Experimental Hematology RSS feed: Current Issue. Experimental Hematology publishes original research reports, reviews, letters to the editor, and abstracts of the annual meeting of the International Society for Experimental Hematology. We welcome manuscripts describing research involving in vivo and ex vivo studies in the following areas: cell cycle regulation, cytokines, erythropoiesis, gene therapy, general hematopoiesis, granulopoiesis, hematological malignancies, immunobiology, immunotherapy, lymphopoiesis, megakaryocytopoiesis, microenvironment, monocyte development, molecular genetics, signal transduction, stem cell biology, stem cell plasticity, and experimental as well as clinical stem cell transplantation.http://www.exphem.org/?rss=yesElsevier Inc.en © 2010 Published by Elsevier Inc. All rights reserved. Experimental Hematology0301-472X382February 2010 © 2010 Published by Elsevier Inc. All rights reserved. healthpermissions@elsevier.comhttp://www.exphem.org/article/PIIS0301472X09004640/abstract?rss=yesEditorial Board10.1016/S0301-472X(09)00464-0Experimental Hematology 38, 2 (2010)2010-02-01Experimental Hematology2010-02-01382S0301-472X(09)X0014-7IFCIFChttp://www.exphem.org/article/PIIS0301472X09004494/abstract?rss=yesObjective: Emerging work has revealed an integral role of the tumor necrosis factor–α (TNF-α) nuclear factor (NF)-κB pathway in the regulation of hematopoiesis. TNF-α inhibition of hematopoietic stem/progenitor cell growth involves type I TNF-α receptor (TNF-RI) and type II TNF-α receptor (TNF-RII). However, the role of TNF-RI vs TNF-RII in mediating this response is less clear. Full induction of NF-κB–dependent gene expression through TNF-RI requires the transcriptional coactivator SIMPL (substrate that interacts with mouse pelle-like kinase). To address the role of SIMPL in TNF-α-dependent signaling in hematopoiesis, endothelial cells and hematopoietic progenitors expressing SIMPL short hairpin RNA were characterized.Material and Methods: In vitro gene expression and progenitor assays employing SIMPL short hairpin RNA were used to examine the requirement for SIMPL in TNF-α–dependent effects upon cytokine gene expression and hematopoietic progenitor cell growth. Competitive repopulation studies were used to extend these studies in vivo.Results: SIMPL is required for full TNF-RI–dependent expression of NF-κB–controlled cytokines in endothelial cells. Hematopoietic progenitor cell expansion is not affected if progenitors lacked SIMPL or if progenitors are treated with human TNF-α, which signals through TNF-RI. In the absence of SIMPL, human TNF-α leads to a dramatic decrease in progenitor cell expansion that is not due to apoptosis. Loss of SIMPL does not affect the activity of transforming growth factor–β1 and interferon-γ, other known suppressors of hematopoiesis.Conclusions: Suppression of myeloid progenitor cell expansion requires signaling through TNF-RI and TNF-RII. Signals transduced through the TNF-α–TNF-RI–SIMPL pathway support hematopoietic progenitor cell survival, growth and differentiation.Loss of SIMPL compromises TNF-α-dependent survival of hematopoietic progenitorsEric A. Benson, Mark G. Goebl, Feng-Chun Yang, Reuben Kapur, Jeanette McClintick, Sonal Sanghani, D. Wade Clapp, Maureen A. Harrington10.1016/j.exphem.2009.11.006Experimental Hematology 38, 2 (2010)2009-11-26Experimental Hematology2009-11-26382S0301-472X(09)X0014-7Cytokines7181http://www.exphem.org/article/PIIS0301472X09004512/abstract?rss=yesObjective: Angiopoietin-1 (Ang-1) plays a critical role in the maintenance of hematopoietic stem cells (HSCs) in the bone marrow (BM) through its binding to the Tie2 receptor. Ang-2, another Tie2 ligand, is known to be an antagonist of Tie2/Ang-1 signaling in angiogenesis; however, its function in regulation of HSCs remains unclear. Here, we investigated the functional differences between Ang-1 and Ang-2 in the maintenance of HSCs.Materials and Methods: We treated mouse BM lineage−Sca-1+c-Kit+ side population+ cells with Ang-1 and/or Ang-2, and evaluated angiopoietin function by gene expression analysis, immunocytochemical staining of phosphorylated Akt, a colony-formation assay, and a long-term BM reconstitution assay.Results: Gene expression analysis and BM transplantation assay revealed that Ang-1 upregulated expression of p57, p18, Itgb1, Alcam, Tie2, Hoxb4, and Bmi1 genes in HSCs, while Ang-2 antagonized the effects of Ang-1. Ang-1 enhanced the phosphorylation of Akt, while Ang-2 again reduced the effect of Ang-1. The colony assay demonstrated that neither Ang-1, nor Ang-2 influenced the colony formation of HSCs. BM transplantation assay, following in vitro cultivation of HSCs with angiopoietins, showed that Ang-1 maintained long-term repopulating activity of HSCs, while the addition of Ang-2 interfered drastically with the effects of Ang-1.Conclusion: Gene expression analysis and BM transplantation assay demonstrated that Ang-1 maintained HSC activity in an in vitro culture. In contrast, Ang-2 reversed the effects of Ang-1/Tie2 signaling in the regulation of long-term HSCs. Our data suggest that Ang-1 is a dominant ligand for the Tie2 receptor in long HSCs in BM.Functional differences between two Tie2 ligands, angiopoietin-1 and -2, in regulation of adult bone marrow hematopoietic stem cellsYumiko Gomei, Yuka Nakamura, Hiroki Yoshihara, Kentaro Hosokawa, Hiroko Iwasaki, Toshio Suda, Fumio Arai10.1016/j.exphem.2009.11.007Experimental Hematology 38, 2 (2010)2009-11-30Experimental Hematology2009-11-30382S0301-472X(09)X0014-7Cytokines8289.e1http://www.exphem.org/article/PIIS0301472X09004299/abstract?rss=yesObjective: Mutations in the CCAAT enhancer binding protein epsilon (C/EBPε) gene have been identified in the cells of patients with neutrophil specific granule deficiency, a rare congenital disorder marked by recurrent bacterial infections. Their neutrophils, in addition to lacking specific granules required for normal respiratory burst activity, also lack normal phagocytosis and chemotaxis. Although the specific granule deficiency phenotype has been replicated in C/EBPε−/− (knockout [KO]) mice, the mechanisms by which C/EBPε mutations act to decrease neutrophil function are not entirely clear.Materials and Methods: In order to determine the role of C/EBPε in neutrophil differentiation and migration, we generated immortalized progenitor cell lines from C/EBPε KO and wild-type mice and performed expression and flow cytometric analysis and functional studies.Results: Expression of lineage-specific cell surface antigens on our in vitro differentiated cell lines revealed persistent expression of monocytic markers on KO granulocytes. We verified this in primary murine peripheral blood and bone marrow cells. In addition, KO bone marrow had an increase in immature myeloid precursors at the common myeloid progenitor and granulocyte/monocyte progenitor levels, suggesting a critical role for C/EBPε not only in granulocyte maturation beyond the promyelocyte stage, but also in the monocyte/granulocyte lineage decision. We found that restoration of Hlx (H2.0-like homeo box 1) expression, which was decreased in C/EBPε KO cells, rescued chemotaxis, but not the other defects of C/EBPε KO neutrophils.Conclusions: We show two new regulatory functions of C/EBPε in myelopoiesis: in the absence of C/EBPε, there is not only incomplete differentiation of granulocytes, but myelopoiesis is disrupted with the appearance of an intermediate cell type with monocyte and granulocyte features, and the neutrophils have abnormal chemotaxis. Restoration of expression of Hlx provides partial recovery of function; it has no effect on neutrophil maturation, but can completely ameliorate the chemotaxis defect in C/EBPε KO cells.C/EBPε directs granulocytic-vs-monocytic lineage determination and confers chemotactic function via HlxStephanie Halene, Peter Gaines, Hong Sun, Theresa Zibello, Sharon Lin, Arati Khanna-Gupta, Simon C. Williams, Archibald Perkins, Diane Krause, Nancy Berliner10.1016/j.exphem.2009.11.004Experimental Hematology 38, 2 (2010)2009-11-18Experimental Hematology2009-11-18382S0301-472X(09)X0014-7Granulopoiesis90103.e4http://www.exphem.org/article/PIIS0301472X09004500/abstract?rss=yesObjective: Canine cyclic hematopoiesis (CH), a model of human cyclic neutropenia and severe congenital neutropenia, is characterized by a periodic reduced neutrophil count and decreased neutrophil elastase (NE) enzymatic activity. Canine CH is caused by a mutation of AP3B1 encoding the β3A subunit of adaptor protein complex–3 (AP-3). It has been proposed that trafficking of elastase is affected by AP-3. The aim of this study was to study intracellular sorting/trafficking of NE in CH dogs using antibodies specific to canine NE.Materials and Methods: Polyclonal and monoclonal antibodies were generated to immunogenic epitopes in the middle (aa85–98) and C-terminal (aa269–282) regions of NE. The antibodies to canine NE were characterized by Western immunoblotting and immunocytochemistry.Results: Antibody ELA85 (antibody to canine NE aa 85–98) specifically recognized mature 28-kD NE. Immunocytochemical analysis using ELA85 and an antibody to myeloperoxidase demonstrated colocalizaton of NE and myeloperoxidase in primary granules of normal dogs. Antibody ELA269 (antibody to canine NE aa 269–282) reacted exclusively with the 33-kD NE presumptive precursor form. Immunocytochemical analysis demonstrated that the NE precursor was not colocalized with myeloperoxidase in the primary granules of normal or CH dogs. Western immunoblotting using these antibodies demonstrated that CH dogs contained reduced mature NE, but accumulated a large amount of the NE precursor protein that was not enzymatically active.Conclusion: Antibodies ELA85 and ELA269 were found to be useful reagents for studying the biosynthesis, processing, and trafficking of NE during normal myelopoiesis. Neutrophils from CH dogs accumulated large amounts of higher molecular weight elastase precursors compared to normal dogs.Neutrophil elastase–processing defect in cyclic hematopoietic dogsRonghua Meng, Roger Bridgman, Maria Toivio-Kinnucan, Glenn P. Niemeyer, William Vernau, Tommy Hock, Clinton D. Lothrop10.1016/j.exphem.2009.09.010Experimental Hematology 38, 2 (2010)2009-11-26Experimental Hematology2009-11-26382S0301-472X(09)X0014-7Granulopoiesis104115http://www.exphem.org/article/PIIS0301472X09004305/abstract?rss=yesObjective: Primary mediastinal B-cell lymphoma (PMBCL) and Hodgkin lymphoma (HL) share many biological and clinical characteristics supporting a common pathogenetic pathway. Interleukin (IL)-13 has an important pathophysiological role in HL. In this study, we asked the question of whether IL-13 is a major contributor to the observed difference in features of inflammation between HL and PMBCL.Materials and Methods: Expression of IL-13 and IL-4 receptors was studied by flow cytometry, expression of a functional cysteinyl leukotriene receptor type 1 (CysLT1R) was investigated by calcium flux measurement, expression and activity of 15-lipoxygenase type 1 (15-LO-1) was determined by Western blot and reversed-phase high-performance liquid chromatography, respectively, and cytokines were quantified by Bioplex detection.Results: Stimulation of the PMBCL cell line Karpas-1106P with IL-13 or IL-4 induced a proinflammatory phenotype similar to that of the HL cell line L1236. Upon interleukin stimulation of the PMBCL cell line, the cellular size increased and cells became multinucleated. Cells also expressed CysLT1R and 15-LO-1, and produced the proinflammatory eoxins. The IL-13 or IL-4 treated PMBCL cell line and the HL cell line secreted a similar set of cytokines such as IL-6, tumor necrosis factor−α, interferon-inducible protein−10, interferon-γ, and RANTES.Conclusions: IL-13 or IL-4 stimulation of the PMBCL cell line Karpas-1106P induced an inflammatory phenotype that resembles that of the HL cell line. Our results suggest that the autocrine release of IL-13 in HL is one critical factor that can at least partly explain the difference in phenotype between these two lymphoma entities.Interleukin-13 stimulation of the mediastinal B-cell lymphoma cell line Karpas-1106P induces a phenotype resembling the Hodgkin lymphoma cell line L1236Erik Andersson, Frida Schain, Jan Sjöberg, Magnus Björkholm, Hans-Erik Claesson10.1016/j.exphem.2009.11.005Experimental Hematology 38, 2 (2010)2009-11-20Experimental Hematology2009-11-20382S0301-472X(09)X0014-7Hematological Malignancies116123http://www.exphem.org/article/PIIS0301472X09004524/abstract?rss=yesObjective: It is generally assumed that plasma cells from multiple myeloma (MM) patients do not express the stem cell marker CD34. This assumption has led to several clinical trials based on autologous CD34+ cell transplantation. However, the results of these trials have been disappointing.Materials and Methods: We investigated the presence of CD34+ cell populations in RPMI 8226, KARPAS 417, and U266 MM cell lines in vitro and during their growth as plasmacytoma tumors in nonobese diabetic severe combined immunodeficient mice, and in plasma cells isolated from the bone marrow of 38 MM patients.Results: We showed that in both patients and cell lines, a small population of plasma cells expresses CD34. These cells display morphological characteristics of MM plasma cells, are CD19-negative, and express B7-H1 (PD-L1), a T-cell inhibitory molecule. In patients, CD34+CD138+ cells expressed Ki67, a marker for proliferation. Moreover, when cells from the human myeloma cell line U266 were injected into nonobese diabetic severe combined immunodeficient mice, the U266-derived plasmacytoma tumors showed a large CD34+CD138+ Ki67+ cell population, indicating that these cells were not quiescent in vivo.Conclusions: MM patients carry a small subpopulation of cycling CD34+CD138+B7-H1+ plasma cells. Their presence may limit the clinical benefits of autologous CD34+ cell transplantation.A subpopulation of malignant CD34+CD138+B7-H1+ plasma cells is present in multiple myeloma patientsKlaudia Kuranda, Céline Berthon, Caroline Dupont, Dariusz Wolowiec, Xavier Leleu, Renata Polakowska, Nathalie Jouy, Bruno Quesnel10.1016/j.exphem.2009.11.008Experimental Hematology 38, 2 (2010)2009-11-30Experimental Hematology2009-11-30382S0301-472X(09)X0014-7Hematological Malignancies124131.e4http://www.exphem.org/article/PIIS0301472X09004275/abstract?rss=yesObjective: Despite improvements in current combinational chemotherapy regimens, the prognosis of the (1;19)(q23;p13) translocation (E2A/PBX1)−positive B-cell precursor acute lymphoblastic leukemia (ALL) is poor in pediatric leukemia patients.Materials and Methods: In this study, we examined the roles of growth arrest−specific-6 (GAS6)/Mer axis in the interactions between E2A/PBX1-positive B-cell precursor ALL cells and the osteoblastic niche in the bone marrow.Results: Data show that primary human osteoblasts secrete GAS6 in response to the Mer-overexpressed E2A/PBX1-positive ALL cells through mitogen-activated protein kinase signaling pathway and that leukemia cells migrate toward GAS6 using pathways activated by Mer. Importantly, GAS6 supports survival and prevents apoptosis from chemotherapy of E2A/PBX1-positive ALL cells by inducing dormancy.Conclusions: These data suggest that GAS6/Mer axis regulates homing and survival of the E2A/PBX1-positive B-cell precursor ALL in the bone marrow niche.GAS6/Mer axis regulates the homing and survival of the E2A/PBX1-positive B-cell precursor acute lymphoblastic leukemia in the bone marrow nicheYusuke Shiozawa, Elisabeth A. Pedersen, Russell S. Taichman10.1016/j.exphem.2009.11.002Experimental Hematology 38, 2 (2010)2009-11-16Experimental Hematology2009-11-16382S0301-472X(09)X0014-7Hematological Malignancies132140http://www.exphem.org/article/PIIS0301472X09004536/abstract?rss=yesObjective: Multiple myeloma (MM) is characterized by a high incidence of osteolytic bone lesions, which have been previously correlated with the gene expression profiles of MM cells. The aim of this study was to investigate the transcriptional patterns of cells in the bone microenvironment and their relationships with the presence of osteolysis in MM patients.Materials and Methods: Both mesenchymal (MSC) and osteoblastic (OB) cells were isolated directly from bone biopsies of MM patients and controls to perform gene expression profiling by microarrays and real-time polymerase chain reaction on selected bone-related genes.Results: We identified a series of upregulated and downregulated genes that were differentially expressed in the MSC cells of osteolytic and nonosteolytic patients. Comparison of the osteolytic and nonosteolytic samples also showed that the MSC cells and OB had distinct transcriptional patterns. No significantly modulated genes were found in the OBs of the osteolytic and nonosteolytic patients.Conclusions: Our data suggest that the gene expression profiles of cells of the bone microenvironment are different in MM patients and controls, and that MSC cells, but not OBs, have a distinct transcriptional pattern associated with the occurrence of bone lesions in MM patients. These data support the idea that alterations in MSC cells may be involved in MM bone disease.Distinct transcriptional profiles characterize bone microenvironment mesenchymal cells rather than osteoblasts in relationship with multiple myeloma bone diseaseKatia Todoerti, Gina Lisignoli, Paola Storti, Luca Agnelli, Francesca Novara, Cristina Manferdini, Katia Codeluppi, Simona Colla, Monica Crugnola, Manuela Abeltino, Marina Bolzoni, Valentina Sgobba, Andrea Facchini, Giorgio Lambertenghi-Deliliers, Orsetta Zuffardi, Vittorio Rizzoli, Antonino Neri, Nicola Giuliani10.1016/j.exphem.2009.11.009Experimental Hematology 38, 2 (2010)2009-12-07Experimental Hematology2009-12-07382S0301-472X(09)X0014-7Microenvironment141153http://www.exphem.org/article/PIIS0301472X09004287/abstract?rss=yesObjective: Cord blood banks provide fully human leukocyte antigen−typed cells, from which a set of standard induced pluripotent stem (iPS) cells for use in allogenic transplantation can be derived. Hence, the ability to generate iPS cells from cord blood cells has the potential to provide a suitable source for clinical transplantation. The aim of this work is to determine the reprogramming methods, culture conditions, and cell fractions that can be used to generate iPS cells from cord blood cells effectively.Materials and Methods: CD34+, mononucleated, and derived adherent cells from cord blood were cultured in hematopoietic medium (X-vivo10 containing 50 ng/mL interleukin-6, 50 ng/mL soluble interleukin-6 receptor, 50 ng/mL stem cell factor, 10 ng/mL thrombopoietin, and 20 ng/mL Flit3/4 ligand) 3 days prior to viral infection. Cells were then infected with retroviral constructs driving the expression of OCT3/4, SOX2, Krüppel-like factor 4, c-MYC, and enhanced green fluorescent protein together with or without the p53 knockdown lentiviral construct Shp53 pLKO.1-puro. Infected cells were then cultured for an additional 4 days in hematopoietic culture medium before being transferred onto mouse embryonic fibroblast (MEF) or SNL76/7 feeder cells in human embryonic stem cell medium (Dulbecco's modified Eagle medium/F-12 containing 20% knockout serum replacement, 200mM l-glutamine, 1% non-essential amino acids (NEAA), 0.1mM 2-mercaptoethanol, and 4 ng/mL basic fibroblast growth factor). Subsequently, the number of embryonic stem cell−like colonies that emerged in the following 4 weeks was scored. Expression of a number of pluripotency makers were examined by immunochemistry and reverse transcriptase polymerase chain reaction. Finally, the differentiation potential of selected colonies was determined by teratoma formation in severe combined immunodeficient mice and in vitro culture.Results: Repression of p53 expression by the addition of a lentiviral p53 short-hairpin RNA expression vector increased the frequency of formation of iPS-like colonies from 1 (on average) to around 100 per 2×104 cells when infected cells were grown on SNL feeder cells.Conclusions: iPS cells can be generated easily from CD34+ cord blood cells through the addition of p53 inhibition to standard reprogramming conditions.Effective generation of iPS cells from CD34+ cord blood cells by inhibition of p53Chiemi Takenaka, Naoki Nishishita, Nozomi Takada, Lars Martin Jakt, Shin Kawamata10.1016/j.exphem.2009.11.003Experimental Hematology 38, 2 (2010)2009-11-16Experimental Hematology2009-11-16382S0301-472X(09)X0014-7Stem Cell Biology154162.e2http://www.exphem.org/article/PIIS0301472X09004597/abstract?rss=yesIn the article entitled “Identification of small Sca-1+, Lin−, CD45− multipotential cells in the neonatal murine retina,” which appeared in the September 2009 issue of Experimental Hematology (Volume 37, Issue 9, pages 1096-1107), the correct spelling of the name of the eighth author is Volker Enzmann.Erratum10.1016/j.exphem.2009.12.002Experimental Hematology 38, 2 (2010)2009-12-30Experimental Hematology2009-12-30382S0301-472X(09)X0014-7Erratum163163