HSPC Mobilization Research Articles

Noncoding GATA2 Genetic Variation As a Determinant of Hematopoietic Stem/Progenitor Cell Activity and Mobilization Efficiency

Germlinecoding and enhancer variants within the hematopoietic regulator GATA2 create a bone marrow failure and leukemia predisposition (Soukup and Bresnick, 2020). We generated a mouse model of GATA2 deficiency syndrome by engineering mice with a single-nucleotide variant in an intronic enhancer (+9.5) from GATA2 deficiency patients combined with deletion of the enhancer from the second allele (compound heterozygous; CH). This system models patients harboring a germline pathogenic allele accompanied by epigenetic silencing of the normal allele. Unlike homozygous Gata2 coding or regulatory deletions that are embryonic lethal, CH represents a unique system to analyze postnatal GATA2 function. We demonstrated that adult germline Gata2 variation alters steady-state HSPCs, blocks HSPC expansion and differentiation upon chemotherapy, attenuates long-term repopulating activity following bone marrow transplantation, leads to bone marrow failure following chronic inflammation by the viral mimetic polyI:C, alters HSPC response to the bacterial cell wall component LPS and attenuates HSPC mobilization in response to granulocyte colony stimulating factor (G-CSF) (Soukup et al., 2019, Soukup et al., 2021). As short-term G-CSF treatment induces HSPC metabolic gene signatures (Fast et al., 2021) and expansion and cell cycle entry (Schuettpelz et al., 2014), and CH have decreased MPPs (LSKCD150 -CD48 -) and common myeloid progenitors (CMPs) (Lin −Sca1 −Kit +FcγR −CD34 +) (Soukup et al., 2021), we asked if mobilization defects reflected decreased functional HSPCs in bone marrow. CH had 2.2-fold fewer colony forming units (CFU) from bone marrow (p = 0.007) than wild type (WT), reflecting a paucity of functional HSPCs. 16 hours following a single G-CSF dose, WT bone marrow immunophenotypic HSCs (LSKCD150 +CD48 -) and MPPs expanded 1.8-fold (p = 0.001 and 0.01, respectively); CH HSCs and MPPs were unchanged. CH led to decreased HSPCs and failed to induce rapid HSPC expansion post-G-CSF. We tested whether Gata2 variation impairs mobilization in response to different stimuli, or if select pathways are impacted via disrupting the CXCR4/CXCL12 chemokine axis with the CXCR4 antagonist plerixafor, stromal/neutrophil CXCR2 activation with rhIL-8, or VLA-4/VCAM1 binding with VLA-4 inhibitor BIO5192. IL-8 or BIO5192 resulted in 3.9- and 2.2-fold increased WT CFU, respectively (p < 0.0001 and 0.0014) without altering CH CFU. By contrast, mobilization with plerixafor resulted in a 4.3-fold increase in WT CFU (p < 0.0001) and 4.5-fold increase in CH CFU (p = 0.004). Targeting these pathways collectively with G-CSF was insufficient to restore normal mobilization in CH. Competitive transplantations with peripheral blood revealed G-CSF + plerixafor increased WT donor-derived hematopoiesis 24- and 20-fold at 8 and 12 weeks, respectively, versus vehicle control. Treatment of CH did not increase donor-derived contributions. Since GATA2 deficiency syndrome and somatic GATA2 variation involve heterozygous alleles, we tested whether monoallelic +9.5 Ets motif variation disrupts mobilization. G-CSF increased CFU 28.5-fold (p < 0.0001) relative to vehicle-treated WT. Mobilization of Gata2 Ets +/- HSPCs were reduced significantly (p = 0.003). CH variation severely disrupted mobilization, with 7.3-fold fewer CFU than Gata2 Ets +/- (p = 0.02). Synergistic G-CSF/plerixafor treatment increased CFU 89-fold in WT versus untreated (p < 0.0001), and Gata2 Ets +/- mobilization was reduced (p = 0.006) and not improved relative to Gata2 Ets -/- or CH, resembling G-CSF/BIO5192. Thus, Gata2 heterozygosity impaired G-CSF-induced HSPC mobilization with all regimens. Although GATA2 noncoding variation is not commonly evaluated, analysis of human data will be performed to assess relationships between variation and poor mobilization outcomes. Gata2 variation depletes functional HSPCs in the bone marrow, reducing the available HSPCs to mobilize and rendering most mobilization regimens inefficient. Ongoing studies are elucidating molecular mechanisms underlying the mobilization blockade. Our mobilization-defective system offers unique utility for elucidating fundamental HSPC mechanisms e.g., in the context of additional environmental and/or genetic insults. Future knowledge linking functionality of mobilization regimens in these contexts may unveil insights into mechanisms operational in poor mobilizers.

IL-1β expression in bone marrow dendritic cells is induced by TLR2 agonists and regulates HSC function

AbstractHematopoietic stem/progenitor cells (HSPCs) reside in localized microenvironments, or niches, in the bone marrow that provide key signals regulating their activity. A fundamental property of hematopoiesis is the ability to respond to environmental cues such as inflammation. How these cues are transmitted to HSPCs within hematopoietic niches is not well established. Here, we show that perivascular bone marrow dendritic cells (DCs) express a high basal level of Toll-like receptor-1 (TLR1) and TLR2. Systemic treatment with a TLR1/2 agonist induces HSPC expansion and mobilization. It also induces marked alterations in the bone marrow microenvironment, including a decrease in osteoblast activity and sinusoidal endothelial cell numbers. TLR1/2 agonist treatment of mice in which Myd88 is deleted specifically in DCs using Zbtb46-Cre show that the TLR1/2-induced expansion of multipotent HPSCs, but not HSPC mobilization or alterations in the bone marrow microenvironment, is dependent on TLR1/2 signaling in DCs. Interleukin-1β (IL-1β) is constitutively expressed in both murine and human DCs and is further induced after TLR1/2 stimulation. Systemic TLR1/2 agonist treatment of Il1r1−/− mice show that TLR1/2-induced HSPC expansion is dependent on IL-1β signaling. Single-cell RNA-sequencing of low-risk myelodysplastic syndrome bone marrow revealed that IL1B and TLR1 expression is increased in DCs. Collectively, these data suggest a model in which TLR1/2 stimulation of DCs induces secretion of IL-1β and other inflammatory cytokines into the perivascular niche, which in turn, regulates multipotent HSPCs. Increased DC TLR1/2 signaling may contribute to altered HSPC function in myelodysplastic syndrome by increasing local IL-1β expression.

Conditionally Pathogenic Genetic Variants of a Hematopoietic Disease-Suppressing Enhancer

Human genetic variants are classified based on potential pathogenicity to guide clinical decisions. However, mechanistic uncertainties often preclude definitive categorization. Germline coding and enhancer variants within the hematopoietic regulator GATA2 create a bone marrow failure and leukemia predisposition (Soukup and Bresnick, 2020). The conserved murine enhancer (+9.5) promotes hematopoietic stem cell (HSC) genesis, and a single-nucleotide human variant in an Ets motif attenuates chemotherapy- and transplantation-induced hematopoietic regeneration in a cell-autonomous manner (Soukup et al., 2019). Since an epigenetically-silenced normal allele can exacerbate phenotypes of a pathogenic heterozygous variant, we engineered a bone marrow failure model harboring the Ets motif variant and a severe E-box:GATA enhancer mutation on the second allele (CH) that renders the enhancer chromatin inaccessible, to determine how perturbations of the hematopoietic system trigger pathogenesis.Given the propensity of GATA2 deficiency syndrome patients to infection, we asked if CH mice could mount an appropriate response to the bacterial cell wall component LPS. In WT, LPS acts on HSCs to induce inflammatory signaling and proliferation (Caiado et al., 2021). Twenty-four hours after LPS (0.5 mg/kg) administration, CH LSK levels were 3.1-fold lower than WT (p = 0.009). Immunophenotypic LT-HSCs (LSKCD150 +CD34 -) were 1.9-fold lower in treated CH vs. WT mice (p = 0.019). LPS reduced all LSK subpopulations: CD34 -CD150 -, CD34 +CD150 -, and CD34 +CD150 + were 3.4-, 5.7-, and 5.3-fold lower, respectively (p = 0.007, 0.029, and 0.006). Thus, the CH mutation attenuated acute inflammation-induced HSPC expansion.G-CSF is among the cytokines/chemokines induced by infection (Quinton et al., 2002). As G-CSF is used to mobilize bone marrow HSCs for collection prior to transplantation, we tested the following models: 1) +9.5 mutation abrogates HSPC expansion and/or mobilization in response to diverse stimuli; 2) +9.5 mutation permits expansion/mobilization, yet compromises function; 3) HSPC expansion, mobilization and function are +9.5 mutation-insensitive. After 8 doses of G-CSF, circulating neutrophil numbers increased 2.6-fold in WT (p = 0.003), while neutrophils did not increase in the CH mice. G-CSF treatment increased bone marrow HSCs 2.6-fold in WT mice, whereas the CH mutation abrogated this response (p < 0.0001). As a metric of mobilization, splenic HSCs were quantified. After G-CSF treatment, WT splenic HSCs expanded 11-fold (p < 0.0001), while CH HSCs were unchanged. To functionally analyze mobilized HSPCs, colony forming unit (CFU) assays were conducted with peripheral blood. CFU increased 11-fold in WT blood post-treatment (p < 0.0001), and CH abrogated the increase.To test whether G-CSF signaling is impacted by the CH mutation, we asked whether G-CSF treatment of bone marrow ex vivo induced STAT3 phosphorylation using a phospho-flow cytometry assay. While G-CSF induced pSTAT3 8.5-fold in WT Lin -Kit + cells (p < 0.0001), the induction was attenuated to 4.9-fold in CH (p = 0.003). Signaling in Gr1 + granulocytes was indistinguishable between genotypes (p = 0.999). Thus, the CH mutation attenuated G-CSF-induced signaling and HSPC mobilization, abrogated HSPC expansion, and compromised HSPC function.The Gata2 CH bone marrow failure model is characterized by relatively normal developmental hematopoiesis and adult steady-state hematopoiesis, yet the mice are vulnerable to stressors. As the CH mice were hypersensitive to myeloablation and acute inflammation and exhibited an attenuated response to a clinical HSC-mobilizing agent, GATA2-driven regenerative hematopoiesis mitigates damage resulting from chemotherapy and inflammation and promotes G-CSF-induced HSC mobilization. As the enhancer variant controlled HSC responsiveness to a therapeutically important HSC-mobilizing drug, these results link non-coding genome variation with mobilization efficacy. Based on the prominent GATA2 activities described herein, we are investigating how the full ensemble of macromolecular components constituting the GATA2 regulatory network generate a vital protective system conferring resilience to the hematopoietic system in the context of diverse stressors and whether genetic variants in genes encoding components of these pathways resemble or differ from those of GATA2 variants. DisclosuresNo relevant conflicts of interest to declare.

Tetraspanin CD82 regulates S1PR1-mediated hematopoietic stem and progenitor cell mobilization.

SummaryHematopoietic stem and progenitor cell (HSPC) mobilization into the blood occurs under normal physiological conditions and is stimulated in the clinic to enable the isolation of HSPCs for transplantation therapies. In the present study, we identify the tetraspanin CD82 as a novel regulator of HSPC mobilization. Using a global CD82 knockout (CD82KO) mouse, we measure enhanced HSPC mobilization after granulocyte-colony stimulating factor (G-CSF) or AMD3100 treatment, which we find is promoted by increased surface expression of the sphingosine 1-phosphate receptor 1 (S1PR1) on CD82KO HSPCs. Additionally, we identify a disruption in S1PR1 internalization in CD82-deficient HSPCs, suggesting that CD82 plays a critical role in S1PR1 surface regulation. Finally, combining AMD3100 and anti-CD82 treatments, we detect enhanced mobilization of mouse HSPCs and human CD34+ cells in animal models. Together, these data provide evidence that CD82 is an important regulator of HSPC mobilization and suggests exploiting the CD82 scaffold as a therapeutic target to enhance stem cell isolation.

Oncostatin M regulates hematopoietic stem cell (HSC) niches in the bone marrow to restrict HSC mobilization.

We show that pro-inflammatory oncostatin M (OSM) is an important regulator of hematopoietic stem cell (HSC) niches in the bone marrow (BM). Treatment of healthy humans and mice with granulocyte colony-stimulating factor (G-CSF) dramatically increases OSM release in blood and BM. Using mice null for the OSM receptor (OSMR) gene, we demonstrate that OSM provides a negative feed-back acting as a brake on HSPC mobilization in response to clinically relevant mobilizing molecules G-CSF and CXCR4 antagonist. Likewise, injection of a recombinant OSM molecular trap made of OSMR complex extracellular domains enhances HSC mobilization in poor mobilizing C57BL/6 and NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ mice. Mechanistically, OSM attenuates HSC chemotactic response to CXCL12 and increases HSC homing to the BM signaling indirectly via BM endothelial and mesenchymal cells which are the only cells expressing OSMR in the BM. OSM up-regulates E-selectin expression on BM endothelial cells indirectly increasing HSC proliferation. RNA sequencing of HSCs from Osmr-/- and wild-type mice suggest that HSCs have altered cytoskeleton reorganization, energy usage and cycling in the absence of OSM signaling in niches. Therefore OSM is an important regulator of HSC niche function restraining HSC mobilization and anti-OSM therapy combined with current mobilizing regimens may improve HSPC mobilization for transplantation.

O-Fucose and Fringe-Modified Recombinant Notch Peptides As Decoy Molecules to Promote Hematopoietic Progenitor Mobilization

Nearly all autologous hematopoietic cell therapy (HCT) procedures are performed using mobilized hematopoietic progenitor cells (HPCs). Successful outcome is dependent on infusing an adequate number of functionally active HPCs. Mobilization strategy of AMD3100, in combination with G-CSF, increases total CD34+ cells mobilized, and often is used for HPC mobilization in myeloma and non-Hodgkin lymphoma patients. Unfortunately, this approach does not always lead to adequate HPC collection in patients who previously have been exposed to extensive cytotoxic therapy. Furthermore, suboptimal number of HPCs mobilized in some patients may result in slower hematopoietic reconstitution and increased risk of complications during the transplant. Hence developing new mobilization strategy is highly desired for patients who are considered poor-mobilizers. Notch is a signaling molecule important for stem cell self-renewal and fate determination in many tissues including the hematopoietic system. An important feature of Notch is its adhesive nature which was first described by cell aggregation assays in Drosophila studies. We previously reported that hematopoietic stem cell and progenitors (HSPCs) with faulty Notch-ligand interaction display increased cell cycling, decreased adhesion to marrow osteoblastic lineage cells, and enhanced egress from the marrow.Since Notch2 is the major Notch isotype expressed on HSPCs, we showed that prior treatment in mice with Notch2 blocking antibody but not Notch1 blocking antibody sensitizes HSPC to the mobilizing stimuli of G-CSF and AMD3100 with a 3~4 - fold increase in mobilization. We showed that transient Notch2 blockade leads to decreased CXCR4 expression by HSPCs with CXCR4 transcription being directly regulated by the Notch transcriptional protein RBPJ. Notch2 blockade also increases cell cycling of HPCs during mobilization temporarily without affecting hematopoietic stem cell (HSC) homeostasis and self-renewal. Moreover, transient Notch2 blockade leads to greater HSPC homing to the marrow that is accompanied with a faster hematopoietic recovery and a competitive repopulating advantage of HSPCs.Herein, to search for alternative agents to Notch blocking antibody, we evaluate the ability of recombinant Notch peptides as decoy molecules to promote disengagement of Notch2-ligand interaction between HSPCs and niche supporting cells. By using OP9 cell-based binding assay and surface plasmon resonance (SPR) analyses, we showed that recombinant Notch111-13, Notch19-13, Notch18-13, Notch211-13, Notch29-13, and Notch28-13 peptide comprising EGF-like repeats 11-13, 8-13, and 9-13, of Notch1 or Notch2, respectively, all bind to Notch ligand Dll4, Dll1 and Jag2. Because EGF-like repeat 12 (EGF 12) has been shown located within the essential ligand binding domain, and the O -fucose attached to the threonine residue of Notch1 EGF12 acts as a surrogate amino acid to make specific and functional contact to residues of Notch ligand, we generated peptides in CHO cells with O -fucose attached to the threonine residue of EGF12 and further extended by Fringe. We found that binding affinities of the glycosylated peptides further increased compared to those of non-glycosylated peptides. Compared to Notch2 peptides, O -fucose and Fringe-modified Notch111-13 peptide shows the highest biding affinity to Notch ligand Dll4 with dissociation constant (Kd) reaching 34 nM. Similar to Notch2 blocking antibody, the O -fucose and Fringe-modified Notch111-13 peptide effectively outcompetes Notch2-ligand binding and thus blocks the adhesion of marrow HSPCs to the primary osteoblasts or OP9 cells expressing Notch ligands. Further, O -fucose and Fringe-modified Notch111-13 peptide can effectively induce HSPCs emigration from a 3D osteoblastic “spheroid niche”, an in vitro model of HSC niche. Compared to Notch2 blocking antibody, O -fucose and Fringe-modified Notch111-13 peptide caused a further decrease of HSPC cells remained in the “spheroid niche” and more HSPC emigration out of the “niche”.In summary, our findings suggest that recombinant Notch decoy peptides may be considered as alternatives to Notch2 blockade to be combined with the mobilizing regimen of AMD3100 and G-CSF for more effective HSPC mobilization. DisclosuresNo relevant conflicts of interest to declare.

Novel Evidence That the Pannexin 1 Channel Is Involved in Adenosine Triphosphate (ATP) Release from Cells for Optimal Mobilization of Hematopoietic Stem Progenitor Cells, and the Pannexin 1 SNP 5 (Rs3020015) T/C Polymorphism Characterizes Poor Mobilizer Status in Patients

Background . Pannexins have been shown to act predominantly as large transmembrane channels connecting the intracellular and extracellular space, allowing the passage of ions and small molecules, such as adenosine triphosphate (ATP), between these compartments. Pannexin 1 channels are involved in the release of ATP from cells and have been shown to be involved in the early stages of the innate immune response through an interaction with the P2X7 purinergic receptor. Recently, we provided evidence that activation of innate immunity in BM after administration of G-CSF produces "sterile inflammation" in the BM microenvironment and activates the pannexin channel to release ATP, which through binding to the P2X7 receptor leads to the mobilization of HSPCs (Leukemia 2018, 32:1920-1931). Corroborating our observation, it has been recently reported that the presence of the Gln460Arg SNP polymorphism within the P2X7 receptor gene in HSPCs, which is always co-inherited with Ala348Thr to form the gain-of-function haplotype 4, resulted in a significant increase in CD34+ HSPC mobilization (Leukemia 2018; 32:2724-2726). To shed more light on the novel concept that pannexin-1-released ATP, which interacts with P2X7, plays a crucial role in the egress of HSPCs from BM into peripheral blood (PB), we focused on the role of pannexin 1 in the mobilization process. Hypothesis. We hypothesized that pannexin 1 deficiency would negatively impact mobilization of HSPCs. Materials and Methods. First, we mobilized mice with G-CSF or AMD3100 in the presence of pannexin-1-blocking peptide. Following mobilization, we measured i) the total number of white blood cells (WBCs) and ii) the number of circulating clonogenic colony-forming unit granulocyte/macrophage (CFU-GM) progenitors and Sca-1+c-kit+lineage- (SKL) cells circulating in PB. Next, we analyzed five types of polymorphisms in the human pannexin 1 gene (SNP1-Rs1138800 A/C, SNP2-Rs7928030 G/C, SNP3-Rs12294985 C/T, SNP4-Rs127933348 A/G, and SNP5-Rs3020015 T/C) and correlated them with good or poor mobilization status of the patients. Patients in our studies were mobilized in the Bone Marrow Transplant Unit, Warsaw Medical University with G-CSF using a standard mobilization protocol, and poor mobilizers were identified as patients who were not able to mobilize the required number of CD34+ cells according to standard criteria. In our studies DNA from patient blood samples was isolated and fragments of DNA amplified and subsequently sequenced. Our patients in the good- and poor-mobilizer groups were matched for age, sex, and basic disorders. Results. We found that mice with blocked pannexin 1 channels mobilized HSPCs significantly less efficiently. More importantly, our patient data revealed that ~60% of patients that turned out to be poor HSPC mobilizers (n=20) displayed the pannexin 1 polymorphism SNP5 (Rs3020015) T/C. This polymorphism was observed in only 1 out of 26 good-mobilizer patients. Conclusions. Our results further support an important role for ATP-mediated purinergic signaling in the mobilization of HSPCs. Moreover, the pannexin 1 SNP5Rs3020015 T/C polymorphism may serve as a diagnostic tool to identify poor mobilizers. This investigation shed more light on the molecular pathways involved in the egress of HSPCs from BM into PB and will help to design better mobilization protocols in the case of poor mobilizers. Disclosures No relevant conflicts of interest to declare.

Quiescence/Mobilization of Hematopoietic Immature Cells Induced By Polyphenols through Modulation of APC/EPCR/PAR-1 Axis

The mechanism underlying quiescence and/or mobilization of hematopoietic stem cells and their bone marrow progenitors (HSPC) into circulation are tightly regulated for the continuous supply of peripheral blood cells; however, non-physiological or stress conditions, such as infections, can accelerate these mechanisms. Our results have shown that polyphenols modulate quiescence/mobilization of HSPC, but do not affect mature populations. Thrombin has been reported to induce the rapid HSPC mobilization through coagulation thrombin/PAR-1 axis, and quiescence is maintained across the APC/EPCR/PAR-1 axis (Nat. Med. 2015, 21:1307-17). Our objective was to investigate the effect of polyphenols on thrombin/PAR-1 and APC/EPCR/PAR-1 axis. C57BL/6J mice (6-8 weeks old) were treated with polyphenols from green tea extract (250 mg/kg body weight) orally (gavage) once every seven days and injected (i.p.) at day 7 with lipopolysaccharide (LPS) (100μg;Sigma) (n=6). The control group received vehicle and was injected with LPS (n=6). After 24h of LPS injection, mice were anesthetized for blood collection, and then sacrificed for bone marrow collection. PAR-1 and EPCR expression, nitric oxide (NO) production and endothelial nitric oxide synthase (eNOS) phosphorylation were evaluated in HSPC by flow cytometry. The functional ability of HSC was assessed by competitive repopulation assay. Vascular permeability was studied using Evans blue. After LPS injection, mice showed reduced expression of EPCR in bone marrow LSK parallel to an increase of PAR-1 expression in circulating immature and mature cells. Treatment of these mice with polyphenols partially prevented the reduced expression of EPCR in bone marrow LSK (13±3 vs 54±12; p&lt;0.05), but did not affect the increased PAR-1 expression in circulating immature and mature cells. Evans blue assay revealed a reduction in the vascular permeability of the bone marrow of LPS-injected mice treated with polyphenols (3.9±0.5 vs 2.1±0.1; p&lt;0.05). To assess whether polyphenols altered NO production, we measured NO levels and eNOS phosphorylation in immature LSK EPCRhigh (or LT-HSC) cells. NO production is activated by eNOS phosphorylation at Ser1177 and negatively regulated by eNOS phosphorylation at Thr495. LPS injection rapidly increased NO levels and eNOS phosphorylation at Ser1177 in bone marrow LSK of mice. Treatment of these mice with polyphenols reduced the percentage of bone marrow LSK EPCRhigh cells with higher intracellular NO (52±2.8 vs 28±5.6; p&lt;0.01) and increased eNOS phosphorylation at Thr495 in immature LSK. In order to evaluate the action of polyphenols on the functional ability of HSC, a competitive bone marrow repopulation assay was performed. Donor mice (C57BL/6J) received or not polyphenols followed by LPS injection (treated group: Polyphenols+LPS; control group: LPS), and bone marrow cells were transplanted (1:1) together with bone marrow cells of competitors (B6.SJL-PtprcaPepcb/BoyJ) in lethally irradiated recipients (B6.SJL-PtprcaPepcb/BoyJ). Mice were followed for 16 weeks and hematological analysis revealed no difference in circulating leukocytes, platelets or hemoglobin levels. Transplanted mice (recipients) presented a higher percentage of CD45+ cells from Polyphenols+LPS donors (33.7±13 vs 78.6±0.9; p&lt;0.05) in the peripheral blood, as well as increased number of T lymphocytes (6.7±4.5 vs 37.2±2.9; p&lt;0.05) and myeloid cells (68.5±1.7 vs 82.5±3.5; p&lt;0.05) from Polyphenols+LPS group. After 16 weeks, mice were euthanized and a higher percentage of LSK (or HSC) and LSK EPCRhigh (or LT-HSC) cells from Polyphenols+LPS donors were detected in the bone marrow, although only the percentage of LSK EPCRhigh was statistically different (0.0014±0.0001 vs 0.0032±0.001; p&lt;0.05). Taken together, our results indicate that polyphenols increased the functional ability of HSC in LPS-injected mice showing increased percentage of bone marrow LSK EPCRhigh cells, which are the most quiescent stem cells with strong self-renewal ability. Polyphenols reduced EPCR expression and NO production in immature cells of LPS-injected mice, exhibiting an anti-inflammatory effect that leads to the maintenance of barrier integrity and quiescence of cells, which was corroborated by reducing vascular permeability in the bone marrow. Thus, polyphenols appear to modulate quiescence/mobilization of HSPC through APC/EPCR/PAR-1 axis. Disclosures No relevant conflicts of interest to declare.

Polyphenols Modulate Quiescence/Mobilization of Hematopoietic Immature Cells through APC/EPCR/PAR-1 Axis

Coordinated arrangement of bone marrow (BM) microenvironment is essential for the maintenance of hematopoietic stem cells and their progenitors (HSPC), and for the continuous supply of peripheral blood (PB) cells. Quiescence and/or mobilization of these BM cells into the circulation are tightly regulated however non-physiological or stress conditions, such as infections, can accelerate them. Our previous findings have shown that polyphenols from green tea modulate the myeloid population (Gr-1+Mac-1+) of BM, spleen and PB of mice challenged by lipopolysaccharide (LPS). We have also observed a reduction in the circulating levels of thrombin-antithrombin complex (TAT) and soluble endothelial protein C receptor (sEPCR) of these mice. It has been reported that thrombin induces the rapid HSPC mobilization through coagulation thrombin/PAR-1 axis, and quiescence is maintained across the APC/EPCR/PAR-1 axis (Nat. Med. 2015, 21:1307-17). In this context, our goal was to investigate the effects of polyphenols on the Lin-Sca-1+c-Kit+ (LSK) primitive stem cell population and mature populations including: T and B lymphocytes, monocytes, granulocytes and erythrocytes (Lin+). The expression of PAR-1 and EPCR in these cells, and the levels of sEPCR in the BM fluid were also evaluated. The 250 mg/kg dose of polyphenol from green tea extract was given once every 7 days orally (gavage) to mice (n=6/group) challenged with i.p. injection of 100µg LPS. Control group received vehicle only. After 24h, mice were sacrificed; BM and PB were collected for the assays. LPS injection induced an increase in circulating LSK, without affecting the pool of these cells in BM. Treatment with polyphenols prevented recruitment of LSK into the PB, and did not alter the percentage of BM LSK. Corroborating these findings, polyphenols also reduced the number of immature progenitors (CFU-C) in the PB, evaluated by the clonal culture assay. In addition, polyphenols did not affect the increase in the number of Lin+ cells and circulating leukocytes (total WBC) induced by LPS injection. We also observed that LPS induced a rapid increase in the number of PB LSK and Lin+ cells expressing PAR-1, while reducing BM LSK expressing EPCR. Polyphenols did not affect PAR-1 expression in both populations, but partially recovered expression of EPCR in BM LSK. Polyphenols also reduced the increased levels of sEPCR induced by LPS in the BM niche. Taken together, our results demonstrate that polyphenols promote an effect on the quiescence/mobilization of both the most primitive hematopoietic cells and their progenitors, but does not affect mature populations. The reduction in vascular permeability observed in the BM of mice using Evans blue reinforces this action of polyphenols. It appears that the effect of polyphenols is thrombin dependent; although polyphenols did not affect the expression of PAR-1, they modulated EPCR, mainly in the medullary microenvironment with maintenance of barrier integrity and consequent cellular quiescence. In addition, polyphenols appear to have an anti-inflammatory effect, possibly triggered by their effect on the EPCR. DisclosuresDe Paula:Hematology and Transfusion Medicine Center, University of Campinas: Employment. Shiraishi:Hematology and Transfusion Medicine Center, University of Campinas: Employment. Queiroz:University of Campinas: Employment.

TGF-β–induced intracellular PAI-1 is responsible for retaining hematopoietic stem cells in the niche

AbstractHematopoietic stem and progenitor cells (HSPCs) reside in the supportive stromal niche in bone marrow (BM); when needed, however, they are rapidly mobilized into the circulation, suggesting that HSPCs are intrinsically highly motile but usually stay in the niche. We questioned what determines the motility of HSPCs. Here, we show that transforming growth factor (TGF)-β–induced intracellular plasminogen activator inhibitor (PAI)-1 activation is responsible for keeping HSPCs in the BM niche. We found that the expression of PAI-1, a downstream target of TGF-β signaling, was selectively augmented in niche-residing HSPCs. Functional inhibition of the TGF-β−PAI-1 signal increased MT1-MMP–dependent cellular motility, causing a detachment of HSPCs from the TGF-β–expressing niche cells, such as megakaryocytes. Furthermore, consistently high motility in PAI-1–deficient HSPCs was demonstrated by both a transwell migration assay and reciprocal transplantation experiments, indicating that intracellular, not extracellular, PAI-1 suppresses the motility of HSPCs, thereby causing them to stay in the niche. Mechanistically, intracellular PAI-1 inhibited the proteolytic activity of proprotein convertase Furin, diminishing MT1-MMP activity. This reduced expression of MT1-MMP in turn affected the expression levels of several adhesion/deadhesion molecules for determination of HSPC localization, such as CD44, VLA-4, and CXCR4, which then promoted the retention of HSPCs in the niche. Our findings open up a new field for the study of intracellular proteolysis as a regulatory mechanism of stem cell fate, which has the potential to improve clinical HSPC mobilization and transplantation protocols.

Myeloid Dendritic Cells Contribute to Hematopoietic Homeostasis

Dendritic cells (DCs) are antigen-presenting cells that are distributed throughout the body, and their main function is thought to be immune-surveillance. There is considerable phenotypic and functional heterogeneity of dendritic cells that tracks, in part, with their tissue localization. Myeloid dendritic cells (mDC), also known as conventional dendritic cells, are DCs with a myeloid origin. A previous study showed that perivascular mDCs in the bone marrow provide signals that regulate the survival of mature B cells (Sapoznikov et al., Nat Immunol, 2008). Using Cx3cr1gfp/+mice, we show that mDCs, defined as CX3CR1-GFP-bright, MHCII-bright cells, represent 0.2 ± 0.08% of bone marrow cells. They are localized to both venous sinusoids and arterioles in the bone marrow, placing them in the perivascular stem niche, along with CXCL12-abundant reticular (CAR) cells and Nestin-GFP+ cells. To assess the contribution of mDCs to the regulation of hematopoiesis, we used two independent mouse models to ablate mDCs: CD11cDTR and Zbtb46DTR mice. We previously reported that ablation of mDCs induces modest hematopoietic stem/progenitor (HSPC) mobilization. We show that mDC ablation also suppresses osteoblast function, with expression of osteocalcin mRNA (a marker of mature osteoblasts) decreasing 3.5-fold after mDC ablation (from 18.7 ± 9.9 to 5.3 ± 3.0; P < 0.05).To our surprise, mDC ablation (in both models) was associated with a significant loss of bone marrow macrophages. Prior studies have shown that macrophage ablation results in a loss of mature osteoblasts and modest HPSC mobilization. Thus, it is not clear whether mDCs have an independent effect on HSPC trafficking and osteoblast function. To address this issue, we first asked whether the macrophage loss after mDC ablation was mediated in a non-cell autonomous fashion. Mixed bone marrow chimeras were established containing both Zbtb46DTR and wild-type hematopoietic cells. Upon treatment with diphtheria toxin, we observed depletion of Zbtb46DTR but not wild-type mDCs (as expected). In contrast, a similar decrease in both Zbtb46DTR and wild-type macrophages was observed. These data show that the decrease in macrophages is an indirect consequence of mDC ablation and suggest that mDCs generate signals that contribute to macrophage retention and/or survival in the bone marrow. To further address the role of macrophages in this phenotype, we generated mice expressing Zbtb46-DTR alone, CD169-DTR alone (previously shown to ablate macrophages), or mice carrying both Zbtb46-DTR and CD169-DTR. As reported previously, ablation of macrophages induces a modest mobilization of Kit+ Sca1+ lineage- (KSL) cells to the spleen (7.1 ± 3.2 x104 versus 3.0 ± 1.2 x104 for PBS treated mice; P =0.06). Ablation of mDCs also induces modest mobilization (9.1 ± 2.5 x104 versus 5.8 ± 3.5 x104 for PBS treated mice; P < 0.05). Preliminary analysis of double mutant mice (n = 4) suggest an additive effect of combined mDC and macrophage ablation on HSPC mobilization with 25.9 ± 18.4 x104 KSL cells per spleen (P < 0.05 compared with macrophage alone ablation). Likewise, preliminary analysis suggests that the magnitude of osteoblast suppression (as measured by osteocalcin expression) is greater in double mutant mice. Collectively, these data suggest that bone marrow mDCs, in addition to a possible role in immune surveillance, contribute to blood homeostasis through multiple mechanisms. Specifically, mDCs appear to generate signals that are required for macrophage retention and/or survival in the bone marrow. mDCs also regulate HSPC trafficking and osteoblast function through a macrophage independent mechanism. Studies are underway to identify signals generated by mDCs that mediate these biological responses. DisclosuresNo relevant conflicts of interest to declare.

Unexpected Evidence That Inducible Nitric Oxide Synthase (iNOS) Is a Negative Regulator of Hematopoietic Stem/Progenitor Cell Mobilization

Background. Nitric oxide (NO) is gaseous free radical molecule involved in several biological processes related to inflammation, tissue damage, and infections. NO is synthesized by three isoforms of nitric oxide synthetase: two constitutively expressed isoforms, neuronal NOS (nNOS) and endothelial NOS (eNOS), and one isoform (iNOS) that is induced in response to inflammation. Interestingly, iNOS activity is enhanced during complement cascade (ComC) activation, which is a crucial element of innate immunity, and its cleavage fragments C3 and C5 (C3a and C5a, respectively) orchestrate the egress of HSPCs from BM into peripheral blood (PB). Mobilization studies in eNOS-/- mice revealed that the lack of constitutively expressed eNOS in endothelium reduces vascular endothelial growth factor (VEGF)-induced mobilization of endothelial progenitor cells (EPCs) and increases murine mortality after myelosuppression (Nature Med. 2003, 9:1370-6). However, no mobilization studies have been performed so far to assess the role of inducible hematopoietic cell-expressed iNOS in the mobilization of HSPCs. Because of this research void, we became interested in the potential role of iNOS in mobilization of HSPCs. This interest had two motivations. First, it was demonstrated in a recent paper that NO inhibits migration of granulocytes and monocytes, and it is known that these cells are crucial in facilitating egress of HSPCs across the bone marrow (BM)-peripheral blood (PB) barrier. Second, NO, which promotes inflammation, may have anti-inflammatory activity, and inflammation itself is one of the driving forces in HSPC mobilization. Aim of the study. To fill in this knowledge gap, we focused on the role of iNOS in the trafficking of hematopoietic cells as well as mobilization and homing of HSPCs. Materials and Methods. iNOS was upregulated or downregulated in established hematopoietic cell lines to study migration and adhesion of these cells. HSPC mobilization studies were performed in iNOS-/- mice, and, in parallel, homing and engraftment of iNOS-/- BM cells was evaluated in wild type animals. Since activation of the ComC is negatively regulated by heme oxygenase 1 (HO-1), and the ComC may downregulate HO-1, we tested the effect of HO-1 on iNOS-mediated cell trafficking. Results. Our results indicate that iNOS is a negative regulator of hematopoietic cell migration and prevents egress of HSPCs into PB during the mobilization process. At the molecular level, downregulation of iNOS resulted in downregulation of HO-1, and, vice versa, upregulation of iNOS enhanced HO-1 activity. Since, as we observed recently, HO-1 is a negative regulator of cell migration, the inhibitory effects of iNOS on the mobilization of HSPCs can be at least partially explained by its enhancing the HO-1 level in BM cells. Moreover, our studies, in which we generated hematopoietic chimeras, revealed that the iNOS effect depends on its expression in hematopoietic cells. Conclusions. We identified iNOS as a negative regulator of cell trafficking and mobilization of HSPCs. Further studies are needed to establish the mutual relationship between activation of iNOS and activation of HO-1 in hematopoietic cells, as both inducible enzymes are potent negative regulators of HSPC trafficking and together and regulate stem cell mobilization and homing. Therefore, inhibition of iNOS or HO-1 in HSPCs by employing small molecular inhibitors could find practical clinical application. DisclosuresNo relevant conflicts of interest to declare.

Neuropeptide Y Induces Hematopoietic Stem/Progenitor Cell Mobilization by Regulating Matrix Metalloproteinase-9 Activity Through Y1 Receptor in Osteoblasts.

Hematopoietic stem/progenitor cell (HSPC) mobilization is an essential homeostatic process regulated by the interaction of cellular and molecular components in bone marrow niches. It has been shown by others that neurotransmitters released from the sympathetic nervous system regulate HSPC egress from bone marrow to peripheral blood. In this study, we investigate the functional role of neuropeptide Y (NPY) on this process. NPY deficient mice had significantly impaired HSPC mobilization due to increased expression of HSPC maintenance factors by reduction of matrix metalloproteinase-9 (MMP-9) activity in bone marrow. Pharmacological or endogenous elevation of NPY led to decrease of HSPC maintenance factors expression by activating MMP-9 in osteoblasts, resulting in HSPC mobilization. Mice in which the Y1 receptor was deleted in osteoblasts did not exhibit HSPC mobilization by NPY. Furthermore, NPY treatment in ovariectomized mice caused reduction of bone loss due to HSPC mobilization. These results suggest a new role of NPY on HSPC mobilization, as well as the potential therapeutic application of this neuropeptide for stem cell-based therapy. Stem Cells 2016;34:2145-2156.

Exercise as an Adjuvant Therapy for Hematopoietic Stem Cell Mobilization.

Hematopoietic stem cell transplant (HSCT) using mobilized peripheral blood hematopoietic stem cells (HSPCs) is the only curative strategy for many patients suffering from hematological malignancies. HSPC collection protocols rely on pharmacological agents to mobilize HSPCs to peripheral blood. Limitations including variable donor responses and long dosing protocols merit further investigations into adjuvant therapies to enhance the efficiency of HSPCs collection. Exercise, a safe and feasible intervention in patients undergoing HSCT, has been previously shown to robustly stimulate HSPC mobilization from the bone marrow. Exercise-induced HSPC mobilization is transient limiting its current clinical potential. Thus, a deeper investigation of the mechanisms responsible for exercise-induced HSPC mobilization and the factors responsible for removal of HSPCs from circulation following exercise is warranted. The present review will describe current research on exercise and HSPC mobilization, outline the potential mechanisms responsible for exercise-induced HSPC mobilization, and highlight potential sites for HSPC homing following exercise. We also outline current barriers to the implementation of exercise as an adjuvant therapy for HSPC mobilization and suggest potential strategies to overcome these barriers.

Changes of angiopoietin 1 expression in G-CSF induced hematopoietic stem progenitor cells mobilization

目的探讨G-CSF诱导的造血干／祖细胞（HSPC）动员过程中血管生成素1(Ang1)在小鼠骨髓的表达变化及导致其变化的机制。方法采用流式细胞术检测小鼠动员前及动员后外周血Lin−Sca1+cKit+(LSK)细胞比例变化，并应用ELISA、免疫组化、RQ-PCR等方法检测G-CSF动员不同时间点小鼠外周血和骨髓标本，观察Ang1、骨钙素（OCN）基因及蛋白水平变化，显微镜下计数骨髓标本成骨细胞数量。结果G-CSF动员5 d小鼠外周血LSK细胞比例为（0.61±0.05)%，相对于对照组的(0.04±0.01)%显著增高(P<0.05)；稳态小鼠OCN及Ang1 mRNA在骨内膜细胞表达高于骨髓有核细胞；动员后骨内膜细胞OCN及Ang1 mRNA相对表达量对照组（28.64±8.61及2.84±0.95)、G-CSF动员3 d组（12.55±7.06及0.93±0.30）及动员5 d组（4.75±1.62及0.92±0.22）均下降；动员后骨内膜成骨细胞数量显著减少，骨髓组织Ang1蛋白表达下降；对照组、动员3 d及5 d组外周血OCN及Ang1浓度分别为（24.11±3.17）及（2.24±0.52)ng/ml、(9.96±2.16）及（1.21±0.38)ng/ml和（8.43±2.62）及(0.90±0.24) ng/ml，动员3 d及5 d组均明显低于对照组，差异均有统计学意义(P值均<0.05)。结论在G-CSF介导的HSPC动员过程中，骨髓微环境中成骨细胞数量下降、功能减低，进而导致骨髓Ang1表达下降，导致骨髓对HSPC的趋化和黏附能力下降，有利于HSPC动员的发生。

EPCR Limits Nitric Oxide Levels, Mediating Human and Murine Stem Cell Adhesion and Retention In The Bone Marrow, By Conjugating PAR1 and CXCR4 Signaling

Long term repopulating hematopoietic stem cells (LTR-HSC) in the murine bone marrow (BM) highly express endothelial protein C receptor (EPCR), yet the function of EPCR in HSC is incompletely defined. EPCR is expressed primarily on endothelial cells and has anti coagulation and anti inflammatory roles. While physiological stress due to injury or bleeding is a strong inducer of HSC mobilization and leukocyte production, a role for the coagulation protease thrombin, and its major receptor PAR1 in regulation of HSC has not yet been identified. We hypothesized that thrombin plays a role in HSC mobilization in the context of injury and that, conversely, signaling involving EPCR and its ligand activated protein C (aPC) play a regulatory role in HSC maintenance. Herein, we report that murine BM EPCRhigh stem cells display enhanced CXCL12 mediated adhesion and reduced migration capacitie, while motile circulating HSC in the murine blood and spleen lack high EPCR expression. Mechanistically, we found that EPCR is a negative regulator of nitric oxide (NO) levels. EPCRhigh stem cells display low intracellular NO levels, low motility, and increased adhesion to BM stroma. Furthermore, EPCRlow transgenic mouse cells displayed reduced stem cell adhesion to BM stroma and increased motility, manifested by reduced EPCRlow HSC in the BM and their corresponding increased levels in the blood. In vitro stimulation with the EPCR ligand, aPC, which we found to be physiologically expressed adjacent to small murine BM blood vessels, augmented EPCRhigh HSC adhesion and further limited their intracellular NO content by increasing eNOS phosphorylation at Thr495 in BM HSC, causing reduced production of NO. Conversely, administration of the pro-coagulant protease thrombin to mice induced PAR1 mediated EPCR shedding from BM HSC, followed by CXCR4 upregulation on HSC, and PAR1-mediated CXCL12 secretion by BM stromal cells. Together, these events lead to loss of retention and rapid stem cell mobilization to the blood. Interestingly, shedding of EPCR was found to be mediated by elevation of intracellular NO content, leading to EPCR co-localization with Caveolin. Correspondingly, thrombin failed to induce EPCR shedding and mobilization in eNOS and PAR1 deficient mice. Additionally, we found that BM LTR-HSC functionally express the metalloproteinase TACE (ADAM17) on the cell membrane, and that in- vitro inhibition of TACE activity by a newly developed selective inhibitor, reduces thrombin- mediated EPCR shedding, suggesting the involvement of TACE in EPCR shedding and HSC mobilization. Moreover, EPCR shedding was also CXCR4 dependent, revealing a crosstalk between EPCR, PAR1 and CXCR4. HSPC mobilized by thrombin possessed increased long-term repopulation capability following transplantation into lethally irradiated recipient mice and re-synthesis of EPCR by donor HSC in the engrafted host BM. In addition, EPCR expression was re-induced on circulating stem cells following in vitro treatment with eNOS inhibitor. Interestingly, bypassing eNOS by directly injecting NO donor, induced EPCR shedding, CXCR4 upregulation and rapid HSPC mobilization in both wild type and eNOS KO mice. Importantly, we found that similar to mice, EPCR was selectively and highly expressed by primitive human BM CD34+CD38- HSC, but not in the blood circulation of clinical G-CSF mobilized stem cells or in motile cord blood stem cells. Human BM CD34+/CD38- HSC are functionally EPCRhigh cells, maintaining low levels of intracellular NO which mediates their increased adhesion, while EPCR shedding was important for their migration and mobilization. In the functional pre-clinical NOD/SCID mouse model, G-CSF mobilization induced EPCR shedding, up-regulation of PAR1 and CXCR4 on human stem and progenitor cells, while NO signaling inhibition blocked G-CSF induced mobilization and increased both murine and human EPCRhigh stem cell accumulation in the murine BM.Our results define functional roles for EPCR, on both human and murine HSC, and suggest that regulation of EPCR expression is linked to NO, PAR1 and CXCR4 signaling as a pivotal mechanism determining HSC localization and function. Our study reveals that activation of coagulation in the context of cell injury controls stem cells retention and motility, and suggests that targeting this system may be useful in improving clinical stem cell mobilization and transplantation protocols. Disclosures:No relevant conflicts of interest to declare.

Fibroblast growth factor signaling promotes physiological bone remodeling and stem cell self-renewal

Fibroblast growth factor (FGF) signaling activates many bone marrow cell types, including various stem cells, osteoblasts, and osteoclasts. However, the role of FGF signaling in regulation of normal and leukemic stem cells is poorly understood. This review highlights the physiological roles of FGF signaling in regulating bone marrow mesenchymal and hematopoietic stem and progenitor cells (MSPCs and HSPCs) and their dynamic microenvironment. In addition, this review summarizes the recent studies which provide an overview of FGF-activated mechanisms regulating physiological stem cell maintenance, self-renewal, and motility. Current results indicate that partial deficiencies in FGF signaling lead to mild defects in hematopoiesis and bone remodeling. However, FGF signaling was shown to be crucial for stem cell self-renewal and for proper hematopoietic poststress recovery. FGF signaling activation was shown to be important also for rapid AMD3100 or post 5-fluorouracil-induced HSPC mobilization. In vivo, FGF-2 administration successfully expanded both MSPCs and HSPCs. FGF-induced expansion was characterized by enhanced HSPC cycling without further exhaustion of the stem cell pool. In addition, FGF signaling expands and remodels the supportive MSPC niche cells. Finally, FGF signaling is constitutively activated in many leukemias, suggesting that malignant HSPCs exploit this pathway for their constant expansion and for remodeling a malignant-supportive microenvironment. The summarized studies, concerning regulation of stem cells and their microenvironment, suggest that FGF signaling manipulation can serve to improve current clinical stem cell mobilization and transplantation protocols. In addition, it may help to develop therapies specifically targeting leukemic stem cells and their supportive microenvironment.

Microrna-155 Promotes Hematopoietic Stem and Progenitor Cell Mobilization and Proliferation

Abstract 214MicroRNAs (miRNAs) are small non-coding RNAs involved in various physiological processes, including hematopoiesis. Although miRNAs are broadly studied with regards to normal and malignant leukocyte development, the role of miRNAs in hematopoietic stem and progenitor (HSPC) migration and mobilization is poorly understood. Currently, induction of HSPC mobilization from the bone marrow (BM) to the peripheral blood (PB) is the major mean to harvest HSPCs for clinical transplantation. Recently, several miRNAs were found to be upregulated in macaque G-CSF-mobilized CD34+ HSPCs, among them the oncogenic miRNA mir-155 (Donahue et al., Blood 2009).To study the involvement of mir-155 in HSPC regulation, we examined hematopoiesis in mir-155 knock out (KO) mice. Of interest, mir-155 KO mice had normal BM and PB levels of mature cells, but reduced levels of immature BM Lineage−/Sca-1+/c-Kit+ (LSK) and primitive BM CD34−LSK HSPCs. Profiling of mir-155 expression in murine hematopoietic BM populations, following G-CSF treatment, revealed differential expression patterns in wild type (WT) mice. G-CSF treatment upregulated mir-155 levels in immature LSK cells, in T-cells and in Mac-1+/Gr-1+ monocyte/macrophages. In contrast, G-CSF downregulated mir-155 levels in common lymphoid progenitors and in B-cells. Suggesting that mature hematopoietic cells may also participate in HSPC mobilization process. G-CSF administration to mir-155 KO mice resulted in reduced HSPC mobilization, as assessed by CFU-C and LSK cell counts in the PB. Surprisingly, G-CSF treatment increased BM LSK cell frequency in mir-155 KO mice to the same levels as in WT mice. On the contrary, G-CSF treatment reduced BM CD34−LSK cell frequency in WT mice and increased it in mir-155 KO mice showing an opposing effect on the more primitive HSPC population. Since mir-155 is involved also in mesenchymal development regulating osteoblast differentiation, we propose that BM HSPC pool reduction could be mediated by the stromal microenvironment. Additionally, osteoblasts and other BM residing cells undergo substantial changes in response to G-CSF that might be mediated by mir-155.To determine whether the mobilization defect is hematopoietic cell-autonomous or due to an abnormal microenvironment, we examined G-CSF-induced mobilization in chimeric mice. Mir-155 KO mice reconstituted with wild type (WT) BM cells had normal mobilization as WT mice reconstituted with WT BM cells. Of interest, WT mice reconstituted with mir-155 KO BM cells showed reduced mobilization as mir-155 KO mice reconstituted with mir-155 KO BM cells. These results indicate that the mobilization defect in mir-155 KO mice is also due to a defect in HSPC motility. Since the CXCL12/CXCR4 axis plays a major role in HSPC mobilization, we examined the ability of mir-155 KO cells to perform CXCL12-induced migration and found reduced migration capacity of HSPCs in vitro. Although having reduced migration potential, mir-155 KO LSK cells had normal CXCR4 expression levels, suggesting that an aberrant intracellular response to SDF-1 is responsible for the observed defect. In support, AMD3100 treatment to mir-155 KO mice resulted in reduced HSPC rapid mobilization. Since SHIP-1 phosphatase mRNA is targeted by mir-155 in hematopoietic cells (Costinean et al., Blood 2009) and SHIP-1 KO hematopoietic cells exhibit increased migration towards CXCL12 (Kim et al., JCI 1999), we examined intracellular SHIP-1 expression during HSPC mobilization. SHIP-1 levels were downregulated in WT BM LSK cells in response to G-CSF or AMD3100 mobilizing treatments. In contrast, mir-155 KO BM LSK cells upregulated SHIP-1 levels in response to the mobilizing treatments. These results suggest that mir-155 may promote HSPC mobilization and increased motility via SHIP-1 downregulation.In summary, our data indicates that mir-155 directly promotes HSPC motility and mobilization by SHIP-1-mediated regulation of intracellular response to CXCL12 signaling. We also propose the mechanism of indirect regulation of BM HSPC pool size during steady state and following G-CSF treatment by mir-155, via stromal BM microenvironment, which is currently under investigation. Deciphering the mechanisms of HSPC migration and maintenance in general and by mir-155 in particular, may potentially improve clinical mobilization protocols and contribute to increased donor BM engraftment following transplantation. Disclosures:No relevant conflicts of interest to declare.

Sinusoidal Endothelial CD26 Cleavage of Neuropeptide Y Regulates Transendothelial Migration and Mobilization of Hematopoietic Stem and Progenitor Cells in Response to G-CSF.

Abstract 2347Hematopoietic stem and progenitor cell (HSPC) mobilization by G-CSF is reduced in CD26 knockout (−/−) mice or mice treated with compounds that inhibit CD26 enzyme activity. However, how CD26 regulates mobilization is not known. CD26 is a surface dipeptidyl peptidase expressed on hematopoietic and non-hematopoietic cells. The fact that CD26 is expressed on HSPC, has suggested that intrinsic HSPC CD26 is involved in their mobilization. We reasoned that if intrinsic HSPC CD26 is crucial for their mobilization, then CD26+ HSPC would mobilize more readily than CD26− HSPC. After G-CSF treatment, CD26+ SLAM LSK and LSK cells increased 2 and 3.7-fold in peripheral blood (PB) whereas CD26− SLAM LSK and LSK cells increased 3.9 and 8.7 fold respectively; with the ratio of CD26+/CD26− HSPC in the bone marrow (BM) remaining unchanged. In chimeric mice where hematopoietic cells were either wild-type or CD26−/− in origin and the stromal environment was wild-type, equivalent CFU-GM and LSK cells were mobilized by G-CSF. These studies suggest that CD26 expression on hematopoietic origin cells including HSPC is not required for the G-CSF induced mobilization of HSPC.The BM stromal environment includes both osteolineage cells and endothelial cells (EC). To explore whether osteolineage cell CD26 is involved in G-CSF mobilization, we quantitated CD45−CD119−CD31−Sca-1+ Alcam− cells enriched for mesenchymal progenitor cells (MPC) and CD45−CD119−CD31− Sca-1- Alcam+ cells enriched for osteoblasts (OB) in the BM of mice treated with G-CSF or G-CSF plus the CD26 inhibitor diprotin A. Total MPC and OB number as well as the retention factors SDF-1, SCF-1 and osteoprotegerin (OPG) were reduced equally in the BM of G-CSF and G-CSF plus diprotin A treated mice, suggesting that osteolineage cell CD26 is not involved in mobilization. Since endothelial cells (EC) express CD26 and HSPC resides proximal to sinusoidal endothelial cells (SEC), we evaluated if SEC CD26 has a role in G-CSF induced HSPC mobilization. G-CSF treatment increased CD26 expression on CD45- Ter119− Sca-1− VEGFR3+ SEC by 2-fold. Because mobilized HSPC must transmigrate across SEC to enter the PB, we determined if EC CD26 regulates HSPC transmigration. Treatment of confluent EC monolayers with G-CSF in vitro, increased transendothelial migration of both wild-type and CD26−/−HSPC, which was significantly reduced by diprotin A, supporting the hypothesis that enhanced expression of CD26 on BM SEC by G-CSF is involved in HSPC mobilization.Since SDF-1 is a BM retention factor for HSPC and CD26 can cleave SDF-1 in vitro, it is possible that SDF-1 degradation by CD26 plays a role in HSPC egress. However, direct evidence supporting proteolytic inactivation of SDF-1 by CD26 in response to G-CSF is not available. Using a novel bio-binding assay and mass spectroscopy, we found similar levels of active SDF-1 in the BM extracellular fluid and on SEC membranes from G-CSF treated wild-type and CD26−/− mice, suggesting that G-CSF mobilization is not linked to SDF-1degradation. We therefore searched protein sequence databases for known EC products containing a CD26 recognition site and implicated in HSPC trafficking. We found that the neurotransmitter neuropeptide Y (NPY), a sympathetic nerve and EC product contains an N-terminal Tyr-Pro sequence and can be cleaved to NPY3–36 by CD26. We hypothesized that if cleavage of NPY by EC CD26 is involved in transendothelial migration of HSPC, then reduced HSPC migration through EC monolayers in the presence of G-CSF plus diprotin A would be reversed in the presence of NPY3–36. As expected, NPY3–36 reversed the diprotin A mediated attenuation in HSPC migration through G-CSF treated EC. In vivo, the attenuated response to G-CSF mobilization in diprotin A treated or CD26−/− mice could be completely reversed by co-administration of NPY3–36, but not full length NPY. In G-CSF mobilized wild-type mice, we found that expression of gap junction proteins on BM SEC, including CD31 and VE-cadherin are reduced coincident with increased EC permeability, which is attenuated by co-administration of diptotin A and restored to normal in mice receiving G-CSF plus diprotin A and NPY3–36, but not full length NPY. In conclusion, we have found that CD26 regulates G-CSF induced mobilization that is independent of the SDF-1 paradigm; rather elevated SEC CD26 in response to G-CSF enhances transendothelial migration and mobilization of HSPC via cleavage of NPY. Disclosures:No relevant conflicts of interest to declare.

Thrombopoietin-Receptor Signalling Induces Proliferation of Dormant HSC.

AbstractAbstract 2343Lifelong blood production is maintained by a very rare population of self-renewing hematopoietic stem cells (HSCs) in bone marrow (BM). Proliferation, differentiation and survival of HSC toward stepwise hematopoietic cell development needs to be tightly controlled by cell intrinsic and extrinsic factors, as excess or insufficient production of mature blood cells potentially leads to neoplasia or aplasia. HSCs and progenitors (HSPCs) are equipped with cell surface receptors for different cytokines or chemokines (Kaushansky, NEJM 2006), and thus can integrate external signals, leading finally to proliferation and subsequent increase of hematopoietic cells in demand. Some of these regulatory pathways are already exploited in clinical settings: CXCR4 antagonists for HSPC mobilization, human granulocyte colony-stimulating factor (hG-CSF) for HSC mobilization and myeloid regeneration, and thrombopoietin agonists (THPO) for improving thrombocytopenia. However, despite their clinical use, little is known about the influence of these molecules on HSC.We established in vivo HSC divisional tracking with CFSE (5(6)-carboxyfluorescein diacetate N-succinimidyl ester). This allows to track single HSC division with high resolution, and subsequently to test biological activity of HSC-containing fractions (LKS) with different divisional history (Takizawa et al., JEM 2011). Using this system we evaluated the effects of systemic administration of human fms-related tyrosine kinase 3 ligand (hFlt3L), hG-CSF (Filgrastim), CXCR4 antagonist (AMD3100), and the THPO receptor (cMpl) agonist (Romiplostim) on HSC division. CFSE-labeled LKS were transferred into non-irradiated steady-state recipients. The non-dividing cell fraction was defined by the CFSE profile of CD4+CD62L+ T cells. One week after transplantation mice were injected with PBS or respective reagents daily or every other day for over one week. Three weeks after transfer, phenotypic BM analysis demonstrated that most of donor LKS had undergone several divisions while a small fraction of LKS remained undivided in PBS treated control mice (Figure 1a), containing long term self-renewing HSCs with at least 20–30% frequency (Takizawa et al., JEM 2011). Administration of hFlt3L increased the percentage of intermediate (1–5x divided) and fast cycling (>5x divided) LKS, which mainly contains CD150- Flt3+ multipotent progenitor cells (Figure 1b). Upon injections of cMpl agonist all donor LKS divided more than once, leaving no cells in quiescent fraction, with substantial expansion of CD150+ cells in the divided fraction. CXCR4 antagonist and hG-CSF administration had little effect on LKS proliferation. These data suggest that cMpl agonist drives dormant cells into proliferation, whereas hG-CSF has little effect on LKS division.To determine whether cMpl agonist increases the turnover of functionally defined, bona fide HSCs, we performed secondary transplantation of 0–1, 2–4, and ≥5x divided LKS. Twenty fast- (≥5x divided cells at 3 weeks), slow-cycling (2–4x divided) or relatively dormant LKS Flt3- cMpl+ cells (0–1x divided) were sorted from mice treated with PBS or cMpl agonist, and transplanted into lethally irradiated mice. Early results demonstrate increased percentage of secondary recipient engrafted with 2–4x divided cells from primary animals treated with cMpl agonist compared to those cells from PBS treated controlOur results thus suggest that cMpl agonists have mitogenic activity not only for megakaryocyte progenitors but also for HSCs. How far this holds true in the human species needs to be determined. However, it should be taken in consideration given clinical data on evolution of pre-existing clonal myeloid diseases under cMpl agonist treatment (Dantoni, ASH abstract 2011), and also when treatment is applied long-term to patients with primary non-clonal hematopoietic diseases as immune thrombocytopenia or aplastic anemia. [Display omitted] Disclosures:No relevant conflicts of interest to declare.