Noncanonical Wnt Signaling Maintains Hematopoietic Stem Cells in Different Zones

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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Microrna 199b Regulates Mouse Hematopoietic Stem Cells Maintenance

Blood ◽

10.1182/blood-2019-126283 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3704-3704

Author(s):

Aldona A Karaczyn ◽

Edward Jachimowicz ◽

Jaspreet S Kohli ◽

Pradeep Sathyanarayana

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Hematopoietic Stem Cells ◽

Conflicts Of Interest ◽

Quiescent State ◽

Differentiation Potential ◽

Self Renewal ◽

Hematopoietic Stem

The preservation of hematopoietic stem cell pool in bone marrow (BM) is crucial for sustained hematopoiesis in adults. Studies assessing adult hematopoietic stem cells functionality had been shown that for example loss of quiescence impairs hematopoietic stem cells maintenance. Although, miR-199b is frequently down-regulated in acute myeloid leukemia, its role in hematopoietic stem cells quiescence, self-renewal and differentiation is poorly understood. Our laboratory investigated the role of miR-199b in hematopoietic stem and progenitor cells (HSPCs) fate using miR-199b-5p global deletion mouse model. Characterization of miR-199b expression pattern among normal HSPC populations revealed that miR-199b is enriched in LT-HSCs and reduced upon myeloablative stress, suggesting its role in HSCs maintenance. Indeed, our results reveal that loss of miR-199b-5p results in imbalance between long-term hematopoietic stem cells (LT-HSCs), short-term hematopoietic stem cells (ST-HSCs) and multipotent progenitors (MMPs) pool. We found that during homeostasis, miR-199b-null HSCs have reduced capacity to maintain quiescent state and exhibit cell-cycle deregulation. Cell cycle analyses showed that attenuation of miR-199b controls HSCs pool, causing defects in G1-S transition of cell cycle, without significant changes in apoptosis. This might be due to increased differentiation of LT-HSCs into MPPs. Indeed, cell differentiation assay in vitro showed that FACS-sorted LT-HSCs (LineagenegSca1posc-Kitpos CD48neg CD150pos) lacking miR-199b have increased differentiation potential into MPP in the presence of early cytokines. In addition, differentiation assays in vitro in FACS-sorted LSK population of 52 weeks old miR-199b KO mice revealed that loss of miR-199b promotes accumulation of GMP-like progenitors but decreases lymphoid differentiation, suggesting that miR199b may regulate age-related pathway. We used non-competitive repopulation studies to show that overall BM donor cellularity was markedly elevated in the absence of miR-199b among HSPCs, committed progenitors and mature myeloid but not lymphoid cell compartments. This may suggest that miR-199b-null LT-HSC render enhanced self-renewal capacity upon regeneration demand yet promoting myeloid reconstitution. Moreover, when we challenged the self-renewal potential of miR-199b-null LT-HSC by a secondary BM transplantation of unfractionated BM cells from primary recipients into secondary hosts, changes in PB reconstitution were dramatic. Gating for HSPCs populations in the BM of secondary recipients in 24 weeks after BMT revealed that levels of LT-HSC were similar between recipients reconstituted with wild-type and miR-199b-KO chimeras, whereas miR-199b-null HSCs contributed relatively more into MPPs. Our data identify that attenuation of miR-199b leads to loss of quiescence and premature differentiation of HSCs. These findings indicate that loss of miR-199b promotes signals that govern differentiation of LT-HSC to MPP leading to accumulation of highly proliferative progenitors during long-term reconstitution. Hematopoietic regeneration via repopulation studies also revealed that miR-199b-deficient HSPCs have a lineage skewing potential toward myeloid lineage or clonal myeloid bias, a hallmark of aging HSCs, implicating a regulatory role for miR-199b in hematopoietic aging. Disclosures No relevant conflicts of interest to declare.

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Fractionated Osteoblastic Niche Cells Enhances the Homing Activity of Hematopoietic Stem Cells.

Blood ◽

10.1182/blood.v114.22.560.560 ◽

2009 ◽

Vol 114 (22) ◽

pp. 560-560

Author(s):

Fumio Arai ◽

Yuka Nakamura ◽

Kentaro Hosokawa ◽

Isao Kobayashi ◽

Hiroko Iwasaki ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Conflicts Of Interest ◽

The Other ◽

Quiescent State ◽

Mesenchymal Progenitor Cells ◽

Differentiation Potential ◽

Hematopoietic Stem ◽

Other Hand

Abstract Abstract 560 Hematopoietic stem cells (HSCs) retain quiescent state in the adult bone marrow (BM), interacting with specialized niches along the endosteum (osteoblastic niche) and in perivascular sites adjacent to endothelial and reticular cells (perivascular niche). We have previously reported that the regulations of HSCs in the osteoblastic niche through the receptor-cytokine, cell-to-cell, and cell-to-extracellular matrix (ECM) interactions are critical for the maintenance of cell cycle quiescence. However, osteoblast population is heterogeneous in terms of the degree of differentiation and function of each cell. In this study, we characterized the cellular components of osteoblastic niche and examined the ability for the maintenance of HSCs. We found that CD45–CD31–Ter119– endosteal population can be subdivided into three fractions, ALCAM+Sca-1–, ALCAM–Sca-1+, and ALCAM–Sca-1– cells. Expression of osteoblast markers and differentiation potential of these three fractions revealed that osteoblasts were enriched in the Sca-1– populations. On the other hand, ALCAM–Sca-1+ cells had characteristics of mesenchymal progenitor cells (MPCs). Microarray analysis showed that ALCAM+Sca-1– fraction tend to highly express the genes related to cell-to-cell or cell-to-ECM adhesions, such as N-cadherin, Osteopontin, and Alcam, compared to other fractions, indicating that this population might regulate HSCs through the physiological interactions. On the other hand, ALCAM–Sca-1+ fraction highly expressed the genes related to growth factor and cytokine that are involved in the regulation of both quiescence and proliferation of long-term-HSCs, such as Angiopoietin-1, Flt3l, Cxcl12, Thrombopoietin, and Kitl. These data rise the possibility that multiple cell populations in endosteum collaborate as a complex to regulate the balance between HSC proliferation and quiescence in the endosteum in adult BM. Next we examined whether these fractionated cells could maintain the long-term repopulation (LTR) activity of HSCs in vitro. BM Lin–Sca-1+Kit+ (LSK) cells were cocultured with each fractionated endosteal population without growth factors. After 2 days of coculture, Ly5.1+ cells were sorted and transplanted into lethally irradiated mice. We found that all three fractions maintained LTR-activity of LSK cells. In particular, LSK cells cocultured with ALCAM+Sca-1– cells showed significantly higher LTR-activity compared to that cocultured with other fractions. Then we analyzed the gene expression implicated in the homing and lodgment of HSCs. Q-PCR array analysis revealed that upregulation of Cxcr4, integrins, Cd44, N-cadherin and Alcam was induced in LSK cells cocultured with Sca-1– populations. In particular, ALCAM+Sca-1– cells significantly upregulated N-cadherin expression in HSCs. These data suggest that osteoblasts increased homing activity of HSCs in culture or enriched cell population that had high homing activity. Furthermore, the cell-to-cell adhesion between HSCs and ALCAM+Sca-1– cells enhanced the interaction of HSCs with niche complex and maintained self-renewal activity of HSCs. Disclosures: No relevant conflicts of interest to declare.

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Stability of Self-Renewal in Hematopoietic Stem Cells

Blood ◽

10.1182/blood.v116.21.sci-42.sci-42 ◽

2010 ◽

Vol 116 (21) ◽

pp. SCI-42-SCI-42

Author(s):

Norman N. Iscove

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Conflicts Of Interest ◽

Expression Profiles ◽

Single Cells ◽

Gene Expression Profiles ◽

Short Term ◽

Blood Leukocytes ◽

Hematopoietic Stem

Abstract Abstract SCI-42 For many years a distinction was drawn between prospectively separable murine HSC populations with long-term, essentially permanent reconstituting potential (LT-HSC), versus HSC populations yielding short-term engraftment lasting only 4 – 6 weeks after transplantation (ST-HSC). Recent work based on transplantation of single cells shows that highly purified populations of LT-HSC prepared by standard sorting parameters consist in fact predominantly of a distinct, newly recognized class of intermediate- term reconstituting cells (IT-HSC) whose grafts endure longer than short-term HSC but also eventually fail (1). IT-HSC are separable from long-term reconstituting cells on the basis of expression of more alpha2 integrin and less SLAM150. Crucial to recognition of the distinction between LT- and IT-HSC are the endpoints used to evaluate reconstitution. If blood erythroid or myeloid reconstitution is measured, IT reconstitution is readily distinguished by the disappearance of these elements by 16 wk post-transplant. If instead reconstitution is measured simply by presence of blood leukocytes of donor origin, which in the mouse are almost entirely lymphocytes, the distinction is not made because lymphoid elements persist even in fading IT clones to 24 wk or beyond. The observations imply a need for reinterpretation of most of the published descriptions of the biology and gene expression profiles previously attributed to LT-HSC but in fact derived from analysis of populations that consisted mainly of IT-HSC. The capacity now to separate LT- from IT-HSC creates new opportunities for probing the mechanisms that specify and sustain long term function in the former but not the latter. 1. Benveniste P, Frelin C, Janmohamed S, Barbara M, Herrington R, Hyam D, Iscove NN. Intermediate-term hematopoietic stem cells with extended but time-limited reconstitution potential. Cell Stem Cell. 2010;6:48–58 Disclosures: No relevant conflicts of interest to declare.

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Hematopoietic Stem Cells Increase Quiescence during Aging

Blood ◽

10.1182/blood-2019-130668 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2484-2484 ◽

Cited By ~ 1

Author(s):

Larisa V. Kovtonyuk ◽

Peter Ashcroft ◽

Gianluca Spaltro ◽

Nageswara Rao Tata ◽

Radek C. Skoda ◽

...

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Steady State ◽

Hematopoietic Stem Cells ◽

Conflicts Of Interest ◽

Quiescent State ◽

Hematopoietic Stem ◽

Cfse Dilution ◽

Turn Over ◽

Old Mice

Introduction: Definitive hematopoietic stem cells (HSCs) sustain blood production from fetal development throughout life. In mice, most of steady state, young adult HSCs are in the G0 phase of cell cycle (quiescence), and are estimated to divide roughly once a month. Daily hematopoietic production is thus mainly sustained by highly proliferative downstream hematopoietic progenitor cells (HPCs). Aged haematopoiesis was demonstrated to be distinct from young haematopoiesis in various aspects such as i) a shift from lymphopoiesis to myelopoiesis, ii) functional decline of HSCs (self-renewal, homing), and iii) HSCs pool expansion. While several studies attempted to address whether changes in HSCs turnover during aging can account for the distinct aging associated phenotype and function, it remained to be determined whether aged HSCs overall cycle more or less frequently than young HSCs. Methods: To construct data-based, quantitative models, we measured turnover rates and compartment sizes of populations of HSCs, HSPCs and granulopoiesis/granulocytes, i.e. a post-mitotic mature hematopoietic linage with a short half-life. We examined four age groups: 3 week, 2 month, 1 year and 2 year old mice. Mice in each group were i.p. injected every 4 hours with 1 mcg EdU up to a maximum time of 48 hours. HSC, HSPC and granulopoiesis/granoulocyte compartment sizes and snapshot cell-cycle analysis was performed by FACS at multiple sampling points in BM and peripheral blood (PB), respectively. Based on this data, we built a mathematical model of HSC turn-over and HSPC differentiation during ageing. Moreover, we evaluated HSC cycling by CFSE dilution in steady-state transplantation experiments (as described before; Takizawa et al., J Exp Med 2011). Results: In line with previous reports, the HSCs compartment size gradually increased with age from 3wk old mice to 2 year old mice. In sharp contrast, cycling activity of HSCs as determined by EdU incorporation decreased gradually and significantly with increasing age. This was driven by decreased activation from the quiescent state, while the time that actively cycling HSCs require to progress through cell-division remains constant with age. Multipotent Progenitor (MPP) cycling showed a non-significant trend towards slower turn-over. These results were confirmed by complementary CFSE-dilution experiments. Mathematical modeling of HSC proliferation and differentiation revealed a higher probability of self-renewing divisions in 3 week old mice as compared to 2 month, 1 and 2 year old mice, with the latter both having nearly equal chances of self-renewing versus differentiating divisions. Conclusions: Our data clarifies the long-standing question, how the HSC pool increases with age. Instead of an increase in active cycling, an increase in HSC quiescence is responsible for the increased size of the HSCs pool in aging. Disclosures No relevant conflicts of interest to declare.

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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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Identification of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment and Self-Renewal.

Blood ◽

10.1182/blood.v114.22.816.816 ◽

2009 ◽

Vol 114 (22) ◽

pp. 816-816

Author(s):

Faiyaz Notta ◽

Sergei Doulatov ◽

John E. Dick

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Cord Blood ◽

Conflicts Of Interest ◽

Single Cells ◽

Human Cord Blood ◽

Hematopoietic Stem ◽

Human Hscs ◽

Insight Into

Abstract Abstract 816 A fundamental tenet that has guided our insight into the biology of hematopoietic stem cells (HSCs) over the past 50 years is the principle that an HSC can only be assayed by functional repopulation of an irradiated host1. In its strictest definition, only a HSC can provide long-term reconstitution of all the major lineages following single cell transplantation. However, the existing strategies for human HSC isolation lack quantitation and do not submit to this rigorous standard, thus precluding further biological analysis. Here, we report the prospective and quantitative analysis of human cord blood (CB) HSCs transplanted into female NOD/SCID/IL-2Rgcnull mice. We identify integrin a6 (CD49f) as a novel marker of cord blood (CB) HSCs and report that single Lin-CD34+CD38-CD90+CD45RA-RholoCD49fhi cells can reconstitute myeloid, B-, and T-cell lineages for 18 weeks. 5 of 29 mice transplanted with single cells gave rise to human cells indicating that approximately 20% of cells in this fraction are HSCs. This advance finally enables utilization of near-homogeneous populations of human HSCs to gain insight into their biology and to harness them for stem cell-based therapeutics. Disclosures: No relevant conflicts of interest to declare.

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PDK1 Controls the Differentiation of Hematopoietic Stem Cells Via Modulating ROS Levels

Blood ◽

10.1182/blood.v126.23.896.896 ◽

2015 ◽

Vol 126 (23) ◽

pp. 896-896

Author(s):

Tianyuan Hu ◽

Cong Li ◽

Le Wang ◽

Yingchi Zhang ◽

Luyun Peng ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Conflicts Of Interest ◽

Conditional Knockout ◽

Quiescent State ◽

Self Renewal ◽

Hematopoietic Stem ◽

Hematopoietic System ◽

Cell Counts

Abstract Hematopoietic stem cells (HSCs) exist as a rare population with two essential properties of self-renewal and differentiation. HSCs can give rise to all hematopoietic progenitor and mature cells. While critical for a full understanding of the hematopoietic process and HSC-related clinical applications, the mechanisms of self-renewal and differentiation of HSCs remain elusive. The PI3K-Akt signaling pathway plays essential roles in the regulation of hematopoiesis. 3-Phosphoinositide-dependent protein kinase 1 (PDK1) activates multiple AGC kinases including Akt and is a pivotal regulator in this pathway. PDK1 phosphorylates Akt at its T308 residue and regulates the functional development of B and T cells during hematopoiesis. However, the role of PDK1 in HSCs has not been fully defined. In this study, we generated PDK1 conditional knockout mice Vav-Cre;PDK1fl/fl (PDK1Δ/Δ) to explore the roles of PDK1 in HSCs. While PDK1Δ/Δ mice have reduced B and T cell counts as previously described, their LT-HSCs and ST-HSCs were significantly increased in comparison with WT mice while MPPs and CMPs were decreased after PDK1 deletion, indicating that the loss of PDK1 perturbed the steady-state hematopoiesis. Furthermore, although deletion of PDK1 increased the frequency of HSCs, PDK1-deficient HSCs fail to reconstitute the hematopoietic system when PDK1-deficient HSCs were used in bone marrow transplantation and competitive transplantation experiments in comparison to the WT HSCs, indicating that PDK1 is vital for hematopoiesis. To explore the mechanisms by which PDK1 regulates HSC function, we examined the cell cycle status and found the percentage of PDK1Δ/Δ HSCs was decreased significantly in G0 stage while increased in G1 and S/G2/M phases. This suggests an increase in HSC exit from a quiescent state. Since MPPs were significantly decreased in bone marrow, we examined the percentage of Annexin V+ DAPI- PDK1Δ/Δ and WT MPPs and found that they are comparable. This indicates that apoptosis did not cause the decrease in MPPs. In addition, a total of 300 LT-HSCs from PDK1Δ/Δ or WT mice and competitor cells were transplanted into lethally irradiated recipient mice to examine whether the decrease in MPPs is due to a defect in HSC differentiation. We found that less than 1% of MPPs arose from PDK1Δ/Δ HSCs 12 weeks after transplantation, indicating that PDK1 is required for the differentiation from LT-HSCs to MPPs. Because the full activation of Akt requires cooperative phosphorylation at its S473 and T308 residues by mTORC2 and PDK1, respectively, we also investigated the function of HSCs in RictorΔ/Δ PDK1Δ/Δ (DKO) mice in conjunction with RictorΔ/Δ or PDK1Δ/Δ mice to explore how mTORC2 and/or PDK1 influence Akt function in HSCs. The flow cytometric analyses of peripheral blood and bone marrow samples revealed very similar parameters of RictorΔ/Δ PDK1Δ/Δ and PDK1Δ/Δ mice. Interestingly, Rictor seemed to exert a minimal impact on HSCs and MPPs. More importantly, in contrast to RictorΔ/Δ, RictorΔ/Δ PDK1Δ/Δ HSCs failed to reconstitute the hematopoietic system after transplantation as PDK1Δ/Δ HSCs, suggesting that PDK1 plays a dominant role in the Akt-mediated regulation of HSC function. To explore the mechanism that leads to the defect in HSCs due to loss of PDK1, we assessed ROS levels in PDK1-deficient HSCs and found that PDK1-deficient LSKs and HSCs exhibit greatly reduced ROS levels when compared with the control HSCs. Treating PDK1-deficient BM cells with BSO in vitro increased cellular ROS levels and the colony counts of PDK1-deficient BM cells significantly. Notably, the recovery effect was only observed with BSO concentrations lower than 0.03 mM. This suggests that ROS levels are precisely controlled in HSCs. Higher or lower ROS levels beyond the normal range are both harmful to normal HSC functions. Since increased SDFα expression is associated with cellular ROS levels in various cells including hematopoietic cells, we also treated PDK1Δ/Δ mice with SDFα and found that it couldpartially rescue the defective differentiation ability of PDK1-deficient HSCs. In addition, we found that PDK1 deletion could significantly prolong the life span and inhibit the leukemia development in murine T-ALL model via altering leukemic cell differentiation and proliferation. Taken together, PDK1 controls HSC differentiation via regulating cellular ROS levels and regulates malignant hematopoiesis. Disclosures No relevant conflicts of interest to declare.

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Long-term, multilineage hematopoiesis occurs in the combined absence of β-catenin and γ-catenin

Blood ◽

10.1182/blood-2007-07-102558 ◽

2008 ◽

Vol 111 (1) ◽

pp. 142-149 ◽

Cited By ~ 161

Author(s):

Grégoire Jeannet ◽

Marina Scheller ◽

Léonardo Scarpellino ◽

Stéphane Duboux ◽

Noemie Gardiol ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Wnt Signaling ◽

Ex Vivo ◽

Wnt Signaling Pathway ◽

Transcriptional Responses ◽

Differentiation Potential ◽

Hematopoietic Stem ◽

Canonical Wnt

The canonical Wnt signaling pathway plays key roles in stem-cell maintenance, progenitor cell expansion, and lineage decisions. Transcriptional responses induced by Wnt depend on the association of either β-catenin or γ-catenin with lymphoid enhancer factor/T cell factor transcription factors. Here we show that hematopoiesis, including thymopoiesis, is normal in the combined absence of β- and γ-catenin. Double-deficient hematopoietic stem cells maintain long-term repopulation capacity and multilineage differentiation potential. Unexpectedly, 2 independent ex vivo reporter gene assays show that Wnt signal transmission is maintained in double-deficient hematopoietic stem cells, thymocytes, or peripheral T cells. In contrast, Wnt signaling is strongly reduced in thymocytes lacking TCF-1 or in nonhematopoietic cells devoid of β-catenin. These data provide the first evidence that hematopoietic cells can transduce canonical Wnt signals in the combined absence of β- and γ-catenin.

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Nuclear Protein DEK Governs Quiescence and Metabolic Homeostasis of Hematopoietic Stem Cells By Shaping Chromatin Accessibility

Blood ◽

10.1182/blood-2020-136859 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 7-7

Author(s):

Zhe Chen ◽

Lei Li ◽

Jieping Chen ◽

Yu Hou

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Conflicts Of Interest ◽

Epigenetic Modification ◽

Regulation Of Gene Expression ◽

Chromatin Accessibility ◽

Hematopoietic Stem ◽

Chromatin Remodelers ◽

G0 Phase

Hematopoietic stem cells (HSCs) must achieve a balance between quiescence and activation that fulfils the demands for hematopoiesis without compromising long-term maintenance of HSCs. DEK, a chromatin architectural factor, is involved in chromatin remodeling, transcriptional regulation and DNA replication, and is implicated in genetic and epigenetic regulation of gene expression. Here, we identified that DEK is a critical regulator of HSCs quiescence. Deletion of DEK in mice resulted in abnormal hematopoiesis with an obvious decreased HSC pool size (~3700 to ~1700 cells/mouse), associated with apparent reduction in the proportion of HSCs in G0 phase as compared to control HSCs (~72% to ~57%). As shown by serial bone marrow transplantation and competitive repopulation assays, deletion of DEK impaired the self-renewal capacity of HSCs. Mechanistically, deficiency of DEK in HSC altered chromatin accessibility landscape, resulting in increased transcription of activation-specific genes (including Akt1/2,Ccnb2, and Rps6) and decreased transcription of quiescence-specific genes (including p21 and Gata2), leading to excessively activated Akt-mTOR signaling and elevated metabolism of HSC. Targeting the Akt-mTOR pathway efficiently abrogated the impaired quiescence and the increased metabolism of HSC in DEK-deficient mice, and partially rescued the long-term functions of HSC. Further, DEK regulated chromatin accessibility of HSC by recruiting the co-repressor NCoR1 to repress acetylation of histone 3 at lysine 27. Collectively, our findings revealed crucial functions of DEK in HSC quiescence maintenance and disclosed a new link between chromatin remodelers, epigenetic modification, gene transcription, and HSC homeostasis. Disclosures No relevant conflicts of interest to declare.

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Mir-99 Regulates Normal and Leukemic Stem Cell Function

Blood ◽

10.1182/blood.v128.22.1469.1469 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1469-1469

Author(s):

Mona Khalaj ◽

Carolien Woolthuis ◽

Wenhuo Hu ◽

Benjamin Heath Durham ◽

Christopher Y. Park

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Peripheral Blood ◽

Cell Function ◽

Conflicts Of Interest ◽

Ectopic Expression ◽

Gene Set Enrichment Analysis ◽

Hematopoietic Stem

Abstract Acute myeloid leukemia (AML) is composed of functionally heterogeneous cells including leukemic stem cells (LSCs), which exhibit the ability to self-renew and propagate disease. It is thought that failure of common chemotherapy regimens is due to insufficient eradication of LSCs. However, the mechanisms that maintain stem cell function in the hematopoietic system are not well understood. MicroRNAs play an important role in the regulation of normal and malignant hematopoietic stem cells. Our studies showed that miR-99, a miRNA highly expressed in AML patient cell populations enriched for LSC activity, is among the most highly expressed miRNAs in hematopoietic stem cells (HSCs), suggesting that miR-99 plays a role in regulating normal HSCs as well as LSCs. To test the role of miR-99 in normal hematopoiesis, we knocked down (KD) miR-99 in mouse HSCs (Lin-cKit+Sca1+CD34-SLAM+), which resulted in ~3 fold reduced methylcellulose colony formation upon secondary plating (P=0.01), as well as accelerated granulopoiesis as demonstrated by increased Gr1+Mac1+ cells 7 days after culture initiation (P<0.01), suggesting that miR-99 functions to suppresses differentiation. Consistent with this model, transplantation assays demonstrated >10-fold reduction in long-term engraftment capacity of miR-99 KD compared to scrambled controls (P=0.0004). In addition, Ki-67/DAPI staining of stably engrafted miR-99 KD hematopoietic stem and progenitor cells (HSPCs) showed increased cell cycling, demonstrating that miR-99 also maintains HSPC quiescence. Gene set enrichment analysis (GSEA) of RNA-sequencing data generated from stably engrafted Lin-Sca-1+c-Kit+ cells revealed that miR-99 KD induces significant depletion of LT-HSC gene signatures (P<0.001) and induction of a late progenitor signature (P<0.001), providing further evidence that miR-99 normally functions to maintain HSPCs in the undifferentiated state. To test whether miR-99 maintains LSCs, we performed miR-99 KD experiments using the MLL-AF9 retroviral mouse model. miR-99 KD resulted in a significant extension in survival in secondary transplants compared to scrambled controls (median 92 days vs. 48 days, P<0.001). Evaluation of the bone marrow at the time of death revealed ~2.5 fold decrease in the frequency of LSCs (P<0.01) and ~2 fold increase in the percentage of cycling LSCs (in SG2M) (P<0.001). Analysis of RNA-seq data from miR-99 KD LSCs revealed induction of a differentiated normal progenitor signature (P<0.001) and depletion of a shared HSC/LSC gene signature (P=0.05). Giemsa staining of peripheral blood showed miR-99 KD also induced a significant increase in the number of differentiated myeloid precursors in the peripheral blood (P<0.001), reminiscent of AML differentiation-inducing agents used in the clinic such as ATRA. Consistent with a role in regulating leukemic blast differentiation, microRNA-Seq data from the 153 AML patients in the TCGA database revealed that miR-99 expression inversely correlated with their French-American-British classifications, with low expression levels associated with M4 and M5 subtypes. Compatible with a role in maintaining LSCs, miR-99 KD in a primary AML sample reduced long-term engraftment upon xenotransplantation into NSG mice, and the engrafting cells displayed increased CD14 expression. Together, these data demonstrate that similar to normal HSPCs, miR-99 maintains LSCs function. As miR-99 functions to maintain both LSCs and HSCs, we asked which miR-99 target genes mediate miR-99 KD phenotypes. To address this question, we performed a shRNA library-based forward genetic screen designed to rescue the reduced HSC function following miR-99 KD. We designed 180 shRNAs against 45 predicted miR-99 targets that we identified as upregulated upon acute miR-99 KD in mouse HSPCs. Among the conserved miR-99 targets, Hoxa1, a member of the Hox family of transcription factors, was among the top hits, with all 4 shRNAs being enriched compared to controls. Ectopic expression of Hoxa1 in MonoMac6 AML cells was sufficient to induce differentiation, a phenotype similar to miR-99 KD. These data indicate that Hoxa1 is an important downstream mediator of miR-99 function. Disclosures No relevant conflicts of interest to declare.

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