Alcam Mediated Cellular Interaction Regulates Hematopoietic Stem Cell Repopulation and Self-Renewal

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1291-1291
Author(s):  
Robin Jeannet ◽  
Qi Cai ◽  
Hongjun Liu ◽  
Hieu Vu ◽  
Ya-Huei Kuo
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Cell Surface ◽  
Hematopoietic Stem Cell ◽  
Cellular Interaction ◽  
Mrna Levels ◽  
Wild Type ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract Abstract 1291 Alcam, which encodes the activated leukocyte cell adhesion molecule (CD166), is a cell surface immunoglobulin superfamily member mediating homophilic adhesion as well as heterotypic interactions with CD6. It has recently been shown that Alcam+ endosteal subset in the bone marrow contain hematopoietic niche cells able to support hematopoietic stem cell (HSC) activity. We examined Alcam mRNA levels and cell surface expression by quantitative RT-PCR and flow cytometry in various hematopoietic stem and progenitor subsets. We found that Alcam is highly expressed in long-term repopulating HSC (LT-HSC), multipotent progenitors (MPP), and granulocyte/macrophage progenitors (GMP). We use an Alcam null mouse allele to assess the function of Alcam in HSC differentiation and self-renewal. Clonogenic colony-forming progenitor serial-replating assay show that the replating potential of Alcam-deficient LT-HSCs is impaired. An in vitro single-cell differentiation assay of phenotypic LT-HSCs reveals that Alcam-deficiency leads to an enhanced granulocytic differentiation. In competitive repopulation transplantation, Alcam-deficient cells show a transient engraftment enhancement, however, the engraftment is significantly lower in secondary transplantation, suggesting that the self-renewal capacity of Alcam-deficient HSC is compromised. We performed a limiting-dilution transplantation assay and determined that the frequency of long-term repopulating cells in Alcam-deficient bone marrow is significantly reduced compared to wild type control. We further assessed the engraftment efficiency of phenotypically purified LT-HSCs. We show that the engraftment efficiency of Alcam-deleted LT-HSCs is significantly reduced compared to wild type LT-HSCs. Since Alcam-deleted HSCs are able to home efficiently to the bone marrow cavity, the engraftment defect is not due to inefficient homing upon transplantation. Collectively, These studies implicate Alcam mediated cell-cell interaction in the regulation of hematopoietic transplantation and recovery. Disclosures: No relevant conflicts of interest to declare.

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

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

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

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

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

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

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

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

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

Superior Impact of p18INK4c over p27kip1 on Long-Term Repopulating Ability of Hematopoietic Stem Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 796-796
Author(s):  
Hui Yu ◽  
Hongmei Shen ◽  
Xianmin Song ◽  
Paulina Huang ◽  
Tao Cheng
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Cell Biology ◽  
Wild Type ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
The Impact

Abstract The G1-phase is a critical window during the cell cycle in which stem cell self-renewal may be balanced with differentiation and apoptosis. Increasing evidence suggests that the cyclin-dependent kinase inhibitors (CKIs) such as p21Cip1/Waf1, p27kip1, p16INK4A, and p18INK4C (p21, p27, p16 and p18 hereafter) are involved in stem cell self-renewal, as largely demonstrated in murine hematopoietic stem cells (HSCs). For example, we have recently demonstrated a significant increase of HSC self-renewal in the absence of p18 (Yuan et al, Nature Cell Biology 2004). But the actual roles of these CKIs in HSCs appear to be distinct as p21 and p18 have opposite effects (Yu H et al, ASH 2004) whereas p16 has a limited effect (Stepanova et al, Blood 2005) on HSC exhaustion after serial bone marrow transfer. Like p18, however, p27 was recently reported to also inhibit HSC self-renewal due to the fact that the competitive repopulating units (CRUs) were increased in p27−/− mouse bone marrow (Walkley et al, Nature Cell Biology 2005) in contrast to the results in a previous report (Cheng T et al, Nature Medicine 2000). To further gauge the impact of p18 versus p27 on the long-term repopulating ability (LTRA) of HSCs, we have generated different congenic strains (CD45.1 and CD45.2) of p18−/− or p27−/− mice in the C57BL/6 background, allowing us to compare them with the competitive repopulation model in the same genetic background. The direct comparison of LTRA between p18−/− and p27−/− HSCs was assessed with the competitive bone marrow transplantation assay in which equal numbers of p18−/− (CD45.2) and p27−/− cells (CD45.1) were co-transplanted. Interestingly, the p18−/− genotype gradually dominated the p27−/− genotype in multiple hematopoietic lineages and p18−/− HSCs showed 4-5 times more LTRA than p27−/− HSCs 12 months after cBMT. Further self-renewal potential of HSCs was examined with secondary transplantation in which primarily transplanted p18−/− or p27−/− cells were equally mixed with wild-type unmanipulated cells. Notably, while the p18−/− cells continued to outcompete the wild-type cells as we previously observed, the p27−/− cells did not behave so in the secondary recipients. Based on the flow cytometric measurement and bone marrow cellularity, we estimated that transplanted p18−/− HSCs (defined with the CD34−LKS immunophenotype) had undergone a 230-fold expansion, while transplanted p27−/− and wild-type HSCs had only achieved a 6.6- and 2.4-fold expansion in the secondary recipients respectively. We further calculated the yield of bone marrow nucleated cells (BMNCs) per HSC. There were approximately 44 x 103, 20.6 x 103, and 15 x 103 BMNCs generated per CD34−LKS cell in p18−/−, p27−/− and wild-type transplanted recipients respectively. Therefore, the dramatic expansion of p18−/− HSCs in the hosts was not accompanied by decreased function per stem cell. Our current study demonstrates that hematopoietic engraftment in the absence of p18 is more advantageous than that in the absence of p27, perhaps due to a more specific role of p18 on self-renewal of the long-term repopulating HSCs.

p16INK4a Is a Key Downstream Mediator of the Deleterious Effects of FoxO Deficiency on Maintenance of the Hematopoietic Stem Cell Compartment.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1405-1405
Author(s):  
Zuzana Tothova ◽  
Stephen M Sykes ◽  
Dena S Leeman ◽  
James W Horner ◽  
Norman Sharpless ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Accelerated Aging ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Hematopoietic System ◽  
Downstream Mediator

Abstract Regulation of oxidative stress in the hematopoietic stem cell (HSC) compartment is critical for the maintenance of HSC self-renewal. A number of reports have previously implicated p16 in aging of HSCs, pancreatic β-islet cells and subventricular zone progenitors in the brain [1–3]. In the context of the hematopoietic system, p16INK4a expression in HSCs increases with age, and correlates with decreased HSC repopulating ability, decreased self-renewal, and increased apoptosis with stress [1]. We and others have recently reported that FoxO play essential roles in the response to physiologic oxidative stress and thereby mediate quiescence and enhanced survival in the HSC compartment [4, 5]. Young mice deficient in FoxO1, FoxO3, and FoxO4 in the adult hematopoietic system, with striking similarity to aging wild-type mice, show a defect in bone marrow repopulating ability, decrease in self-renewal, myeloid skewing in differentiation and increased levels of apoptosis. Furthermore, young FoxO-deficient HSC show increased levels of p16 when compared to their wildtype counterparts. These collective findings suggested the possibility that FoxO loss could result in accelerated aging of HSC due to increased expression of p16 as a consequence of increased ROS. To test the hypothesis that p16 is one of the key mediators of FoxO loss responsible for accelerated aging of HSC, we deleted FoxO1, FoxO3, and FoxO4 in the adult hematopoietic system of mice deficient in p16INK4a. Young mice deficient in FoxO and p16 shared the same characteristics of their HSC(Lin−Sca1+c-kit+) compartment as mice deficient in FoxO only, including decreased number of HSC, increased percentage of HSC entering S/G2/M and apoptosis, and increased levels of ROS as compared to their wildtype counterparts. However, in a setting of long-term repopulation studies, bone marrow isolated from mice deficient in p16 and FoxO demonstrated a rescue of long-term repopulation for up to 20 weeks, as compared to FoxO deficient bone marrow that showed a severe defect in long-term repopulation. p16 deficiency in the setting of FoxO deficiency did not result in reduction of ROS levels in the HSC compartment. Taken together, these findings indicate that p16 is a critical downstream mediator of FoxO in the maintenance of the HSC compartment, and that it can dissociate the detrimental effects of ROS on HSC self-renewal in a setting of FoxO deficiency.

Pivotal Role for Gsk3 in Hematopoietic Stem Cell Homeostasis

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1292-1292
Author(s):  
Jian Huang ◽  
Peter S. Klein
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Signaling Pathways ◽  
Hematopoietic Stem Cell ◽  
Pivotal Role ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Gsk3 Inhibitor

Abstract Abstract 1292 Hematopoietic stem cells (HSCs) maintain the ability to self-renew and to differentiate into all lineages of the blood. The signaling pathways regulating hematopoietic stem cell (HSCs) self-renewal and differentiation are not well understood. We are very interested in understanding the roles of glycogen synthase kinase-3 (Gsk3) and the signaling pathways regulated by Gsk3 in HSCs. In our recent study (Journal of Clinical Investigation, December 2009) using loss of function approaches (inhibitors, RNAi, and knockout) in mice, we found that Gsk3 plays a pivotal role in controlling the decision between self-renewal and differentiation of HSCs. Disruption of Gsk3 in bone marrow transiently expands HSCs in a μ-catenin dependent manner, consistent with a role for Wnt signaling. However, in long-term repopulation assays, disruption of Gsk3 progressively depletes HSCs through activation of mTOR. This long-term HSC depletion is prevented by mTOR inhibition and exacerbated by μ-catenin knockout. Thus GSK3 regulates both Wnt and mTOR signaling in HSCs, with opposing effects on HSC self-renewal such that inhibition of Gsk3 in the presence of rapamycin expands the HSC pool in vivo. These findings identify unexpected functions for GSK3 in HSC homeostasis, suggest a therapeutic approach to expand HSCs in vivo using currently available medications that target GSK3 and mTOR, and provide a compelling explanation for the clinically prevalent hematopoietic effects of lithium, a widely prescribed GSK3 inhibitor. In the following study, we found that the combination of Gsk3 inhibitor and mTOR inhibitor can expand phenotypic HSCs in vivo and maintain functional HSC in ex vivo culture. This study will provide the basis for a new clinical approach to improve the efficiency of bone marrow transplantation. Disclosures: Klein: Follica: Consultancy.

Enhanced Notch Signaling Skews Hematopoietic Stem Cell Differentiation in Fanconi Anemia Murine Models

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1191-1191
Author(s):  
Wei Du ◽  
Jared Sipple ◽  
Jonathan Schick ◽  
Qishen Pang
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Stem Cell ◽  
Notch Signaling ◽  
Hematopoietic Stem Cell ◽  
Fanconi Anemia ◽  
Gfp Expression ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract Abstract 1191 Objective: Hematopoietic stem cells (HSCs) can either self-renew or differentiate into various types of cells of the blood lineage. Little is known about the signaling pathways that regulate this choice of self-renewal versus differentiation. We studied the effect of altered Notch signaling on HSC differentiation in mouse models of Fanconi anemia (FA), a genetic disorder associated with bone marrow failure and progression to leukemia and other cancers. Methods: The study used a Notch reporter mouse, in which Notch-driven GFP expression acts as a sensor for HSC differentiation. Long-term hematopoietic stem cell (LT-HSC) and multipotential progenitor (MPP) cell compartments, as well as GFP expression in different cell populations were detected by Flow Cytometry analysis using primary bone marrow cells from Notch-eGFP-WT, Notch-eGFP-Fanca−/− or Notch-eGFP-Fancc−/− mice. Cell Cycle analysis was performed to distinguish the difference of quiescent state in GFP-gated LSK cells from these Notch-eGFP reporter mice. Colony forming units (CFU) assay and bone marrow transplantation (BMT) were utilized to determine HSC self-renew capacity. Gene arrays for pathways involved in DNA repair, cell cycle control, anti-oxidant defense, inflammatory response and apoptotic signaling were employed to define the gene expression signatures of the MPP population. Results and conclusions: In mice expressing a transgenic Notch reporter, deletion of the Fanca or Fancc gene enhanced Notch signaling in MPPs, which was correlated with decreased phenotypic long-term HSCs and increased formation of MPP1 progenitors. Furthermore, we found a functional correlation between Notch signaling and self-renewal capacity in FA hematopoietic stem and progenitor cells (HSPCs). Significantly, we show that FA deficiency in MPPs deregulates a complex network of genes in the Notch and canonical NF-kB pathways. Specifically, enhanced Notch signaling in FA MPPs was associated with the unregulation of genes involved in inflammatory and stress responses (including Rela, Tnfrsf1b, Gadd45b, Sod2, Stat1, Irf1 and Xiap), cell-cycle regulation (including Ccnd1, Cdc16, Cdkn1a, Gsk3b, Notch2 and Nr4a2), and transcription regulation (including Rela, Stat1, Hes1, Hey1, Hoxb4, Notch1 and Notch2). Consequently, TNF-a stimulation enhanced Notch signaling of FA LSK cells, leading to decreased HSC quiescence and compromised HSC self-renewal. Finally, genetic ablation of NF-kB reduced Notch signaling in FA MPPs to nearly wide-type level, and blocking either NF-kB or Notch signaling partially restored FA HSC quiescence and self-renewal capacity. Translational Applicability: The study identifies a functional interaction between the FA pathway and Notch signaling in HSC differentiation and establishes a role of FA proteins in the control of balance between renewal and lineage commitment, hence contributing to hematopoiesis. These findings indicate that the Notch signaling pathway may represent a novel and therapeutically accessible pathway in FA. Disclosures: No relevant conflicts of interest to declare.

Disruption of the Osteoblast Niche by G-CSF Is Associated with Hematopoietic Stem Cell Quiescence and Loss of Long-Term Repopulating Activity: Role of Cdkn1a

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2447-2447
Author(s):  
Priya K. Gopalan ◽  
Matthew J. Christopher ◽  
Adam M. Greenbaum ◽  
Daniel C. Link
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Bone Marrow Microenvironment ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Stem Cell Quiescence ◽  
A Cell ◽  
Rna Expression Profiling

Abstract The bone marrow microenvironment plays a key role in regulating hematopoietic stem cell (HSC) function. In particular, bone marrow stromal signals contribute to the maintenance of HSC quiescence, a property that is thought to be associated with long-term repopulating activity. We previously reported that G-CSF treatment disrupts the osteoblast niche by inducing osteoblast apoptosis and inhibiting osteoblast differentiation. In this altered bone marrow microenvironment, we also showed that the number of HSCs in the bone marrow after G-CSF treatment (as defined by CD34− Kit+ Sca+ lineage-cells or CD150+ CD48− CD41− lineage-[SLAM] cells) was unchanged and that the HSCs were more quiescent than HSCs from untreated mice. However, despite the quiescent phenotype, there was a marked loss of HSC long-term repopulating activity. To define mechanisms for this phenotype, we first asked whether G-CSF acts directly on HSCs to inhibit their long-term repopulating activity. Bone marrow chimeras containing wild type and G-CSFR−/− cells were established and treated with G-CSF. The contribution of G-CSFR−/− cells to hematopoiesis remained stable for at least 3 months after G-CSF treatment, demonstrating that the effects of G-CSF on HSC function are not direct. We next performed RNA expression profiling on sorted SLAM cells, a cell population highly enriched for HSCs. These data showed that expression of Cdkn1a (p21cip1/waf1) was increased in HSCs harvested from G-CSF treated mice. To define the contribution of Cdkn1a to HSC quiescence and loss of repopulating activity following treatment with G-CSF, Cdkn1a−/− mice (inbred on a C57BL/6 background) were studied. Wild-type or Cdkn1a−/− mice were treated with G-CSF for 7 days and pulse labeled with bromo-deoxyuridine (BrdU), and the percentage of SLAM cells that labeled with BrdU was determined. Consistent with our previous observations, treatment of wild-type mice with G-CSF resulted in a significant decrease in the percentage of BrdU+ SLAM cells in the bone marrow. In contrast, in Cdkn1a−/− mice, no change in the percentage of BrdU+ SLAM cells after G-CSF treatment was observed [10.08 ± 2.26% (untreated); 10.96 ± 2.80% (G-CSF treated); p = NS]. To assess HSC function, competitive repopulation assays were performed using untreated or G-CSF treated bone marrow from wild type or Cdkn1a−/− mice. Surprisingly, G-CSF had a similar deleterious effect on HSC repopulating activity in both wild type and Cdkn1a−/− mice. Collectively, these data show G-CSF treatment, possibly through disruption of the osteoblast niche, induces HSC quiescence and loss of long-term repopulating activity. HSC quiescence, but not loss of repopulating activity, is dependent upon Cdkn1a−/−. The mechanisms by which G-CSF treatment results in a loss of HSC function are under investigation.

The p110α Catalytic Isoform of PI3 Kinase Is Important for Erythropoiesis, but Has a Minimal Role in Hematopoietic Stem Cell Self-Renewal.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3620-3620
Author(s):  
Kira Gritsman ◽  
Tulasi Khandan ◽  
Rachel Okabe ◽  
Maricel Gozo ◽  
Mahnaz Paktinat ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Hematopoietic Cells ◽  
Pi3 Kinase ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cell Counts

Abstract Abstract 3620 Poster Board III-556 PIK3CA, which encodes the p110α catalytic isoform of PI3 kinase (PI3K), is mutated in many human cancers, and is an attractive therapeutic target. However, PI3K may also be important during hematopoiesis, as it is activated by hematopoietic growth factor receptors which control hematopoietic stem cell (HSC) and progenitor proliferation, differentiation, and self-renewal, such as erythropoietin receptor (epoR), c-kit receptor, and fms-like tyrosine kinase 3 (FLT3). In hematopoietic cells, receptor tyrosine kinases signal through the catalytic p110 subunit of PI3K, which has 3 isoforms (α, β, δ). However, the roles of PI3K and its specific catalytic isoforms in normal HSC function are poorly understood. We hypothesized that signaling through the p110α isoform is important for hematopoiesis and HSC self-renewal. We have used the Cre-loxP system to delete p110α in the HSCs of adult mice by breeding p110αF/F mice to Mx1-Cre transgenic mice. p110αF/F;Mx1-Cre+ (Cre+) mice and their p110αF/F (Cre-) littermates were injected with PolyI-PolyC (pIpC) at 4-6 weeks of age to induce Cre-mediated excision at the PIK3CA locus specifically in hematopoietic cells. Deletion of p110α in the bone marrow (BM) was verified by PCR and by immunoblot. We observed that, by four weeks after pIpC treatment, Cre+ mice developed microcytic anemia compared with Cre- littermates, characterized by a decreased mean hemoglobin (p<0.0001) and decreased mean corpuscular volume, while white blood cell counts and platelet counts were unaffected. Cre+ mice also had significantly decreased spleen, liver, and thymus weights. Flow cytometry analyses of bone marrow and spleen cells revealed a relative block in erythropoiesis in the spleens of Cre+ mice, with expansion of the basophilic erythroblast population, and a decrease in the most mature nucleated erythroblast population. Colony assays of splenocytes in erythropoietin-containing methylcellulose medium revealed a 52% decrease in BFU-E colony formation by p110α-deleted cells in response to erythropoietin, suggesting that loss of p110α may lead to blunted epoR signaling (p=0.009). Multiparameter flow cytometry revealed that the overall number of Lin-Sca1+ckit+ (LSK) cells, which contains the HSC population, was increased two-fold in the BM of Cre+ mice compared with Cre- littermates (p=0.01). To determine whether loss of p110α affects long-term HSC self-renewal in vivo, we performed competitive repopulation assays, in which CD45.2+ BM cells from PIPC-treated Cre+ mice or Cre- controls were transplanted together with CD45.1+CD45.2+ competitor BM cells into lethally irradiated CD45.1+ recipient mice. Donor BM chimerism (%CD45.2+ cells) at 16 weeks was mildly reduced in the absence of p110α, but Cre+ cells were still capable of long-term reconstitution. In summary, we have found that the p110α catalytic isoform is specifically required for erythropoiesis, but has only a small role in HSC homeostasis and in differentiation of the other hematopoietic lineages. This suggests that pharmacologic targeting of p110α in cancer therapy may result in mild anemia, but should otherwise have minimal hematologic toxicity. Disclosures: Gilliland: Merck Research Laboratories: Employment. Roberts:Novartis Pharmaceuticals, Inc.: Consultancy. Zhao:Novartis Pharmaceuticals, Inc.: Consultancy.

SIRT1 Deacetylase Is Essential for Hematopoietic Stem Cell Activity Via Regulation of Foxo3.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2315-2315 ◽  
Author(s):  
Pauline Rimmele ◽  
Carolina L. Bigarella ◽  
Valentina d'Escamard ◽  
Brigitte Izac ◽  
David Sinclair ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Bone Marrow Cells ◽  
Leukemic Stem Cells ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Marrow Cells

Abstract Abstract 2315 SIRT1 is a member of the NAD-dependent family of sirtuin deacetylases with critical functions in cellular metabolism, response to stress and aging. Although SIRT1 is clearly a regulator of embryonic stem cells, reports on the function of SIRT1 in adult hematopoietic stem cell (HSC) have been conflicting. While SIRT1 was positively associated with HSC activity on a genetic screen, using a germline deletion of SIRT1 three groups found SIRT1 to be dispensable for adult HSC. Here, we first showed that nuclear SIRT1 expression is enriched in bone marrow-derived Lin−Sca1+cKit+ (LSK) cells, as compared to total bone marrow cells. Germline deletion of SIRT1 is associated with developmental defects and high perinatal mortality resulting in only 10% of mice reaching adulthood. To circumvent the potential developmental adaptation of these mice, we used an adult-tamoxifen inducible SIRT1 knockout mouse model. Full-length SIRT1 protein was nearly undetectable in the bone marrow and spleen of SIRT1−/− mice. Analysis of wild type and SIRT1−/− bone marrow cells, 4 weeks after tamoxifen treatment, showed that loss of SIRT1 increased the size and frequency of the LSK compartment. Interestingly, this was associated with a significant decrease in the frequency of long-term repopulating HSC as determined by SLAM markers (CD48−CD150+LSK) within LSK cells. This decrease was even more pronounced with time. In agreement with these results, the long-term repopulation ability of CD48−CD150+LSK cells is severely compromised in SIRT1−/− mice as measured 16 weeks after transplantation, strongly suggesting that SIRT1 is essential for long-term HSC function. Thus, loss of SIRT1 results in loss of long-term repopulating stem cells in favor of total LSK cells that is a more heterogeneous population of stem cells. SIRT1 has several substrates with a potential function in HSC. Among these, we focused on Foxo3 Forkhead transcription factor which is essential for the maintenance of hematopoietic and leukemic stem cell pool. Despite the importance of Foxo3 to the control of HSC function, mechanisms that regulate Foxo3 activity in HSC remain unknown. Negative regulation of FoxOs by AKT phosphorylation promotes their cytosolic localization in response to growth factors stimulation. Interestingly, Foxo3 is constitutively nuclear in bone marrow LSK and in leukemic stem cells, strongly suggesting that negative phosphorylation may not be the sole Foxo3 regulatory mechanism in these stem cells. FoxO proteins are regulated by several post-translational modifications including acetylation in addition to phosphorylation, although the impact of acetylation on Foxo3 function remains unresolved. Therefore, we asked whether regulation of adult HSC activity by SIRT1 deacetylase is mediated by Foxo3. The in vivo injection of sirtinol, a SIRT1 inhibitor, for 3 weeks compromised significantly the long-term repopulation capacity of wild type but not Foxo3−/− HSC as measured by the repopulation ability of CD48−CD150+LSK cells in lethally irradiated mice after 16 weeks. These results suggest that Foxo3 is likely to be required for SIRT1 regulation of HSC activity. In agreement with this, we showed that in contrast to wild type LSK cells, Foxo3 is mostly cytoplasmic in SIRT1−/− LSK cells, indicating that loss of SIRT1 is sufficient to translocate Foxo3 to the cytosol and presumably inhibit its activity. We further showed that ectopically expressed acetylation-mimetic mutant of Foxo3 where all putative acetyl-lysine residues are mutated to glutamine, in bone marrow mononuclear cells, is mostly localized in the cytosol in contrast to wild type Foxo3 protein and results in significant decrease of colony-forming unit-spleen (CFU-S) activity. Using pharmacological antagonism as well as conditional deletion of SIRT1 in adult HSC, we identified a critical function for SIRT1 in the regulation of long-term HSC activity. Our results contrast with previously published data obtained from germline deleted SIRT1 mice, and suggest that the use of a conditional approach is essential for unraveling SIRT1 function in adult tissues. Our data also suggest that SIRT1 regulation of HSC activity is through activation of Foxo3. These findings are likely to have an important impact on our understanding of the regulation of hematopoietic and leukemic stem cells and may be of major therapeutic value for hematological malignancies and disorders of stem cells and aging. Disclosures: No relevant conflicts of interest to declare.

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