The histone lysine acetyltransferase HBO1 (KAT7) regulates hematopoietic stem cell quiescence and self-renewal

Blood ◽  
2021 ◽  
Author(s):  
Yuqing Yang ◽  
Andrew J Kueh ◽  
Zoe Grant ◽  
Waruni Abeysekera ◽  
Alexandra L Garnham ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Hematopoietic Stem Cell ◽  
Cell Function ◽  
Bone Marrow Cells ◽  
High Rate ◽  
Embryonic Patterning ◽  
Self Renewal ◽  
Hematopoietic Stem

The histone acetyltransferase HBO1 (MYST2, KAT7) is indispensable for postgastrulation development, histone H3 lysine 14 acetylation (H3K14Ac) and the expression of embryonic patterning genes. In this study, we report the role of HBO1 in regulating hematopoietic stem cell function in adult hematopoiesis. We used two complementary cre-recombinase transgenes to conditionally delete Hbo1 (Mx1-Cre and Rosa26-CreERT2). Hbo1 null mice became moribund due to hematopoietic failure with pancytopenia in the blood and bone marrow two to six weeks after Hbo1 deletion. Hbo1 deleted bone marrow cells failed to repopulate hemoablated recipients in competitive transplantation experiments. Hbo1 deletion caused a rapid loss of hematopoietic progenitors (HPCs). The numbers of lineage-restricted progenitors for the erythroid, myeloid, B-and T-cell lineages were reduced. Loss of HBO1 resulted in an abnormally high rate of recruitment of quiescent hematopoietic stem cells (HSCs) into the cell cycle. Cycling HSCs produced progenitors at the expense of self-renewal, which led to the exhaustion of the HSC pool. Mechanistically, genes important for HSC functions were downregulated in HSC-enriched cell populations after Hbo1 deletion, including genes essential for HSC quiescence and self-renewal, such as Mpl, Tek(Tie-2), Gfi1b, Egr1, Tal1(Scl), Gata2, Erg, Pbx1, Meis1 and Hox9, as well as genes important for multipotent progenitor cells and lineage-specific progenitor cells, such as Gata1. HBO1 was required for H3K14Ac through the genome and particularly at gene loci required for HSC quiescence and self-renewal. Our data indicate that HBO1 promotes the expression of a transcription factor network essential for HSC maintenance and self-renewal in adult hematopoiesis.

The F-Box Protein NIPA Regulates the Hematopoietic Stem Cell Pool

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2330-2330
Author(s):  
Stefanie Kreutmair ◽  
Anna Lena Illert ◽  
Rouzanna Istvanffy ◽  
Melanie Sickinger ◽  
Christina Eckl ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Bone Marrow Cells ◽  
Wild Type ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cell Pool ◽  
F Box Protein ◽  
Stem Cell Pool

Abstract Abstract 2330 Hematopoietic stem cells (HSCs) are characterized by their ability to self-renewal and multilineage differentiation. Since mostly HSCs exist in a quiescent state re-entry into cell cycle is essential for their regeneration and differentiation and the expression of numerous cell cycle regulators must be tightly controlled. We previously characterized NIPA (Nuclear Interaction Partner of ALK) as a F-Box protein that defines an oscillating ubiquitin E3 ligase targeting nuclear cyclin B1 in interphase thus contributing to the timing of mitotic entry. To examine the function of NIPA on vivo, we generated NIPA deficient animals, which are viable but sterile due to a defect in recombination and testis stem cell maintenance. To further characterize the role of NIPA in stem cell maintenance and self-renewal we investigated hematopoiesis in NIPA deficient animals. Peripheral blood counts taken at different ages revealed no apparent difference between NIPA knockout and wild type mice in numbers and differentiation. In contrast, looking at the hematopoietic stem cell pool, FACS analyses of bone marrow showed significantly decreased numbers of Lin-Sca1+cKit+ (LSK) cells in NIPA deficient animals, where LSKs were reduced to 40% of wild type littermates (p=0,0171). This effect was only apparent in older animals, where physiologically higher LSK numbers have to compensate for the exhaustion of the stem cell pool. Additionally, older NIPA deficient mice have only half the amount of multi myeloid progenitors (MMPs) in contrast to wild type animals. To examine efficient activation of stem cells to self-renew in response to myeloid depression, we treated young and old mice with the cytotoxic drug (5-FU) four days before bone marrow harvest. As expected, 5-FU activated hematopoietic progenitors in wild type animals, whereas NIPA deficient progenitors failed to compensate to 5-FU depression, e.g. LSKs of NIPA knockout mice were reduced to 50% of wild type levels (p<0.001), CD150+CD34+ Nipa deficient cells to 20% of wild type levels (p<0.0001). Interestingly, these effects were seen in all NIPA deficient animals independent of age, allowing us to trigger the self-renewal phenotype by activating the hematopoietic stem cell pool. Using competitive bone marrow transplantation assays, CD45.2 positive NIPA deficient or NIPA wild type bone marrow cells were mixed with CD45.1 positive wild type bone marrow cells and transplanted into lethally irradiated CD45.2 positive recipient mice. Thirty days after transplantation, FACS analysis of peripheral blood and bone marrow showed reduced numbers of NIPA knockout cells in comparison to NIPA wild type bone marrow recipient mice. This result was even more severe with aging of transplanted mice, where NIPA deficient cells were reduced to less than 10% of the level of wild type cells in bone marrow of sacrificed mice 6 months after transplantation, pointing to a profound defect in repopulation capacity of NIPA deficient HSCs. Taken together our results demonstrate a unique and critical role of NIPA in regulating the primitive hematopoietic compartment as a regulator of self-renewal, cycle capacity and HSC expansion. Disclosures: No relevant conflicts of interest to declare.

Chromatin Remodeling Enzyme CHD7 Negatively Regulate Hematopoietic Stem Cell Function

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2413-2413 ◽  
Author(s):  
Jingmei Hsu ◽  
Hsuan-Ting Huang ◽  
Chung-Tsai Lee ◽  
Shuqian Yu ◽  
Leonard I. Zon ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Hematopoietic Stem Cell ◽  
Chromatin Remodeling ◽  
Cell Function ◽  
Charge Syndrome ◽  
Hematopoietic Stem ◽  
Morpholino Knockdown ◽  
Stem And Progenitor Cells

Abstract ATP-dependent chromatin remodeling enzymes alter histone/DNA interactions, and are involved in the regulation of transcription, chromosome segregation, DNA replication and repair. We identified Chromodomain Helicase DNA binding protein 7 (CHD7) as a negative regulator of hematopoietic stem cell function. Autosomal dominant CHD7 mutations are associated with CHARGE syndrome (Coloboma of the eye, Heart defects, Atresia of the choanae, Retardation of growth and/or development, Genital and/or urinary abnormalities, and Ear abnormalities and deafness). Although several cases of T and B cell immunodeficiency in a subset of CHARGE syndrome patients have been reported, no previous role for CHD7 in hematopoiesis had been proposed. We show that morpholino knockdown of Chd7 in zebrafish embryos results in an increased number of runx1 and c-myb expressing hematopoietic stem and progenitor cells in the aorta/gonad/mesonephros (AGM) region, and this effect is cell autonomous as determined by blastula transplantation. Heterozygous germline Chd7 deletion in mice also results in an increased number of phenotypic hematopoietic stem and progenitor cells (Runx1+c-kit+CD31+) in the AGM region. Downstream lineages such as myeloid and erythroid cells are expanded in zebrafish, and similarly conditional pan-hematopoietic deletion of Chd7 in mice with Vav1-Cre results in myeloid lineage expansion and increased granulocyte/monocyte progenitors. Consistent with these results, microarray analysis of murine CD48- CD150+ Lin- Sca-1+ c-kit+ (SLAM LSK) phenotypic long term repopulating HSCs shows up-regulation of genes in several subclasses of the myeloid lineages. Interestingly, although CHD7 deficient mouse bone marrow had a normal frequency of SLAM LSK cells, it had a two-fold higher frequency of functional LT-HSCs as determined by whole bone marrow, purified LSK and SLAM LSK limiting dilution transplants. ChIP-seq performed in human CD34+ hematopoietic stem and progenitor cells show CHD7 localizes to genes encoding many key hematopoietic transcription factors including MYB and RUNX1. The most abundant transcription factor motif under CHD7 genomic binding sites is a RUNX motif, indicating that CHD7 and Runx1 function together. We show CHD7, Runx1 and c-Myb interact both physically and genetically. CHD7 function in hematopoietic stem cells is dependent on both Runx1 and c-Myb since the increase in hematopoiesis in fish upon morpholino knockdown of Chd7 is abolished when either Runx1 or c-Myb is mutated. In summary, our study identifies CHD7 as a novel evolutionarily conserved negative epigenetic regulator of HSCs and progenitors through its interaction with Runx1 and c-Myb. Disclosures: No relevant conflicts of interest to declare.

Hematopoietic Stem Cell Expansion by HOXB4 Is Greatly Enhanced in p21 Deficient Stem Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1688-1688 ◽  
Author(s):  
Noriko Miyake ◽  
Ann C.M. Brun ◽  
Mattias Magnusson ◽  
David T. Scadden ◽  
Stefan Karlsson
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Hematopoietic Stem Cell ◽  
Bone Marrow Cells ◽  
Ex Vivo ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract Hox transcription factors have emerged as important regulators of hematopoiesis. In particular, enforced expression of HOXB4 is a potent stimulus for murine hematopoietic stem cell (HSC) self-renewal. Murine HSCs engineered to overexpress HoxB4 expand significantly more than control cells in vivo and ex vivo while maintaining a normal differentiation program. HSCs are regulated by the cell proliferation machinery that is intrinsically controlled by cyclin-dependent kinase inhibitors such as p21Cip1/Waf1(p21) and p27Kip1 (p27). The p21 protein restricts cell cycling of the hematopoietic stem cell pool and maintains hematopoietic stem cell quiescence. In order to ask whether enhanced proliferation due to HOXB4 overexpression is mediated through suppression of p21 we overexpressed HOXB4 in hematopoietic cells from p21−/− mice. First, we investigated whether human HOXB4 enhances in vitro expansion of BM cells from p21−/− mice compared to p21+/+ mice. 5FU treated BM cells from p21−/− or p21+/+ mice were pre-stimulated with SCF, IL-6, IL-3 for 2 days followed by transduction using retroviral vector expressing HOXB4 together with GFP (MIGB4) or the control GFP vector (MIG). The cells were maintained in suspension cultures for 13 days and analyzed for GFP positive cells by flow-cytometry. Compared to MIG transduced BM cells from p21+/+ mice (MIG/p21+), the numbers of GFP positive cells were increased 1.1-fold in MIG/p21−, 3.2-fold in MIGB4/p21+ and 10.0-fold in MIGB4/p21− respectively (n=5). CFU assays were performed after 13 days of culture. The numbers of CFU were increased 4.8-fold in MIG/p21−, 19.5-fold in MIG/p21+ and 33.9 -fold in MIGB4/p21− respectively. Next, we evaluated level of HSCs expansion by bone marrow repopulation assays. After 12-days of culture, 1.5 x 105 MIGB4 or MIG-transduced cells (Ly5.2) were transplanted into lethally irradiated mice in combination with 8 x 105 fresh Ly5.1 bone marrow cells. Sixteen weeks after transplantation, no Ly5.2 cells could be detected in MIG/p21+ or MIG/p21− transplanted mice (n=6). In contrast, Ly5.2 positive cells were detected in both MIGB4/p21+/+ and MIGB4/p21−/− cells. The % of Ly5.2 positive cells in MIGB4/p21− transplanted mice was 9.9-fold higher than MIGB4/p21+ transplanted mice. (38.4 % vs 3.9 %, P<0.02, n=5). These Ly5.2 positive cells differentiated into all lineages, as determined by proportions of Mac-1, B-220, CD3 and Ter119 positive populations. Currently, we are enumerating the expansion of HOXB4 transduced HSCs in p21 deficient BM cells using the CRU assay. Our findings suggest that HOXB4 increases the self-renewal of hematopoietic stem cells by a mechanism that is independent of p21. In addition, the findings demonstrate that deficiency of p21 in combination with enforced expression of HOXB4 can be used to rapidly and effectively expand hematopoietic stem cells.

scholarly journals Hematopoietic Stem Cell Function in a Murine Model of Sickle Cell Disease

Anemia ◽  
2012 ◽  
Vol 2012 ◽  
pp. 1-9 ◽  
Author(s):  
Elisabeth H. Javazon ◽  
Mohamed Radhi ◽  
Bagirath Gangadharan ◽  
Jennifer Perry ◽  
David R. Archer
Keyword(s):  
Cell Cycle ◽  
Lipid Peroxidation ◽  
Bone Marrow ◽  
Stem Cell ◽  
Sickle Cell Disease ◽  
Hematopoietic Stem Cell ◽  
Cell Function ◽  
Cell Disease ◽  
Hematopoietic Progenitor ◽  
Hematopoietic Stem

Previous studies have shown that the sickle environment is highly enriched for reactive oxygen species (ROS). We examined the oxidative effects of sickle cell disease on hematopoietic stem cell function in a sickle mouse model.In vitrocolony-forming assays showed a significant decrease in progenitor colony formation derived from sickle compared to control bone marrow (BM). Sickle BM possessed a significant decrease in the KSL (c-kit+, Sca-1+, Lineage−) progenitor population, and cell cycle analysis showed that there were fewer KSL cells in the G0phase of the cell cycle compared to controls. We found a significant increase in both lipid peroxidation and ROS in sickle-derived KSL cells.In vivoanalysis demonstrated that normal bone marrow cells engraft with increased frequency into sickle mice compared to control mice. Hematopoietic progenitor cells derived from sickle mice, however, demonstrated significant impairment in engraftment potential. We observed partial restoration of engraftment by n-acetyl cysteine (NAC) treatment of KSL cells prior to transplantation. Increased intracellular ROS and lipid peroxidation combined with improvement in engraftment following NAC treatment suggests that an altered redox environment in sickle mice affects hematopoietic progenitor and stem cell function.

scholarly journals Osteoblast ablation reduces normal long-term hematopoietic stem cell self-renewal but accelerates leukemia development

Blood ◽  
2015 ◽  
Vol 125 (17) ◽  
pp. 2678-2688 ◽  
Author(s):  
Marisa Bowers ◽  
Bin Zhang ◽  
Yinwei Ho ◽  
Puneet Agarwal ◽  
Ching-Cheng Chen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Hematopoietic Stem Cell ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Key Points ◽  
Leukemia Development

Key Points Bone marrow OB ablation leads to reduced quiescence, long-term engraftment, and self-renewal capacity of hematopoietic stem cells. Significantly accelerated leukemia development and reduced survival are seen in transgenic BCR-ABL mice following OB ablation.

Involvement of the Integrin Alpha 6 Receptor in the Homing and Engraftment of Hematopoietic Stem and Progenitor Cells to Bone Marrow.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1293-1293
Author(s):  
Hong Qian ◽  
Sten Eirik W. Jacobsen ◽  
Marja Ekblom
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Progenitor Cell ◽  
Bone Marrow Cells ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells ◽  
Functional Blocking

Abstract Within the bone marrow environment, adhesive interactions between stromal cells and extracellular matrix molecules are required for stem and progenitor cell survival, proliferation and differentiation as well as their transmigration between bone marrow (BM) and the circulation. This regulation is mediated by cell surface adhesion receptors. In experimental mouse stem cell transplantation models, several classes of cell adhesion receptors have been shown to be involved in the homing and engraftment of stem and progenitor cells in BM. We have previously found that integrin a6 mediates human hematopoietic stem and progenitor cell adhesion to and migration on its specific ligands, laminin-8 and laminin-10/11 in vitro (Gu et al, Blood, 2003; 101:877). Using FACS analysis, the integrin a6 chain was now found to be ubiquitously (>95%) expressed in mouse hematopoietic stem and progenitor cells (lin−Sca-1+c-Kit+, lin−Sca-1+c-Kit+CD34+) both in adult bone marrow and in fetal liver. In vitro, about 70% of mouse BM lin−Sca-1+c-Kit+ cells adhered to laminin-10/11 and 40% adhered to laminin-8. This adhesion was mediated by integrin a6b1 receptor, as shown by functional blocking monoclonal antibodies. We also used a functional blocking monoclonal antibody (GoH3) against integrin a6 to analyse the role of the integrin a6 receptor for the in vivo homing of hematopoietic stem and progenitor cells. We found that the integrin a6 antibody inhibited the homing of bone marrow progenitors (CFU-C) into BM of lethally irradiated recipients. The number of homed CFU-C was reduced by about 40% as compared to cells incubated with an isotype matched control antibody. To study homing of long-term repopulating stem cells (LTR), antibody treated bone marrow cells were first injected intravenously into lethally irradiated primary recipients. After three hours, bone marrow cells of the primary recipients were analysed by competitive repopulation assay in secondary recipients. Blood analysis 16 weeks after transplantation revealed an 80% reduction of stem cell activity of integrin a6 antibody treated cells as compared to cells treated with control antibody. These results suggest that integrin a6 plays an important role for hematopoietic stem and progenitor cell homing in vivo.

Multilineage Expansion of Lymphoid and Myeloid Cells in Mice Expressing PML-RARα under the Control of the Murine Cathepsin G Locus.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2551-2551
Author(s):  
Geoffrey L. Uy ◽  
Jacqueline E. Payton ◽  
Timothy James Ley
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
T Cells ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Cell Function ◽  
Cathepsin G ◽  
Pcr Analysis ◽  
Self Renewal ◽  
Marrow Cells

In one of our laboratory’s models of acute promyelocytic leukemia (APL), the PML-RARα fusion cDNA is knocked into the 5′ UT of the azurophil granule protease, cathepsin G. Nearly all mCG-PML-RARα mice develop lethal leukemia with promyelocytic features following a 150–400 day latent period. We originally chose the cathepsin G gene for the targeting locus because its expression was believed to be restricted to promyelocytes. However, we have now observed high levels of cathepsin G expression with gene expression profiling of leukemic bone marrow samples from patients with AML FAB M1 or M2, i.e. blasts with minimal or no differentiation (M1: n=21, mean raw expression value for cathepsin G=23,885 ± 31,737; M2: n=22, mean=24,088 ± 26,585; M3: n=14, mean 116,029 ± 47,017). To examine whether cathepsin G is normally expressed in murine hematopoietic progenitor cells, we performed gene expression arrays with highly purified Sca-1+/lin− bone marrow cells; cathepsin G mRNA was easily detected in these cells (n=4, mean cathepsin G raw expression value=9,324 ± 5,082; array average normalized to 1,500). We therefore decided to determine whether the early progenitor compartment in mCG-PML-RARα mice was expanded due to unexpected expression of the transgene in KLS (c-kit+, Lin−, Sca-1+) cells; however, no difference was detected in the frequency of marrow-derived KLS cells between mCG-PML-RARα and WT mice that were age, strain, and gender matched (0.074% vs 0.071%, p=0.89). The GMP compartment (Lin−, c-kit+, Sca1−, CD34+, FcRγ+) showed a tendency towards expansion in mCG-PML-RARα mice (0.10% vs 0.032%) but the difference was not significant (p=0.067). To better assess stem cell function in these mice, we performed a competitive repopulation study using marrow derived from Ly 5.2/mCG-PML-RARα mice mixed with Ly5.1/WT marrow cells at various ratios (1:1, 9:1, and 1:9) that were transplanted into genetically compatible hosts. Lineage markers in the peripheral blood were tested at 6, 12, and 24 weeks to assess the contribution of mCG-PML-RARα-derived progenitor and stem cells to the B, T, and myeloid lineages. As expected, we observed a highly reproducible increase in the proportion of Gr-1+ cells derived from mCG-PML-RARα donors at all time points (78.8% ± 11% donor-derived cells at a 1:1 donor: recipient ratio at 6 months, p=0.0006). Surprisingly, we also observed a reproducible increase in B220+/CD19+ cells (66.7% ± 4%, p<0.0001) and CD3+ cells from the mCG-PML-RARα donors (60.4% ± 3% p=0.0010) at 6 months, suggesting that mCG-PML-RARα also confers a growth or survival advantage to B and T cells. To determine whether cathepsin G was expressed in these compartments, we purified CD19+ B and CD3+ T cells by flow cytometry (>99% purity) and did not detect cathepsin G mRNA using a sensitive, knockout-proven qRT-PCR analysis. These data strongly suggest that both the WT cathepsin G gene and the mutant mCG-PML-RARα allele are unexpectedly expressed in the stem cell compartment, with both myeloid and lymphoid progeny of these cells displaying a growth advantage in vivo. However, lymphoid malignancies have not been detected in these mice (n>400), suggesting that PML-RARα is unable to initiate transformation in lymphocytes. Our data imply that the PML-RARα gene is activated in a multipotent compartment in this mouse model, raising the possibility that PML-RARα may not actually ‘reprogram’ progenitor cells to undergo self-renewal, but may rather initiate transformation in pluripotent cells with intrinsic self-renewal capabilities.

DNMT3b’s Role in Hematopoietic Stem Cell Self-Renewal.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1406-1406
Author(s):  
Matthew J Boyer ◽  
Feng Xu ◽  
Hui Yu ◽  
Tao Cheng
Keyword(s):  
Dna Methylation ◽  
Bone Marrow ◽  
Stem Cell ◽  
Bone Marrow Transplantation ◽  
Peripheral Blood ◽  
Marrow Transplantation ◽  
Bone Marrow Cells ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Marrow Cells

Abstract DNA methylation is an epigenetic means of gene regulation and is carried out by a family of methyltransferases of which DNMT1 acts to maintain methylation marks following DNA replication and DNMT3a and DNMT3b methylate DNA de novo. DNMT3b has been shown to be essential for mammalian development and necessary for differentiation of germline and neural progenitor cells. Mutations of DNMT3b in humans lead to a rare autosomal recessive disorder characterized by immunodeficiency, centromeric instability, and facial abnormalities. We have shown by real-time, RT-PCR that DNMT3b mRNA is uniquely over-expressed by approximately 30-fold in immunophenotypically-defined longterm repopulating hematopoietic stem cells (HSCs) that are CD34−lineage−c-kit+Sca-1+ as compared to progenitor and differentiated cell types within the bone marrow and with respect to the other members of the DNMT family, namely DNMT1 and DNMT3a. To determine DNMT3b’s function in HSCs competitive bone marrow transplantation was undertaken. Isolated lineage− enriched bone marrow cells were transduced with a retroviral backbone based on the Murine Stem Cell Virus (MSCV) carrying either GFP and a short, hairpin RNA (shRNA) targeting DNMT3b or GFP alone. Following transduction 1×105 GFP+ cells along with 1×105 competitor cells were transplanted into 9.5 Gray irradiated congenic recipients. Two months following transplantation mice receiving bone marrow cells transduced with DNMT3b shRNA showed a significantly lower engraftment of donor cells as a percentage of total competitor cell engraftment in the peripheral blood as compared to those receiving cells transduced with GFP alone (24.8 vs 3.7, p&lt;0.05) which persisted at 3 months (22.8 vs 1.5, p&lt;0.05). Similarly, within the donor derviced cells in the peripheral blood there was a lower percentage of myeloid (CD11b+) cells at 2 and 3 months in the recipients of DNMT3b shRNA transduced cells as compared to controls. However there was no observed difference in the percentage of peripheral B (CD45R+) or T (CD3+) cells within the donor-derived cells. To determine the mechanism behind the observed engraftment defect with DNMT3b knockdown we cultured GFP+ transduced bone marrow cells in vitro with minimal cytokine support. As a control for our targeting methodology we also transduced bone marrow cells from mice harboring two floxed DNMT3b alleles with a MSCV carrying Cre recombinase and GFP. While lineage− bone marrow cells transduced with GFP alone increased 10-fold in number over two weeks of culture, cells in which DNMT3b was down regulated by shRNA or Cre-mediated recombination only doubled. Culture of lineage− bone marrow cells in methylcellulose medium by the colony-forming cell (CFC) assay revealed increases in the granulocytic and total number of colonies with DNMT3b knockdown or Cre-mediated recombination of DNMT3b similar to the increased myeloid engraftment of DNMT3b shRNA transduced cells observed 1 month following competitive bone marrow transplantation. However when 5,000 of these cells from the first CFC assay were sub-cultured there was a significant loss of colony forming ability within all lineages when DNMT3b was targeted by shRNA or Cre-mediated recombination. Taken together with the decreased engraftment of DNMT3b shRNA cells following competitive bone marrow transplantation, the observed limited proliferation in liquid culture and loss of colony forming ability during serial CFC assays is suggestive of a self-renewal defect of HSCs in the absence of DNMT3b, that was previously only reported in the absence of both DNMT3a and DNMT3b. Further elucidation of this proposed self-renewal defect is being undertaken and results of ongoing studies including long-term culture initiating cell (LTC-IC) assays and identification of genomic sites of DNA methylation within different hematopoietic subsets will also be presented.

G-CSF Treatment Induces Hematopoietic Stem Cell Quiescence but Loss of Repopulating Activity

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1206-1206
Author(s):  
Joshua N. Borgerding ◽  
Priya Gopalan ◽  
Matthew Christopher ◽  
Daniel C. Link ◽  
Laura G. Schuettpelz
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Hematopoietic Stem Cell ◽  
Inflammatory Signaling ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
A Cell

Abstract Abstract 1206 There is accumulating evidence that systemic signals, such as inflammatory cytokines, can affect hematopoietic stem cell (HSC) function. Granulocyte colony stimulating factor (G-CSF), the principal cytokine regulating granulopoiesis, is often induced in response to infection or inflammation. Additionally, G-CSF is the most commonly used agent for HSC mobilization prior to stem cell transplantation. Recently there has been a renewed interest in the use of “G-CSF primed bone marrow” for stem cell transplantation, so understanding the affect of G-CSF on bone marrow HSCs is clinically relevant. Because the G-CSF receptor is expressed on HSCs, and G-CSF creates biologically relevant modifications to the bone marrow microenvironment, we hypothesized that increased signaling through G-CSF may alter the repopulating and/or self-renewal properties of HSCs. Due to G-CSF's role as an HSC mobilizing agent, we predicted that the number of HSCs in the bone marrow would be reduced after 7 days of G-CSF treatment. Surprisingly, we observe that stem cell numbers markedly increase, regardless of which HSC-enriched population is analyzed. C-kit+lineage−sca+CD34− (KLS-34−), KLS CD41lowCD150+CD48− (KLS-SLAM), and KLS-SLAM CD34− increase by 6.97±2.25 fold, 1.79±0.29 fold, and 2.08±0.39 fold, respectively. To assess HSC repopulating activity, we conducted competitive bone marrow transplants. Donor mice were treated with or without G-CSF for 7 days, and bone marrow was transplanted in a 1:1 ratio with marrow from untreated competitors into lethally irradiated congenic recipients. Compared to untreated HSCs, we found that G-CSF treated cells have significantly impaired long-term repopulating and self-renewal activity in transplanted mice. In fact, on a per cell basis, the long-term repopulating activity of KLS-CD34− cells from G-CSF treated mice was reduced approximately 13 fold. The loss of repopulating activity per HSC was confirmed by transplanting purified HSCs. Homing experiments indicate that this loss of function is not caused by an inability to home from the peripheral blood to the bone marrow niche. As HSC quiescence has been positively associated with repopulating activity, we analyzed the cell cycle status over time of KLS-SLAM cells treated with G-CSF. This analysis revealed that after a brief period of enhanced cycling (69.8±5.0% G0 at baseline; down to 55.9±4.1% G0after 24 hours of G-CSF), treated cells become more quiescent (86.8±2.8% G0) than untreated HSCs. A similar increase in HSC quiescence was seen in KLS-34− cells. Thus our data show that G-CSF treatment is associated with HSC cycling alterations and function impairment. Because G-CSF is associated with modifications to the bone marrow microenvironment, and the microenvironment is known to regulate HSCs at steady state, we asked whether the G-CSF induced repopulating defect was due to a cell intrinsic or extrinsic (secondary to alterations in the microenvironment) mechanism. To do this, we repeated the competitive transplantation experiments using chimeric mice with a mixture of wild-type and G-CSF receptor knockout (Csf3r−/−) bone marrow cells. We find that only the repopulating activity of HSCs expressing the G-CSF receptor is affected by G-CSF, suggesting a cell-intrinsic mechanism. To identify targets of G-CSF signaling that may mediate loss of stem cell function, we performed RNA expression profiling of sorted KSL-SLAM cells from mice treated for 36 hours or seven days with or without G-CSF. The profiling data show that G-CSF treatment is associated with activation of inflammatory signaling in HSCs. Studies are in progress to test the hypothesis that activation of specific inflammatory signaling pathways mediates the inhibitory effect of G-CSF on HSC function. In summary, G-CSF signaling in HSCs, although associated with increased HSC quiescence, leads to a marked loss of long-term repopulating activity. These data suggest that long-term engraftment after transplantation of G-CSF-primed bone marrow may be reduced and requires careful follow-up. Disclosures: No relevant conflicts of interest to declare.

Dnmt3a is Required For Aberrant Self-Renewal Driven by PML-RARA

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 477-477
Author(s):  
Christopher B Cole ◽  
Angela M. Verdoni ◽  
David H Spencer ◽  
Timothy J. Ley
Keyword(s):  
Dna Methylation ◽  
Bone Marrow ◽  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Ex Vivo ◽  
Myeloid Cells ◽  
Cd11b Expression ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Marrow Cells

We previously identified recurrent mutations in the DNA methyltransferase DNMT3A in patients with acute myeloid leukemia (AML). DNMT3A and the highly homologous gene DNMT3B encode the two methyltransferases that are primarily responsible for mediating de novo methylation of specific CpG residues during differentiation. Loss of Dnmt3a in hematopoietic stem cells impairs their ability to differentiate into committed progenitors (Challen et al Nat Gen 44:23, 2011). Importantly, DNMT3A mutations are mutually exclusive of the favorable prognosis AML-initiating translocations, including the t(15;17) translocation (which creates the PML-RARA fusion gene), and translocations involving MLL. PML-RARA has been shown to interact with DNMT3A in vitro (Di Croce et al Science 295:1079,2002), and to require DNMT3A to induce methylation and transcriptional silencing of a subset of specific target genes. These findings, and the lack of DNMT3A mutations in APL patients, suggest that PML-RARA may require functional DNMT3A to initiate leukemia. To investigate this possibility, we utilized a well-characterized transgenic mouse model (in a pure B6 background) in which expression of PML-RARA is driven in hematopoietic stem/progenitor cells by the mouse Cathepsin G locus (Ctsg-PML-RARA+/- mice). These mice spontaneously develop acute promyelocytic leukemia (APL) with high penetrance and long latency, and also exhibit a preleukemic phenotype marked by the accumulation of myeloid cells in bone marrow and spleen. In addition, myeloid progenitor cells derived from these mice have the ability to serially replate in methylcellulose cultures, demonstrating aberrant self-renewal. We generated Ctsg-PML-RARA+/- mice lacking Dnmt3a (PML-RARA+/- x Dnmt3a-/-) as well as mice in which conditional ablation of Dnmt3b in hematopoietic cells is driven by Vav-Cre (PML-RARA+/- x Dnmt3b fl/fl x Vav-Cre+). Loss of Dnmt3a completely abrogated the ex vivo replating ability of PML-RARA bone marrow (Figure 1). Although colonies from both PML-RARA+/- and PML-RARA+/- x Dnmt3a-/- mice appeared similar in morphology and number on the first plating, PML-RARA+/- x Dnmt3a-/- marrow ceased to form colonies with subsequent replating (see Figure), and cultured cells lost the expression of the myeloid marker CD11b. The same phenotype was also observed using bone marrow from both genotypes that was secondarily transplanted into wild type recipients, indicating that it is intrinsic to transplantable hematopoietic progenitors. Reintroduction of DNMT3A into bone marrow cells derived from PML-RARA+/- x Dnmt3a-/- mice with retroviral transduction restored replating ability and CD11b expression. Competitive repopulation experiments with PML-RARA+/- x Dnmt3a-/- marrow revealed a decreased contribution to peripheral lymphoid and myeloid cells at 4 weeks, relative to PML-RARA+/- or WT control animals. Finally, 12 weeks after transplantation, recipients of PML-RARA+/- x Dnmt3a-/- bone marrow did not display an accumulation of myeloid cells in the bone marrow and spleen. Importantly, bone marrow from PML-RARA+/- x Dnmt3b fl/fl x Vav-Cre+/- mice displayed no replating deficit or loss of CD11b expression ex vivo, indicating different functions for Dnmt3a versus Dnmt3b in this model. Finally, we interrogated the effect of Dnmt3a loss on bone marrow DNA methylation patterns using a liquid phase DNA capture technique that sampled ∼1.9 million mouse CpGs at >10x coverage. Loss of Dnmt3a caused a widespread loss of DNA methylation in whole bone marrow cells, with 36,000 CpGs that were highly methylated (methylation value >0.7) in the PML-RARA+/- and WT mice, but hypomethylated (methylation value <0.4) in Dnmt3a-/- and PML-RARA+/- x Dnmt3a-/- mice. Characterization of the effect of Dnmt3a loss on leukemia latency, penetrance, and phenotype in PML-RARA+/- mice is currently being defined in a tumor watch. In summary, we have demonstrated that PML-RARA requires functional Dnmt3a (but not Dnmt3b) to drive aberrant self-renewal of myeloid progenitors ex vivo, and that loss of Dnmt3a leads to widespread DNA hypomethylation in bone marrow cells, and abrogates preleukemic changes in mice expressing PML-RARA. This data may explain why DNMT3A mutations are not found in patients with APL initiated by PML-RARA. Disclosures: No relevant conflicts of interest to declare.

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