Ott1(Rbm15)-Deficient Hematopoietic Stem Cells Are Unable to Maintain Quiescence During Replicative Stress and Display Features of Premature Aging,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3433-3433
Author(s):  
Nan Xiao ◽  
Kaushal Jani ◽  
Jonathan L Jesneck ◽  
Glen D Raffel
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Bone Marrow ◽  
Steady State ◽  
Hematopoietic Stem Cells ◽  
Bone Marrow Failure ◽  
Gene Set Enrichment Analysis ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Replicative Stress

Abstract Abstract 3433 With age, hematopoietic stem cells (HSCs) have numerical expansion, skewing towards myeloid development, loss of lymphoid potential, an underlying pro-inflammatory state and loss of self-renewal potential thus severely limiting responses to hematopoietic stress, ultimately leading to bone marrow failure. The mechanisms and pathways responsible for these changes in aged HSCs are incompletely understood. Using a conditional allele of Ott1, a gene originally isolated as the 5' fusion partner in t(1;22) acute megakaryocytic leukemia, we previously found a global regulatory role for the gene in hematopoiesis. Deletion of Ott1 in adult mice utilizing Mx1-cre recapitulated certain aspects of aging hematopoiesis including increased Lin−Sca1+c-Kit+ (LSK) population, myeloid expansion and decreased lymphopoiesis. The LSK compartment was further characterized using SLAM and CD34/Flk2 markers and demonstrated normal levels of LT-HSCs and increased ST-HSCs. Despite sufficient LT-HSC numbers, Ott1-deleted bone marrow was unable to competitively or non-competitively repopulate irradiated recipients. To exclude a homing or engraftment effect, Ott1flox/null Mx1-cre bone marrow was transplanted with competitor then excised post-engraftment. The rapid loss of the Ott1-deficient graft demonstrated Ott1 is required for maintenance under competitive stress. In contrast, primary mice undergoing Ott1 excision lived a normal lifespan and were able to maintain sufficient hematopoiesis although with a partial reduction in bone marrow clonagenicity showing loss of Ott1 is not limiting under steady state conditions. To test the HSC requirement for Ott1 under replicative stress, Ott1 knockout mice were challenged with 5-fluorouracil (5-FU). Ott1-deleted mice treated with 5-FU displayed delayed peripheral blood neutrophil recovery and showed accelerated bone marrow failure. Cell cycle analysis of steady state Ott1 knockout HSCs showed a similar profile to wild type controls, however, after 5-FU treatment, the G0 fraction was dramatically reduced. The G0 fraction is associated with the quiescent, self-renewing HSC population, therefore, Ott1 is required for maintaining HSC quiescence during replicative stress but not steady state hematopoiesis. To more specifically assess whether the functional hematopoietic changes seen after loss of Ott1 were accompanied by alterations in known aging-associated pathways, Gene Set Enrichment Analysis comparing Ott1-deleted HSCs in steady state to aged HSCs was performed and showed a highly enriched gene expression signature (NES 2.02 p<0.0001). Physiologic sequelae of HSC aging were observed after Ott1 excision including activation of NFκβ, elevation of reactive oxygen species (ROS), increase in DNA damage (γH2A.X levels) and activation of p38Mapk. Although ROS was elevated under steady state conditions, neither apoptosis, senescence or proliferation was significantly different from wild type control HSCs. Furthermore, anti-oxidant treatment with N-acetyl-cysteine was unable to rescue the HSC maintenance defect of the Ott1 knockout, signifying additional requirements in HSCs for Ott1 beyond regulation of ROS. An observed increase of mitochondrial mass in Ott1-deleted HSCs suggests an upstream function for Ott1 in metabolic control, potentially contributing to ROS generation or degradation. In summary, we have demonstrated an essential role for Ott1 in maintaining HSC quiescence during replicative stress and shown loss of Ott1 leads to the acquisition of key gene expression patterns and pathophysiologic changes associated with aging. These data suggest Ott1 functions in part to oppose specific consequences of aging in the hematopoietic compartment. Ott1 and Ott1-dependent pathways therefore represent a potential therapeutic target to prevent the morbidity and mortality arising from age-related defects in hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

Erg is required for self-renewal of hematopoietic stem cells during stress hematopoiesis in mice

Blood ◽  
2011 ◽  
Vol 118 (9) ◽  
pp. 2454-2461 ◽  
Author(s):  
Ashley P. Ng ◽  
Stephen J. Loughran ◽  
Donald Metcalf ◽  
Craig D. Hyland ◽  
Carolyn A. de Graaf ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Steady State ◽  
Hematopoietic Stem Cells ◽  
Blood Cells ◽  
Bone Marrow Failure ◽  
Mutant Mice ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract Hematopoietic stem cells (HSCs) are rare residents of the bone marrow responsible for the lifelong production of blood cells. Regulation of the balance between HSC self-renewal and differentiation is central to hematopoiesis, allowing precisely regulated generation of mature blood cells at steady state and expanded production at times of rapid need, as well as maintaining ongoing stem cell capacity. Erg, a member of the Ets family of transcription factors, is deregulated in cancers; and although Erg is known to be required for regulation of adult HSCs, its precise role has not been defined. We show here that, although heterozygosity for functional Erg is sufficient for adequate steady-state HSC maintenance, Erg+/Mld2 mutant mice exhibit impaired HSC self-renewal after bone marrow transplantation or during recovery from myelotoxic stress. Moreover, although mice functionally compromised for either Erg or Mpl, the receptor for thrombopoietin, a key regulator of HSC quiescence, maintained sufficient HSC activity to sustain hematopoiesis, Mpl−/−Erg+/Mld2 compound mutant mice displayed exacerbated stem cell deficiencies and bone marrow failure. Thus, Erg is a critical regulator of adult HSCs, essential for maintaining self-renewal at times of high HSC cycling.

scholarly journals p38α Is Required for Proper Proliferation of Hematopoietic Stem Cells during Stress

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4317-4317
Author(s):  
Daiki Karigane ◽  
Keiyo Takubo ◽  
Shinichiro Okamoto ◽  
Toshio Suda
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Steady State ◽  
Hematopoietic Stem Cells ◽  
Cellular Senescence ◽  
Expression Level ◽  
Wild Type ◽  
Post Transplantation ◽  
Hematopoietic Stem ◽  
Deficient Mice

Abstract Hematopoietic stem cells (HSCs) are capable of self-renewal and multilineage-differentiation during lifespan. HSCs are maintained in a quiescent state to avoid cellular senescence. Previous reports utilizing pharmacological inhibitors or shRNAs against p38MAPK suggest a pivotal role of p38MAPK-pRb-Ink4a signaling in induction of HSC senescence by hematological stress or chronological aging. However, no genetic evidence exists for p38MAPK-mediated cellular senescence in vivo. Here we report unexpected roles of the dominant isoform of p38MAPK family, p38α, in adult hematological system. p38MAPK has four isozymes, α, β, γ and δ. Among them, p38α isozyme was highly expressed in various bone marrow hematopoietic cells, and the expression level of p38α in HSCs was higher than differentiated cells (p<0.01). Phosphorylation of p38MAPK was mainly observed in multipotent progenitors but not in HSCs in steady-state hematopoiesis, in addition, physiological aging (1 year old mouse bone marrow) did not affect phosphorylation status of p38MAPK in steady state. In contrast, p38MAPK was phosphorylated in HSCs after transplantation or 5-FU treatment. Mean fluorescence index (MFI) of phosphorylation of p38MAPK in HSCs is significantly higher at day 3 post 5-FU treatment (250 mg/kg) than steady-state. MFI of phosphorylation of p38MAPK in HSCs was higher at day 1 post transplantation than steady state, and returned to normal at day 7 post transplantation. These results showed phosphorylation of p38α was immediately induced after hematopoietic demand. p38α-deficient embryos die due to defective erythropoiesis in a non-cell-autonomous manner. Thus, we used a conditional knockout model; CAG-CreERT2:p38αfl/fl mouse to analyze the effects of p38α on adult hematopoiesis and HSCs. Expression level of p16Ink4a, one of the cellular senescence markers, was not significantly different between p38α-deficient mice and wild-type mice. Treatment of p38α-deficient mice with 5-FU exhibited defective recovery of hematopoiesis, and the survival rate were lower in p38α-deficient mice than wild-type (42.9%, N=7, p38α-deficient mice, vs 100%, wild-type, N=6, p<0.05). Loss of p38α in HSCs showed a defective transplantation capacity. Inducible loss of p38α in bone marrow chimera resulted in a gradual loss of peripheral blood chimerism of p38α-deficient cells. In addition, short-term BrdU incorporation assay showed that the cell cycle progression of p38α-deficient HSCs was suppressed (BrdU positive rate; 3.5±2.2%, N=9, p38α-deficient cells vs 6.5±2.6%, N=5, wild-type, p<0.05). Therefore, hematopoietic function was obviously lowered in p38α-deficient HSCs during hematopoietic stresses. These observations collectively support the requirement of p38α for proper proliferation of HSCs during stress hematopoiesis. Disclosures No relevant conflicts of interest to declare.

scholarly journals Hematopoietic Stem Cells Are Endowed with Erythroid Signature in Beta-Thalassemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 31-31
Author(s):  
Maria Rosa Lidonnici ◽  
Giulia Chianella ◽  
Francesca Tiboni ◽  
Matteo Barcella ◽  
Ivan Merelli ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Gene Set Enrichment Analysis ◽  
Lineage Commitment ◽  
Erythroid Cell ◽  
Beta Thalassemia ◽  
Hematopoietic Stem ◽  
Multipotent Progenitors

Background Beta-thalassemia (Bthal) is a genetic disorder due to mutations in the ß-globin gene, leading to a reduced or absent production of HbA, which interferes with erythroid cell maturation and limits normal red cell production. Patients are affected by severe anemia, hepatosplenomegaly, and skeletal abnormalities due to rapid expansion of the erythroid compartment in bone marrow (BM) caused by ineffective erythropoiesis. In a classical view of hematopoiesis, the blood cell lineages arise via a hierarchical scheme starting with multipotent stem cells that become increasingly restricted in their differentiation potential through oligopotent and then unipotent progenitors. In human, novel purification strategies based on differential expression of CD49f and CD90 enrich for long-term (49f+) and short-term (49f−) repopulating hematopoietic stem cells (HSCs), with distinct cell cycle properties, but similar myeloid (My) and lymphoid (Ly) potential. In this view, it has been proposed that erythroid (Ery) and megakaryocytic (Mk) fates branch off directly from CD90-/49f− multipotent progenitors (MPPs). Recently, a new study suggested that separation between multipotent (Ery/My/Ly) long-term repopulating cells (Subset1, defined as CLEC9AhighCD34low) and cells with only My/Ly and no Ery potential (Subset2, defined as CLEC9AlowCD34high)occurs within the phenotypic HSC/MPP and CD49f+ HSCs compartment. Aims A general perturbed and stress condition is present in the thalassemic BM microenvironment. Since its impact on the hematopoietic cell subpopulations is mostly unknown, we will investigate which model of hematopoiesis/erythropoiesis occurs in Bthal. Moreover, since Beta-Thalassemia is an erythropoietic disorder, it could be considered as a disease model to study the 'erythroid branching' in the hematopoietic hierarchy. Methods We defined by immunophenotype and functional analysis the lineage commitment of most primitive HSC/MPP cells in patients affected by this pathology compared to healthy donors (HDs). Furthermore, in order to delineate the transcriptional networks governing hematopoiesis in Beta-thalassemia, RNAseq analysis was performed on sorted hematopoietic subpopulations from BM of Bthal patients and HDs. By droplet digital PCR on RNA purified from mesenchymal stromal cells of Bthal patients, we evaluated the expression levels of some niche factors involved in the regulation of hematopoiesis and erythropoiesis. Moreover, the protein levels in the BM plasma were analyzed by performing ELISA. Results Differences in the primitive compartment were observed with an increased proportion of multipotent progenitors in Bthal patients compared to HDs. The Subset1 compartment is actually endowed with an enhanced Ery potential. Focusing on progenitors (CD34+ CD38+) and using a new sorting scheme that efficiently resolved My, Ery, and Mk lineage fates, we quantified the new My (CD71-BAH1-/+) and Ery (CD71+ BAH1-/+) subsets and found a reduction of Ery subset in Bthal samples. We can hypothesize that the erythroid-enriched subsets are more prone to differentiate quickly due to the higher sensitivity to Epo stimuli or other bone marrow niche signals. Gene set enrichment analysis, perfomed on RNAseq data, showed that Bthal HSC/MPP presented negative enrichment of several pathways related to stemness and quiescence. Cellular processes involved in erythropoiesis were found altered in Bthal HSC. Moreover, some master erythroid transcription factors involved were overrepresented in Bthal across the hematopoietic cascade. We identified the niche factors which affect molecular pathways and the lineage commitment of Bthal HSCs. Summary/Conclusions Overall, these data indicate that Bthal HSCs are more cycling cells which egress from the quiescent state probably towards an erythroid differentiation, probably in response to a chronic BM stimulation. On the other hand,some evidences support our hypothesis of an 'erythroid branching' already present in the HSC pool, exacerbated by the pathophysiology of the disease. Disclosures No relevant conflicts of interest to declare.

The Mll-AF9 Fusion Gene Results in Overexpression of Homeobox Genes and Deregulation of Granulocyte-Monocyte Progenitors.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 645-645
Author(s):  
Ashish Kumar ◽  
Weili Chen ◽  
John H. Kersey
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Young Adult ◽  
Hematopoietic Stem Cells ◽  
Fusion Gene ◽  
Homeobox Genes ◽  
Specific Cell ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Over Expression

Abstract Our understanding of the biology of MLL fusion gene leukemias is limited by the lack of knowledge of the effects of the different MLL fusion genes on expression of specific homoebox genes and the specific cell compartment(s) that are subsequently deregulated. In this study we investigated whether cellular deregulation was present in committed myeloid precursors and/or the multi-potent hematopoietic stem cells derived from Mll-AF9 knock-in mice. We used the murine knock-in model since it offers the advantage of a single copy of the Mll fusion gene under the control of the endogenous promoter that is present in every hematopoietic stem/progenitor cell. The Mll-AF9 knock-in mice display expansion of the myeloid compartment as early as 6 weeks of age (young adult) and develop myeloid leukemia at approximately 6 months. We purified hematopoietic stem cells (HSCs) and granulocyte-monocyte progenitors (GMPs) from wild type and Mll-AF9 young adult bone marrow. We depleted lineage positive cells using a magnetic separation system and purified the respective populations using fluorescence activated cell sorting with specific panels of antibodies (HSC=Li−/Thy1.1lo/IL-7R−/C-kit+/Sca-1+; GMP=Lin−/IL-7R−/Sca-1+/C-kit+/CD34+/CD16/32hi). We cultured these cells in methylcellulose supplemented with GM-CSF, IL-3, SCF and IL-6, conditions that promote the growth of myeloid colonies. We assessed growth deregulation by increased colony numbers at the end of 7 days of culture and by the predominance of dense, compact colony morphology, the latter comprised of immature myeloid cells. Culture of HSCs from Mll-AF9 and wild type mice yielded an identical number of colonies (1102 and 1315 colonies per 104 cells respectively, average). In contrast, GMPs from Mll-AF9 mice yielded almost four times the number of colonies compared to wild type GMPs (3331 and 920 colonies per 104 cells respectively, average). Additionally, Mll-AF9 GMPs formed a higher number of dense, compact colonies compared to Mll-AF9 HSCs (1314 and 352 colonies per 104 cells respectively, average). Neither HSCs nor GMPs from wild type mice formed dense, compact colonies. These results indicate a greater deregulation of GMPs compared to HSCs in Mll-AF9 mice. MLL fusion gene leukemias are characterized by over-expression of specific homeobox genes, and we have previously shown that Mll-AF9 bone marrow cells display increased expression of 5′ Hox-a genes and of the Hox co-factor Meis1 compared to wild type counterparts. We hypothesized that these genes are over-expressed in Mll-AF9 GMPs compared to wild type GMPs. Real time quantitative RT-PCR showed that expression levels of Hoxa7, Hoxa9 and Meis1 were increased in Mll-AF9 GMPs compared to wild type (2.7 ± 0.8, 11.7 ± 7.8 and 19 ± 11.3 fold respectively, mean ± SEM). Overall, these data support the hypothesis that the Mll-AF9 gene is “instructive” at the molecular level at least in part via specific homeobox gene over-expression, resulting in deregulation and expansion of specific progenitor/stem cells such as the GMP population. This expanded GMP population then becomes a target for secondary mutations and later development of leukemia. Future studies focused on understanding the biology of this compartment in Mll-AF9 mice will help in our understanding of the pathogenesis of leukemia and aid in the development of newer, more effective therapies.

Profilin 1 Is Essential for Retention and Metabolism of Hematopoietic Stem Cells in Bone Marrow

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1224-1224
Author(s):  
Junke Zheng ◽  
Chengcheng Zhang
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Actin Binding ◽  
Wild Type ◽  
Cell Fates ◽  
Hematopoietic Stem ◽  
Multiple Cell

Abstract Abstract 1224 How stem cells interact with the microenvironment to regulate their cell fates and metabolism is largely unknown. Here we show that, in a hematopoietic stem cell (HSC) -specific inducible knockout model, the cytoskeleton-modulating protein profilin 1 (pfn1) is essential for the maintenance of multiple cell fates and metabolism of HSCs. The deletion of pfn1 in HSCs led to bone marrow failure, loss of quiescence, increased apoptosis, and mobilization of HSCs in vivo. In reconstitution analyses, pfn1-deficient cells were selectively lost from mixed bone marrow chimeras. By contrast, pfn1 deletion did not significantly affect differentiation or homing of HSCs. When compared to wild-type cells, levels of expression of Hif-1a, EGR1, and MLL were lower and an earlier switch from glycolysis to mitochondrial respiration with increased ROS level was observed in pfn1-deficient HSCs. This switch preceded the detectable alteration of other cell fates. Importantly, treatment of pfn1-deficient mice with the antioxidant N-acetyl-l-cysteine reversed the ROS level and loss of quiescence of HSCs, suggesting that pfn1 maintained metabolism is required for the quiescence of HSCs. Furthermore, we demonstrated that expression of wild-type pfn1 but not the actin-binding deficient or poly-proline binding-deficient mutants of pfn1 rescued the defective phenotype of pfn1-deficient HSCs. This result indicates that actin-binding and proline-binding activities of pfn1 are required for its function in HSCs. Thus, pfn1 plays an essential role in regulating the retention and metabolism of HSCs in the bone marrow microenvironment. Disclosures: No relevant conflicts of interest to declare.

Sqstm1 Is Required to Retain Hematopoietic Stem Cell/ Progenitors As a Negative Regulator of Macrophage-Dependent Inflammatory Signaling in the Bone Marrow Osteoblastic Niche

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 350-350
Author(s):  
Kyung-Hee Chang ◽  
Amitava Sengupta ◽  
Ramesh C Nayak ◽  
Angeles Duran ◽  
Sang Jun Lee ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Research Funding ◽  
Negative Regulator ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
P Retention

Abstract In the bone marrow (BM), hematopoietic stem cells and progenitors (HSC/P) reside in specific anatomical niches. Among these niches, a functional osteoblast (Ob)-macrophage (MΦ) niche has been described where Ob and MΦ (so called "osteomacs") are in direct relationship. A connection between innate immunity surveillance and traffic of hematopoietic stem cells/progenitors (HSC/P) has been demonstrated but the regulatory signals that instruct immune regulation from MΦ and Ob on HSC/P circulation are unknown. The adaptor protein sequestosome 1 (Sqstm1), contains a Phox bemp1 (PB1) domain which regulates signal specificities through PB1-PB1 scaffolding and processes of autophagy. Using microenvironment and osteoblast-specific mice deficient in Sqstm1, we discovered that the deficiency of Sqstm1 results in macrophage contact-dependent activation of Ob IKK/NF-κB, in vitro and in vivo repression of Ccl4 (a CCR5 binding chemokine that has been shown to modulate microenvironment Cxcl12-mediated responses of HSC/P), HSC/P egress and deficient BM homing of wild-type HSC/P. Interestingly, while Ccl4 expression is practically undetectable in wild-type or Sqstm1-/- Ob, primary Ob co-cultured with wild-type BM-derived MΦ strongly upregulate Ccl4 expression, which returns to normal levels upon genetic deletion of Ob Sqstm1. We discovered that MΦ can activate an inflammatory pathway in wild-type Ob which include upregulation of activated focal adhesion kinase (p-FAK), IκB kinase (IKK), nuclear factor (NF)-κB and Ccl4 expression through direct cell-to-cell interaction. Sqstm1-/- Ob cocultured with MΦ strongly upregulated p-IKBα and NF-κB activity, downregulated Ccl4 expression and secretion and repressed osteogenesis. Forced expression of Sqstm1, but not of an oligomerization-deficient mutant, in Sqstm1-/- Ob restored normal levels of p-IKBα, NF-κB activity, Ccl4 expression and osteogenic differentiation, indicating that Sqstm1 dependent Ccl4 expression depends on localization to the autophagosome formation site. Finally, Ob Sqstm1 deficiency results in upregulation of Nbr1, a protein containing a PB1 interacting domain. Combined deficiency of Sqstm1 and Nbr1 rescues all in vivo and in vitro phenotypes of Sqstm1 deficiency related to osteogenesis and HSC/P egression in vivo. Together, this data indicated that Sqstm1 oligomerization and functional repression of its PB1 binding partner Nbr1 are required for Ob dependent Ccl4 production and HSC/P retention, resulting in a functional signaling network affecting at least three cell types. A functional ‘MΦ-Ob niche’ is required for HSC/P retention where Ob Sqstm1 is a negative regulator of MΦ dependent Ob NF-κB activation, Ob differentiation and BM HSC/P traffic to circulation. Disclosures Starczynowski: Celgene: Research Funding. Cancelas:Cerus Co: Research Funding; P2D Inc: Employment; Terumo BCT: Research Funding; Haemonetics Inc: Research Funding; MacoPharma LLC: Research Funding; Therapure Inc.: Consultancy, Research Funding; Biomedical Excellence for Safer Transfusion: Research Funding; New Health Sciences Inc: Consultancy.

Regulation of Hematopoietic Stem Cells by Interferons and by DNA Methylation: Implications for Bone Marrow Failure

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. SCI-20-SCI-20
Author(s):  
Margaret A. Goodell
Keyword(s):  
Dna Methylation ◽  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Bone Marrow Failure ◽  
Stem Cell Differentiation ◽  
Primary Myelofibrosis ◽  
Differentiation Potential ◽  
Hematopoietic Stem

Bone marrow failure (BMF), the inability to regenerate the differentiated cells of the blood, has a number of genetic and environmental etiologies, such as mutation of telomere-associated protein genes and immune-related aplastic anemia. Recently, mutations in DNA methyltransferase 3A (DNMT3A) have been found to be associated with approximately 15% of cases of primary myelofibrosis (MF), which can be a cause of BMF. The role of DNMT3A more broadly in hematopoiesis, and specifically in BMF, is currently poorly understood. DNMT3A is one of two de novo DNA methylation enzymes important in developmental fate choice. We showed that Dnmt3a is critical for normal murine hematopoiesis, as hematopoietic stem cells (HSCs) from Dnmt3a knockout (KO) mice displayed greatly diminished differentiation potential while their self-renewal ability was markedly increased1, in effect, leading to failure of blood regeneration or BMF. Combined with loss of Dnmt3b, HSCs exhibited a profound differentiation block, mediated in part by an increase of stabilized b-catenin. While we did not initially observe bone marrow pathology or malignancy development in mice transplanted with Dnmt3a KO HSCs, when we aged a large cohort of mice, all mice succumbed to hematologic disease within about 400 days. Roughly one-third of mice developed frank leukemia (acute lymphocytic leukemia or acute myeloid leukemia), one-third developed MDS, and the remainder developed primary myelofibrosis or chronic myelomonocytic leukemia. The pathological characteristics of the mice broadly mirror those of patients, suggesting the Dnmt3a KO mice can serve as a model for human DNMT3A-mutation associated disease. Strikingly, bone marrow of mice with different disease types exhibit distinct DNA methylation features. These will findings and the implications for disease development will be discussed. We are currently investigating the factors that drive different outcomes in the mice, including stressors such as exposure to interferons. We have hypothesized that HSC proliferation accelerates the Dnnmt3a-associated disease phenotypes. We have previously shown that interferons directly impinge on HSCs in the context of infections. Interferons activate HSCs to divide, generating differentiated progeny and cycling HSCs. Repeated interferon stimulation may permanently impair HSC function and bias stem cell output. When combined with loss of Dnmt3a, interferons may promote BMF. We will discuss broadly how external factors such as aging and infection may collaborate with specific genetic determinants to affect long-term hematopoiesis and malignancy development. Reference: Challen GA, Sun D, Jeong M, et al. Dnmt3a is essential for hematopoietic stem cell differentiation. Nat Genet 2012; 44: 23-31 Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals Bclaf1 Promotes Maintenance and Self-Renewal of Fetal Hematopoietic Stem Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1269-1269 ◽  
Author(s):  
Lynn S. White ◽  
Deepti Soodgupta ◽  
Rachel L. Johnston ◽  
Jeffrey A. Magee ◽  
Jeffrey J. Bednarski
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Fetal Liver ◽  
Liver Cells ◽  
Lower Percentage ◽  
Wild Type ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Fetal Liver Cells

Abstract Hematopoietic stem cells (HSC) persist throughout life by undergoing extensive self-renewal divisions while maintaining an undifferentiated state. The mechanisms that support HSC self-renewal change throughout the course of development as temporal changes in transcriptional regulators coordinate distinct genetic programs in fetal, post-natal and adult HSCs. These self-renewal programs are often ectopically activated in leukemia cells to drive neoplastic proliferation and high expression of HSC-associated genes predicts a poor prognosis in acute myelogenous leukemia (AML). In this regard, it was recently shown that expression of the transcriptional regulator BCLAF1 (Bcl2 associated transcription factor 1) is increased in AML blasts relative to normal precursor populations and suppression of BCLAF1 causes reduced proliferation and induction of differentiation to a dendritic cell fate. These findings raise the question of whether BCLAF1 may regulate normal as well as neoplastic self-renewal programs. We find that Bclaf1 is highly expressed in HSCs versus committed bone marrow populations consistent with a potential role for this gene in HSC functions. To test whether BCLAF1 regulates HSC development and hematopoiesis, we used germline loss of function mice. Bclaf1-/- mice succumb to pulmonary failure shortly after birth due to poor lung development, so we assessed prenatal hematopoiesis. Bclaf1-deficient mice had significantly reduced HSC and hematopoietic progenitor cell (HPC) frequencies and numbers despite normal fetal liver cellularity. To determine if Bclaf1 is required for HSC function during fetal development, we performed competitive reconstitution assays. Fetal liver cells from Bclaf1+/+or Bclaf1-/-mice were transplanted along with wild-type competitor bone marrow cells into lethally irradiated recipient mice. Compared to recipients of Bclaf1+/+fetal liver cells, recipients of Bclaf1-/-cells had a significantly lower percentage of donor-derived leukocytes at all time points after transplantation as well as reduced percentage of donor HSCs at 16 weeks post-transplant. Notably, all leukocyte populations (B cells, T cells, granulocytes and macrophages) from Bclaf1-/-donors were reduced consistent with an abnormality in HSC repopulating activity rather than a defect in a specific differentiation pathway. Consistent with these findings, Bclaf-deficient cells did not engraft in competitive transplants with limiting numbers of sorted fetal liver HSCs whereas sorted wild-type Bclaf1+/+cells effectively reconstituted hematopoiesis in recipient mice. In addition, Vav-cre:Bclaf1flox/floxmice, which have selective deletion of Bclaf1 in hematopoietic cells, have reduced frequencies and numbers of fetal liver HSCs identical to the findings observed in germline Bclaf1-/-mice. These results show that loss of Bclaf1 leads to defective development and repopulating activity of fetal HSCs. Interestingly, when adult mice are successfully engrafted with Bclaf1-deficient HSCs, the donor HSCs suffer no additional functional impairment. Furthermore, in secondary transplant experiments Bclaf1-deficient HSCs maintain long-term repopulating activity. Thus, Bclaf1 may have distinct functions in fetal versus adult HSC self-renewal. Collectively, our findings reveal Bclaf1 is a novel regulator of fetal HSC function and suggest that it may have distinct functions in different developmental contexts. Disclosures No relevant conflicts of interest to declare.

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