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

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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Id2 Is Required for the Self-Renewal and Proliferation of Hematopoietic Stem Cells

Blood ◽

10.1182/blood.v126.23.1173.1173 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1173-1173 ◽

Cited By ~ 1

Author(s):

Lei Sun

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Cell Biology ◽

Conflicts Of Interest ◽

Bone Marrow Failure ◽

Conditional Knockout ◽

The Self ◽

Self Renewal ◽

Hematopoietic Stem

Abstract The production of mammalian blood cells is sustained throughout life by the self-renewal and differentiation of hematopoietic stem cells (HSCs). Dysregulation in this system leads to different pathologies including anemia, bone marrow failure and hematopoietic malignancies. The Helix-Loop-Helix transcriptional regulator Id2 plays essential roles in regulating proliferation and cell fate of hematopoietic progenitors; however, its role in regulating HSC development remains largely unknown. To assess the function of Id2 in HSCs, we developed two mouse models, including an Id2 conditional knockout model and an Id2-EYFP model, in which EYFP expression is driven by endogenous Id2 promoter. When we examined HSC function by serial transplantation, we found that mice transplanted with Id2F/F Mx1-Cre+ conditionally deleted bone marrow cells became moribund more rapidly after primary and secondary transplantation, compared to those transplanted with Id2+/F Mx1-Cre+ bone marrow, suggesting that HSC self-renewal is impaired when Id2 is deleted. To further determine if self-renewal and maintenance of HSCs depends on the expression level of Id2, we purified HSCs with different levels of Id2 expression using Id2-EYFP mice to specifically address the role of Id2 in HSCs. First, we confirmed Id2 is highly expressed in HSCs in this model. Second, when HSCs with either low or high levels of Id2-EYFP were transplanted into irradiated mice, cells with high levels of Id2 reconstituted transplanted recipients faster than those with low levels of Id2 at 3 weeks and longer, suggesting that Id2 expression is associated with repopulation advantage. Furthermore, Ki-67 staining showed that HSCs with high levels of Id2 have 15-fold more cells in G2/M phase, and fewer cells in G0. BrdU staining also suggested that there are 5-fold more BrdU+ cells in HSCs with high levels of Id2, indicating that Id2 expression correlates with cell cycle progression in HSCs. In addition, p57 has been reported to be required for quiescence of HSCs. Our preliminary data showed that p57 is downregulated in HSCs with high levels of Id2, and p57 is correspondingly upregulated in Id2-null HSCs. Altogether, our data demonstrate that Id2 is required for the self-renewal and proliferation of HSCs, and suggest a link between Id2 and the transcriptional regulatory networks that regulate the functional hematopoietic system. Since Id2 is also expressed in other adult stem cells including muscle and neuronal stem cells, as well as cancer cells, we believe our results can improve our understanding of stem cell biology and cancer development, and contribute to the identification of novel molecules that may be targeted to eliminate cancer stem cells. 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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Genetic Uncoupling of the Regulation of Hematopoietic Stem Cell Quiescence and Self-Renewal.

Blood ◽

10.1182/blood.v108.11.1347.1347 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1347-1347

Author(s):

Yan Liu ◽

Yasuhiko Miyata ◽

Goro Sashida ◽

Anthony Debalsio ◽

Yuhui Liu ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Hematopoietic Stem Cell ◽

Quiescent State ◽

Double Knockout ◽

Ki 67 ◽

Self Renewal ◽

Hematopoietic Stem

Abstract It is usually stated that HSCs must choose to either self-renew or to differentiate and lose some of their multi potentiality. Recently, we demonstrated that MEF, an ETS family of transcription factor, played an important role in regulating HSC quiescence, illustrating a third choice for the HSC, namely to make an “active” choice and remain quiescent, without undergoing either self-renewal, or differentiation. MEF null HSCs are more quiescent than normal HSCs. In addition, MEF null mice exhibit greater numbers of hematopoietic stem cells and show resistance to chemotherapy and radiation. Little is known about the regulation of self-renewal vs. quiescence of HSCs, however the cdk inhibitor p21 has been implicated in regulating both HSC quiescence and proliferation. In the absence of p21, hematopoietic stem cell numbers are reported to be increased, but so is proliferation, leading to stem cell exhaustion. This implies that while p21 may maintain HSCs in their quiescent state, MEF functions to facilitate the entry of quiescent HSCs into the cycle, To investigate the potential opposing roles of MEF and p21 in HSC quiescence and self-renewal and to test whether the quiescent state of MEF null HSCs is dependent on the presence of p21, we have generated MEF / p21 double-knockout (DKO) mice. These mice are viable and born at normal mendelian frequency. MEF / p21 DKO mice have a higher than normal proportion of HSCs in the G0 phase, based on Pyronin Y/Hoechst staining and staining for the proliferation antigen Ki-67. Thus, the increased quiescence is not dependent on the presence of p21. However, by measuring LSK cells, we have observed a normal number of HSCs in the bone marrow of MEF / p21 DKO mice, in contrast to the increased number of HSCs in the bone marrow of MEF null mice. This suggests that the increased number of hematopoietic stem cells in MEF null mice is dependent on p21. Ongoing studies will further address the unique mechanisms that control HSC vs. stem cell expansion.

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Stress-Mediated Activation of Dormant Hematopoietic Stem Cells In Vivo

Blood ◽

10.1182/blood.v120.21.sci-41.sci-41 ◽

2012 ◽

Vol 120 (21) ◽

pp. SCI-41-SCI-41

Author(s):

Andreas Trumpp ◽

Marieke Essers

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Conflicts Of Interest ◽

Negative Regulator ◽

Strong Increase ◽

Interferon Α ◽

Self Renewal ◽

Hematopoietic Stem

Abstract Abstract SCI-41 Maintenance of the blood system is dependent on dormant hematopoietic stem cells (HSCs), which are characterized by pluripotency and lifelong self-renewal capacity. In order to both maintain a supply of mature blood cells and not exhaust HSCs throughout the lifespan of the organism, most adult HSCs remain deeply quiescent during homeostasis, and only a limited number are cycling at any given time. The balance between self-renewal and differentiation of HSCs is controlled by external factors such as chemokines and cytokines, as well as by interactions of HSCs with their niche environment. The transcriptome of dormant CD34-CD150+CD48-LSK- HSCs significantly differs from that of active HSCs with the same phenotype, while the latter are highly similar to MPP1 progenitors which express CD34. One of the genes differentially expressed is the cylindromatosis (CYLD) gene, which encodes a negative regulator of the NF-κB signaling pathway. HSCs failing to express functional CYLD show various defects associated with a disturbed balance between dormant and active HSCs, suggesting a role for NF-κB signaling in establishing dormancy in HSCs. In addition, our studies have recently shown that the cytokine interferon-α (IFNα) very efficiently activates dormant HSCs in vivo. Within hours after treatment of mice with IFNα, HSCs exit G0 and enter an active cell cycle. In general, IFNα is produced in response to viral infections by cells of the immune system, and plays an important role in the antiviral host defense. We now questioned whether endogenous IFNα is also produced in response to other types of bone marrow stress and whether this affects the proliferation rate of HSCs. To monitor IFNα production in the bone marrow in vivo, we have generated MxCre ROSA-R26-EYFP mice and found that treatment with both the chemotherapeutic agent 5-FU as well as the endotoxin LPS leads to the production of IFNα in the vicinity of HSCs and progenitors. In addition, LPS treatment in vivo induced a strong increase in HSC cycling. Surprisingly, since mice lacking the IFNα receptor (Ifnar−/−) still respond to LPS, this effect is independent of IFNAR signaling. Strikingly, LPS-induced HSC activation correlated with increased expression of Sca-1, similar to what occurs upon IFNα treatment. Moreover, as for IFNα, the upregulation of SCA-1 is required for LPS-induced proliferation, since Sca-1−/− mice fail to respond to LPS stimulation. In summary, these data suggest that not only virus-inducible IFNα, but also infections by gram-negative-bacteria-produced LPS induce cycling of progenitors and otherwise dormant HSCs. Disclosures: No relevant conflicts of interest to declare.

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Growth Factor Independence 1b (Gfi1b) Regulates The Commitment, Differentiation and Expansion Of Hematopoietic Stem Cells

Blood ◽

10.1182/blood.v122.21.2433.2433 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2433-2433

Author(s):

Tarik Moroy ◽

Cyrus Khandanpour ◽

Joseph Krongold

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Mouse Bone Marrow ◽

Paracrine Factors ◽

Self Renewal ◽

Hematopoietic Stem

Abstract The efficacy of bone marrow stem cell transplantation is the therapy of choice for many hematopoietic diseases, in particular leukemia and lymphoma. This therapy is critically dependent on the transfer of sufficient numbers of hematopoietic stem cells (HSCs), which possess the capacity for self-renewal and can fully reconstitute the hematopoietic system. As such, the development of techniques for the expansion of fully functional HSCs is of significant clinical interest. By transiently manipulating the factors that govern HSC homeostasis it has been proposed that HSCs can be expanded without the loss of essential stem cell characteristics. Previously we have observed that ablation of the gene encoding the transcription factor Gfi1b in-vivo results in a dramatic expansion and mobilization of hematopoietic stem cells in the bone marrow and periphery. More recent data suggest that the blood mobilization of Gfi1b deficient HSCs is very likely mediated by a deregulation of the integrin expression. These data led us to hypothesize that Gfi1b could be a potential target for ex-vivo treatment and expansion of HSCs. Indeed, when deletion of Gfi1b was induced in whole bone marrow ex-vivo, HSCs showed a significant expansion in both in absolute number and in terms of proportion of bone marrow. We followed HSCs in ex-vivo expansion cultures from mouse bone marrow by tracking expression of the surface marker CD48, which indicates whether an HSC has transitioned to a differentiation committed multi-potent progenitor. We observed that Gfi1b null HSCs expanded without up-regulating CD48 in contrast to wt HSCs. This suggests that Gf11b deficient HSCs underwent symmetric self-renewal type cell divisions at a significantly increased frequency, when compared to wt HSCs. We had previously shown that HSCs lacking Gfi1b cycle at a faster rate than control HSCs. The combination of increased cell division and preferential self-renewal of Gfi1b-/- HSCs indicates that inhibition of Gfi1b may be the ideal strategy for ex-vivo HSC expansion. As well, in accordance with this preference for self-renewal, Gfi1b null HSCs that were cultured under myeloid differentiation conditions remained primarily in an undifferentiated state as defined by a lack of the myeloid surface markers Gr1 and Mac1. These cultures also demonstrated increased long term colony forming capacity versus controls, further supporting an undifferentiated phenotype in Gfi1b-/- cells. Because the stem cell niche is a highly complex and heterogeneous environment we also investigated whether bone marrow in which Gfi1b has been deleted exerts paracrine effects that contributed to HSC expansion. Co-Culture assays demonstrated that Gfi1b-/- bone marrow was able to induce an expansion of progenitors in wild-type bone marrow of more than 10 fold compared to Gfi1b-/+ bone marrow. Interestingly cells co-cultured with Gfi1b null bone marrow also exhibited an overall proliferation advantage after short-term cultures. This suggests that not only does Gfi1b deletion induce HSC expansion via cell intrinsic mechanisms, but also points to the possibility that this occurs through paracrine factors that alter bone marrow homeostasis. Disclosures: No relevant conflicts of interest to declare.

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Adult hematopoietic stem cells require NKAP for maintenance and survival

Blood ◽

10.1182/blood-2010-02-268391 ◽

2010 ◽

Vol 116 (15) ◽

pp. 2684-2693 ◽

Cited By ~ 22

Author(s):

Anthony G. Pajerowski ◽

Michael J. Shapiro ◽

Kimberly Gwin ◽

Rhianna Sundsbak ◽

Molly Nelson-Holte ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Time Course ◽

Kinase Inhibitors ◽

Conditional Knockout ◽

Similar Time ◽

Self Renewal ◽

Hematopoietic Stem ◽

Radiation Chimeras

Abstract Steady-state hematopoiesis is sustained through differentiation balanced with proliferation and self-renewal of hematopoietic stem cells (HSCs). Disruption of this balance can lead to hematopoietic failure, as hematopoietic differentiation without self-renewal leads to loss of the HSC pool. We find that conditional knockout mice that delete the transcriptional repressor NKAP in HSCs and all hematopoietic lineages during embryonic development exhibit perinatal lethality and abrogation of hematopoiesis as demonstrated by multilineage defects in lymphocyte, granulocyte, erythrocyte and megakaryocyte development. Inducible deletion of NKAP in adult mice leads to lethality within 2 weeks, at which point hematopoiesis in the bone marrow has halted and HSCs have disappeared. This hematopoietic failure and lethality is cell intrinsic, as radiation chimeras reconstituted with inducible Mx1-cre NKAP conditional knockout bone marrow also succumb with a similar time course. Even in the context of a completely normal bone marrow environment using mixed radiation chimeras, NKAP deletion results in HSC failure. NKAP deletion leads to decreased proliferation and increased apoptosis of HSCs, which is likely due to increased expression of the cyclin-dependent kinase inhibitors p21Cip1/Waf1 and p19Ink4d. These data establish NKAP as one of a very small number of transcriptional regulators that is absolutely required for adult HSC maintenance and survival.

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Increasing C/Ebpα Expression Regulates the Transition of Hematopoietic Stem Cells During Development.

Blood ◽

10.1182/blood.v114.22.1506.1506 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1506-1506

Author(s):

Min Ye ◽

Hong Zhang ◽

Pu Zhang ◽

Daniel G. Tenen

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Conflicts Of Interest ◽

Induced Pluripotent Stem Cell ◽

Gene Profiling ◽

Self Renewal ◽

Hematopoietic Stem ◽

Adult Mice ◽

Induced Pluripotent

Abstract Abstract 1506 Poster Board I-529 During ontogeny, hematopoietic stem cells (HSCs) undergo a change from rapid dividing cells with high reconstitution ability to mainly quiescent cells with lower repopulation capacity. However, little is known about how this switch is regulated. Here we report that levels of C/EBPα, a transcription factor that is frequently disrupted in human acute myeloid leukemia, regulate the proliferation and self-renewal transition of HSCs during development. Loss of C/EBPα in adult mice resulted in a profound expansion of phenotypic HSCs and elevated proliferation rates. Limiting dilution transplantation to measure the frequency of competitive repopulation units (CRU) revealed an increase in the number of functional HSC in C/EBPα-/- mice. Serial transplantation of C/EBPα-/- bone marrow showed advanced reconstitution ability, indicating enhanced self-renewal ability. Interestingly, levels of C/EBPα in HSCs were significantly up-regulated 3 weeks after birth during which HSCs change from an actively cycling state to quiescence in bone marrow. When we conditionally inactivated C/EBPα in mice of different age, we observed a tight correlation between the age-specific levels of C/EBPα expression and the expansion of HSCs. Gene profiling analysis of C/EBPα-/- adult HSCs showed the up-regulation of oncogenes c-myc and n-myc, whose expression can regulate pluripotency and self-renewal of stem cells, as shown by recent induced pluripotent stem cell studies. Knocking down n-myc and c-myc expression in C/EBPα-/- HSCs using shRNA, we observed reduced proliferation and decreased colony formation in serial replating assay, which assesses the preservation of “self-renewal” in the progenitor cell compartment. Consistently, we observed down-regulation of n-myc in HSCs during the transition time, which is reciprocal to C/EBPα expression. Together, our data indicate C/EBPα as a key regulator of HSC self-renewal and proliferation during development, whose levels of expression might control the fetal to adult switch of HSC properties through regulating myc genes. Disclosures No relevant conflicts of interest to declare.

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Ang1 Functions as An Autocrine Activating Factor of Tie2 Signaling In Hematopoietic Stem Cells.

Blood ◽

10.1182/blood.v116.21.1548.1548 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1548-1548

Author(s):

Haruka Momose ◽

Kazuya Takizawa ◽

Madoka Kuramitsu ◽

Takuo Mizukami ◽

Atsuko Masumi ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Molecular Mechanisms ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

Lentiviral Vector ◽

Down Regulation ◽

Self Renewal ◽

Hematopoietic Stem

Abstract Abstract 1548 Hematopoietic stem cells (HSCs) are clonogenic cells that possess the self-renewal capacity to produce more HSCs, as well as the multilineage potential that gives rise to a defined set of mature differentiated progeny for maintenance or repair of the whole blood system. HSCs lie in the hematopoietic niches located along the inner surface of the bone or the sinusoidal endothelium, and are in contact with niche cells. The cell-cell interactions with niche cells are believed to be an important prerequisite to trigger signaling events in HSCs, thereby controlling the balance between HSC self-renewal and differentiation. However, the precise molecular mechanisms regulating niche cell-HSC interactions are not well understood. One of the key molecules for those interactions is Angiopoietin-1 (Ang1). Ang1 is expressed by the niche cells and has been identified as an activating ligand for Tie2 (tyrosine kinase with Ig-like loops and epidermal growth factor homology domains 2). The expression of Tie2 is dominant in HSCs, and Tie2 in HSCs is supposed to be stimulated by Ang1 derived from niche cells. However, Ang1 is also expressed in HSCs. Detailed analysis has shown that Ang1 expression was found to be restricted in long-term HSCs (CD34-lineage-Sca-1+c-Kit+), indicating that Ang1 derived from HSCs plays a role in regulating HSCs. We attempted to elucidate a novel regulating system for HSCs through Ang1-Tie2 signaling by utilizing a hematopoietic cell line in which Tie2 was stably expressed (Ba/F3-Tie2). In Ba/F3-Tie2 cells, Tie2 was found to be phosphorylated on tyrosine residues, even without exogenous addition of Ang1. In the same cells, the expression level of endogenous Ang1 was increased four-fold. When Ang1 expression was down-regulated by transduction with a lentiviral vector expressing short hairpin RNA (shRNA) for Ang1 (shAng1), the phosphorylation of Tie2 was suppressed, suggesting that Tie2 expressed in Ba/F3-Tie2 cells could be stimulated by endogenous Ang1. To mimic the physiological circumstances of the bone marrow, Ba/F3-Tie2 cells were cultured on OP9 stromal cells. Under these culture conditions, the effect of endogenous Ang1 was investigated. Down-regulation of Ang1 by shAng1 demonstrated an approximate 50% reduction in the proliferation of Ba/F3-Tie2 cells on the OP9 cell layer. A HSC-rich population of cells prepared from bone marrow (lineage-Sca-1+c-Kit+; LSK) was also analyzed on OP9 cell layers. Similar to the results obtained from the analysis of Ba/F3-Tie2 cells, down-regulation of Ang1 by shAng1 resulted in an approximately 70% decrease in the proliferation of LSK cells cultured on OP9 monolayers. We confirmed that the suppressive effect on HSC proliferation was due to the lack of Ang1 from HSCs by culturing on Ang1-defective OP9 cells. Finally, we performed in vivo analysis to confirm the importance of endogenous Ang1 to HSCs. Ly5.2 LSK cells transduced with the shAng1 expressing vector were transplanted along with Ly5.1xLy5.2 bone marrow cells into lethally irradiated Ly5.1 mice. The Ly5.2 donor-derived cells in the recipient's peripheral blood were monitored every 2 weeks. As expected, shAng1-introduced donor cells were at decreased ratios at week four (mean ratios, 31.5% for control vs. 17.5% for shAng1), and were reduced to an even lower level at week 12 (mean ratios, 27.1% for control vs. 6.79% for shAng1). This phenomenon was also confirmed by histochemical results, where statistically fewer HSCs existed in the bone marrow of recipient mice in which shAng1-introduced HSCs were transplanted, as compared to the control. Altogether, our data suggested that Tie2 in HSCs could be stimulated by the Ang1 produced by the surrounding HSCs, and this possible autocrine regulation might control the functions of HSCs. Disclosures: No relevant conflicts of interest to declare.

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The Role of Kdm6a in Normal Hematopoiesis

Blood ◽

10.1182/blood.v128.22.1467.1467 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1467-1467

Author(s):

Ling Tian ◽

Lukas D. Wartman

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Conflicts Of Interest ◽

Young Female ◽

Conditional Knockout ◽

Transplant Experiment ◽

Self Renewal ◽

Hematopoietic Stem ◽

Aged Mice

Abstract The role of the X-linked H3K27 demethylase KDM6A in normal hematopoiesis remains unclear. We generated Kdm6a conditional knockout mice (with LoxP sites flanking the 3rd exon) and crossed these mice with Vav1-Cre mice to inactivate Kdm6a in hematopoietic stem/progenitor cells. We characterized normal hematopoiesis from young (6 to 8 week old) and aged (50 to 55 week old) Kdm6a conditional KO mice. We included female and male animals. The inactivation of Kdm6a alone, both in male and female mice, results in a myeloid replating phenotype indicative of aberrant self-renewal. This is present in both young and aged mice with an increased number of colonies at week 2 in the female KO mice as compared to the hemizygous male KO mice. Interestingly, heterozygous female KO mice only acquire this abnormal replating phenotype with age. Next, we found that the genetic inactivation of Kdm6a has age- and gender-dependent effects on primitive hematopoietic cell populations and progenitors (Figure 1). The phenotype outlined in Figure 1 is specific to female Kdm6a-null mice and is not present in male hemizygous or female heterozygous mice at any time point. We went on to show that the decrease in the KLS compartment seen in the young female Kdm6a-null mice is associated with an increase in the fraction of these cells in G0/G1 along with increased apoptosis. The deletion of Kdm6a in young female homozygous mice causes mild thrombocytopenia but in aged mice is associated with anemia and thrombocytopenia. There is also a myeloid skewing with an increased number of neutrophils and a B-cell lymphopenia in the bone marrow, spleen and peripheral blood that becomes more pronounced with age in these mice. The female homozygous KO mice also have mild splenomegaly and an increased number of red blood cell precursors in the spleen. We went on to perform a competitive transplant experiment mixing donor marrow from all cohorts of young Kdm6a conditional KO x Vav1-Cre mice (CD 45.2) with WT competitor marrow (CD 45.1 x 45.2) in a 1:1 ratio, which was then transplanted into lethally-irradiated CD45.1 recipients so that we could easily follow donor versus recipient chimerism. It has been shown that hematopoietic stem cells (HSCs) from female Kdm6a-null mice had a cell migration defect (Thieme S et al., Blood, 2013). We have now shown a decreased repopulation potential for all three cohorts of Kdm6a KO mice (homozygous and heterozygous females and hemizygous males). The finding is most pronounced in the female homozygous Kdm6a KO mice. Strikingly, and in keeping with results noted in the non-transplanted aged mice, there is a selective preservation of the donor SLAM compartment from all cohorts of the Kdm6a conditional KO mice, which is most pronounced in the female Kdm6a-null mice. The competitive repopulation disadvantage was observed when the bone marrow from primary recipients was transplanted into secondary mice. Again, however, relative preservation of the SLAM compartment was sustained. To circumvent the effects of a migration/engraftment defect of female Kdm6a-null stem cells, we repeated a competitive transplant experiment using young, female homozygous Kdm6a conditional KO x ERT2-Cre mice (and appropriate controls). After engraftment (6-weeks post-transplant), these mice were given tamoxifen to activate the Cre locus and to inactivate Kdm6a. In the bone marrow, we achieved approximately 50% floxing of the Kdm6a conditional allele 2-weeks after the last dose of drug. Again, we observed decreased repopulation potential in all lineages with the exception of the relative preservation of the SLAM compartment. We also repeated serial replating assays and showed that the floxed Kdm6a allele increased with serial replating as expected. Finally, we performed gene expression profiling via exon arrays on flow-sorted SLAM cells from aged female Kdm6a-null mice and aged controls. Hierarchical clustering revealed a clear distinction between cohorts. In sum, our data shows that female Kdm6a KO mice have a gender-specific phenotype that emerges with aging and is similar to human myelodysplastic syndrome (MDS). The female KO aged mice have an expansion of their HSCs with aberrant self-renewal, but these HSCs do not differentiate into downstream progeny as in normal hematopoiesis. As such, these mice become anemic and thrombocytopenic-but do not develop overt leukemia or die of these abnormalities. Disclosures No relevant conflicts of interest to declare.

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