Differential H4K16ac levels ensure a balance between quiescence and activation in hematopoietic stem cells

Hematopoietic stem cells (HSCs) are able to reconstitute the bone marrow while retaining their self-renewal property. Individual HSCs demonstrate heterogeneity in their repopulating capacities. Here, we found that the levels of the histone acetyltransferase MOF (males absent on the first) and its target modification histone H4 lysine 16 acetylation are heterogeneous among HSCs and influence their proliferation capacities. The increased proliferative capacities of MOF-depleted cells are linked to their expression of CD93. The CD93+ HSC subpopulation simultaneously shows transcriptional features of quiescent HSCs and functional features of active HSCs. CD93+ HSCs were expanded and exhibited an enhanced proliferative advantage in Mof+/− animals reminiscent of a premalignant state. Accordingly, low MOF and high CD93 levels correlate with poor survival and increased proliferation capacity in leukemia. Collectively, our study indicates H4K16ac as an important determinant for HSC heterogeneity, which is linked to the onset of monocytic disorders.

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TIMP-3 Inhibition of Angiopoietin-1 Signaling Recruits Hematopoietic Stem Cells from the Bone Marrow Niche.

Blood ◽

10.1182/blood.v110.11.1255.1255 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1255-1255

Author(s):

Hideaki Nakajima ◽

Miyuki Ito ◽

David Smookler ◽

Fumi Shibata ◽

Yumi Fukuchi ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Critical Role ◽

Quiescent State ◽

Hematopoietic Stem ◽

Bm Niche ◽

Quiescent Hscs ◽

Angiopoietin 1

Abstract Regulating transition of hematopoietic stem cells (HSCs) between quiescent and cycling states is critical for maintaining homeostasis of blood cell production in the adult bone marrow. Quiescent HSCs are rapidly recruited into the cell cycle when they face hematopoietic demands such as myelosuppression, returning to quiescence once they produce enough progenitors. It was previously shown that quiescent HSCs express Tie2 and that Tie2/angiopoietin-1 (Ang-1) signaling plays a critical role for maintaining HSC quiescence. However, molecular cues for recruiting HSCs from a quiescent state into cycle remain poorly understood. Extracellular signals are often regulated by the extracellular matrix environment, which is modulated by metalloproteinase (MMP) activities. TIMP-3 is an endogenous inhibitor of MMPs, and we have previously proposed that TIMP-3 may play a critical role in HSC physiology. In addition, TIMP-3 has been reported to suppress angiogenesis by inhibiting vascular endothelial growth factor (VEGF) signaling. By analogy with VEGF inhibition, we reasoned that TIMP-3 might suppress Ang-1 signaling in HSC and act as a molecular cue for HSC recruitment. In order to investigate a role of TIMP-3 in the HSC recruitment, we first examined whether TIMP-3 is regulated in the BM upon myelosuppression. Analyses by reverse transcription polymerase chain reaction (RT-PCR) and immunostaining revealed that the injection of 5-fluorouracil (5-FU) or irradiation induced TIMP-3 at the endosteal surface of the BM after 3-days of treatment. We next tested the hypothesis that TIMP-3 might be regulating Ang-1 signals by using cell line models. This revealed that the pre-treatment of cells with TIMP-3 suppressed autophosphorylation of Tie-2 in response to Ang-1. BIAcore and in vitro binding assay revealed that TIMP-3 directly interacted with Ang-1 and Tie-2, indicating that TIMP-3 suppressed Ang-1 signaling through interfering ligand-receptor interaction. Next we examined the effect of TIMP-3 on HSC physiology. TIMP-3 promoted the proliferation of CD34-KSL cells in vitro by approximately 2–3 fold. This was mainly due to the enhanced production of multipotential progenitors from CD34-KSL cells, which was accomplished by an enhanced symmetrical cell division of multipotential progenitors as revealed by paired-daughter cell analysis. Bone marrow transplantation study of TIMP-3-treated CD34-KSL cells showed that they sustained long-term repopulating potential comparable to the control-treated cells. Furthermore, in vivo administration of TIMP-3 into mice accelerated recovery and protected mice from myelosuppression, and in turn, the bone marrow recovery after myelosuppression was delayed in TIMP-3-deficient animals. In summary, TIMP-3 is induced by myelosuppression in the BM niche, stimulates HSC proliferation by inhibiting Ang-1 signaling, and thereby promotes production of multipotential progenitors from HSCs. These results demonstrate that TIMP-3 acts as a molecular cue for recruiting quiescent HSCs from the BM niche.

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Noncanonical Wnt Signaling Maintains Hematopoietic Stem Cells in Different Zones

Blood ◽

10.1182/blood.v120.21.639.639 ◽

2012 ◽

Vol 120 (21) ◽

pp. 639-639

Author(s):

Ryohichi Sugimura ◽

Linheng Li

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Wnt Signaling ◽

Conflicts Of Interest ◽

Quiescent State ◽

Perivascular Niche ◽

Hematopoietic Stem ◽

Knockout Mouse Model ◽

Quiescent Hscs

Abstract Abstract 639 Hematopoietic stem cells (HSCs) are maintained in a balance between quiescent state and proliferating state. While proliferating HSCs are critical for supporting the routine blood production, quiescent HSCs are essential for long term maintenance and can also be activated to replenish lost proliferating or active HSCs. How the different states of HSCs are regulated is a fundamental question. Accumulated evidence supports a model that quiescent HSCs are located in the endosteal zone and active HSCs are in the perivascular zone. The underlying signaling to regulate the quiescence and activation in different niches remains largely unknown. To address this question, we have analyzed the expression profile of Wnt receptors, Frizzleds, in HSCs. We found that noncanonical Wnt signaling via receptor Frizzled8 (Fz8) and co-receptor Flamingo presents in and functionally maintains quiescent HSCs in the endosteal zone (Sugimura, et al., Cell 2012). However, it has not been clear whether and how active HSCs in the perivascular zone are regulated by Wnt signaling. Recently, we detected that another noncanonical Wnt receptor, Frizzled5 (Fz5), is expressed in metabolically active (indicated by Mitotracker) HSCs and also in Nestin-GFP+ mesenchymal stem cells (MSCs) in the perivascular zone of central marrow. Fz5 expresses neither in H2B-GFP label-retaining quiescent HSCs nor in endosteal cells and nor in sinusoidal cells as well. Using an Mx1-Cre:Fz5 knockout mouse model, we found a 60% decrease of HSCs isolated from central marrow, but no change in the number of HSCs isolated from endosteum. Functionally, hematopoietic reconstitution was not affected in the primary transplantation, but was substantially decreased (by 80%) in the secondary transplantation compared to the control. This indicates that Fz5 maintains HSCs in the perivascular zone. To examine the role of Fz5 in Nestin+ MSCs for HSC maintenance, we examined the Nestin-Cre:Fz5 model. We observed a large loss of CD49hiHSCs that were reported to represent intermediate (IT) HSCs. We further found a correlation of the quiescent vs. active HSCs respectively to long term (LT) HSCs vs. IT-HSCs with the latter population sensitive to 5FU treatment. Mechanistically we observed that Fz5 inactivation also led to a loss of Cdc42 polarity in the HSCs residing in the perivascular niche. The results suggest that Fz5-mediated noncanonical Wnt signaling regulates polarity of active HSCs in the perivascular zones. Future study is required to see whether the Fz5-Cdc42 mediated polarity in HSCs is associated with symmetric vs. asymmetric division. We propose that noncanonical Wnt signaling maintains quiescent and active HSCs reside respectively in the endosteal zone and the perivascular zone. In these zones, Fz8 and Fz5 are differentially expressed and mediate noncanonical Wnt signaling for HSC maintenance in the endosteal niche and to regulate active HSC action in the perivascular niche. Disclosures: No relevant conflicts of interest to declare.

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Implication of AML1/RUNX1 Function in the Homeostasis and Leukemic Transformation of Hematopoietic Stem Cells.

Blood ◽

10.1182/blood.v110.11.4117.4117 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4117-4117

Author(s):

Motoshi Ichikawa ◽

Masataka Takeshita ◽

Susumu Goyama ◽

Takashi Asai ◽

Eriko Nitta ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Bone Marrow Cells ◽

Hematopoietic Cells ◽

Chimeric Protein ◽

Chromosomal Translocation ◽

Hematopoietic Stem ◽

Quiescent Hscs ◽

Marrow Cells

Abstract Transcription factor AML1/RUNX1, initially isolated from the t(8;21) chromosomal translocation in human leukemia, is essential for the development of multilineage hematopoiesis in mouse embryos. AML1 negatively regulates the number of immature hematopoietic cells in adult hematopoiesis, while it is required for megakaryocytic maturation and lymphocytic development. However, it remains yet to be determined how AML1 contributes to homeostasis of hematopoietic stem cells (HSCs). To address this issue, we analyzed in detail HSC function in the absence of AML1. Notably, cells in the Hoechst 33342 side population fraction and c-Kit-positive cells in the G0 cell cycle status were increased in number in AML1-deficient bone marrow, which suggests enrichment of quiescent HSCs. We also found an increase in HSC number within the AML1-deficient bone marrow using limiting dilution bone marrow transplantation assays. Thus, the number of quiescent HSCs is negatively regulated by AML1, loss of which may result in accumulation of leukemic stem cell pool in AML1-related leukemia. To identify mechanisms through which functional loss of AML1 exerts leukemogenic potential, we focused on the AML1-Evi-1 chimeric protein, which is generated by the t(3;21) chromosomal translocation and disturbs the normal function of AML1. We introduced AML1-Evi-1 and its mutants into murine bone marrow cells, and evaluated hematopoietic cell transformation by colony replating assays. The transforming activity of AML1-Evi-1 was impaired when any of the major functional domains of AML1-Evi-1 was lost. Moreover, overexpression of Evi-1 could not transform AML1-deleted bone marrow cells, suggesting that fusion of AML1 and Evi-1, rather than AML1 suppression and Evi-1 overexpression, is essential for AML1-Evi-1 leukemogenesis. Intriguingly, among the hematopoietic progenitor cell fractions, AML1-Evi-1 could transform only the uncommitted, immature hematopoietic cells, which contrasts with MLL-ENL, a chimeric protein generated in t(11;19) leukemia. AML1-Evi-1 transformed cells show a surface marker profile different from that of the cells transformed by AML1-MTG8/ETO, another leukemic gene product that also perturbs AML1 function. These results provide a valuable clue to a distinct mechanism determined by the Evi-1 moiety in the AML1-Evi-1 leukemogenesis and to a role of AML1 loss in the self-renewal of leukemic stem cells.

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Abstract 3890: MicroRNA-551b is highly expressed in hematopoietic stem cells and expression in acute myeloid leukemia is associated with relapse and poor survival

10.1158/1538-7445.am2014-3890 ◽

2014 ◽

Author(s):

David C. de leeuw ◽

Fedor Denkers ◽

Peter Valk ◽

Iris de Rink ◽

Ron Kerkhoven ◽

...

Keyword(s):

Stem Cells ◽

Acute Myeloid Leukemia ◽

Hematopoietic Stem Cells ◽

Myeloid Leukemia ◽

Poor Survival ◽

Hematopoietic Stem ◽

Acute Myeloid

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TIMP-3 recruits quiescent hematopoietic stem cells into active cell cycle and expands multipotent progenitor pool

Blood ◽

10.1182/blood-2010-01-266528 ◽

2010 ◽

Vol 116 (22) ◽

pp. 4474-4482 ◽

Cited By ~ 22

Author(s):

Hideaki Nakajima ◽

Miyuki Ito ◽

David S. Smookler ◽

Fumi Shibata ◽

Yumi Fukuchi ◽

...

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Ex Vivo ◽

Active Cell ◽

Tissue Inhibitor Of Metalloproteinase ◽

Hematopoietic Stem ◽

Hematopoietic Recovery ◽

Quiescent Hscs

Regulating transition of hematopoietic stem cells (HSCs) between quiescent and cycling states is critical for maintaining homeostasis of blood cell production. The cycling states of HSCs are regulated by the extracellular factors such as cytokines and extracellular matrix; however, the molecular circuitry for such regulation remains elusive. Here we show that tissue inhibitor of metalloproteinase-3 (TIMP-3), an endogenous regulator of metalloproteinases, stimulates HSC proliferation by recruiting quiescent HSCs into the cell cycle. Myelosuppression induced TIMP-3 in the bone marrow before hematopoietic recovery. Interestingly, TIMP-3 enhanced proliferation of HSCs and promoted expansion of multipotent progenitors, which was achieved by stimulating cell-cycle entry of quiescent HSCs without compensating their long-term repopulating activity. Surprisingly, this effect did not require metalloproteinase inhibitory activity of TIMP-3 and was possibly mediated through a direct inhibition of angiopoietin-1 signaling, a critical mediator for HSC quiescence. Furthermore, bone marrow recovery from myelosuppression was accelerated by over-expression of TIMP-3, and in turn, impaired in TIMP-3–deficient animals. These results suggest that TIMP-3 may act as a molecular cue in response to myelosuppression for recruiting dormant HSCs into active cell cycle and may be clinically useful for facilitating hematopoietic recovery after chemotherapy or ex vivo expansion of HSCs.

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AML1/Runx1 Negatively Regulates the Number of Quiescent Hematopoietic Stem Cells in Adult Hematopoiesis.

Blood ◽

10.1182/blood.v108.11.4204.4204 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4204-4204

Author(s):

Motoshi Ichikawa ◽

Takashi Asai ◽

Masahiro Nakagawa ◽

Masahito Kawazu ◽

Susumu Goyama ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Side Population ◽

Hematopoietic Cells ◽

Myelogenous Leukemia ◽

Human Leukemia ◽

Myeloproliferative Disease ◽

Hematopoietic Stem ◽

Quiescent Hscs

Abstract Transcription factor AML1 (also called Runx1), which was initially isolated from the t(8;21) chromosomal translocation frequently found in the acute myelogenous leukemia FAB M2 subtype, is essential for the development of multilineage hematopoiesis in mouse embryos. By analyzing conditional AML1 knockout mice, we have previously shown that AML1 negatively regulates the number of immature hematopoietic cells defined as lineage-negative, CD34− Sca-1+ c-Kit+ (34KSL) cells in adult hematopoiesis, while it is required for megakaryocytic maturation and lymphocytic development. The former is a significant observation because an increase in hematopoietic stem/progenitor cells due to defective AML1 function may be closely related to the development of human leukemia. In support of this is the fact that mice in which leukemic chimeric protein AML1/ETO is expressed in hematopoietic cells are subject to myeloproliferative disease and develop leukemia after additional mutation. However, it has remained yet to be determined how AML1 contributes to homeostasis of hematopoietic stem cells (HSCs). To address this issue, we analyzed in detail HSC function in the absence of AML1. Notably, cells in the Hoechst 33342 side population (SP) fraction are increased in number in AML1-deficient bone marrow, which suggests enrichment of quiescent HSCs. We quantitatively evaluated HSCs by bone marrow transplantation assays using limiting dilution and found a significant increase in HSC number within the AML1-deficient bone marrow. These results indicate that the number of quiescent HSCs is negatively regulated by AML1, loss of which may result in accumulation of leukemic stem cell pool in AML1-related leukemia.

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Senescence and apoptosis block hematopoietic activation of quiescent hematopoietic stem cells with short telomeres

Blood ◽

10.1182/blood-2014-04-568055 ◽

2014 ◽

Vol 124 (22) ◽

pp. 3237-3240 ◽

Cited By ~ 32

Author(s):

Jianwei Wang ◽

Xin Lu ◽

Vadim Sakk ◽

Christoph A. Klein ◽

Karl Lenhard Rudolph

Keyword(s):

Stem Cells ◽

Dna Damage ◽

Hematopoietic Stem Cells ◽

Telomere Shortening ◽

Hematopoietic Stem ◽

Key Points ◽

Quiescent Hscs

Key Points DNA damage induced by telomere shortening resides in most quiescent HSCs. Senescence and apoptosis compromise the activation of HSCs with dysfunctional telomeres.

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Activated but Not Quiescent Hematopoietic Stem Cells Rely Readily on Glycolysis As Their Main Source of Energy

Blood ◽

10.1182/blood-2019-129099 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 271-271

Author(s):

Tasleem Arif ◽

Raymond Liang ◽

Maio Lin ◽

Svetlana Kalmikova ◽

Artem Kasianov ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Glucose Uptake ◽

Glucose Transporter ◽

Tricarboxylic Acid Cycle ◽

Embryonic Stem ◽

Hematopoietic Stem ◽

Glucose Transporter 1 ◽

Quiescent Hscs

Elucidating mechanisms that regulate hematopoietic stem cells (HSCs) quiescence is critical for improving bone marrow transplantation. It is postulated that quiescent HSCs rely mostly on glycolysis rather than mitochondrial oxidative phosphorylation (OXPHOS) as their energy source. We have identified a population of HSCs with low mitochondrial activity (LSKCD150+CD48- within the 25% lowest mitochondrial membrane potential; MMP-low) that we show are highly enriched in quiescent HSCs (~95% ±2.65 in G0) as measured by pyronin Y/Hoechst staining in contrast to LSKCD150+CD48- within 25% highest MMP (MMP-high) that are in majority in G1/S/G2/M phase of cell cycle (55.4%±16; p<0.05; n=3). MMP-low HSCs exhibit greater in vivo competitive repopulation ability (3.7 fold; p=0.021; n=10 mice) at 16 weeks as compared to MMP-high fractions, show higher self renewal ability and are enriched in label-retaining H2B-GFP+ cells (~3 fold; p<0.01; n=3). Conversely, label-retaining GFP+HSCs maintain lower MMP than non-label-retaining cells. Altogether these results reinforce the notion that MMP-low HSCs are quiescent whereas MMP-high HSCs are primed/activated. Using single cell RNA-sequencing (scRNA-seq) analysis to interrogate the transcriptome by a Fluidigm C1 platform within MMP-high versus MMP-low HSCs we found major metabolic pathways including OXPHOS, exhibit significantly greater expression within the MMP-high than in the MMP-low HSC fraction. Seahorse analysis confirmed that MMP-high but not -low hematopoietic stem and progenitor cells (HSPCs) use OXPHOS as their source of energy (3.9 fold; n=16 mice). Unexpectedly, glycolytic gene expression was also enriched in the primed MMP-high HSCs and relatively low in quiescent MMP-low HSCs. qRT-PCR analysis further confirmed that the expression of glycolytic enzymes and other genes including glucose transporter 1 (Glut1, slc2a1) that is the main glucose transporter on HSCs is greater in MMP-high relative to -low HSCs. These unforeseen findings raised the potential that despite the current consensus in HSC biology, glycolysis may more readily support activated rather than quiescent HSCs. We thus measured the glucose uptake in MMP-low vs -high HSCs under metabolic (pyruvate, glucose and glutamine)-restricted conditions. Using 2NBDG, a fluorescently tagged glucose analog, we found that MMP-high HSCs uptake 3.3-fold more glucose and contained three times more 2NBDG+ cells as compared to MMP-low HSCs (p<0.001 for each; n=3). Pharmacological inhibition of Glut1 reduced glucose uptake in MMP-high but not -low HSCs suggesting the specific sensitivity of MMP-high HSCs to the glucose inhibition. In addition, activation of tricarboxylic acid cycle TCA cycle with combined methyl-pyruvate and dimethyl-alpha-ketoglutarate led to a greater glucose uptake (~3.5 fold) in MMP-high as compared to MMP low HSCs (~2.2 fold; p<0.0001). To address the degree to which glycolysis is necessary, FACS-purified MMP-low and -high HSCs were incubated with 2-Deoxy D-Glucose (2DG) - a glucose analog - that inhibits glycolysis via its action on hexokinase. While glucose deprivation with 50 mM dose of 2DG within 12 hours did not have much effect on MMP-low HSCs, over 60% of MMP-high HSCs died (p<0.001; n=3), suggesting that MMP-high but not -low HSCs rely readily on glycolysis for their survival. Also, the inhibition of mitochondrial transport of pyruvate that is the end product of glycolysis (α-cyano-4-hydroxycinnamate (CHC)), decreased survival in MMP-high HSCs by 80% with negligible effect on MMP-low HSCs (p<0.01; n=3). Finally 50 FACS-purified HSCs (LSKCD150+CD48-MMP-low or -high) were transplanted in lethally irradiated mice along with 200,000 unfractionated bone marrow cells in a competitive repopulation assay and mice were subsequently treated with 2DG or control vehicle every other day for two weeks. Two months later, 2DG treatment led to significantly enhanced reconstitution levels in MMP-high HSCs with no or little effect on MMP low reconstitution levels (ongoing). These combined results suggest that primed MMP-high rather than quiescent MMP-low HSCs rely on glycolysis as their main source of energy. These findings are consistent with the concept that glycolysis is key in sustaining rapidly dividing cells such as embryonic stem cells and cancer cells. Disclosures Ghaffari: Rubius Therapeutics: Consultancy.

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Kindlin-3–mediated integrin adhesion is dispensable for quiescent but essential for activated hematopoietic stem cells

Journal of Experimental Medicine ◽

10.1084/jem.20150269 ◽

2015 ◽

Vol 212 (9) ◽

pp. 1415-1432 ◽

Cited By ~ 19

Author(s):

Raphael Ruppert ◽

Markus Moser ◽

Markus Sperandio ◽

Emanuel Rognoni ◽

Martin Orban ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Progenitor Cells ◽

Leukocyte Adhesion ◽

Wild Type ◽

Hematopoietic Stem ◽

Cell Lineages ◽

Stem And Progenitor Cells ◽

Deficiency Type ◽

Quiescent Hscs

Hematopoietic stem cells (HSCs) generate highly dividing hematopoietic progenitor cells (HPCs), which produce all blood cell lineages. HSCs are usually quiescent, retained by integrins in specific niches, and become activated when the pools of HPCs decrease. We report that Kindlin-3–mediated integrin activation controls homing of HSCs to the bone marrow (BM) and the retention of activated HSCs and HPCs but not of quiescent HSCs in their BM niches. Consequently, Kindlin-3–deficient HSCs enter quiescence and remain in the BM when cotransplanted with wild-type hematopoietic stem and progenitor cells (HSPCs), whereas they are hyperactivated and lost in the circulation when wild-type HSPCs are absent, leading to their exhaustion and reduced survival of recipients. The accumulation of HSPCs in the circulation of leukocyte adhesion deficiency type III patients, who lack Kindlin-3, underlines the conserved functions of Kindlin-3 in man and the importance of our findings for human disease.

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