Experimental Hematology RSS feed: Current Issue. Experimental Hematology publishes original research reports (regular and fast-track submissions), reviews, perspectives, letters to the editor, and abstracts of the annual meeting of ISEH- Society for Hematology and Stem Cells. We welcome manuscripts describing basic in vitro and in vivo research centered on normal and malignant hematopoiesis as well as non-malignant hematologic diseases. Submissions focused on non-hematopoietic stem cells (e.g. mesenchymal stem cells, embryonic stem cells and induced pluripotent stems) with potential relevance to hematopoiesis are also welcome, as are studies involving experimental or early phase clinical cell transplantation. Studies employing genomic and systems biology approaches to the study of normal and malignant hematopoiesis are strongly encouraged, as are those employing model organisms. http://www.exphem.org/?rss=yesElsevier Inc.en © 2012 Published by Elsevier Inc. All rights reserved. Experimental Hematology0301-472X406June 2012 © 2012 Published by Elsevier Inc. All rights reserved. healthpermissions@elsevier.comhttp://www.exphem.org/article/PIIS0301472X12001403/abstract?rss=yes See Liu et al., pages 487–498.Inside this issue10.1016/j.exphem.2012.04.006Experimental Hematology 40, 6 (2012)2012-04-30Experimental Hematology2012-04-30406S0301-472X(11)X0018-8429430http://www.exphem.org/article/PIIS0301472X12000161/abstract?rss=yesTwenty-six patients with recurrent CD20+ B-cell lymphoid malignancies received fludarabine, cyclophosphamide, and rituximab–based nonablative conditioning followed by either matched related (n = 18) or unrelated (n = 8) donor allogeneic stem cell transplantation (allo-SCT) between March 2008 and May 2011. Median age of patients at transplantation was 59 years (range, 41–64 years). At diagnosis, 20 (77%) had stage IV disease; 23 (88%) received ≥3 regimens, 14 (54%) received ≥4 regimens, and 4 (15%) had earlier autologous-SCT. All patients had either chemosensitive or stable disease and nine (35%) were in complete remission before transplantation. At the time of analysis, 17 patients were alive with an estimated 2-year overall survival and progression-free survival rate of 63% and nonrelapse mortality of 25%. Grade II to IV acute graft-vs-host-disease occurred in 8 (31%) and chronic graft-vs-host-disease in 6 (23%) patients (extensive, n = 3). Causes of death include progressive disease in four, acute graft-vs-host-disease in two (both after receiving donor lymphocyte infusion for mixed chimerism with residual disease), infection in one, and other (e.g., substance abuse, leukoencephalopathy) in two. Six patients required rehospitalization within 100 days of SCT (mean = 10 days; range, 3–18 days). Our data support fludarabine, cyclophosphamide, and rituximab–based nonablative conditioning allo-SCT in CD20+ B-cell lymphoid malignancies and it is time to compare this regimen with an alternative reduced-intensity conditioning regimen in B-cell malignancies.Immunomodulatory nonablative conditioning regimen for B-cell lymphoid malignanciesWichai Chinratanalab, Nishitha Reddy, John P. Greer, David Morgan, Brian Engelhardt, Adetola Kassim, Stephen J. Brandt, Madan Jagasia, Stacey Goodman, Bipin N. Savani10.1016/j.exphem.2012.01.014Experimental Hematology 40, 6 (2012)2012-01-24Experimental Hematology2012-01-24406S0301-472X(11)X0018-8Clinical Investigations431435http://www.exphem.org/article/PIIS0301472X12000471/abstract?rss=yesIn utero transplantation (IUT) of human hematopoietic stem cells has been conducted in sheep, which are used as large animal models of human hematopoietic reconstitution and models for clinical IUT; however, the levels of engraftment have generally been low. Busulfan (BU), a myeloablative agent, is often administered to patients before hematopoietic stem cells transplantation to improve the engraftment. In this study, hematopoietic activity was evaluated in adult sheep after administering BU at different doses. Next, pregnant ewes were administered BU, and dams as well as their fetuses were evaluated, as BU readily crosses the sheep placenta. Then, the BU dose with the desired outcomes was selected and administered to pregnant ewes at 2 or 6 days before performing IUT using human cord blood CD34+ cells. The engraftment was evaluated in recipients that underwent IUT in the presence or absence of BU. As a result, hematopoietic activity was safely and transiently suppressed in adult sheep treated with 5 to 7.5 mg/kg BU. BU crossed the sheep placenta, and fetal sheep were indeed conditioned by administering 3 mg/kg BU to pregnant ewes. Engraftment of human CD34+ cells in fetal recipients was enhanced when IUT was carried out 6 days post-BU. Up to 3.3% engraftment levels (in terms of bone marrow colony-forming units) were achieved with the IUT of 0.72 to 2.4 million CD34+ cells when BU was used. BU can be administered to pregnant ewes to effectively condition the fetal recipient for IUT with enhanced engraftment of donor cells.Maternal administration of busulfan before in utero transplantation of human hematopoietic stem cells enhances engraftments in sheepTomoyuki Abe, Shigeo Masuda, Yujiro Tanaka, Suguru Nitta, Yoshihiro Kitano, Satoshi Hayashi, Yutaka Hanazono, Yoshikazu Nagao10.1016/j.exphem.2012.01.018Experimental Hematology 40, 6 (2012)2012-02-03Experimental Hematology2012-02-03406S0301-472X(11)X0018-8Experimental Stem Cell Transplantation436444http://www.exphem.org/article/PIIS0301472X12000446/abstract?rss=yesDelayed engraftment remains a major hurdle after cord blood (CB) transplantation. It may be due, at least in part, to low fucosylation of cell surface molecules important for homing to the bone marrow microenvironment. Because fucosylation of specific cell surface ligands is required before effective interaction with selectins expressed by the bone marrow microvasculature can occur, a simple 30-minute ex vivo incubation of CB hematopoietic progenitor cells with fucosyltransferase-VI and its substrate (GDP-fucose) was performed to increase levels of fucosylation. The physiologic impact of CB hematopoietic progenitor cell hypofucosylation was investigated in vivo in NOD-SCID interleukin (IL)-2Rγnull (NSG) mice. By isolating fucosylated and nonfucosylated CD34+ cells from CB, we showed that only fucosylated CD34+ cells are responsible for engraftment in NSG mice. In addition, because the proportion of CD34+ cells that are fucosylated in CB is significantly less than in bone marrow and peripheral blood, we hypothesize that these combined observations might explain, at least in part, the delayed engraftment observed after CB transplantation. Because engraftment appears to be correlated with the fucosylation of CD34+ cells, we hypothesized that increasing the proportion of CD34+ cells that are fucosylated would improve CB engraftment. Ex vivo treatment with fucosyltransferase-VI significantly increases the levels of CD34+ fucosylation and, as hypothesized, this was associated with improved engraftment. Ex vivo fucosylation did not alter the biodistribution of engrafting cells or pattern of long-term, multilineage, multi-tissue engraftment. We propose that ex vivo fucosylation will similarly improve the rate and magnitude of engraftment for CB transplant recipients in a clinical setting.Ex vivo fucosylation improves human cord blood engraftment in NOD-SCID IL-2Rγnull miceSimon N. Robinson, Paul J. Simmons, Michael W. Thomas, Nathalie Brouard, Jeannie A. Javni, Suprita Trilok, Jae-Seung Shim, Hong Yang, David Steiner, William K. Decker, Dongxia Xing, Leonard D. Shultz, Barbara Savoldo, Gianpietro Dotti, Catherine M. Bollard, Leonard Miller, Richard E. Champlin, Elizabeth J. Shpall, Patrick A. Zweidler-McKay10.1016/j.exphem.2012.01.015Experimental Hematology 40, 6 (2012)2012-02-03Experimental Hematology2012-02-03406S0301-472X(11)X0018-8Experimental Stem Cell Transplantation445456http://www.exphem.org/article/PIIS0301472X12000458/abstract?rss=yesThis study found that levels of thrombomodulin (TM) were downregulated in freshly isolated leukemia cells from patients with acute promyelocytic leukemia (APL, n = 7) and acute myelogenous leukemia (n = 14), as compared with CD34+/CD38− hematopoietic stem/progenitor cells and CD34−/CD33+/CD11b− promyelocytes isolated from healthy volunteers (n = 3). Exposure of APL NB4 cells to recombinant human soluble TM (rTM, 1500 ng/mL) inhibited clonogenic growth of these cells by approximately 30%, and induced expression of CD11b, a marker of myeloid differentiation, on their surfaces, in association with upregulation of nuclear levels of myeloid-specific transcription factor CCAAT/enhancer binding protein ε. These antileukemic effects of rTM were mediated by thrombin/activated protein C–dependent mechanisms, as hirudin, an inhibitor of thrombin and a blocking antibody against endothelial receptor for protein C to which activated protein C binds, hampered the ability of rTM to induce expression of CD11b in NB4 cells. This study also found that rTM downregulated expression of Annexin II, a receptor for both plasminogen and tissue plasminogen activator, and inhibited plasmin activity in APL cells. Interestingly, rTM significantly enhanced the ability of all–trans retinoic acid to induce growth arrest, differentiation and apoptosis, and inhibited plasmin activity in APL cells. Taken together, these results suggest that administration of rTM should be considered for treatment of individuals with disseminated intravascular coagulation associated with APL.Thrombomodulin enhances the antifibrinolytic and antileukemic effects of all–trans retinoic acid in acute promyelocytic leukemia cellsTakayuki Ikezoe, Jing Yang, Chie Nishioka, Mayuka Isaka, Naomi Iwabu, Mizu Sakai, Ayuko Taniguchi, Goichi Honda, Akihito Yokoyama10.1016/j.exphem.2012.01.016Experimental Hematology 40, 6 (2012)2012-02-10Experimental Hematology2012-02-10406S0301-472X(11)X0018-8Malignant Hematopoiesis457465http://www.exphem.org/article/PIIS0301472X12000926/abstract?rss=yesThe relevance of viral infections to the onset and progression of human hematologic malignancies and other blood diseases is still a matter of active investigation. Purified human T lymphocytes isolated from the peripheral blood mononuclear cells of healthy blood donors were experimentally infected with simian virus 40 (SV40), a small DNA tumor virus. SV40-positive T lymphocytes extended their lifespan up to day 80 postinfection (PI). Expression of viral antigens, such as the large T antigen and the viral capsid protein VP1 from the early and late regions, respectively, was detected up to day 40 PI. SV40 viral progeny were continuously produced from day 10 to 40 PI. SV40 DNA sequences were detected in infected T cells for up to 80 days. Our data indicate that human T lymphocytes can be efficiently infected with SV40. Although T cells infected by SV40 were not immortalized, 30% of these lymphocytes appeared to be morphologically transformed with an enlarged T-cell shape. Our investigation provides a simple model for studying the interactions of human T lymphocytes with this small DNA tumor virus and it might represent an experimental tool for investigating new biomarkers and targets for innovative therapeutic approaches.Simian virus 40 efficiently infects human T lymphocytes and extends their lifespanElisa Mazzoni, Gian Matteo Rigolin, Franca Nneka Alaribe, Cecilia Pancaldi, Stefania Maniero, Manola Comar, Fernanda Martini, Mauro Tognon10.1016/j.exphem.2012.02.008Experimental Hematology 40, 6 (2012)2012-03-14Experimental Hematology2012-03-14406S0301-472X(11)X0018-8Malignant Hematopoiesis466476http://www.exphem.org/article/PIIS0301472X1200015X/abstract?rss=yesErythroid-specific 5-aminolevulinate synthase (ALAS2) is essential for hemoglobin production, and a loss-of-function mutation of ALAS2 gene causes X-linked sideroblastic anemia. Human ALAS2 protein consists of 587 amino acids and its carboxyl(C)-terminal region of 33 amino acids is conserved in higher eukaryotes, but is not present in prokaryotic ALAS. We explored the role of this C-terminal region in the pathogenesis of X-linked sideroblastic anemia. In vitro enzymatic activity was measured using bacterially expressed recombinant proteins. In vivo catalytic activity was evaluated by comparing the accumulation of porphyrins in eukaryotic cells stably expressing each mutant ALAS2 tagged with FLAG, and the half-life of each FLAG-tagged ALAS2 protein was determined by Western blot analysis. Two novel mutations (Val562Ala and Met567Ile) were identified in patients with X-linked sideroblastic anemia. Val562Ala showed the higher catalytic activity in vitro, but a shorter half-life in vivo compared to those of wild-type ALAS2 (WT). In contrast, the in vitro activity of Met567Ile mutant was about 25% of WT, while its half-life was longer than that of WT. However, in vivo catalytic activity of each mutant was lower than that of WT. In addition, the deletion of 33 amino acids at C-terminal end resulted in higher catalytic activity both in vitro and in vivo with the longer half-life compared to WT. In conclusion, the C-terminal region of ALAS2 protein may function as an intrinsic modifier that suppresses catalytic activity and increases the degradation of its protein, each function of which is enhanced by the Met567Ile mutation and the Val562Ala mutation, respectively.The carboxyl-terminal region of erythroid-specific 5-aminolevulinate synthase acts as an intrinsic modifier for its catalytic activity and protein stabilitySenkottuvelan Kadirvel, Kazumichi Furuyama, Hideo Harigae, Kiriko Kaneko, Yoshiko Tamai, Yoji Ishida, Shigeki Shibahara10.1016/j.exphem.2012.01.013Experimental Hematology 40, 6 (2012)2012-01-24Experimental Hematology2012-01-24406S0301-472X(11)X0018-8Normal Hematopoiesis477486.e1http://www.exphem.org/article/PIIS0301472X1200046X/abstract?rss=yesUbiquitously transcribed tetratricopeptide repeat, X chromosome (UTX), an H3K27Me2/3 demethylase, has been implicated in development, self-renewal, and differentiation of various organs and embryonic stem cells through chromatin modifications and transcriptional regulation of important developmentally related genes, such as Hox genes. However, the function of UTX in hematopoiesis is not well understood. To study the role of UTX in the mammalian hematopoietic system, we used lentiviral short hairpin RNA constructs to knockdown UTX in the murine hematopoietic progenitor cell line EML, in primary murine bone marrow cells and in leukemic cell lines. We report that Utx is highly expressed in the hematopoietic compartment and that it plays an important role in cell proliferation and homeostasis of hematopoietic cells in vitro. Knockdown of UTX in EML and primary murine bone marrow cells impairs their colony-forming ability. Moreover, knockdown of UTX affects expression of key genes that regulate hematopoietic differentiation such as Mll1, Runx1, and Scl in primary murine bone marrow cells. And we further demonstrate that UTX directly associates with the promoters of the Mll1, Runx1, and Scl genes and modulate their transcription by controlling H3K27me3 marks on respective promoter regions. In addition, UTX depletion severely impaired proliferation of several human leukemia cell lines. Together, these data demonstrate a functional role for UTX in normal and malignant hematopoiesis.A functional role for the histone demethylase UTX in normal and malignant hematopoietic cellsJianing Liu, Thomas Mercher, Claudia Scholl, Kristina Brumme, D. Gary Gilliland, Nan Zhu10.1016/j.exphem.2012.01.017Experimental Hematology 40, 6 (2012)2012-02-03Experimental Hematology2012-02-03406S0301-472X(11)X0018-8Stem Cells487498.e3http://www.exphem.org/article/PIIS0301472X12000495/abstract?rss=yesThe senescence accelerated-prone mouse variant 6 (SAMP6) shows normal growth followed by rapid aging, development of osteopenia, and shortened lifespan, compared with control R1 mice. Because oxidative stress is a fundamental mechanism of tissue aging, we tested whether cellular parameters that are associated with oxidative stress are impaired with marrow from SAMP6 mice. We compared in vitro hematopoiesis, irradiation sensitivity, proliferative potential, and osteoblastogenesis with marrow cells from SAMP6 and R1 mice. Marrow cells from SAMP6 mice showed shortened in vitro hematopoiesis; their stromal cells showed greater radiation sensitivity and decreased proliferation. Consistent with those properties, there was constitutive upregulation of transforming growth factor–β1, an inhibitor of hematopoiesis, and of cell cycle inhibitory genes, p16INK4A and p19ARF. Paradoxically, there was constitutive expression of osteoblast genes in stromal cells from SAMP6 mice, but in vitro matrix mineralization was impaired. These studies and data included in other reports indicate that impaired proliferation of osteoblast progenitors in SAMP6 marrow may be a major factor contributing to accelerated loss of bone mass. In sum, marrow from SAMP6 mice had diminished capacity for long-term hematopoiesis, increased radiosensitivity, and reduced proliferative capacity.Dysregulated in vitro hematopoiesis, radiosensitivity, proliferation, and osteoblastogenesis with marrow from SAMP6 miceRegina P. O'Sullivan, Joel S. Greenberger, Julie Goff, Shaonan Cao, Kiera A. Kingston, Shuanhu Zhou, Tracy Dixon, Frank D. Houghton, Michael W. Epperly, Hong Wang, Julie Glowacki10.1016/j.exphem.2012.01.019Experimental Hematology 40, 6 (2012)2012-02-10Experimental Hematology2012-02-10406S0301-472X(11)X0018-8Stem Cells499509