient NK cells. Therefore, besides controlling NK cytotoxicity, we showed here that C/ebpg also plays a role in the regulation of HSCs by NK-mediated cytokine production. Next, we investigated whether depletion of NK cells from human BM samples would improve transplantation efficiency. NK cells were removed using CD56 antibody and transplanted into sublethally irradiated NSG mice. Sixteen weeks after transplantation, animals were sacrificed and the percentage of human CD45 cells in blood, BM, and spleen demonstrated that NK depletion from human BM favors engraftment. Altogether, these findings provide new insights to the knowledge of HSC regulation by NK cells, which are present in BM transplantation (BMT) grafts. Although the alloreactive effect of NK cells against non-identical tumor cells from BMT recipients is well known, its cytokine-mediated effects over identical progenitor cells from the graft were not previously explored. We show that NK-secreted cytokines harm stem cell function, thus suggesting that depletion of NK cells from BM donor cells preparations can improve stem cell engraftment, particularly in the setting of alternative transplants with limiting cell numbers or non-myeloablative conditioning regimens. Disclosures No relevant conflicts of interest to declare.

scholarly journals CD70/CD27 signaling promotes blast stemness and is a viable therapeutic target in acute myeloid leukemia

2016 ◽  
Vol 214 (2) ◽  
pp. 359-380 ◽  
Author(s):  
Carsten Riether ◽  
Christian M. Schürch ◽  
Elias D. Bührer ◽  
Magdalena Hinterbrandner ◽  
Anne-Laure Huguenin ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Myeloid Leukemia ◽  
Hematopoietic Stem ◽  
Cell Divisions ◽  
Cell Gene ◽  
Stem Cell Gene ◽  
Acute Myeloid

Aberrant proliferation, symmetric self-renewal, increased survival, and defective differentiation of malignant blasts are key oncogenic drivers in acute myeloid leukemia (AML). Stem cell gene signatures predict poor prognosis in AML patients; however, with few exceptions, these deregulated molecular pathways cannot be targeted therapeutically. In this study, we demonstrate that the TNF superfamily ligand–receptor pair CD70/CD27 is expressed on AML blasts and AML stem/progenitor cells. CD70/CD27 signaling in AML cells activates stem cell gene expression programs, including the Wnt pathway, and promotes symmetric cell divisions and proliferation. Soluble CD27, reflecting the extent of CD70/CD27 interactions in vivo, was significantly elevated in the sera of newly diagnosed AML patients and is a strong independent negative prognostic biomarker for overall survival. Blocking the CD70/CD27 interaction by mAb induced asymmetric cell divisions and differentiation in AML blasts and AML stem/progenitor cells, inhibited cell growth and colony formation, and significantly prolonged survival in murine AML xenografts. Importantly, hematopoietic stem/progenitor cells from healthy BM donors express neither CD70 nor CD27 and were unaffected by blocking mAb treatment. Therefore, targeting CD70/CD27 signaling represents a promising therapeutic strategy for AML.

scholarly journals New genetic tools for the in vivo study of hematopoietic stem cell function

2018 ◽  
Vol 61 ◽  
pp. 26-35 ◽  
Author(s):  
Samik Upadhaya ◽  
Boris Reizis ◽  
Catherine M. Sawai
Keyword(s):  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Cell Function ◽  
In Vivo Study ◽  
Hematopoietic Stem ◽  
Genetic Tools

A Novel MBT-Containing Protein, Hemp, Plays Essential Roles In Skeletal Formation and Hematopoietic Stem Cell Function.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1597-1597
Author(s):  
Phyo Wai Htun ◽  
Keiyo Takubo ◽  
Hideaki Oda ◽  
Feng Ma ◽  
Kenjiro Kosaki ◽  
...  
Keyword(s):  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Cell Function ◽  
Conflicts Of Interest ◽  
Conditional Knockout ◽  
Thoracic Vertebrae ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Epigenetic Regulator ◽  
Skeletal Formation

Abstract Abstract 1597 Hemp (hematopoietic expressed mammalian polycomb, also denoted as mbt-containing 1) gene was originally identified in the hematopoietic stem cell (HSC)-enriched fraction of the mouse fetal liver (FL). It encodes a protein containing a putative Cys2-Cys2 zinc-finger region, followed by four tandem malignant brain tumor (MBT) repeats, which is frequently observed in polycomb gene (PcG) proteins. The structural characteristics strongly suggest that Hemp functions as an epigenetic regulator, but its biological role remains unknown. To address this issue, we generated hemp-deficient (hemp–/–) mice. Hemp–/– mice died soon after birth. Although no abnormalities were detected in internal organs, skeletal analysis exhibited a variety of malformations. Severe deformities were observed in the thoracic cavity, strongly suggesting that hemp–/– mice died of respiratory failure. Interestingly, they showed malformations of cervical and thoracic vertebrae, which were different from typical homeotic transformations observed in PcG-deficient mice. These results suggest that Hemp governs downstream target genes in distinct manners from conventional PcG proteins. The hematopoietic analysis of hemp in the FL showed that hemp is preferentially expressed in CD150+LSK and CD150–LSK HSC fractions in the hematopoietic hierarchy. Hemp–/– FL contained a significantly reduced number of hematopoietic cells and produced fewer number of hematopoietic colonies as compared to hemp+/+ FL. The decreases correlated with reduced number of CD150+LSK HSCs in hemp–/– FL, which generated much fewer hematopoietic colonies in the HPP-CFC assay. In addition, the competitive repopulation assay exhibited that the hematopoietic reconstitution ability of hemp–/– FL CD150+LSK HSCs was significantly impaired. Moreover, microarray analysis revealed that expression levels of several genes, such as Prdm16, Sox4, and Erdr1 were altered in hemp–/– FL HSCs. Since hemp–/– mice died at neonate, the role of Hemp in adult hematopoiesis remains to be elucidated. To address this issue, we generated hemp conditional knockout (cKO) mice. Acquired deletion of Hemp in the hematopoietic tissues was successfully achieved by crossing hempflox/flox mice with MxCre mice and stimulating the compound mice with pIpC. Analysis of the hematopoietic tissues revealed that the cell numbers of Mac+Gr1– and Mac+Gr1+ fractions in the hemp cKO bone marrow (BM) were significantly increased and decreased, respectively, as compared to those of the wild-type BM. However, no apparent differences have so far been observed between hemp cKO and wild-type littermates in functional analyses, such as colony forming activity and competitive repopulation ability of the BM cells. Here, we report that a novel MBT-containing protein, Hemp, plays essential roles in skeletal formation and HSC function during embryogenesis and also contributes to myeloid differentiation in adult hematopoiesis. Since Hemp likely functions as an epigenetic regulator, further studies will be required to clarify whether and what methylated lysine residues Hemp interacts with through the MBT repeats, what kind of genes are direct targets of Hemp, and how Hemp exerts its biological activity. Disclosures: No relevant conflicts of interest to declare.

Role of Notch Signaling in Hematopoietic Stem Cell Function

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. SCI-15-SCI-15
Author(s):  
Lluis Espinosa ◽  
Anna Bigas
Keyword(s):  
Stem Cell ◽  
Cell Function ◽  
Conflicts Of Interest ◽  
Notch Pathway ◽  
Cell Types ◽  
Loss Of Function ◽  
Hematopoietic Stem ◽  
On Chip ◽  
Stem Cell Populations

Abstract Abstract SCI-15 The Notch pathway controls the generation of different cell types in most tissues including blood, and dysregulation of this pathway is strongly associated with oncogenic processes. In many systems, Notch is also required for the maintenance of the stem cell populations. However, in the adult hematopoietic system this link between Notch and stemness has not been established. Instead, work of several groups, including ours, has clearly demonstrated that Notch has a prominent role in the generation of hematopoietic stem cells (HSC) during embryonic development. Although the first wave of blood cells appears in the mouse embryo around day 7.5 of development and is independent of Notch function, embryonic HSC are formed around day 10 of development from endothelial-like progenitors that reside in the embryonic aorta surrounded by the gonad and mesonephros, also called AGM region. By analyzing different Notch pathway mutant mouse embryos, we have demonstrated the involvement of the Jagged1-Notch1-GATA2 axis in this event. However, the formal demonstration that Notch regulates the GATA2 gene during HSC generation is still lacking. We have now found that GATA2 is a direct Notch target in vivo during embryonic HSC generation. However, whereas Notch positively activates GATA2 transcription in the HSC precursors, it simultaneously activates hes1 transcription, which acts a repressor of the same GATA2 gene. This finding directly implicates hes1 in the regulation of HSC development although further studies using loss-of-function mutant embryos are still needed. Altogether, our results indicate that both Notch and hes1 are required to finely regulate the levels, distribution, and likely the timing of GATA2 expression through an incoherent feed-forward loop. In parallel, we have identified other downstream targets of Notch in the AGM region by ChIP-on-chip and expression microarray analysis that we are currently characterizing. Disclosures: No relevant conflicts of interest to declare.

Disturbed Endothelial Blood-Bone Marrow-Barrier In Nox4 Deficient Mice

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1169-1169
Author(s):  
Maren Weisser ◽  
Kerstin B. Kaufmann ◽  
Tomer Itkin ◽  
Linping Chen-Wichmann ◽  
Tsvee Lapidot ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Endothelial Cells ◽  
Stem Cell ◽  
Steady State ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Knock Out ◽  
Deficient Mice ◽  
Permeability State

Abstract Reactive oxygen species (ROS) have been implicated in the regulation of stemness of hematopoietic stem cells (HSC). HSC with long-term repopulating capabilities are characterized by low ROS levels, whereas increased ROS levels correlate with lineage specification and differentiation. Several tightly regulated sources of ROS production are well known among which are the NADPH oxidases (Nox). HSC are known to express Nox1, Nox2 and Nox4, however, their role in maintenance of stem cell potential or in the activation of differentiation programs are poorly understood. While Nox2 is activated in response to various extrinsic and intrinsic stimuli, mainly during infection and inflammation, Nox4 is constitutively active and is considered to be responsible for steady-state ROS production. Consequently, Nox4 deficiency might lower ROS levels at steady-state hematopoiesis and thereby could have an impact on HSC physiology. In this work we studied HSC homeostasis in Nox4 knock-out mice. Analysis of the hematopoietic stem and progenitor cell (HSPC) pool in the bone marrow (BM) revealed no significant differences in the levels of Lineage marker negative (Lin-) Sca-1+ ckit+ (LSK) and LSK-SLAM (LSK CD150+ CD48-) cells in Nox4 deficient mice compared to wild type (WT) C57BL/6J mice. HSPC frequency upon primary and secondary BM transplantation was comparable between Nox4 deficient and WT mice. In addition, the frequency of colony forming cells in the BM under steady-state conditions did not differ between both mouse groups. However, Nox4 deficient mice possess more functional HSCs as observed in in vivo competitive repopulating unit (CRU) assays. Lin- cells derived from Nox4 knock out (KO) mice showed an increased CRU frequency and superior multilineage engraftment upon secondary transplantation. Surprisingly, ROS levels in different HSPC subsets of NOX4 KO mice were comparable to WT cells, implying that the absence of Nox4 in HSCs does not have a major intrinsic impact on HSC physiology via ROS. Therefore, the increased levels of functional HSCs observed in our studies may suggest a contribution of the BM microenvironment to steady-state hematopoiesis in the BM of Nox4 KO animals. Recent observations suggest a regulation of the BM stem cell pool by BM endothelial cells, in particular by the permeability state of the blood-bone marrow-barrier (Itkin T et al., ASH Annual Meeting Abstracts, 2012). Endothelial cells interact with HSCs predominantly via paracrine effects and control stem cell retention, egress and homing as well as stem cell activation. As Nox4 is highly expressed in endothelial cells and is involved in angiogenesis, we reasoned that the absence of NOX4 could affect HSC homeostasis through altered BM endothelium properties and barrier permeability state. Indeed, in preliminary assays we found reduced short-term homing of BM mononuclear cells into the BM of Nox4 deficient mice as compared to wild type hosts. Furthermore, in vivo administration of Evans Blue dye revealed reduced dye penetration into Nox4-/- BM compared to wild type mice upon intravenous injection. Taken together, these data indicate a reduced endothelial permeability in Nox4 KO mice. Ongoing experiments aim at further characterization of the Nox4-/- phenotype in BM sinusoidal and arteriolar endothelial cells, the impact of Nox4 deletion on BM hematopoietic and mesenchymal stem cells, and in deciphering the role of Nox4 in the bone marrow microenvironment. Disclosures: No relevant conflicts of interest to declare.

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