Usp18 Is Required for Normal Stem Cell Maintenance

Abstract Stable and permanent hematopoiesis is established from the most primitive long-term self-renewing hematopoietic stem cells (LT-HSC), which can give rise to more differentiated short-term (ST-HSC) and multi-potent progenitors (MPP). Progenitors further differentiate into more committed cells that can generate the mature lymphoid and myeloid lineages. In order to maintain a normal hematopoietic system, HSCs must be properly regulated. We previously cloned Ubiquitin Specific Protease 18 (USP18/UBP43) during analysis of hematopoietic cells of t(8;21) AML fusion protein AML1-ETO knock-in mice (Liu et al, 1999 Mol Cell Biol 19:3029-3038; Schwer et al, 2000 Genomics 65, 44-52). However, its function in hematopoiesis, especially in hematopoietic stem cells, has not been carefully examined. We show here that depletion of Usp18 in C57/BL6 mice leads to death at embryonic days 13.5-14.5 with less fetal liver cellularity. To examine the precise role of Usp18 in vivo, we generated Usp18 conditional knockout mice (Usp18f/f). Survival analyses of Usp18f/- crossed with Usp18f/+Vav-iCre revealed that the embryonic lethality of Usp18 -deficient mice is due to defects in hematopoiesis. To examine the hematopoietic potential of fetal liver cells of Usp18-deficient mice, we conducted a colony forming assay using the E12.5 fetal livers. All types of colonies as well as the number of total cells from colonies were substantially reduced in Usp18-/- fetal liver compared to control, indicating that the blood progenitor cells of Usp18-/- fetal liver are not fully functional. To assess whether Usp18 is required for fetal liver HSC maintenance, we determined the frequency of HSCs in the fetal liver of Usp18+/+, Usp18+/-, and Usp18-/-. We detected the Lin- Sca-1+ c-Kit+ (LSK) cell population, which is HSC-enriched population in fetal livers, in mice of all three genotypes. Recent studies indicate that the most primitive LT-HSC population in fetal livers includes ESAM positive (LSK CD48- CD150+ ESAM+) stem cells (Ooi et al, 2009 Stem Cells 27:653-661; Pietras et al, 2014 JEM 211:245-262). Both the frequency and absolute numbers of the LT-HSC population in Usp18 -/- fetal livers were appreciably reduced compared to wild-type. Taken together, we conclude that Usp18 is indispensable for fetal liver HSC maintenance. We then addressed whether Usp18 is required for the HSC maintenance in adult mice by analyzing the frequency of HSCs in UBCER-Cre negative or positive Usp18 f/- bone marrow cells. After tamoxifen injections, we observed a significant reduction in the frequency of the LT-HSC population in Usp18f/-UBCER-Cre positive bone marrow cells compared to Usp18 f/-UBCER-Cre negative ones. Consistent with these results, Usp18 f/-UBCER-Cre positive bone marrow cells were much less competitive than Cre negative cells by competitive bone marrow transplantation assay. Finally, to examine whether the suppression of Usp18 in the leukemic cells provides a survival benefit, we used secondary-transplanted mice receiving Usp18f/fUBCER-Cre positive AML1-ETO9a leukemia cells (5 × 10 5 EGFP+ cells) isolated from primary transplanted mice. The tamoxifen treatment was initiated 3 weeks after transplantation. All the mice in the vehicle injected group (n = 7) succumbed to leukemia within a week after treatment started. However, mice treated with tamoxifen (n = 7) showed a longer survival time. Five of seven mice are still alive after 5 weeks of bone marrow transplantation, demonstrating the critical role of USP18 in maintenance of leukemia stem cells. Collectively, we conclude that Usp18 is essential for hematopoietic stem cell maintenance, and specific modulating activity of USP18 in leukemic cells may be considered as an effective therapeutic approach. Disclosures No relevant conflicts of interest to declare.

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Role Of Sf3b1 On Hematopoiesis

Blood ◽

10.1182/blood.v122.21.600.600 ◽

2013 ◽

Vol 122 (21) ◽

pp. 600-600

Author(s):

Manabu Matsunawa ◽

Ryo Yamamoto ◽

Masashi Sanada ◽

Aiko Sato ◽

Yusuke Shiozawa ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Bone Marrow Transplantation ◽

Hematopoietic Stem Cells ◽

Marrow Transplantation ◽

Bone Marrow Cells ◽

Hematopoietic Stem ◽

Ring Sideroblasts ◽

Marrow Cells

Abstract Frequent pathway mutation involving multiple components of the RNA splicing machinery is a cardinal feature of myeloid neoplasms showing myeloid dysplasia, in which the major mutational targets include U2AF35, ZRSR2, SRSF2 and SF3B1. Among these, SF3B1 mutations were strongly associated with MDS subtypes characterized by increased ring sideroblasts, such as refractory anemia and refractory cytopenia with multiple lineage dysplasia with ring sideroblasts, suggesting the critical role of SF3B1 mutations in these MDS subtypes. However, currently, the molecular mechanism of SF3B1mutation leading to the ring sideroblasts formation and MDS remains unknown. The SF3B1 is a core component of the U2-small nuclear ribonucleoprotein (U2 snRNP), which recognizes the 3′ splice site at intron–exon junctions. It was demonstrated that Sf3b1 null mice were shown to be embryonic lethal, while Sf3b1 +/- mice exhibited various skeletal alterations that could be attributed to deregulation of Hox gene expression due to haploinsufficiency of Sf3b1. However, no detailed analysis of the functional role of Sf3b1 in hematopoietic system in these mice has been performed. So, to clarify the role of SF3B1 in hematopoiesis, we investigated the hematological phenotype of Sf3b1 +/- mice. There was no significant difference in peripheral blood counts, peripheral blood lineage distribution, bone marrow total cellularity or bone marrow lineage composition between Sf3b1 +/+ and Sf3b1 +/- mice. Morphologic abnormalities of bone marrow and increased ring sideroblasts were not observed. However, quantitative analysis of bone marrow cells from Sf3b1 +/- mice revealed a reduction of the number of hematopoietic stem cells (CD34 neg/low, cKit positive, Sca-1 positive, lineage-marker negative: CD34-KSL cells) measured by flow cytometry analysis, compared to Sf3b1 +/+ mice. Whereas examination of hematopoietic progenitor cells revealed a small decrease in KSL cell populations and megakaryocyte - erythroid progenitors (MEP) in Sf3b1 +/- mice, and common myeloid progenitors (CMP), granulocyte - monocyte progenitors (GMP) and common lymphoid progenitors (CLP) remained unchanged between Sf3b1 +/+ and Sf3b1 +/- mice. In accordance with the reduced number of hematopoietic stem cells in Sf3b1 +/- mice, the total number of colony-forming unit generated from equal number of whole bone marrow cells showed lower colony number in Sf3b1 +/- mice in vitro. Competitive whole bone marrow transplantation assay, which irradiated recipient mice were transplanted with donor whole bone marrow cells from Sf3b1 +/+ or Sf3b1 +/- mice with an equal number of competitor bone marrow cells, revealed impaired competitive whole bone marrow reconstitution capacity of Sf3b1 +/- mice in vivo. These data demonstrated Sf3b1 was required for hematopoietic stem cells maintenance. To further examine the function of hematopoietic stem cells in Sf3b1 +/- mice, we performed competitive transplantation of purified hematopoietic stem cells from Sf3b1 +/+ or Sf3b1 +/- mice into lethally irradiated mice together with competitor bone marrow cells. Sf3b1 +/- progenitors showed reduced hematopoietic stem cells reconstitution capacity compared to those from Sf3b1 +/+ mice. In serial transplantation experiments, progenitors from Sf3b1 +/- mice showed reduced repopulation ability in the primary bone marrow transplantation, which was even more pronounced after the second bone marrow transplantation. Taken together, these data demonstrate that Sf3b1 plays an important role in normal hematopoiesis by maintaining hematopoietic stem cell pool size and regulating hematopoietic stem cell function. To determine the molecular mechanism underlying the observed defect in hematopoietic stem cells of Sf3b1 +/- mice, we performed RNA-seq analysis. We will present the results of our biological assay and discuss the relation of Sf3b1 and hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

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Loss of Gfi1 Impedes Development of T-Cell Lymphoma upon Exposure to N-ethyl-N-nitrosourea but Predisposes to Severe Myleodysplastic Changes.

Blood ◽

10.1182/blood.v110.11.2221.2221 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2221-2221

Author(s):

Cyrus Khandanpour ◽

Ulrich Duehrsen ◽

Tarik Möröy

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Dna Damage ◽

T Cell ◽

Bone Marrow Cells ◽

Cell Lymphoma ◽

Wild Type ◽

Hematopoietic Stem ◽

Deficient Mice

Abstract Exogenous toxic substances often cause the initiation and development of leukemia and lymphoma by acting as mutagens. N-ethyl-N-nitrosourea (ENU) is a paradigmatic example for such a substance, which introduces point mutations in the genome through DNA damage and repair pathways. ENU is widely used to experimentally induce T-cell lymphomas in mice. We have used ENU to investigate whether the hematopoietic transcription factor Gfi1 is required for lymphomagenesis. The Gfi1 gene was originally discovered as a proviral target gene and a series of experiments with transgenic mice had suggested a role of Gfi1 as a dominant oncogene with the ability to cooperate with Myc and Pim genes in the generation of T-cell lymphoma. In addition, Gfi1 deficient mice showed a defect in T-cell maturation but also aberration in myeloid differentiation and an accumulation of myelomonocytic cells. ENU was administered i.p. once a week for three weeks with a total dose of 300mg/kg to wild type (wt) and Gfi1 null mice. Wild type mice (12/12) predominantly developed T-cell tumors and rarely acute myeloid leukemia, as expected. However, only 2/8 Gfi1 −/− mice succumbed to lymphoid neoplasia; they rather showed a severe dysplasia of the bone marrow that was more pronounced than in wt controls. These changes in Gfi1 null mice were accompanied by a dramatic decrease of the LSK (Lin-, Sca1- and c-Kit+) bone marrow fraction that contains hematopoietic stem cells and by a higher percentage (18%) of bone marrow cells, not expressing any lineage markers (CD4, CD 8, Ter 119, Mac1, Gr1, B220, CD3). In particular, we found that the LSK subpopulation of Gfi1 deficient mice showed a noticeable increase in cells undergoing apoptosis suggesting a role of Gfi1 in hematopoietic stem cell survival. In addition, Gfi1−/− bone marrow cells and thymic T-cells were more sensitive to DNA damage such as radiation and exposure to ENU than their wt counterparts pointing to a role of Gfi1 in DNA damage response. Our results indicate that Gfi1 is required for development of T-cell tumors and that a loss of Gfi1 may sensitize hematopoietic cells and possibly hematopoietic stem cells for programmed cell death. Further studies have to show whether interfering with Gfi1 expression or function might represent a tool in the therapy of leukemia.

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Mutant p53 Drives the Development of Pre-Leukemic Hematopoietic Stem Cells through Modulating Epigenetic Regulators

Blood ◽

10.1182/blood.v126.23.307.307 ◽

2015 ◽

Vol 126 (23) ◽

pp. 307-307

Author(s):

Sarah C Nabinger ◽

Michihiro Kobayashi ◽

Rui Gao ◽

Sisi Chen ◽

Chonghua Yao ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Bone Marrow Cells ◽

Leukemia Stem Cells ◽

Mutant P53 ◽

Self Renewal ◽

Hematopoietic Stem ◽

Tp53 Mutations ◽

Marrow Cells

Abstract AML is thought to arise from leukemia stem cells (LSCs); however, recent evidence suggests that the transforming events may initially give rise to pre-leukemic hematopoietic stem cells (pre-leukemic HSCs), preceding the formation of fully transformed LSCs. Pre-leukemic HSCs have been shown to contribute to normal blood development and harbor a selective growth advantage compared to normal HSCs. Pre-leukemic HSCs can acquire subsequent mutations, and once differentiation capacity is impaired, leukemia emerges. Recently, acquired somatic TP53 mutations, including p53R248W and p53R273H, were identified in healthy individuals as well as AML patients, suggesting that TP53 mutations may be early events in the pathogenesis of AML. We found that p53R248W HSCs showed a multi-lineage repopulation advantage over WT HSCs in transplantation experiments, demonstrating that mutant p53 confers a pre-leukemic phenotype in murine HSCs. Although TP53 mutations are limited in AML, TP53 mutations do co-exist with mutations of epigenetic regulator, ASXL-1, or receptor tyrosine kinase, FLT3, in AML. Mutations in Asxl-1 are present in ~10-30% of patients with myeloid malignancies and confer poor prognosis. Loss of Asxl-1 in the hematopoietic compartment leads to a myelodysplastic-like syndrome in mice and reduced stem cell self-renewal. Internal tandem duplications in Flt3 (Flt3-ITD) occur in ~30% of AML patients and are associated with adverse clinical outcome. Flt3-ITD-positive mice develop a myeloproliferative neoplasm (MPN) and HSCs expressing Flt3-ITD have decreased self-renewal capabilities. We hypothesize that mutant p53 drives the development of pre-leukemic HSCs with enhanced self-renewal capability, allowing clonal expansion and subsequent acquisition of Asxl-1 or Flt3 mutations leading to the formation of fully transformed leukemia stem cells. To define the role of mutant p53 in Asxl-1+/- HSCs, we generated p53R248W/+ Asxl-1+/- mice and performed in vitro serial replating assays as well as in vivo competitivebone marrow transplantation experiments. We found that p53R248W significantly enhanced the serial replating ability of Asxl-1-deficient bone marrow cells. Interestingly, while bone marrow from Asxl-1+/- mice had very poor engraftment compared to wild type bone marrow cells 16 weeks post-transplantation, the expression of p53R248W in Asxl-1+/- bone marrow rescued the defect. To examine the role of mutant p53 in Flt3-ITD-positive HSCs, we generated p53R248W/+ Flt3ITD/+ mice. We found that p53R248W enhanced the replating ability of Flt3ITD/+ bone marrow cells. Despite the fact that Flt3ITD/+ bone marrow cells displayed decreased repopulating ability compared to wild type cells 16 weeks post-transplant, expression of p53R248W in Flt3ITD/+ cells rescued the defect. We are monitoring leukemia development in primary and secondary transplant recipients as well as in de novo p53R248W/+ Asxl-1+/- and p53R248W/+ Flt3ITD/+ animals and predict that mutant p53 may cooperate with Asxl-1 deficiency or Flt3-ITD in the formation of LSCs to accelerate leukemia development in Asxl-1 deficient or Flt-ITD-positive neoplasms. Mechanistically, dysregulated epigenetic control underlies the pathogenesis of AML and we discovered that mutant p53 regulates epigenetic regulators, including Ezh1, Ezh2, Kdm2a, and Setd2, in HSCs. H3K27me3 is catalyzed by EZH1 or EZH2 of the Polycomb repressing complex 2 (PRC2). Both Ezh1 and Ezh2 are important for HSC self-renewal. SETD2 is a histone H3K36 methyltransferase and mutations in SETD2 have been identified in 6% of patients with AML. SETD2 deficiency resulted in a global loss of H3K36me3 and increased self-renewal capability of leukemia stem cells. We found that there were increased levels of H3K27me3 and decreased levels of H3K36me3 in p53R248W/+ HSCs compared to that of the WT HSCs. In ChIP experiments, we found that p53R248W, but not WT p53, was associated with the promoter region of Ezh2 in mouse myeloid progenitor cells, suggesting that p53R248W may directly activate Ezh2 expression in hematopoietic cells. Given that Asxl-1 has been shown to regulate H3K27me3 in HSCs, the synergy between mutant p53 and Asxl-1 deficiency on LSC self-renewal could be due to changes in histone modifications. Overall, we demonstrate that mutant p53 promotes the development of pre-leukemic HSCs by a novel mechanism involving dysregulation of the epigenetic pathways. Disclosures No relevant conflicts of interest to declare.

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MEIS1 Is Required for the Maintenance of Long Term Hematopoietic Stem Cells Wherein It Regulates Quiescence

Blood ◽

10.1182/blood.v118.21.551.551 ◽

2011 ◽

Vol 118 (21) ◽

pp. 551-551

Author(s):

Zeenath Unnisa ◽

Jason P Clark ◽

Elizabeth Wojtowicz ◽

Lino Tessarollo ◽

Neal G. Copeland ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Peripheral Blood ◽

Bone Marrow Cells ◽

Self Renewal ◽

Hematopoietic Stem ◽

Bone Marrow Analysis ◽

Marrow Cells

Abstract Abstract 551 Normal hematopoiesis is maintained by long-term hematopoietic stem cells (LT-HSCs) that are defined by their extensive self-renewal and multipotency. Self-renewal of LT-HSCs in turn is regulated by a complex network of intrinsic and extrinsic factors. The transcription factor MEIS1 is highly expressed in hematopoietic stem and progenitor cells and also in several leukemias, suggesting that MEIS1 might be important in regulating self-renewal. However, the role of MEIS1 in normal hematopoiesis has not been defined. To determine the role of MEIS1 in hematopoiesis, we studied conditional knockout mice. We generated transgenic mice bearing loxp sites flanking the homeodomain of MEIS1. The MEIS1-floxed mice were then bred to Rosa26-CreERT2 mice, the latter expressing cre-recombinase ubiquitously, that can be activated by estrogen or its analog Tamoxifen (Tam). Efficient, complete recombination was achieved in vivo by treating MEIS1-f/f-Cre (homozygous for MEIS1-flox) mice with Tam and in vitro by treating bone marrow cells with 4-hydroxy tamoxifen. Loss of MEIS1 expression was detected by QRT-PCR and western blotting. To determine the role of MEIS1 in the maintenance of adult hematopoiesis, MEIS1-f/f-Cre and control mice were treated with Tam and MEIS1 deletion confirmed by PCR. At three weeks post deletion, bone marrow analysis showed a significant reduction in the number of LT-HSCs defined as lin-/c-Kit+/Sca1+/CD48−/CD150+ in the MEIS1-depleted mice compared to controls (0.012% compared to 0.037%, N=6, p<0.05, t-test). However, the progenitor populations were unaffected by MEIS1 deletion. Over a period of 12 weeks of observation, the mice did not show any signs of distress and the peripheral blood counts of the experimental and control mice remained normal, indicating that short term hematopoiesis was not affected. Cell cycle analysis of LT-HSCs showed that MEIS1 deletion resulted in a significant shift of cells from G0 to G1 phase (G0 and G1 proportions respectively, 81.75±3.25% and 9.40±3% for control and 56.10±0.873% and 31.17±1.5% for MEIS1-deleted). To determine the effects of MEIS1 loss on intrinsic hematopoietic stem cell function, we performed competitive repopulation assays. Bone marrow cells harvested from MEIS1-f/f-Cre or MEIS1-f/+-Cre (control) mice were combined with equal numbers of bone marrow cells from BoyJ mice and transplanted via tail vein injection into lethally irradiated BoyJ mice. Four weeks after transplant, recipients were treated with Tam or vehicle for 5 days and deletion of MEIS1 confirmed by PCR on peripheral blood. Peripheral blood of recipient mice was analyzed at 1, 4, 8, 12 and 16 weeks after treatment and relative chimerism assessed by flow cytometry. At 1 and 4 weeks after treatment, the chimerism in the MEIS1 deleted group (Tam treated MEIS1-f/f-CreER) and the control groups (Tam treated MEIS1-f/+-CReER and vehicle treated MEIS1-f/f-CreER) was comparable (41%, 40.5% and 41.5% respectively, average, N=5 to 8). However, by 8 weeks after treatment, the MEIS1 deleted group showed a significant decline in chimerism compared to controls (18.2% compared to 43.1% and 35.1% respectively, p<0.02, t-test) and at 16 weeks the chimerism in the MEIS1-deleted group declined further (11.1% compared to 40.2% and 35.0% respectively, p<0.001). Subpopulation analysis showed loss of chimerism in granulocytes and in B and T lymphocytes. The latency and breadth of the effect of MEIS1 loss suggested an effect on the hematopoietic stem cell population. Indeed, bone marrow analysis of transplant recipients showed near complete loss of LT-HSC chimerism (3% compared to 70.25% and 75.6% respectively, p<0.001). Finally, we performed gene expression profiling on lineage negative bone marrow cells with and without MEIS1 deletion. Results showed that loss of MEIS1 was associated with decreased expression of hypoxia-responsive genes. Collectively, these results indicate that MEIS1 is required for the maintenance of the pool of LT-HSCs. Loss of MEIS1 promotes cycling and exhaustion of LT-HSCs. Further, we propose that activation of the hypoxia-response pathway may be one of the mechanisms by which MEIS1 exerts its effects on hematopoietic stem cells. Disclosures: No relevant conflicts of interest to declare.

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Pannexin-1 channel “fuels” by releasing ATP from bone marrow cells a state of sterile inflammation required for optimal mobilization and homing of hematopoietic stem cells

Purinergic Signalling ◽

10.1007/s11302-020-09706-1 ◽

2020 ◽

Vol 16 (3) ◽

pp. 313-325

Author(s):

Monika Cymer ◽

Katarzyna Brzezniakiewicz-Janus ◽

Kamila Bujko ◽

Arjun Thapa ◽

Janina Ratajczak ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Bone Marrow Cells ◽

Sterile Inflammation ◽

Hematopoietic Stem ◽

Extracellular Adenosine ◽

Pannexin 1 ◽

Molecular Pattern ◽

Channel Blockage

Abstract An efficient harvest of hematopoietic stem/progenitor cells (HSPCs) after pharmacological mobilization from the bone marrow (BM) into peripheral blood (PB) and subsequent proper homing and engraftment of these cells are crucial for clinical outcomes from hematopoietic transplants. Since extracellular adenosine triphosphate (eATP) plays an important role in both processes as an activator of sterile inflammation in the bone marrow microenvironment, we focused on the role of Pannexin-1 channel in the secretion of ATP to trigger both egress of HSPCs out of BM into PB as well as in reverse process that is their homing to BM niches after transplantation into myeloablated recipient. We employed a specific blocking peptide against Pannexin-1 channel and noticed decreased mobilization efficiency of HSPCs as well as other types of BM-residing stem cells including mesenchymal stroma cells (MSCs), endothelial progenitors (EPCs), and very small embryonic-like stem cells (VSELs). To explain better a role of Pannexin-1, we report that eATP activated Nlrp3 inflammasome in Gr-1+ and CD11b+ cells enriched for granulocytes and monocytes. This led to release of danger-associated molecular pattern molecules (DAMPs) and mitochondrial DNA (miDNA) that activate complement cascade (ComC) required for optimal egress of HSPCs from BM. On the other hand, Pannexin-1 channel blockage in transplant recipient mice leads to a defect in homing and engraftment of HSPCs. Based on this, Pannexin-1 channel as a source of eATP plays an important role in HSPCs trafficking.

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Long-term monoclonal reconstitution of erythropoiesis in genetically anemic W/Wv mice by injection of 5-fluorouracil-treated bone marrow cells of Pgk-1b/Pgk-1a mice

Blood ◽

10.1182/blood.v70.6.1758.1758 ◽

1987 ◽

Vol 70 (6) ◽

pp. 1758-1763 ◽

Cited By ~ 12

Author(s):

T Nakano ◽

N Waki ◽

H Asai ◽

Y Kitamura

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Bone Marrow Cells ◽

Electrophoretic Pattern ◽

Phosphoglycerate Kinase ◽

X Chromosomes ◽

Hematopoietic Stem ◽

Colony Forming Units ◽

Marrow Cells

Abstract The spleen colony-forming assay does not represent the number of hematopoietic stem cells with extensive self-maintaining capacity because five to 50 spleen colony-forming units (CFU-S) are necessary to rescue a genetically anemic (WB X C57BL/6)F1-W/Wv(WBB6F1-W/Wv) mouse. We investigated which is more important for the reconstitution of erythropoiesis, the transplantation of multiple CFU-S or that of a single stem cell with extensive self-maintaining potential. The electrophoretic pattern of hemoglobin was used as a marker of reconstitution and that of phosphoglycerate kinase (PGK), an X chromosome-linked enzyme, as a tool for estimating the number of stem cells. For this purpose, we developed the C57BL/6 congeneic strain with the Pgk-1a gene. Bone marrow cells were harvested after injection of 5- fluorouracil from C57BL/6-Pgk-1b/Pgk-1a female mice in which each stem cell had either A-type PGK or B-type PGK due to the random inactivation of one or two X chromosomes. When a relatively small number of bone marrow cells (ie, 10(3) or 3 X 10(3] were injected into 200-rad- irradiated WBB6F1-W/Wv mice, the hemoglobin pattern changed from the recipient type (Hbbd/Hbbs) to the donor type (Hbbs/Hbbs) in seven of 150 mice for at least 8 weeks. Erythrocytes of all these WBB6F1-W/Wv mice showed either A-type PGK alone or B-type PGK alone during the time of reconstitution, which suggests that a single stem cell with extensive self-maintaining potential may sustain the whole erythropoiesis of a mouse for at least 8 weeks.

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Prdm16 is a physiologic regulator of hematopoietic stem cells

Blood ◽

10.1182/blood-2010-08-300145 ◽

2011 ◽

Vol 117 (19) ◽

pp. 5057-5066 ◽

Cited By ~ 82

Author(s):

Francesca Aguilo ◽

Serine Avagyan ◽

Amy Labar ◽

Ana Sevilla ◽

Dung-Fang Lee ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Fetal Liver ◽

Feedback Loops ◽

Hematopoietic Stem ◽

Hematopoietic System ◽

Number Of Genes ◽

Adult Bone ◽

Deficient Mice

Abstract Fetal liver and adult bone marrow hematopoietic stem cells (HSCs) renew or differentiate into committed progenitors to generate all blood cells. PRDM16 is involved in human leukemic translocations and is expressed highly in some karyotypically normal acute myeloblastic leukemias. As many genes involved in leukemogenic fusions play a role in normal hematopoiesis, we analyzed the role of Prdm16 in the biology of HSCs using Prdm16-deficient mice. We show here that, within the hematopoietic system, Prdm16 is expressed very selectively in the earliest stem and progenitor compartments, and, consistent with this expression pattern, is critical for the establishment and maintenance of the HSC pool during development and after transplantation. Prdm16 deletion enhances apoptosis and cycling of HSCs. Expression analysis revealed that Prdm16 regulates a remarkable number of genes that, based on knockout models, both enhance and suppress HSC function, and affect quiescence, cell cycling, renewal, differentiation, and apoptosis to various extents. These data suggest that Prdm16 may be a critical node in a network that contains negative and positive feedback loops and integrates HSC renewal, quiescence, apoptosis, and differentiation.

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Kruppel-Like Factor 10 (KLF10)-Deficient Mice Have Marked Defects In EPC Differentiation, Function, and Angiogenesis

Blood ◽

10.1182/blood.v116.21.4314.4314 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4314-4314

Author(s):

Akm Khyrul Wara ◽

Kevin Croce ◽

ShiYin Foo ◽

Xinghui Sun ◽

Basak Icli ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

Hindlimb Ischemia ◽

Hematopoietic Progenitor ◽

Hematopoietic Stem ◽

Deficient Mice ◽

Tgf Β1 ◽

Impaired Angiogenesis

Abstract Abstract 4314 Background: Emerging evidence demonstrates that endothelial progenitor cells (EPCs) may originate from the bone marrow and are capable of being recruited to sites of ischemic injury and contribute to neovascularization. However, the identities of these bone marrow cells and the signaling pathways that regulate their differentiation into functional EPCs remain poorly understood. Methods and Results: We previously identified that among hematopoietic progenitor stem cells, common myeloid progenitors (CMPs) and granulocyte-macrophage progenitors (GMPs) can preferentially differentiate into EPCs and possess high angiogenic activity under ischemic conditions compared to megakaryocyte-erythrocyte progenitors (MEPs), hematopoietic stem cells (HSCs), and common lymphoid progenitors (CLPs). Herein, we identify that a TGF-β1-responsive Kruppel-like Factor, KLF10, is robustly expressed in EPCs derived from CMPs and GMPs, compared to progenitors lacking EPC markers. KLF10–/– mice have marked defects in circulating EPCs (–23.6% vs. WT, P<0.004). In addition, EPC differentiation and TGF-β induced KDR responsiveness is markedly impaired (CMPs: WT 22.3% vs. KO 8.64%, P<0.0001; GMPs: WT 32.8% vs. KO 8.97%, P<0.00001). Functionally, KLF10–/– EPCs derived from CMPs and GMPs adhered less to fibronectin-coated plates (CMPs: WT 285 vs. KO 144.25, P< 0.0004; GMPs: WT 275.25 vs. KO 108.75, P <0.0003) and had decreased rates of migration in transwell Boyden chambers (CMPs: WT 692 vs. KO 298.66, P<0.00004; GMPs: WT 635.66 vs. KO 263.66, P<0.00001). KLF10–/– mice displayed impaired blood flow recovery after hindlimb ischemia (day 14, WT 0.827 vs. KO 0.640, P <0.009), an effect completely rescued by WT EPCs, but not KLF10–/– EPCs. Matrigel plug implantation studies demonstrated impaired angiogenesis in KLF10–/– mice compared to WT mice (WT 158 vs. KO 39.83, P<0.00000004). Overexpression studies revealed that KLF10 rescued EPC formation from TGF-β1+/– CMPs and GMPs. Mechanistically, TGF-β1 and KLF10 target the VEGFR2 promoter in EPCs which may underlie these effects. Background: Collectively, these observations identify that TGF-β1 signaling and KLF10 are part of a key signaling pathway that regulates EPC differentiation from CMPs and GMPs and may provide a therapeutic target during cardiovascular ischemic states. Disclosures: No relevant conflicts of interest to declare.

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

Blood ◽

10.1182/blood.v118.21.859.859 ◽

2011 ◽

Vol 118 (21) ◽

pp. 859-859 ◽

Cited By ~ 1

Author(s):

Chen Zhao ◽

Yan Xiu ◽

John M Ashton ◽

Lianping Xing ◽

Yoshikazu Morita ◽

...

Keyword(s):

Bone Marrow ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

Wild Type ◽

Double Knockout ◽

Self Renewal ◽

Hematopoietic Stem ◽

Physiological Processes ◽

Marrow Cells

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

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Leukemia-Associated Mutations Of DNMT3A Inhibit Differentiation Of Hematopoietic Stem Cells and Promote Leukemic Transformation Through Abberant Recruitment Of Bmi1

Blood ◽

10.1182/blood.v122.21.469.469 ◽

2013 ◽

Vol 122 (21) ◽

pp. 469-469

Author(s):

Junji Koya ◽

Keisuke Kataoka ◽

Takako Tsuruta-Kishino ◽

Hiroshi Kobayashi ◽

Kensuke Narukawa ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Research Funding ◽

Bone Marrow Cells ◽

Cpg Island ◽

Hematopoietic Stem ◽

Cell Accumulation ◽

Exogenous Expression ◽

Marrow Cells

Whole genome sequencing has revealed DNMT3A mutation is present in over 20% of cytogenetically normal acute myeloid leukemia (CN-AML) and R882 is the most frequent and recurrent mutated site. Cumulating clinical data have emphasized the importance of the mutation as a poor prognostic factor of AML. Since the functional role of DNMT3A mutation in leukemogenesis remains largely unknown, we aimed to elucidate the impact of DNMT3A mutation on the development and maintenance of AML. To investigate the effect of exogenous expression of DNMT3A R882 mutant (Mut) in hematopoiesis, we transplanted 5-FU primed mouse bone marrow cells transduced with empty vector (EV), DNMT3A wild type (WT), or DNMT3A Mut to lethally irradiated mice. Recipients transplanted with DNMT3A Mut-transduced cells exhibited hematopoietic stem cell (CD150+CD48-Lin-Sca1+c-Kit+) accumulation and enhanced repopulating capacity compared with EV and DNMT3A WT recipients. To identify the downstream target genes of DNMT3A Mut that evoked hematopoietic stem cell accumulation, we sorted vector-transduced LSK cells from transplanted mice and conducted quantitative PCR (Q-PCR) of various hematopoiesis-related genes. Q-PCR revealed that Hoxb cluster expression was up-regulated and differentiation-associated genes, such as PU.1 and C/ebpa, were down-regulated in DNMT3A Mut-transduced LSK cells. Targeted bisulfite sequencing showed hypomethylation of the Hoxb2 promoter-associated CpG island in DNMT3A Mut-transduced cells compared with EV-transduced cells, which suggests dominant-negative effect of DNMT3A R882 mutation. DNMT3A Mut caused no change in methylation status of PU.1 promoter-associated CpG island, indicating that DNA methylation-independent mechanism underlies PU.1 downregulation. Given that DNMT3A interacts with several histone modifiers to regulate target gene transcription, we performed co-immunoprecipitation to investigate whether these interactions are altered by DNMT3A mutation. We found that DNMT3A Mut has the emhanced capacity to interact with polycomb repressive complex 1 (PRC1), which is thought to be a potential mechanism of the DNMT3A Mut-induced differentiation defect. Co-immunoprecipitation experiments showed that DNMT3A R882H and R882C mutant exhibited augmented interaction with BMI1 and MEL18, respectively. In addition, RING1B, an essential component of PRC1, co-localized with DNMT3A Mut more frequently than WT, irrespective of the type of amino acid substitution. Furthermore, heterozygosity of Bmi1 restored the PU.1 mRNA to the normal level and canceled the effect of stem cell accumulation in mice transplanted with DNMT3A Mut bone marrow cells. Chromatin immunoprecipitation in AML cell lines showed that BMI1 and RING1B were more efficiently recruited to the upstream regulatory element of PU.1 upon expression of DNMT3A Mut than WT, while the amount of DNMT3A recruited were comparable between DNMT3A WT and Mut. In the murine transplantation model, we found that exogenous PU.1 expression impaired repopulating capacity in both EV and R882H-transduced cells to the similar level. Exogenous expression of DNMT3A WT inhibited proliferation and induced terminal myeloid differentiation, whereas DNMT3A Mut-transduced cells remained immature in AML cell lines. DNMT3A Mut-transduced cells were resistant to ATRA-induced differentiation compared to EV-transduced cells. Furthermore, R882 mutation promoted blastic transformation of murine c-Kit+ bone marrow cells in vitro in combination with HOXA9 which is highly expressed in clinical cases harboring DNMT3A mutation. Morphological and surface marker analysis revealed these cells were F4/80+ monocytic blasts, consistent with clinical observation that DNMT3A mutation is found frequently in FAB M4/M5 leukemia. These results indicate a distinct role for DNMT3A Mut as well as a potential collaboration between DNMT3A Mut and HOXA9 in malignant transformation of hematopoietic cells. Interestingly, Bmi1 heterozygosity impaired this monoblastic transformation of R882H and HOXA9 co-transduced progenitors. Taken together, our results highlight the functional role of DNMT3A mutation in differentiation block of hematopoietic stem cells and in promoting leukemic transformation via aberrant recruitment of Bmi1 and other PRC1 components. Disclosures: Kurokawa: Celgene: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Bristol-Myers Squibb: Research Funding.

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