Level Of Hematopoietic Progenitor Cells Research Articles

Eosinophil Progenitors in Patients With Non-Asthmatic Eosinophilic Bronchitis, Eosinophilic Asthma, and Normal Controls.

ObjectiveThis study aims to explore the potential of in situ airway differentiation of eosinophil progenitors (EoPs) and hematopoietic progenitor cells (HPCs) in sputum and peripheral blood from patients with non-asthmatic eosinophilic bronchitis (NAEB), eosinophilic asthma (EA), and healthy controls (HC).MethodsUsing flow cytometry, we enumerated sputum and blood HPCs and EoPs in patients with NAEB (n=15), EA (n=15), and HC (n=14) at baseline. Patients with NAEB and EA were then treated for 1 month with budesonide (200 μg, bid) or budesonide and formoterol (200/6 μg, bid), respectively. HPCs and EoPs in both compartments were re-evaluated.ResultsAt baseline, NAEB and EA both had significantly greater numbers of sputum but not blood HPCs and EoPs (p<0.05) compared to HC. There were no differences between NAEB and EA. After 1 month of inhaled corticosteroid (ICS) treatment, NAEB patients showed a significant improvement in cough symptoms, but the attenuation of sputum HPC and EoP levels was not significant.ConclusionsNAEB patients have increased airway levels of HPCs and EoPs. One-month treatment with ICS did not fully suppress the level of EoPs in NAEB. Controlling in situ airway differentiation of EoPs may control airway eosinophilia and provide long-term resolution of symptoms in NAEB.

The ability of bone marrow progenitor cells to form colonies in ex vivo culture of MDS patients

The results of in vitro hematopoiesis studies have provided most of the knowledge about the organization, regulation, and development of the human hematopoietic system over the past three to four decades. However, due to the impossibility of an appropriate assessment of hematopoietic stem cells (HSC) in humans and because of the shortcomings of methodological approaches to determining the role of hematopoietic progenitor cells in the pathogenesis of MDS and to predicting the course of the pathological process, semiliquid agar cultures of bone marrow from patients with myelodysplastic syndrome were used. Myelodysplastic syndrome (MDS) refers to a clinically, morphologically, and genetically heterogeneous group of diseases characterized by clonalism and arising from mutations at the level of hematopoietic progenitor cells. Proliferation of such a mutated stem cell progenitors leads to ineffective maturation of myeloid lineage cells and dysplastic changes in the bone marrow (BM). The aim of the study was to establish the relationship between the functional activity of hematopoietic progenitor cells in the ex vivo culture and the activity of the pathological process in the myelodysplastic syndrome. We studied bone marrow samples from patients with the myelodysplastic syndrome, namely refractory anemia with excess blasts I (MDS RAEB I) and refractory anemia with excess blasts II (MDS RAEB II) and AML under conditions in vitro, as well as their clinical laboratory data. It was found that the percentage of blasts and myeloblasts in the samples of patients with AML and MDS RAEB II increased, compared to the samples of patients with MDS RAEB I (63.5±3.9 %, 18.05±1.01 % and 9.49±1.53 % respectively). An increase in the number of erythrocytes and hemoglobin content was noted in the group of patients with MDS RAEB I compared with MDS RAEB II (2.9±1.4×1012 / l and 105.04±3.6 g / l versus 9±0.8×1012 / l and 84.5±4.8 g / l, respectively). The analysis of the results of BM studies of patients with MDS in in vitro culture indicated a significant lag in the formation of cell aggregates during cultivation and a pronounced inhibition of the colony-forming ability of progenitor cells, compared to the control. A noticeable decrease in the colony-forming ability was observed in patients with MDS RAEB I, MDS RAEB II and AML in this sequence – 4.1±1.2 per 1×105 explanted cells, 3.2±0.9 per 1×105 explanted cells and 2.0±0.6 per 1×105 explanted cells, respectively. The analysis of hematological parameters and the results of BM cells cultivation at different stages of MDS indicates that the colony-forming ability of progenitor cells correlates with the depth of the pathological process.

Histidine decarboxylase deficiency inhibits NBP-induced extramedullary hematopoiesis by modifying bone marrow and spleen microenvironments.

Histidine decarboxylase (HDC), a histamine synthase, is expressed in various hematopoietic cells and is induced by hematopoietic cytokines such as granulocyte colony-stimulating factor (G-CSF). We previously showed that nitrogen-containing bisphosphonate (NBP)-treatment induces extramedullary hematopoiesis via G-CSF stimulation. However, the function of HDC in NBP-induced medullary and extramedullary hematopoiesis remains unclear. Here, we investigated changes in hematopoiesis in wild-type and HDC-deficient (HDC-KO) mice. NBP treatment did not induce anemia in wild-type or HDC-KO mice, but did produce a gradual increase in serum G-CSF levels in wild-type mice. NBP treatment also enhanced Hdc mRNA expression and erythropoiesis in the spleen and reduced erythropoiesis in bone marrow and the number of vascular adhesion molecule 1 (VCAM-1)-positive macrophages in wild-type mice, as well as increased the levels of hematopoietic progenitor cells and proliferating cells in the spleen and enhanced expression of bone morphogenetic protein 4 (Bmp4), CXC chemokine ligand 12 (Cxcl12), and hypoxia inducible factor 1 (Hif1) in the spleen. However, such changes were not observed in HDC-KO mice. These results suggest that histamine may affect hematopoietic microenvironments of the bone marrow and spleen by changing hematopoiesis-related factors in NBP-induced extramedullary hematopoiesis.

Germline Mutations Predispose to Familial Myeloproliferative Neoplasms

Background: Myeloproliferative neoplasms (MPNs), commonly polycythemia vera (PV), primary myelofibrosis (PMF) and essential thrombocythemia (ET), are a group of malignant hematologic disorders characterized by proliferation and accumulation of terminally differentiated blood cells of erythroid, myeloid and megakaryocytic origin (Cazzola, Blood 2014).Somatic driver mutations, frequently JAK2V617F, MPLW515L/K or mutant CALR in sporadic MPNs affect signaling by the thrombopoietin (THPO) and/or the erythropoietin (EPO) receptor (Pickman, PLoS 2016). MPL is the receptor of THPO and JAK2 is the key downstream mediator of THPO/MPL signaling (Nangalia NEJM 2013). In familial MPNs (families with more than 1 member developing a MPN) a germline mutation predisposes to disease development. These mutations can reveal pathways or processes involved in the pathogenesis of the disease, independent of, or cooperating with, the somatic driver mutations (Rumi, JCO 2007). We identified 3 putative predisposing germline mutations in 1 family, 3 affected generations developed MPN (PMF, ET and PV): RBSN frameshift,JAK2R340Q &amp; PRKAA2Q425P missense variants. RBSN encodes Rabenosyn-5 protein (RBSN) which plays a role in early growth-factor receptor clathrin-dependent endocytosis (Nielsen, J Cell Biol 2000) (Fig.1).We discovered that wild type (WT) Rabenosyn-5 regulates cell surface MPL levels in hematopoietic progenitor cells and predict that MPL levels on the cell surface are negatively associated with WT Rabenosyn-5 levels. We hypothesize that the RBSN mutation predisposes to MPN development in this family, leading to abnormal activation of THPO signaling by reducing MPL turnover. Methods: Peripheral blood DNA from patient samples were analyzed for potential putative mutations using Agilent SureSelect Human ALL Exon V5+UTRs exome capture kit followed by parallel sequencing with Illumina HiSeq 2000. In vitro studies involved using the BaF3 system and CRISPR-Cas9 gene editing technique. We generated BaF3-MPL (BaF3 cells transduced with human CD4-MPL fusion) cell lines that rely on IL3 or human THPO cytokines for growth &amp; proliferation (Fig.2&amp;3). We transduced RBSN into the BaF3-MPL cell lines and produced RBSN heterozygous (HT) and homozygous (HM) mutants. We first generated LentiCRISPRV2GFP-mRBSN vector by inserting RBSN specific guide RNA into the LentiCRISPRV2GFP vector and generated lentivirus expressing both Cas9 and guide RNA by co-transfecting 293T cells with LentiCRISPRV2GFP vector and viral packaging vectors. The final virus was used to infect BaF3-MPL cells. Transduced cells were purified by FACS to select GFP+ cells and individual cells were grown in culture. RBSN gene mutations were examined using PCR and sequenced accordingly. To study whether the RBSN mutation alters the surface MPL levels, we stained BaF3-MPL, HT-RBSN-BaF3 and HM-RBSN-BaF3 cells with APC-CD4 antibody and the surface levels of MPL were detected by FACS by comparing the mean fluorescence intensity (MFI) of APC. Results: We identified several colonies with either HT-RBSN-BaF3 or HM-RBSN-BaF3. The guide RNA we designed specifically targeted the 12th exon of the RBSN gene which best recapitulates the mutations observed in our patients, thus, the frameshift mutations in HT-RBSN-BaF3 and HM-RBSN-BaF3 cell lines we predict will make truncated forms of RBSN which will be verified by Western Blotting for future experiments. Surface levels of MPL in the HT-RBSN-BaF3 and HM-RBSN-BaF3 cells are greater than that in BaF3-MPL cells, suggesting RBSN negatively regulates THPO-MPL signaling by regulating the cell surface MPL levels (Fig. 4). Conclusions: RBSN is a regulator of receptor trafficking, which mediates the fusion of the endocytosed receptor to the early endosome and then lysosome for degradation. We propose that genetic inactivation of RBSN might prevent the endocytosed MPL from endosome-degradation and enhance the reuse of MPL by recycling. Future experiments are planned to include in vivo studies to study whether inactivation of RBSN promoted MPN-like disease development into NSG mice and transducing our established cell lines to label individual cell compartments to study THPO-stimulated signaling. Our findings, if confirmed, will further clarify aspects of familial and somatic MPN pathogenesis and may inform efforts to devise new therapeutic and diagnostic strategies for this disease complex. Disclosures No relevant conflicts of interest to declare.

Reduced Expression of the DNA-Binding Protein ARID3a, a Mediator of Human Hematopoiesis, in Hematopoietic Progenitors Contributes to Age-Related Transcriptional Changes

Abstract Population studies estimate that 40% of European and US populations will be over the age of 60 by 2050. Aged individuals exhibit impaired immune responses consisting of poor responses to vaccines and increases in myeloid lineage cells. The reasons for decreased immunity in the elderly haven’t been completely elucidated, making studies of the mechanisms leading to these poor responses important. One possibility is that defects in early hematopoiesis may contribute to poor immune responses in the elderly. Previous studies in our lab indicated that inhibition of the DNA-binding protein ARID3a in hematopoietic cultures of human cord blood led to hematopoietic developmental skewing toward myeloid lineage rather than lymphoid lineage progenitors. Furthermore, we previously reported that numbers of ARID3a-expressing hematopoietic stem cells (HSCs) can be highly variable in some patient subsets as well as in healthy individuals. ARID3a expression levels in hematopoietic progenitor cells have not previously been analyzed in relationship to age. We hypothesized that alterations in ARID3a expression levels in early hematopoietic progenitors could contribute to poor immune responses observed in elderly people. Our data suggest that hematopoietic progenitors obtained from peripheral blood of aged healthy persons show decreased frequencies of ARID3a-expressing HSCs in comparison to HSCs from young individuals. Furthermore, lineage committed progenitor numbers were also altered in aged persons compared young people. Ongoing analyses to investigate the gene expression data in young and aged HSCs in relation to ARID3a expression will assess potential genes regulated by ARID3a that contributes to lineage commitment and development.

A Novel PU.1 - Caspase 8/cFLIP Axis in Neutrophil and Macrophage Differentiation of AML Cells

The transcription factor PU.1 (SPI-1) plays an important role in numerous cellular processes of myeloid cells, such as cell survival, proliferation and differentiation. PU.1 is expressed at intermediate levels in hematopoietic progenitor cells, whereas progenitors expressing low amounts of PU.1 differentiate towards the lymphoid lineage, while increased amounts of PU.1 levels promoted macrophage or granulocyte differentiation. Additionally, low PU.1 expression levels contribute to the immature myeloid phenotype in acute myeloid leukemia (AML).We recently linked PU.1 expression to TRAIL and chemotherapy sensitivity in AML cells. To further molecularly dissect the functions of PU.1 in myeloid cells, we focused on putative PU.1 targets associated with cell death responses emerging from a gene expression profiling experiment using Pu.1-null and Pu.1-restored 503 murine myeloid cells. We identified Caspase 8 (Casp8) and its paralogous gene c-Flip (aka FADD-like apoptosis regulator; Cflar) as PU.1-regulated genes, and showed putative PU.1-binding sites in their proximal promoter regions.CASP8 and c-FLIP are known for their function downstream of death-receptor mediated apoptosis. Yet, it has been reported that CASP8 has non-apoptotic functions involving cell proliferation, differentiation, and inflammation. C-FLIP is the enzymatically inactive homolog of CASP8 and exists in three isoforms: a long isoform (c-FLIPL), that partially inhibits CASP8 activity, and two short forms (c-FLIPR and c-FLIPS) that are anti-apoptotic.To assess if CASP8 and/or c-FLIP are involved in PU.1-regulated cellular processes, we took advantage of the NB4 APL cell line model. These cells can be differentiated towards granulocytes with all-trans retinoic acid (ATRA) in a PU.1-dependent manner. We found an 8-10-fold induction of CASP8 and c-FLIP mRNA expression upon granulocytic differentiation of NB4 cells. Underlining a possible function of these two genes in granulocyte differentiation, we detected a markedly increased mRNA expression of both genes in human CD34+ hematopoietic cells differentiated towards neutrophils using G-CSF. Furthermore, knocking down PU.1 in NB4 cells significantly impaired CASP8 and cFLIP mRNA upregulation. Importantly, the anti-apoptotic cFLIPs isoform was exclusively induced after prolonged ATRA-treatment in PU.1 knockdown cells, whereas cFLIPL and CASP8 mRNA levels were reduced. The binding of PU.1 to the CFLAR promoter region together with altered cFLIP isoform ratio upon PU.1 expression indicates direct transcriptional activation of cFLIP and possibly an involvement of PU.1 in alternative splicing of cFLIP.Based on previous reports linking non-apoptotic CASP8 functions to macrophage differentiation and our findings of PU.1-dependent CASP8 regulation, we next studied the role of CASP8 in more detail during monocyte and macrophage differentiation by knocking down CASP8 expression in HL60 cells. HL60 CASP8 knockdown cells, generated using two independent shRNAs, were treated with vitamin D3 (VitD3) or PMA to induce monocyte or macrophage differentiation, respectively. Surprisingly, knocking down CASP8 led to increased CD11b expression together with increased pseudopodia formation. Furthermore, analysis of secreted cytokines in HL60 CASP8 knockdown cells suggests activation of macrophages towards an M2 phenotype.Our findings extend the role of PU.1 function to cell survival during granulocytic differentiation of APL cells. This occurs via distinct regulation of the pro-apoptotic CASP8, and anti-apoptotic cFLIP gene programs, respectively. Our findings suggest that increased expression of the anti-apoptotic, shorter cFLIP isoforms later in neutrophil differentiation may support short-term neutrophil cell survival. Lastly, our results implicate a novel PU.1-CASP8 pathway that may be necessary for alternative activation of M2 macrophages. DisclosuresNo relevant conflicts of interest to declare.

The Yiqi and Yangyin Formula ameliorates injury to the hematopoietic system induced by total body irradiation.

In this study, we examined whether the Yiqi and Yangyin Formula (YYF), used in traditional Chinese medicine, could ameliorate damage to the hematopoietic system induced by total body irradiation (TBI). Treatment with 15 g/kg of YYF increased the survival rate of Institute of Cancer Research (ICR) mice exposed to 7.5 Gy TBI. Furthermore, YYF treatment increased the white blood cell (WBC), red blood cell (RBC), hemoglobin (HGB) and hematocrit (HCT) counts in ICR mice exposed to 2 Gy or 4 Gy TBI. Treatment with YYF also increased the number of bone marrow cells, hematopoietic progenitor cells (HPCs), hematopoietic stem cells (HSCs) and the colony-forming ability of granulocyte–macrophage cells. YYF alleviated TBI-induced suppression of the differentiation ability of HPCs and HSCs and decreased the reactive oxygen species (ROS) levels in bone marrow mononuclear cells (BMMNCs), HPCs and HSCs from mice exposed to 2 Gy or 4 Gy TBI. Overall, our data suggest that YYF can ameliorate myelosuppression by reducing the intracellular ROS levels in hematopoietic cells after TBI at doses of 2 Gy and 4 Gy.

Feasibility and kinetics of CD34+hematopoietic progenitor cell mobilization in response to a single administration of docetaxel chemotherapy and pegfilgrastim in a contemporary cohort of patients with metastatic breast cancer

Autologous hematopoietic stem cell transplantation (auto-HSCT) remains an experimental therapy for metastatic breast cancer (MBC) and there is no established protocol for cluster of differentiation 34+ (CD34+ ) hematopoietic progenitor cell (HPC) mobilization with historic studies using growth factors with or without chemotherapy. This study describes the feasibility and kinetics of CD34+ HPC mobilization following a single administration of docetaxel and the pegylated form of recombinant human granulocyte colony-stimulating factor analogue filgrastim (pegfilgrastim). The study design was serial measurement of peripheral blood CD34+ HPC in patients with MBC following a single administration of intravenous (IV) docetaxel 100 mg/m2 on day 1 and subcutaneous (SC) pegfilgrastim 6 mg on day 2. Eight patients with MBC were enrolled. The median age was 56 years (range 51-75 years). All patients had human epidermal growth factor receptor 2 (HER2) negative disease and either hormone refractory or negative disease. Three patients had bone only disease, four had visceral organ disease with or without bone involvement and one had locally unresectable disease only. All patients had prior therapy for early-advanced stage disease and prior therapy for MBC included seven patients receiving at least one line of hormone therapy and three having palliative chemotherapy. Six patients recorded a rise in the CD34+ count greater than 20 cells/μL. The median peak level was 40.2 cells/μL (standard deviation = 28.7) occurring on day 9 and with an average duration of 4 days. Overall, treatment was well tolerated with manageable side-effects. The single administration of docetaxel and pegfilgrastim was effective in mobilization of CD34+ HPC and peak levels followed a predictable course. This approach needs validation in prospective studies by preparation of auto-HSCT by leukapheresis and quantification of total CD34+ HPC yields.

Circulating Hematopoietic Progenitor Cells are Decreased in COPD

Rationale: Bone marrow derived progenitor cells participate in the repair of injured vessels. The lungs of individuals with emphysema have reduced alveolar capillary density and increased endothelial apoptosis. We hypothesized that circulating levels of endothelial and hematopoietic progenitor cells would be reduced in this group of patients. Objectives: The goal of this study was to measure circulating levels of endothelial progenitor cells (EPCs) and hematopoietic progenitor cells (HPCs) in subjects with COPD and to determine if progenitor levels correlated with disease severity and the presence of emphysema. Methods: Peripheral blood mononuclear cells were isolated from 61 patients with COPD and 32 control subjects. Levels of EPCs (CD45dim CD34+) and HPCs (CD45+ CD34+ VEGF-R2+) were quantified using multi-parameter flow cytometry. Progenitor cell function was assessed using cell culture assays. All subjects were evaluated with spirometry and CT scanning. Measurements and Main Results: HPC levels were reduced in subjects with COPD compared to controls, whereas circulating EPC levels were similar between the two groups. HPC levels correlated with severity of obstruction and were lowest in subjects with severe emphysema. These associations remained after correction for factors known to affect progenitor cell levels including age, smoking status, the use of statin medications and the presence of coronary artery disease. The ability of mononuclear cells to form endothelial cell colony forming units (EC-CFU) was also reduced in subjects with COPD. Conclusions: HPC levels are reduced in subjects with COPD and correlate with emphysema phenotype and severity of obstruction. Reduction of HPCs may disrupt maintenance of the capillary endothelium, thereby contributing to the pathogenesis of COPD.

Microarray and Proteomic Analysis of Myeloproliferative Neoplasms,

Abstract 3856The gene and protein expression profiles in myeloproliferative neoplasms (MPN) may reveal gene markers of a potential clinical function in diagnosis and prediction of response to therapy. Using cDNA microarray analysis, involving 25,100 unique genes, we studied the gene expression profile of hematopoietic CD34+ progenitor cells and granulocytes obtained from peripheral blood of patients with essential thrombocythemia (ET), polycythemia vera (PV) and primary myelofibrosis (PMF) compared with healthy individuals. The microarray analyses of the hematopoietic progenitor cells and granulocytes have been performed on 9 patients with ET, 8 patients with PV, 4 patients with PMF and 8 healthy donors. The granulocytes for proteomic studies have been pooled in 4 groups: PV with JAK2 mutant allele burden above 80%, ET with JAK2 mutation, PMF with JAK2 mutation and ET/PMF with no JAK2 mutation. We focused our analysis to hematopoiesis related genes. In the collected patient samples, the increased number of granulocytes allowed for further validation by protein analysis of microarray gene expression suggested from less differentiated hematopoietic progenitor cells. Folate receptor 3 (FOLR3), constitutively secreted in hematopoietic tissues, has increased protein levels in granulocytes of JAK2V617F homozygous PV as well as mRNA levels in hematopoietic progenitor cells of patients with PV. The enzyme matrix metallopeptidase 9 (MMP9), involved in IL-8-induced mobilization of hematopoietic progenitor cells from bone marrow, also has significantly increased protein levels in granulocytes of PV patients with increased JAK2 mutation allele burden. In addition, Ras-related C3 botulinum toxin substrate 2 (RAC2) protein level, essential for erythropoiesis, is increased specifically in PV granulocytes with JAK2V617F homozygosity. Moreover, RAC2 gene expression is significantly increased in hematopoietic progenitor cells of PV, with no changes in its granulocytes. Although, like PV, RAC2 gene expression was also increased in ET and PMF hematopoietic progenitor cells compared to healthy individuals, in granulocytes of ET and PMF patients with JAK2 mutation RAC2 protein levels were decreased, contrary to the elevated level in PV. Furthermore, inconsistent with JAK2V617F homozygous PV patients, granulocytes of ET and PMF with the JAK2 mutation exhibit FOLR3 protein at levels lower than the ET and PMF with no JAK2 mutation. Investigating the extent to which these genes participate in the complex molecular and cellular mechanisms of MPN will likely lead to new insights of malignancy development. In conclusion, molecular profiling of hematopoietic progenitor cells and granulocytes of MPN patients revealed gene expression patterns that are beyond their recognized function in disease pathogenesis and can be related to patients' clinical characteristics with imminent prognostic relevance. Disclosures:No relevant conflicts of interest to declare.

Clearance of Senescent Neutrophils Maintains Homeostatic Levels of Hematopoietic Progenitor Cells in the Circulation

Abstract 863Neutrophils (PMN) are short-lived cells generated in the bone marrow (BM). After just a few hours in the circulation, aged PMN upregulate the expression of the chemokine receptor CXCR4 and acquire tropism to the BM, where they are cleared by resident macrophages. We have found that a population of “senescent” PMN (sPMN), identified by low expression of L-selectin and high expression of CXCR4, is detectable in the blood of wild-type (WT) mice and that their levels fluctuate circadianally in a way similar to that reported for hematopoietic progenitor cells (HPC). In addition, clearance of sPMN by resident phagocytes has been reported to alter the levels of cytokines involved in HSPC trafficking. In this study, we have asked whether clearance of sPMN modulates the hematopoietic niche and thus controls the physiological trafficking of HSPC.We initially found that mice deficient in endothelial P- and E- selectins (PE mice) or selectin ligands (deficient in Fucosyltransferase 7, Fut7−/−) present granulocytosis and elevated HPC in blood, as detected by clonogenic assays and flow cytometry. To determine whether this phenotype could be corrected by components present in WT blood, we generated parabiotic pairs of these mice with fluorescently-tagged WT partners. Neutrophilia and HSPC levels in the blood of Fut7−/− mice were corrected in WT:Fut7−/− chimeras, whereas in WT:PE parabiotic chimeras we observed neutrophilia and increased circulating levels of HPC derived from the WT partner. To determine whether sPMN were the blood component responsible for modulating HPC trafficking, we prepared and injected BM-derived sPMN into WT mice. This treatment resulted in a 2.5-fold increase in circulating HPC, and this correlated with reduced levels of the chemokine CXCL12 in BM. sPMN treated with pertussis toxin or deficient in Fut7−/− were unable to efficiently home to the BM and completely lost their capacity to modulate HPC trafficking. We next investigated whether sPMN clearance by resident macrophages was required for the modulation of the hematopoietic niche and HPC egress. For this purpose, we treated mice with clodronate-loaded liposomes and injected sPMN 10 days later, a time in which macrophages but not monocytes or PMN were still depleted in the BM. Under these conditions, sPMN lost their capacity to reduce CXCL12 levels in BM and to induce HPC egress. When analyzing transcriptional alterations in the BM of Fut7−/− mice, where sPMN trafficking to BM is impeded, we found reduced expression of genes directly regulated by the nuclear liver × receptors (LXR) α and β, two receptors involved in signalling after phagocytosis. We thus speculated that ingestion of sPMN by BM-macrophages induced signals through LXRαβ which might be important for modulating the hematopoietic niche and triggering HPC egress. Indeed, in vivo administration of the LXR agonist GW3965 in WT mice mimicked sPMN treatment by inducing a 3-fold increase in circulating HPC levels and a reduction of CXCL12 expression in BM. Together, these results demonstrate that sPMN extravasation and clearance by tissue-resident macrophages trigger signals that modulate the hematopoietic niche and induce the egress of HPC, and suggest that LXR-mediated signals are required for this process. Given that the relatively modest changes in HPC levels in blood induced by sPMN were comparable to those found during normal light-dark cycles, we reasoned that this phenomenon might be important to maintain these physiological fluctuations. Indeed, normal HSPC fluctuations in blood were lost in mice in which PMN were depleted with an anti-Ly6G antibody or in mice deficient in LXRαβ.In summary, we uncover a mechanism that links the homeostasis of the immune and hematopoietic systems and that may have important implications for understanding the behavior and function of circulating HPC. Disclosures:No relevant conflicts of interest to declare.

Remobilization with High Dose Methotrexate and Cytarabine in Patients with Non-Hodgkin Lymphoma and Multiple Myeloma Previously Failing Mobilization with Chemotherapy and Cytokine Treatment

Abstract 4402 BackgroundHigh-dose chemotherapy supported by autologous stem cell transplantation is a widely used therapeutic modality especially in patients with non-Hodgkin lymphomas (NHL) and multiple myeloma (MM). However, mobilization failure rates following first attempt are estimated to be between 5% and 30% and remobilization is required in those patients. Although plerixafor, a selective CXCR4 antagonist, is a promising agent for these purpose, mobilization with chemotherapeutic agents would be a hands-on option.°° Therefore we investigated the feasibility of high dose methotrexate and cytarabine with G-CSF as a remobilization regimen in those who failed a prior mobilization and collection with chemotherapy and G-CSF. Patients and methodsMobilization failure was defined as a collection of less than 5 × 106 CD34+ cells after 3–5 apheresis procedures. Methotrexate (3,500 mg/m2 in a 120 min infusion) on day 1 and cytarabine (3,000 mg/m2 infusion for 120 min) on day 4 and day 5 were followed by G-CSF. Supportive care and leucovorine rescue were done as standard protocol. Peripheral blood (PB) hematopoietic progenitor cells (HPCs) were counted with the Sysmex SE9000. PB progenitor cell harvest was initiated when HPC levels reached at least 5 per mm3. ResultsTotal 8 patients (6 NHL and 2 MM, median age: 55 years) who have failed in previous mobilization with conventional chemotherapy and G-CSF were tried again with high dose methotrexate, cytarabine and G-CSF. (table 1) Successful collection of CD34+ cells (> 5 × 106/kg) was achieved in six patients (75%). The total yield of CD34+ cells per kg of body weight harvested by 3 to 5 apheresis procedures was 6.28 × 106/kg (median; range, 1.53– 10.09 × 106/kg). (table 2) ConclusionHigh dose-MTX and cytarabine plus G-CSF may be a feasible chemotherapeutic regimen for stem cell mobilization in NHL and MM patients who have failed in previous mobilization with other type of chemotherapy and G-CSF.Table 1Patients characteristics and mobilization outcome (1st & 2nd °°mobilization)NAgeDiagnosisPrevious Treatmentprevious Mobilization regimen(1st)No. Of apheresis in 1st mobilizationTotal CD34+ yield (x106/kg ) in 1st mobilizationNo. Of apheresis in 2nd mobilizationTotal CD34+ yield (x106/kg ) in 2nd mobilizationCumulative CD34+ yield (x106/kg ) in 1st & 2nd mobilization148PTCLCHOP SMILESMILE44.0333.77.73257MZLRCVP ESHAPESHAP41.8845.337.21359MCLCHOP ESHAPESHAPHD Ara-C3,40.67, 1.5331.533.73454MMVAD PADCYC44.73410.0914.82564PTCLCHOP ESHAPESHAP20.647.017.61645DLBCLRCHOP ESHAPESHAP31.1257.738.85762MMVAD PADCYC43.7639.2112.97842EN NK/T cell lymphomaVIPDCYC53.1645.558.71Abbreviations: PTCL=Peripheral T-cell Lymphoma; MZL=Marginal Zone Lymphoma; MCL=Mantle Cell Lymphoma; MM=Multiple Myeloma; EN=extra nodal; DLBCL=Diffuse Large B-cell Lymphoma; CHOP=cyclophosphamide, doxorubicin, vincristine, prednisolone; SMILE=dexamethasone, methotrexate, ifosfamide, L-asparaginase, etoposide; RCVP=rituximab, cyclophosphamide, vincristine, prednisolone; ESHAP=etoposide, methylprednisolone, cytarabine, cisplatin; VAD=vincristine, doxorubicin, dexamethasone; PAD=bortezomib, doxorubicin, dexamethasone; RCHOP=rituximab+CHOP; VIPD=etoposide, ifosfamide, cisplatin, dexamethasone; HD Ara-C=high dose cytarabine; CYC=cyclophosphamide; No.=number;Table 2Remobilization of CD34+ cells after HD-MTX and cytarabine plus G-CSFNApheresis 1Apheresis 2Apheresis 3Apheresis 4Apheresis 5Cumulative CD34+ yield (x106/kg)10.121.542.043.720.671.890.891.885.3330.290.760.481.5341.831.293.693.1810.0950.410.953.262.397.0160.390.471.871.543.467.7371.353.094.779.2181.121.272.021.145.55Median (range)0.54 (0.12−0.83)1.28 (0.47−3.09)2.03 (0.48−4.77)1.88 (1.14−3.18)3.46 (3.46)6.28 (1.53 – 10.09) Disclosures:No relevant conflicts of interest to declare.

Differential Regulation of MicroRNA Expression In Polycythemia Vera CD34+ Cells

AbstractAbstract 4785 Introduction:Polycythemia vera (PV) is a clonal stem cell disorder characterized by unregulated red cell, white cell and platelet production, a phenotype attributable to an activating point mutation (V617F) in JAK2, a tyrosine kinase utilized for signaling by Type 1 hematopoietic growth factor receptors. However, it is generally conceded that JAK2 V617F is not the initiating mutation in PV, nor are any of the other gene mutations, deletions and amplifications discovered to date. Deregulated gene transcription is also a feature of PV but a unified mechanism for this also cannot be ascribed to the currently known genetic defects. MicroRNAs (miRs) are non-coding 18–22nt RNAs that function as posttranscriptional gene repressors by binding to complementary target sequences in the mRNA 3′UTR to inhibit protein translation. There is growing evidence that miRs regulate hematopoiesis not only at the committed hematopoietic progenitor cell level but also at the level of the hematopoietic stem cell (HSC). Studies of miR behavior in PV have documented differential expression during erythroid differentiation as well as differential expression in granulocytes and platelets. However, PV is a stem cell disorder and to date there has been no analysis of miR behavior in PV HSC. We report here differential miR expression in PV peripheral blood (pb) CD34+ cells. Methods:We studied pb CD34+ cells from eight PV patients (four females and four males, all JAK2 V617F-positive) and six normal donors (three females and three males) in conjunction with a study of mRNA expression in PV pb CD34+ cells by oligonucleotide microarray. Using total cell RNA, microRNA expression was analyzed with the Agilent Human microRNA Microarray Kit Version 3, which contains probes for 866 human miRs. The TaqMan MicroRNA Assay (Applied Biosystems) was used for quantitative reverse-transcription PCR to verify differential miR expression. The Student unpaired t test was used for statistical analysis and P values £ 0.05 were considered significant. Results:Overall, in the 8 PV patients as a group, there were 130 positive calls with differential regulation of 81 miRs (p <0.05) (23 up regulated and 58 down regulated) and there was no correlation between miR expression and the JAK2 V617F allele burden. Given the phenotypic and genotypic differences between men and women PV patients, we analyzed differential miR expression separately by gender to identify gender-specific effects. 75 miRs (26 up and 49 down regulated) were differentially expressed in the male PV patients and 32 (7 up and 25 down regulated) in the women, confirming similar observations of gender-related differences in global mRNA expression in PV pb CD34+ cells. Amongst the differentially regulated miRs in the group as a whole were the miR-H1 group, miR-125a-3p and 196b. The miR-H1 group, also known as miR-17-92, is an oncomir with a unique gene structure from which one primary transcript yields six distinct miRs. This cluster was only up regulated in male PV patients. Likewise, miR-125a-3p was also up regulated only in males. Its expression is dependent on DICER, a gene also only up regulated in male patients. MiR-125a-3p has been identified as being preferentially expressed in HSC, acts to expand HSCs by preventing apoptosis and favors a myeloid fate. MiR-196b, which was concordantly down regulated in both men and women, is normally up regulated in HSC. Altogether, consistent with their different phenotypes, 31 miRs were differently regulated solely in the male patients (16 up regulated and 15 down), while only 12 miRs were differently regulated solely in the women (2 up and 10 down regulated) Seven miRs were concordantly up (miR-575) or down regulated (miR-150; miR-181c; miR-196b; miR-551b; miR-769-5p and miR-874) by both the men and women patients. Notably, down regulation of miR-150 at the HSC level is consistent with its role in lineage-specific progenitor cell commitment. Since there appears to be little overlap with respect to target genes for this core group of miRs, multiple venues for translational control are likely to be involved in the pathogenesis of PV. Conclusion:These data establish the presence of differential miR expression in PV HSC and provide insight into potential mechanisms for PV HSC expansion, confirm a genetic basis for the gender-specific differences that characterize PV and identify novel therapeutic targets for drug discovery. Disclosures:No relevant conflicts of interest to declare.

The mouse mutation “thrombocytopenia and cardiomyopathy” (trac) disrupts Abcg5: a spontaneous single gene model for human hereditary phytosterolemia/sitosterolemia

AbstractThe spontaneous mouse mutation “thrombocytopenia and cardiomyopathy” (trac) causes macrothrombocytopenia, prolonged bleeding times, anemia, leukopenia, infertility, cardiomyopathy, and shortened life span. Homozygotes show a 20-fold decrease in platelet numbers and a 3-fold increase in platelet size with structural alterations and functional impairments in activation and aggregation. Megakaryocytes in trac/trac mice are present in increased numbers, have poorly developed demarcation membrane systems, and have decreased polyploidy. The thrombocytopenia is not intrinsic to defects at the level of hematopoietic progenitor cells but is associated with a microenvironmental abnormality. The trac mutation maps to mouse chromosome 17, syntenic with human chromosome 2p21-22. A G to A mutation in exon 10 of the adenosine triphosphate (ATP)–binding cassette subfamily G, member 5 (Abcg5) gene, alters a tryptophan codon (UGG) to a premature stop codon (UAG). Crosses with mice doubly transgenic for the human ABCG5 and ABCG8 genes rescued platelet counts and volumes. ABCG5 and ABCG8 form a functional complex that limits dietary phytosterol accumulation. Phytosterolemia in trac/trac mice confirmed a functional defect in the ABCG5/ABCG8 transport system. The trac mutation provides a new clinically significant animal model for human phytosterolemia and provides a new means for studying the role of phytosterols in hematologic diseases and testing therapeutic interventions.

Limited Regeneration but Functional Preservation of Hematopoietic Stem Cells in the Mice Developing Leukemia via Constitutive Expression of Notch1.

Leukemia development is a complex process involving both intrinsic and extrinsic factors. While many environmental factors have been studied, the impact of leukemic environment on normal hematopoietic stem cell (HSC) and hematopoietic progenitor cell (HPC) has not been definitively investigated. In this study, we have formally addressed this important issue by examining the potential functional alterations of HSC and HPC in the mice bearing Notch1-induced T acute lymphoblastic leukemia (T-ALL). The MSCV retrovirus vector containing cDNA encoding oncogenic intracellular domain of Notch1 (ICN1) pseudotyped with VSV-G was used to infect Lin−Sca-1+ cells in order to induce leukemic development. Normal hematopoietic cells from the B6.SJL strain (CD45.1+) were co-transplanted with Notch1 transduced Lin−Sca-1+ cells (CD45.2+) into lethally irradiated recipients. In this robust leukemia model with 100% penetrance, the normal hematopoietic cell compartment marked by CD45.1 in the leukemic marrow was sorted for phenotypic analyses and functional assays at different time points. Same numbers of the normal hematopoietic cells without Notch1-transduced cells were transplanted into the irradiated recipients as controls. As expected, progressive hematopoietic suppression was observed at both HSC and HPC levels in the leukemic mice. The frequency of HSC enriched population (Lin−c-Kit+Sca-1+, LKS) in the leukemic group was 7 times lower than that in the control at the 4th week of leukemogensis. When normalized to the bone marrow cellularity, the absolute yield of each population was 246 times lower in the leukemic group than that in the control group. These data were highly consistent with significantly lower yields of colony forming unit (CFU) and cobblestone area forming cell (CAFC). To measure the long-term engraftment of HSCs from leukemic environment, we performed the competitive bone marrow transplantation (cBMT), in which equal numbers of CD45.1+ cells isolated from leukemic or control mice and competitor cells (CD45.1/.2) at the 2nd week of leukemogenesis were co-transplanted into lethally irradiated C57BL/6J recipients. Unexpectedly, the multilineage engraftment of the hematopoietic cells isolated from the leukemic mice was 3 times more than that of the control group. Moreover, HSCs from the leukemic environment remained functional in serial transplant recipients. Finally, to explore the underlying molecular mechanisms for the enhanced function of normal HSC in the cBMT model, we examined a number of cell cycle and self-renewal regulators in HSC and HPC from leukemic marrow and control group at the time of harvest prior to transplantation by qRT-PCR. There was a significant decrease in p18 expression when compared with the control, whereas p21 expression was significantly increased. Notch1, Gfi1 and c-myc signalings were also elevated in the HSCs from leukemic environment. In summary, our current work provides the first definitive evidence for the reversible inhibition of normal HSC growth by the leukemic environment, thereby having important implications for HSC transplantation as well as leukemogenesis.

A randomized comparison of peripheral blood hematopoietic progenitor cell level of 5/mm3 versus 50/mm3 as a surrogate marker to initiate efficient autologous blood stem cell collection

We previously showed that at least 5/mm(3) hematopoietic progenitor cells (HPCs) could be used as a marker for initiating autologous blood stem cell collection (ABSCC). However, the timing of efficient ABSCC following mobilization is still to be determined. We conducted a prospective, randomized comparison of 5/mm(3) versus 50/mm(3) peripheral blood (PB) HPCs as a surrogate marker to initiate efficient ABSCC. Forty-five consecutive patients, 26 with multiple myeloma (MM) and 19 with non-Hodgkin's lymphoma (NHL), were enrolled between October 2004 and October 2006. Chemotherapy was cyclophosphamide 4 g/m(2) for MM and ESHAP (etoposide, methylprednisolone, high-dose cytarabine, and cisplatin), with or without Rituximab, for NHL. Circulating HPCs were monitored daily with the Sysmex SE9000 automated hematology analyzer, and harvested CD34+ cells were counted by flow cytometry. ABSCC was initiated when HPC levels reached at least 5/mm(3) (HPC5 group) or 50/mm(3) (HPC50 group). The median number of harvested CD34+ cells was 15.0 x 10(6)/kg and 21.0 x 10(6)/kg in the HPC5 and HPC50 groups, respectively (P = 0.23). Optimal collection (>5 x 10(6) CD34+ cells/kg) in a single session (day 1) was attained in 15 HPC5 patients (63%) and in 14 HPC50 patients (67%), and targeted collection of 5 x 10(6) CD34+ cells/kg was achieved in 100 and 95% of HPC5 and HPC50 patients, respectively (P = 0.47), with a median number of 1 apheresis in both groups (P = 0.58). There were no between group differences in optimal collection rate on day 1, median number of aphereses to achieve optimal collection, and overall optimal collection rate. HPC > or = 5/mm(3) and > or =50/mm(3) are both reliable indices for the timing of ABSCC.

A new model for predicting the timing of leukapheresis on the basis of CD34+ cell and hematopoietic progenitor cell levels

We developed a model (depending on peripheral CD34(+) cell count and hematopoietic progenitor cell count) to determine the optimal timing of 3-day leukapheresis in patients pretreated with chemotherapy and G-CSF. Marrow potentials were identified on the basis of three patterns of leukapheretic yield. Pattern 1 predicted good marrow potential. The positive predictive value of a first-day leukapheretic yield of >1 x 10(6) CD34(+) cells/kg (mean 3-day yield = 8.18 x 10(6) CD34(+) cells/kg, n = 11) was 100%. Pattern 2 predicted poor marrow potential. The negative predictive value of a 3-day leukapheretic yield of >1 x 10(6) CD34(+) cells/kg (3-day yield = 0.26 x 10(6) CD34(+) cells/kg, n = 1) was 100%. Pattern 3 met neither of the above criteria (mean 3-day yield = 1.37 x 10(6) CD34(+) cells/kg, n = 19). The marrow potential was borderline and patients could be further divided into two subgroups according to peripheral CD34(+) cell counts when WBC reached >10,000/microl. The mean yield differed significantly between pattern 1 and 3 (P < 0.001). For patients with good marrow potential, leukapheresis should begin as soon as the WBC count is >5,000/microl. Patients with borderline marrow potential may benefit from delaying leukapheresis until the WBC level is >10,000/microl and leukapheresis extended more than 3 days.

Initiation of peripheral blood progenitor cell harvest based on peripheral blood hematopoietic progenitor cell counts enumerated by the Sysmex SE9000

The most reliable index for timing peripheral blood progenitor cell (PBPC) collection following mobilization is still to be determined. The techniques to enumerate peripheral blood (PB) CD34+ cells are expensive and time-consuming. The SE9000 (Sysmex) provides an estimate of immature cells, called hematopoietic progenitor cells (HPCs). The aim of this study was to prospectively evaluate the efficacy of PB HPC levels for timing PBPC harvest. Thirty-five patients (15 non-Hodgkin's lymphoma and 20 multiple myeloma) were enrolled. PB HPCs and harvested CD34+ cells were counted with the SE9000 and flow cytometry, respectively. Circulating HPCs were monitored daily. PBPC harvest was initiated when HPC levels reached at least 5 per mm(3). HPC levels reached 5 per mm(3) or more on Median Day 12 (range, days 9 to 16) of mobilizing chemotherapy. The median number of CD34+ cells collected per patient was 19.40 x 10(6) per kg (range, 1.94 x 10(6)-52.55 x 10(6) per kg). Both successful and optimal harvest was achieved in 97 percent of patients. PBPCs were successfully harvested in 25 patients (71%) in one session. An optimal harvest in a single session was attained in 16 patients (46%). This might be the first prospective study showing the PB HPC level for timing PBPC harvest.

Clinical and Biological Significance of CD56 Antigen Expression in Acute Promyelocytic Leukemia

The biological significance of CD56 antigen expression in patients with acute promyelocytic leukemia (APL) has been under investigation. We investigated the clinical and biologic features of CD56+APL. In our series, CD56 antigen was positive in 4 of 28 (14%) APL patients. No differences were found regarding age, gender, performance status (PS), initial leukocyte and platelet counts, lactate dehydrogenase (LDH) and fibrinogen (Fbg) levels according to CD56 expression. CD34 antigen was co-expressed in 3 of the 4 patients with CD56+ APL, in contrast to 2 of the 24 patients with CD56- APL (P = .01). Extramedullary relapse occurred in 3 of the 4 patients with CD56+ APL, in contrast to none of the 24 patients with CD56- APL (P = .001). Median remission duration was 4 months in CD56+ APL and was not reached in CD56- APL. The CD56+ population had a shorter remission duration (P < .0001) and disease-free survival (P < .0001). In contrast, no difference was found in overall survival. These results suggested that CD56 expression was associated with the leukemogenetic mutation at the primitive hematopoietic progenitor cell level and extramedullary relapse in APL patients treated with ATRA and chemotherapy.

Bestatin selectively suppresses the growth of leukemic stem/progenitor cells with BCR/ABL mRNA transcript in patients with chronic myelogeneous leukemia

The in vitro effect of bestatin on Philadelphia (Ph) chromosome positive chronic myeloid leukemia (CML) was investigated using mature clonogenic cells and primitive stem cells derived from long-term culture-initiating cells (LTC-ICs). Individual colonies were grown in methycellulose culture (clonogenic cells) and after 5 weeks, LTC (colonies derived from LTC-ICs) were individually isolated. DNA isolated from these clonogenic colonies was studied by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis to detect BCR/ABL mRNA transcripts b3a2 and b2a2. At the mature hematopoietic progenitor cell level, almost all (20/21) colonies, including both erythroid and myeloid progenitors, were leukemic, i.e. BCR/ABL mRNA positive. Although normal progenitors were able to grow in the presence of bestatin, even at the most primitive progenitor cell level (LTC-ICs), the number of leukemic clones gradually decreased. Furthermore, bestatin suppressed the outgrowth of leukemic clones more frequently than control LTC without any effect on the growth of normal clones. These results indicate that bestatin, at levels that can be obtained by per os administration clinically, suppresses only Ph-positive leukemic clones without affecting normal hematopoiesis. Based on these results, we suggest that bestatin has the potential to provide another treatment for patients with CML.