Osteocytes Embedded Inside Bone Are Essential for G-CSF-Induced Hematopoietic Stem/Progenitor Mobilization

Abstract Abstract 721 Hematopoietic stem/progenitor cells (HSPCs) are released from the bone marrow (BM) to the circulation by granulocyte-colony stimulating factor (G-CSF) via sympathetic nervous system (SNS)-mediated osteoblast suppression (Katayama et al. Cell 2006). We further elucidated that vitamin D receptor is essential for this neuronal control of endosteal niche (Kawamori et al. Blood 2010). Osteoblasts are known to adopt three fates: die by apoptosis, become bone-lining cells, or become embedded in osteoid and then in mineralized bone matrix to terminally differentiate into osteocytes, which constitute more than 95% of bone cells. Osteocytes have been shown to control the functional balance between osteoblast and osteoclast via mechanotransduction. In order to address the role of bone-embedded osteocytes in HSPCs niche function, we first quantified mRNA expression of bone-related genes in the femur of wild-type (WT) mice to examine if osteocytic function changes during G-CSF treatment (125μg/kg/dose, 8 divided doses, every 12 hours). Whereas markers relating to osteoblast function, osteocalcin and osteopontin, started to decrease late at 6 doses of G-CSF administration when mild mobilization of HSPCs had occurred, osteocyte-specific genes, including neuropeptide y, SOST, MEPE, E11/gp38 and Phex, were rapidly suppressed at 1 dose when no mobilization was observed. These data suggest that osteocytes respond to G-CSF with altered gene expression much earlier than osteoblasts. Further, the number and thickness of osteocyte projections extending toward the endosteal surface were markedly reduced, as assessed by fluorescently labeled phalloidin, at 8 doses of G-CSF treatment when full mobilization was achieved; these morphological changes were observed specifically in newly-embedded osteoid osteocytes, but not in mature osteocytes embedded deep inside mineralized bone. These findings suggest that osteoid osteocytes may sense the signal triggered by G-CSF. We confirmed the presence of β2-adrenergic receptor in osteoid osteocytes and tyrosine hydroxylase-positive nerve fibers in the vicinity by immunofluorecence staining, suggesting that osteoid osteocytes may be regulated by SNS. To directly address osteocyte involvement in G-CSF-induced mobilization, we utilized a transgenic (TG) mice in which inducible and specific ablation of osteocytes is achieved through targeted expression of diphtheria toxin (DT) receptor under DMP-1 promoter. A single injection of DT in TG mice generates “osteocyte-less (OL)” mice. We found that mobilization by G-CSF was drastically impaired in OL mice for progenitors (CFU-Cs, mean±SEM, WT vs Tg: 1673±271 vs 242±94/ml blood, n=6-13, p<0.01; lineage-Sca-1+c-kit+ (LSK) cells, WT vs Tg: 6878±1209/ml vs 1763±502/ml, n=6-13, p<0.01) and stem cells (repopulating units at 4 months, WT vs Tg: 2.5±0.7 vs 0.5±0.2, n=6-7, p<0.05), while the OL BM showed normal HSPC number. The levels of CXCL12 mRNA and protein in BM and bone were markedly decreased during G-CSF treatment even in OL mice despite the mobilization defect, and a CXCR4 antagonist AMD3100 induced mobilization normally in the absence of osteocytes. Thus, osteocytes embedded within the bone are indispensable for G-CSF-induced mobilization through a CXCL12-independent mechanism. Although most of bone-related genes exhibited drastic decreases following G-CSF treatment, we found that fibroblast growth factor 23 (fgf23) mRNA displayed a 4-fold increase at 6 doses of G-CSF. FGF23 is mainly produced by osteocytes and Klotho is an obligate coreceptor for FGF23 to bind and activate FGF receptors. Since we confirmed that klotho hypomorphic (kl/kl) mice showed remarkably disrupted osteocyte network, we injected G-CSF into these mice. As we expected, G-CSF induced virtually no mobilization in kl/kl mice while the number of HSPCs in the BM remained comparable to control mice. Collectively, our results demonstrate a novel function of bone-embedded osteocytes as a critical regulator of HSPC trafficking perhaps by controlling the endosteal niche and establish the important physiologic function of skeletal tissue for hematopoietic microenvironment. Disclosures: No relevant conflicts of interest to declare.

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Disruption of CXCR4 Utilizing AMD3100 Bypasses Mobilization Deficiency Exhibited by CD26−/− Mice and Results in Efficient Mobilization of Myeloid Progenitors

Blood ◽

10.1182/blood.v112.11.3492.3492 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3492-3492

Author(s):

Laura A. Paganessi ◽

Andrew L. Walker ◽

Stephanie A. Gregory ◽

Henry C. Fung ◽

Kent W. Christopherson

Keyword(s):

Bone Marrow ◽

Blood Cell ◽

Progenitor Cells ◽

Peripheral Blood ◽

Fold Increase ◽

Cxcr4 Antagonist ◽

Hematopoietic Stem ◽

Genetically Engineered Mice ◽

Colony Forming Units ◽

Myeloid Progenitors

Abstract The exopeptidase CD26 (also known as DPPIV/dipeptidylpeptidase IV) cleaves dipeptides from the N-terminus of proteins that contain the required X-Pro or X-Ala motif. We have previously reported that inhibition or loss of CD26 activity results in a deficiency in normal granulocyte-colony stimulating factor (G-CSF) induced mobilization, suggesting that CD26 is a necessary component of mobilization (Christopherson, et al Blood 2003 and Christopherson, et al Exp Hematol 2003). The chemokine CXCL12 (SDF-1, stromal cell derived factor-1) contains the appropriate recognition sequence for CD26 induced cleavage. This combined with the importance of CXCL12 in the trafficking of hematopoietic stem and progenitor cells (HSC/HPC) suggests CXCL12 as a likely functional target of CD26 during G-CSF induced mobilization. For this reason we therefore decided to investigate whether genetically engineered mice lacking CD26 (CD26−/−) could be mobilized utilizing the CXCR4 antagonist, AMD3100. To evaluate this, ten week old C57BL/6 and CD26−/− mice (also on a C57BL/6 background) received a single subcutaneous injection of AMD3100 (1mg/1kg). One hour following injection the mice were euthanized by CO2 inhalation. Peripheral blood was then obtained by heart stick with a 1.2 ml syringe containing EDTA as an anticoagulant. A complete blood count was taken for each peripheral blood sample. Following red blood cell lysis, cells were plated for myeloid colony formation in a standard 1% methylcellulose colony assay containing the appropriate cytokines. Following 7 days of incubation at 5% O2, 5% CO2 and 37°C plates were scored for colony-forming units-granulocyte macrophage (CFU-GM), burst-forming units-erythroid (BFU-E), and colony-forming units-granulocyte, erythroid, macrophage, and megakaryocytic (CFU-GEMM). Data is presented as the number of colonies per femur for the bone marrow and as the number of colonies per ml of whole blood for the peripheral blood. AMD3100 treatment resulted in an increase in white blood cell (WBC) counts from 5.05±0.48 × 106/ml in untreated mice to 10.21±0.88×106/ml in treated mice (p≤0.01). An increase in WBC counts was also observed during AMD3100 treatment in CD26−/− mice from 7.77±1.28×106/ml in untreated mice to 16.7 ±2.11 × 106/ml in treated mice (p<0.01). AMD3100 treatment resulted in an increase in circulating myeloid progenitors in the peripheral blood of C57BL/6 and CD26−/− mice as compared to untreated C57BL/6 and CD26−/− mice respectively (p≤0.01). Specifically, a 2.38, 3.75, 12.33 fold increase in CFU-GM, BFU-E, and CFU-GEMM were observed in the peripheral blood of C57BL/6 mice after treatment. A 2.63, 5.48, 14.29 fold increase in CFU-GM, BFU-E, and CFU-GEMM were observed in the peripheral blood of CD26−/− mice after treatment. Existing pre-clinical and clinical data suggest that the CXCR4 antagonist, AMD3100, rapidly mobilizes hematopoietic progenitor cells from the bone marrow into the periphery. The results presented here provide pre-clinical evidence that disruption of the interaction between the CXCR4 chemokine receptor and CXCL12, via sub-cutaneous injection of AMD3100, mobilizes significant numbers of myeloid progenitors in mice, even in the absence of CD26. These results support the notion that CD26 is downstream of G-SCF treatment. Additionally, these results support the potential use of AMD3100 to treat patients that may have an altered ability to respond to G-CSF treatment as a result of a reduction or loss in CD26 activity. Future studies are warranted to evaluate potential variations in CD26 levels or activity in the general population, in differing patient populations, and during different treatment regimens.

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Mobilization Studies in Complement-Deficient Mice Reveal That AMD3100 Mobilization Depends On Complement Cascade Activation and Suggest Involvement of “Membrane Attack Complex - MAC” in Egress of Hematopoietic Stem/Progenitor Cells.

Blood ◽

10.1182/blood.v114.22.367.367 ◽

2009 ◽

Vol 114 (22) ◽

pp. 367-367

Author(s):

Marcin Wysoczynski ◽

HakMo Lee ◽

Rui Liu ◽

Wan Wu ◽

Janina Ratajczak, ◽

...

Keyword(s):

Progenitor Cells ◽

Conflicts Of Interest ◽

Membrane Attack Complex ◽

Classical Pathway ◽

Compensatory Mechanism ◽

Complement Cascade ◽

Cxcr4 Antagonist ◽

Hematopoietic Stem ◽

Deficient Mice

Abstract Abstract 367 We reported that complement cascade (CC) becomes activated in bone marrow (BM) during mobilization of hematopoietic stem/progenitor cells (HSPCs) by immunoglobulin (Ig)-dependent pathway and/or by alternative Ig-independent pathway as seen during G-CSF- or Zymosan mobilization, respectively. As a result, several potent bioactive CC anaphylatoxins (C3 and C5 cleavage fragments) are released that regulate egress of HSPCs (Blood 2003;101,3784; Blood 2004;103,2071; Blood 2005;105,40, Leukemia 2009; in press.). This explains why: i) NOD/SCID and RAG-/- animals that do not activate the Ig-dependent CC classical pathway; ii) C2fB-/- and C3-/- mice that do not activate the classical and alternative CC pathways; and iii) C5-/- mice that do not activate the distal pathway of CC are all poor G-CSF- and/or Zymosan mobilizers. In this study, we evaluated the role of CC in mobilization induced by CXCR4 antagonist AMD3100. We noticed that all CC activation-deficient mice mentioned above, except C5-/- mice, mobilize normally in response to AMD3100 administration. Accordingly, the number of mobilized CD34- SKL cells, leucocytes, and CFU-GM clonogeneic progenitors in mutant mice was similar to wt littermates. More important we observed that AMD3100 mobilization of HSPCs was preceded by a massive egress of leucocytes from BM and that AMD3100 was able to stimulate in these cells i) phosphorylation of MAPKp42/44 and ii) secretion of MMP-9. At the same time, ELISA data to detect CC activation revealed that serum levels of CC cleavage fragments, which were low in the initial phase of AMD3100 mobilization during granulocyte egress, become elevated later during HSPC egress. Thus, our data show that despite a fact that G-CSF and AMD3100 mobilize HSPCs by involving different mechanisms, activation of CC is a common phenomenon occurring during mobilization induced by both compounds. This further supports a pivotal role of CC activation in the egress of HSPCs from BM; however, both compounds activate CC differently. While G-CSF administration initiates CC activation at its proximal C1q-C3 level, AMD3100 induces CC activation at the distal C5 level, pointing to a crucial role of C5 cleavage in executing mobilization. To support this, all mice employed in our studies that display defects in activation of proximal stages of CC (NOD/SCID, RAG, C2fB-/-, and C3-/-) are normal AMD3100 mobilizers. However, C5 is cleavage required for mobilization occurs in the plasma of these animals latter on - directly by proteases released from AMD3100-stimulated granulocytes that egress from the BM as a first wave of mobilized cells. This compensatory mechanism cannot occur from obvious reasons in C5-/- mice. We conclude that AMD3100-directed mobilization similarly as G-CSF-induced one depends on activation of CC; however, AMD3100 in contrast to G-CSF activates CC at distal stages – directly by proteases released from mobilized/activated granulocytes. Cleavage of C5 and release of C5a and desArgC5a create a sinusoid-permissive environment in BM for HSPCs egress. This suggests involvement of both C5 cleavage fragments as well as a potential role of downstream elements of CC activation - membrane attack complex - MAC (C5b-C9) in stem cell mobilization. Therefore, some poor AMD3100 patient responders could possess a defect in activation of the distal steps of CC. Disclosures: No relevant conflicts of interest to declare.

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Crispra: A Promising Tool for β-Thalassemia Treatment

Blood ◽

10.1182/blood-2020-141196 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 8-8

Author(s):

Xiaoqing Jia ◽

Qi Yao ◽

Hui Li ◽

Jieping Chen

Keyword(s):

Conflicts Of Interest ◽

Gene Activation ◽

Fold Increase ◽

293T Cell ◽

Genomic Locus ◽

Hematopoietic Stem ◽

Nb4 Cells ◽

Guide Rnas ◽

Promising Tool

Induction of hemoglobin γ expression is a reliable strategy to treat β-thalassemia. Gene editing using CRISPR/Cas9 technology has been widely used. However, application in vivo is limited due to the uncertainty on genomic cleavages of Cas9. In contrast, CRISPR/Cas9-based gene activation (CRISPRa) can only locate genomic locus but not interrupt sequence. Here, we use SAM system of CRISPRa to locate and activate HBG1 and HBG2, exploring the great potential of CRISPRa for β-thalassemia treatment. WWe designed 8 single-guide RNAs (sgRNAs) online and cloned into vector SAM V2, which fused dCas9 and VP64. To test the over-expression efficiency, vector containing sgRNA and MPH (fused HSF1, p65 and MS2) were transfected into 293T cell. After 72h transfection, 293T cells were collected. Q-PCR data showed that two sgRNAs were excellent on activating HBG expression with over 1000-fold increase. WTo test the activating function in hematological cell and the persistence of hemoglobin γexpression, two screened sgRNA were transfected into NB4 cells using lentivirus system. We harvested NB4 cells at different time-point (3 day, 1 week and 2 weeks), and implemented q-PCR assay. HBG expression were increased 50-hold and 1000-hold, respectively. However, the expression were reducing over time and the intrinsic mechanism is unknown. WThis study set out to increase HBG without interrupt genome using CRISPRa system. This study has found two sgRNA to activate the expression of HBG in 293T cell and NB4 cell. Further research is required to vertify the efficiency of sgRNA in hematopoietic stem cells and prolong the expression time. Figure Disclosures No relevant conflicts of interest to declare.

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Early Clonal Hematopoiesis Detected in Gata2 heterozygosity By Color-Barcoding of Hematopoietic Stem Cells in Zebrafish

Blood ◽

10.1182/blood-2018-99-118236 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 748-748

Author(s):

Serine Avagyan ◽

William P. Mannherz ◽

Leonard I. Zon

Keyword(s):

Conflicts Of Interest ◽

Myeloid Cell ◽

Fold Increase ◽

Shannon Index ◽

Hematopoietic Stem ◽

Guide Rnas ◽

Clonal Hematopoiesis ◽

Children And Young Adults ◽

Epigenetic Regulators ◽

Different Color

Abstract Germline GATA2 haploinsufficiency underlies an inherited syndrome of predisposition to myeloid malignancies. The mechanism by which GATA2 mutations lead to leukemia in children and young adults remains to be determined. We generated gata2b heterozygous mutant zebrafish using CRISPR/Cas9 technology to study this process. We used a color-barcoding system in zebrafish called Zebrabow that labels each hematopoietic stem cell (HSC) born during embryogenesis with a different color. Color-barcoding was induced by a blood-specific promoter-driven Cre recombinase during development at 24 hours post fertilization when there are on average 20 HSC clones. We then examined adult hematopoiesis in wildtype and gata2b+/- zebrafish. Surprisingly, at steady state the marrow of gata2b+/- fish showed color dominance with a single color clone contributing to over 30% of granulocytes, while the myeloid output in wildtype clutchmates was polyclonal. This suggested a baseline oligoclonal state in gata2b+/- hematopoiesis. Gata2b+/- fish also had a concomitant mild myelocytopenia with 20% reduction of myeloid cells in the marrow as early as 2 months post fertilization (mpf), reminescent of monocytopenia often present in GATA2 patients. Myelocytopenia with concurrent expansion of progenitor population was more profound in gata2b-/- fish with up to 4-fold increase of progenitor to myeloid cell ratio at 3mpf. Gata2b-/- fish are adult viable, possibly due to partial compensation by a duplicated gata2a gene. Germline GATA2-associated MDS and AML often harbor additional acquired mutations in epigenetic regulators. To study the effect of somatic mutations in GATA2 heterozygosity, we induced mosaic mutagenesis in wild-type or gata2b+/-embryos by injecting 1-cell embryos with Cas9 mRNA and guide RNAs targeting zebrafish orthologs of ASXL1 and STAG2, both of which have been associated with GATA2 mutations in MDS or AML. The marrow analysis of injected gata2b+/- fish showed an increase of hematopoietic progenitor cells and worse myelocytopenia at 7 mpf compared to control injected wildtype fish (progenitor/myeloid ratio of 1.7, p < 0.05). Single color expansion was observed in over 60% of injected gata2b+/-fish as measured by Shannon index of diversity, and correlated with stag2a and stag2b frame-shift mutations with variant allele fraction of >20%. This mutations result in the predicted loss of at least 2 of the 4 total stag2 alleles. In summary, gata2b deficiency alone results in aberrant hematopoiesis with reduced HSC diversity and myelocytopenia. Mutations in stag2 lead to early enhanced clonal dominance in gata2b+/- but not wildtype fish. Our studies suggest a model in which germline GATA2 mutations lead to a state of reduced HSC diversity during development. Such a state predisposes to clonal events due to acquired mutations in epigenetic regulators resulting in clonal hematopoiesis and myeloid leukemias, establishing a link between abnormal developmental hematopoiesis and leukemia risk later in life. Disclosures No relevant conflicts of interest to declare.

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Mobilization and Chemosensitization of AML with the CXCR4 Antagonist Plerixafor (AMD3100): A Phase I/II Study of AMD3100+MEC in Patients with Relapsed or Refractory Disease.

Blood ◽

10.1182/blood.v112.11.1944.1944 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1944-1944 ◽

Cited By ~ 7

Author(s):

Geoffrey L. Uy ◽

Michael P. Rettig ◽

Kyle M. McFarland ◽

Lindsay M. Hladnik ◽

Shashikant Kulkarni ◽

...

Keyword(s):

Phase I ◽

Leukemic Cell ◽

Fold Increase ◽

Complete Response ◽

Peripheral Circulation ◽

Cxcr4 Expression ◽

Leukemic Cells ◽

Cxcr4 Antagonist ◽

Hematopoietic Stem ◽

Dose Levels

Abstract The interaction of leukemic blasts with the bone marrow microenvironment is postulated to be an important mediator of chemoresistance in AML. Although a number of receptor/ligand pairs have been implicated, the CXCR4/SDF-1 axis functions as the principal regulator of homing and retention of both normal and malignant hematopoietic cells in the marrow. Plerixafor (AMD3100) is a bicyclam molecule which reversibly blocks CXCR4 binding to SDF-1 and is being developed clinically as a mobilization agent for hematopoietic stem cell transplantation. Preclinical data from our group has demonstrated that in murine models, plerixafor can disrupt the interaction of leukemic cells with the marrow microenvironment and sensitize blasts to the effect of chemotherapy. Based on these data, we have initiated a phase I/II study in patients with relapsed or refractory AML in which plerixafor is administered prior to salvage chemotherapy. Subjects were required to have AML which is primary refractory to at least 2 induction regimens, in 1st relapse with an initial remission duration of < 12 months, in 1st relapse having failed ≥ 1 salvage regimens, or in 2nd relapse or higher. Plerixafor is administered by SQ injection followed by a 24 hour observation period to analyze its effects on leukemic cell mobilization. Then plerixafor is given daily 4 hours prior to chemotherapy consisting of mitoxantrone 8mg/m2/d, etoposide 100 mg/m2/d and cytarabine 1000 mg/m2/d x 5 days. To date, 19 patients have been treated at 3 dose levels of plerixafor: 80, 160 and 240 mcg/kg/day. We find that plerixafor can modestly mobilize leukemic cells (~ 2-fold increase) into the peripheral circulation at a peak of 6–8 hours after administration. FISH studies performed from informative samples demonstrates that this mobilization occurs equally in both non-leukemic and leukemic populations. While CXCR4 expression is increased on the surface of mobilized blasts, no clear relationship has been observed between CXCR4 expression or plerixafor dose and mobilization. At the 80 and 160 mcg/kg dose levels, a complete response (CR+CRi) was observed in 2 of 6 patients (33%). At the plerixafor 240 mcg/kg dose level, a complete response (CR+CRi) was achieved in 6 of 8 evaluable patients (75%) in this historically chemorefractory population. Plerixafor was well tolerated with no evidence of hyperleukocytosis or significant delays in neutrophil recovery (median 30 days, range 24–40). Based on this encouraging evidence of safety and efficacy, expansion of a phase II cohort is ongoing.

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Growth Factor Independence 1 b (Gfi1b) as a New Regulator of Hematopoietic Stem Cell Fate

Blood ◽

10.1182/blood.v116.21.837.837 ◽

2010 ◽

Vol 116 (21) ◽

pp. 837-837

Author(s):

Cyrus Khandanpour ◽

Lothar Vassen ◽

Marie-Claude Gaudreau ◽

Christian Kosan ◽

Tarik Moroy

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Cell Fate ◽

Conflicts Of Interest ◽

Fold Increase ◽

Surface Proteins ◽

Expression Array ◽

Hematopoietic Stem

Abstract Abstract 837 Donor matched transplantation of bone marrow or hematopoietic stem cells (HSCs) are widely used to treat hematological malignancies, but are associated with high mortality. Methods for expansion of HSC numbers and their mobilization into the bloodstream of a donor could significantly improve therapy. We show here that the zinc finger transcriptional repressor Gfi1b is highly expressed in hematopoietic stem cells (defined as CD 150+, CD 48-, Lin-, Sca1+ and c-kit+) cells and is down-regulated more than 10 fold upon differentiation into multipotential progenitors (defined as CD 150+ or CD150-, CD 48+, Lin-, Sca1+ and c-kit+). Constitutive germline deletion of Gfi1b is lethal at midgestation due to impaired development of erythrocytes and megakaryocytes. We have therefore developed a conditional knock-out of Gfi1b to study its role specifically in the adult hematopoietic system. Deletion of Gfi1b leads to a 30-fold increase of HSC numbers in bone marrow and around a100 fold increase in spleen and peripheral blood. This was due to a higher rate of HSCs undergoing cell cycling. Concomitantly, the number of quiescent HSCs was reduced 5–6 times. We then performed an gene expression array of wt and Gfi1b deficient HSCs and observed that loss of Gfi1b leads to an altered RNA expression of integrins and adhesion molecules, for instance CXCR4, VCAM-1 and Tenascin C, which usually retain HSCs in a dormant state in the endosteal niche. These changes were also confirmed on protein level. Finally, we could observe a higher levels of Reactive Oxygen Species (ROS) in the Gfi1b deficient HSCs compared to wt HSCs. We verified whether elevated level of ROS are causative for the expansion of HSCs and noticed that application of N-Acetyl-Cystein, which counteracts the effects of ROS, limits significantly the expansion of HSCs, underscoring the important role of ROS in the expansion of Gfi1b deficient HSCs. Despite markedly increased proliferation, Gfi1b-/- HSCs can reconstitute lymphoid and myeloid lineages to the same extent as wt HSCs when transplanted in competition with wt HSCs. Furthermore, Gfi1b deficient HSCs also feature an expansion after transplantation and expand 5–10 fold more than wt HSC when transplanted initially in equal numbers with wt HSCs. It is possible that lower expression of CXCR4, VCAM-1 and other surface proteins leads to release and egression of Gfi1b deficient HSCs from the hypoxic endosteal stem cell niche and exposes the HSCs to more oxygen which in turn increases ROS levels. Elevated ROS could promote entry of Gfi1b-/- HSCs into cell cycle. In conclusion Gfi1b regulates HSC dormancy, pool size and potentially also the egress and mobilization of HSCs and might offer a new therapeutic approach to improve human HSC transplantation. Disclosures: No relevant conflicts of interest to declare.

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Osteocytes Support Hematopoiesis by Altering the Bone Marrow Microenvironment Through Gsα Signaling

Blood ◽

10.1182/blood.v118.21.219.219 ◽

2011 ◽

Vol 118 (21) ◽

pp. 219-219

Author(s):

Daniela S. Krause ◽

Keertik Fulzele ◽

Kevin Barry ◽

Sutada Lotinun ◽

Roland Baron ◽

...

Keyword(s):

Bone Marrow ◽

Bone Remodeling ◽

Conflicts Of Interest ◽

Bone Matrix ◽

Myeloid Cells ◽

Hematopoietic Stem ◽

Inflammatory Protein ◽

G Protein Coupled

Abstract Abstract 219 Osteocytes, the most abundant and long living cells of bone embedded in the bone matrix, coordinate bone remodeling by regulating osteoblast and osteoclast activity, at least in part, via G-protein coupled receptor signaling. Osteoblasts and osteoclasts control hematopoiesis primarily by influencing self-renewal, differentiation, and mobilization of hematopoietic stem cells in their endosteal bone niche. A role for osteocytes in hematopoiesis has previously not been demonstrated. We engineered mice lacking Gsα in osteocytes (DMP1-GsαKO) using the Cre-loxP recombination technique. Consistent with the previously established role of osteocytes in regulation of bone remodeling, DMP1-GsαKO mice showed severe osteopenia and a decrease in cortical thickness. The osteopenia in the KO mice was due to a dramatic decrease in osteoblast numbers whereas the number and activity of osteoclasts was unaffected. In addition, DMP1-GsαKO mice displayed hematopoietic abnormalities that resembled a myeloproliferative syndrome (MPS) characterized by leukocytosis and neutrophilia. Myeloid cells were increased in the peripheral blood, bone marrow (BM), and spleen in DMP1-GsαKO mice compared to controls (p<0.01 in blood, BM and spleen, N≥6) as assessed by CBC and immunophenotypical flow cytometry analysis. Lineage- negative c-kit-positive and Sca-1+ (LKS) cells and LKS CD150-positive CD48-negative (LKS SLAM) cells were significantly increased in DMP1-GsαKO spleen compared to controls whereas there was no change in the bone marrow suggesting mobilization from the bone marrow in mutant mice. Surprisingly, the number of colonies formed in in-vitro methylcellulose assays from BM cells from DMP1-GsαKO mice were not changed indicating the requirement of the bone microenvironment to induce MPS. Co-culture of osteocyte-enriched bone explants from DMP1-GsαKO mice with control BM cells significantly increased the number of colonies compared to control explants. Transplantation of BM from control to DMP1-GsαKO mice rapidly recapitulated the MPS whereas converse transplantation completely normalized the hematopoietic abnormality. Protein expression of CXCL2 (macrophage inflammatory protein 2 alpha; MIP2-alpha), a chemotactic cytokine known to mobilize hematopoietic stem and myeloid cells, was markedly increased in Gsa deficient osteocytes as assessed by immunohistochemistry. Furthermore, CXCL2 secretion in conditioned media from osteocyte explants cultures was also increased 3-fold in Gsa deficient osteocytes as compared to controls. In summary, our results represent the first evidence for osteocyte-mediated regulation of hematopoiesis via Gsα-signaling-induced alteration of the BM microenvironment, possibly through CXCL2 signaling. Disclosures: No relevant conflicts of interest to declare.

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Screen for Small Molecules Capable of Expanding Human Hematopoietic Stem Cell Ex Vivo

Blood ◽

10.1182/blood.v118.21.1919.1919 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1919-1919

Author(s):

Iman Hatem Fares ◽

Jalila Chagraoui ◽

Jana Krosl ◽

Denis-Claude Roy ◽

Sandra Cohen ◽

...

Keyword(s):

Stem Cell ◽

Hematopoietic Stem Cell ◽

Mononuclear Cells ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Fold Increase ◽

Hematopoietic Stem ◽

Cd34 Cells

Abstract Abstract 1919 Hematopoietic stem cell (HSC) transplantation is a life saving procedure whose applicability is restricted by the lack of suitable donors, by poor responsiveness to mobilization regimens in preparation of autologous transplantations, by insufficient HSC numbers in individual cord blood units, and by the inability to sufficiently amplify HSCs ex vivo. Characterization of Stemregenin (SR1), an aryl hydrocarbon receptor (AHR) antagonist that promotes HSC expansion, provided a proof of principle that low molecular weight (LMW) compounds have the ability to promote HSC expansion. To identify novel putative agonists of HSC self-renewal, we initiated a high throughput screen (HTS) of a library comprising more than 5,000 LMW molecules using the in vitro maintenance of the CD34+CD45RA- phenotype as a model system. Our study was based on the fact that mobilized peripheral blood-derived CD34+CD45RA- cells cultured in media supplemented with: stem cell factor, thrombopoietin, FLT3 ligand and interleukin 6, would promote the expansion of mononuclear cells (MNC) concomitant with a decrease in CD34+CD45RA- population and HSC depletion. LMW compounds preventing this loss could therefore act as agonists of HSC expansion. In a 384-well plate, 2000 CD34+cells were initially cultured/well in 50μl medium comprising 1μM test compounds or 0.1% DMSO (vehicle). The proportions of CD34+CD45RA− cells were determined at the initiation of experiment and after a 7-day incubation. Six of 5,280 LMW compounds (0.11%) promoted CD34+CD45RA− cell expansion, and seventeen (0.32%) enhanced differentiation as determined by the increase in proportions of CD34−CD45RA+ cells compared to control (DMSO). The 6 LMW compounds promoting expansion of the CD34+CD45RA− cell population were re-analyzed in a secondary screen. Four out of these 6 molecules suppressed the transcriptional activity of AHR, suggesting that these compounds share the same molecular pathway as SR1 in stimulating HSC expansion, thus they were not further characterized. The remaining 2 compounds promoted, similar to SR1 or better, a 10-fold and 35-fold expansion of MNC during 7 and 12-day incubations, respectively. The expanded cell populations comprised 65–75% of CD34+ cells compared to 12–30% determined for DMSO controls. During 12-day incubation with these compounds, the numbers of CD34+ cells increased ∼25-fold over their input values, or ∼ 6-fold above the values determined for controls. This expansion of CD34+ cells was associated with a ∼5-fold increase in the numbers of multilineage CFC (granulocyte, erythroid, monocyte, and megakaryocyte, or CFU-GEMM) compared to that found in DMSO control cultures. The ability of the 2 newly identified compounds to expand functional HSCs is currently being evaluated in vivo usingimmunocompromised mice. In conclusion, results of our initial screen suggest that other mechanism, besides inhibition of AhR, are at play for expansion of human HSC. Disclosures: No relevant conflicts of interest to declare.

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Regulatory T Cells of G-CSF Mobilized Stem Cell Donors Qualify for Clinical Applications,

Blood ◽

10.1182/blood.v118.21.4055.4055 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4055-4055

Author(s):

Sya N. Ukena ◽

Sarvari Velaga ◽

Goudeva Lilia ◽

Philipp Ivanyi ◽

Arnold Ganser ◽

...

Keyword(s):

Clinical Trials ◽

T Cells ◽

Stem Cell ◽

Regulatory T Cells ◽

Conflicts Of Interest ◽

Interleukin 2 ◽

Fold Increase ◽

Hematopoietic Stem ◽

Cell Numbers

Abstract Abstract 4055 Recent clinical studies demonstrate the high potency of human regulatory T cells (Tregs) to control graft-versus-host disease following hematopoietic stem cell transplantation (SCT). Isolation of Tregs after recombinant G-CSF induced mobilization of stem cells would simplify the design of clinical trials in allogeneic SCT. However, there is growing evidence that G-CSF also exerts profound immune modulatory effects in the adaptive immune system. Therefore, we analyzed Tregs isolated by FACS based cell sorting from stem cell donors before (n=8) and after (n=13) G-CSF administration regarding their phenotype, stability, functional and expansion properties. Absolute CD4+ T cell (3.2 fold increase of mean after G-CSF; p<0.05) and CD4+CD25hi Treg cell numbers (4.1 fold increase of mean after G-CSF; p<0.01) were significantly increased after G-CSF administration. Analysis of the Foxp3 TSDR demethylation level displays a stable Foxp3 phenotype of G-CSF encountered Tregs (mean value 97.1% vs. 95.0 % after G-CSF administration). Moreover, the CD4+CD25hi Tregs of G-CSF treated SC donors suppress the proliferation of effector T cells with no significant differences to Tregs isolated from healthy donors before G-CSF treatment (mean values 42.1% vs. 49.9% after G-CSF administration at a Treg/ T effector cell ratio of 1:1). In vitro expansion of Tregs isolated after G-CSF application with anti-CD3 and anti-CD28 dynabeads in the presence of interleukin-2 led to comparable cell numbers as for the stimulation of control Treg cells. However, differences could be detected for the thymic derived marker molecule CD31 and those associated with activation (LAP, CD69, CD62L) and migration (CxCR3) as detected by FACS analysis. Our results show no significant differences regarding suppressive function and stability of Tregs isolated from stem cell donors before and after G-CSF administration. These data support the application of G-CSF mobilized Tregs for clinical trials which in turn opens up new possibilities for the adoptive transfer of Tregs in HSCT. Disclosures: No relevant conflicts of interest to declare.

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Thrombopoietin-Receptor Signalling Induces Proliferation of Dormant HSC.

Blood ◽

10.1182/blood.v120.21.2343.2343 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2343-2343

Author(s):

Larisa V. Kovtonyuk ◽

Markus G. Manz ◽

Hitoshi Takizawa

Keyword(s):

Conflicts Of Interest ◽

Cxcr4 Antagonist ◽

Extrinsic Factors ◽

Subsequent Increase ◽

Hematopoietic Stem ◽

Regulatory Pathways ◽

Surface Receptors ◽

Early Results

Abstract Abstract 2343 Lifelong 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 control Our 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. Disclosures: No relevant conflicts of interest to declare.

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