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BACKGROUND Central to appropriate thrombin formation at sites of vascular injury is the concerted assembly of plasma- and/or platelet-derived factor (F) Va and FXa on the activated platelet surface. While the plasma-derived procofactor,FV,must be proteolytically activated by α-thrombin to FVa to function in prothrombinase,the platelet molecule is released from α-granules in a partially activated state,obviating the need for proteolytic activation. OBJECTIVES The current study was performed to test the hypothesis that subsequent to its endocytosis by megakaryocytes,plasma-derived FV is proteolytically processed to form the platelet-derived pool. METHODS & RESULTS Subsequent to FV endocytosis,a time-dependent increase in FV proteolytic products was observed in megakaryocyte lysates by SDS-PAGE followed by phosphorimaging or western blotting. This cleavage was specific and resulted in the formation of products similar in size to FV/Va present in a platelet lysate as well as to the α-thrombin-activated FVa heavy chain and light chain,and their respective precursors. Other proteolytic products were unique to endocytosed FV. The product/precursor relationships of these fragments were defined using anti-FV heavy and light chain antibodies with defined epitopes. Activity measurements indicated that megakaryocyte-derived FV fragments exhibited substantial FVa cofactor activity that was comparable to platelet-derived FV/Va. CONCLUSIONS Taken together,these observations suggest that prior to its packaging in α-granules endocytosed FV undergoes proteolysis by one or more specific megakaryocyte protease(s) to form the partially activated platelet-derived pool. View Publication

Developing novel therapies that suppress self-renewal of leukemia stem cells may reduce the likelihood of relapses and extend long-term survival of patients with acute myelogenous leukemia (AML). AML1-ETO (AE) is an oncogene that plays an important role in inducing self-renewal of hematopoietic stem/progenitor cells (HSPCs),leading to the development of leukemia stem cells. Previously,using a zebrafish model of AE and a whole-organism chemical suppressor screen,we have discovered that AE induces specific hematopoietic phenotypes in embryonic zebrafish through a cyclooxygenase (COX)-2 and β-catenin-dependent pathway. Here,we show that AE also induces expression of the Cox-2 gene and activates β-catenin in mouse bone marrow cells. Inhibition of COX suppresses β-catenin activation and serial replating of AE(+) mouse HSPCs. Genetic knockdown of β-catenin also abrogates the clonogenic growth of AE(+) mouse HSPCs and human leukemia cells. In addition,treatment with nimesulide,a COX-2 selective inhibitor,dramatically suppresses xenograft tumor formation and inhibits in vivo progression of human leukemia cells. In summary,our data indicate an important role of a COX/β-catenin-dependent signaling pathway in tumor initiation,growth,and self-renewal,and in providing the rationale for testing potential benefits from common COX inhibitors as a part of AML treatments. View Publication

The comparative evaluation of different 3D matrices-Matrigel,Puramatrix,and inverted colloidal crystal (ICC) scaffolds-provides a perspective for studying the pathology and potential cures for many blood and bone marrow diseases,and further proves the significance of 3D cultures with direct cell-cell contacts for in vitro mimicry of the human stem cell niche. View Publication

BACKGROUND Glycophorin C (GPC) is necessary in the maintenance of red blood cell structure. Severe autoimmune hemolytic anemia and hemolytic disease of the fetus and newborn (HDFN) have been associated with Gerbich (Ge) blood group system antigens expressed on GPC. Previous in vitro studies with cord blood progenitor cells have shown that anti-Ge suppresses erythropoiesis. STUDY DESIGN AND METHODS Here,we evaluated the K562 erythroleukemic cell line to study the cellular effects of a murine anti-GPC. Cell proliferation was evaluated after treatment with anti-GPC. Flow cytometry was used to evaluate exofacial phosphatidylserine (PS) expression and cell viability (propidium iodide binding). Cell morphology was evaluated under light microscopy with cytospin preparations stained with May-Grünwald Giemsa. RESULTS Anti-GPC dramatically inhibited K562 proliferation and increased PS expression,consistent with cytoplasmic blebbing,suggesting evidence of apoptosis. Z-VAD-FMK,an inhibitor of classical apoptosis,was unable to reverse the suppressive effect of anti-GPC. However,hemin was able to attenuate growth suppression. CONCLUSION Together,the data suggest that anti-GPC suppresses erythroid proliferation through the induction of nonclassical apoptosis. View Publication

Extrinsic signaling cues in the microenvironment of acute myelogenous leukemia (AML) contribute to disease progression and therapy resistance. Yet,it remains unknown how the bone marrow niche in which AML arises is subverted to support leukemic persistence at the expense of homeostatic function. Exosomes are cell membrane-derived vesicles carrying protein and RNA cargoes that have emerged as mediators of cell-cell communication. In this study,we examined the role of exosomes in developing the AML niche of the bone marrow microenvironment,investigating their biogenesis with a focus on RNA trafficking. We found that both primary AML and AML cell lines released exosome-sized vesicles that entered bystander cells. These exosomes were enriched for several coding and noncoding RNAs relevant to AML pathogenesis. Furthermore,their uptake by bone marrow stromal cells altered their secretion of growth factors. Proof-of-concept studies provided additional evidence for the canonical functions of the transferred RNA. Taken together,our findings revealed that AML exosome trafficking alters the proliferative,angiogenic,and migratory responses of cocultured stromal and hematopoietic progenitor cell lines,helping explain how the microenvironmental niche becomes reprogrammed during invasion of the bone marrow by AML. View Publication

Lifelong blood cell production is governed through the poorly understood integration of cell-intrinsic and -extrinsic control of hematopoietic stem cell (HSC) quiescence and activation. MicroRNAs (miRNAs) coordinately regulate multiple targets within signaling networks,making them attractive candidate HSC regulators. We report that miR-126,a miRNA expressed in HSC and early progenitors,plays a pivotal role in restraining cell-cycle progression of HSC in vitro and in vivo. miR-126 knockdown by using lentiviral sponges increased HSC proliferation without inducing exhaustion,resulting in expansion of mouse and human long-term repopulating HSC. Conversely,enforced miR-126 expression impaired cell-cycle entry,leading to progressively reduced hematopoietic contribution. In HSC/early progenitors,miR-126 regulates multiple targets within the PI3K/AKT/GSK3β pathway,attenuating signal transduction in response to extrinsic signals. These data establish that miR-126 sets a threshold for HSC activation and thus governs HSC pool size,demonstrating the importance of miRNA in the control of HSC function. View Publication

Haematopoietic stem cells (HSCs) regenerate blood cells throughout the lifespan of an organism. With age,the functional quality of HSCs declines,partly owing to the accumulation of damaged DNA. However,the factors that damage DNA and the protective mechanisms that operate in these cells are poorly understood. We have recently shown that the Fanconi anaemia DNA-repair pathway counteracts the genotoxic effects of reactive aldehydes. Mice with combined inactivation of aldehyde catabolism (through Aldh2 knockout) and the Fanconi anaemia DNA-repair pathway (Fancd2 knockout) display developmental defects,a predisposition to leukaemia,and are susceptible to the toxic effects of ethanol-an exogenous source of acetaldehyde. Here we report that aged Aldh2(-/-) Fancd2(-/-) mutant mice that do not develop leukaemia spontaneously develop aplastic anaemia,with the concomitant accumulation of damaged DNA within the haematopoietic stem and progenitor cell (HSPC) pool. Unexpectedly,we find that only HSPCs,and not more mature blood precursors,require Aldh2 for protection against acetaldehyde toxicity. Additionally,the aldehyde-oxidizing activity of HSPCs,as measured by Aldefluor stain,is due to Aldh2 and correlates with this protection. Finally,there is more than a 600-fold reduction in the HSC pool of mice deficient in both Fanconi anaemia pathway-mediated DNA repair and acetaldehyde detoxification. Therefore,the emergence of bone marrow failure in Fanconi anaemia is probably due to aldehyde-mediated genotoxicity restricted to the HSPC pool. These findings identify a new link between endogenous reactive metabolites and DNA damage in HSCs,and define the protective mechanisms that counteract this threat. View Publication

Aldehyde dehydrogenase (ALDH) activity is a widely used marker for human hematopoietic stem cells (HSCs),yet its relevance and role in murine HSCs remain unclear. We found that murine marrow cells with a high level of ALDH activity as measured by Aldefluor staining (ALDH(br) cells) do not contain known HSCs or progenitors. In contrast,highly enriched murine HSCs defined by the CD48(-)EPCR(+) and other phenotypes contain two subpopulations,one that stains dimly with Aldefluor (ALDH(dim)) and one that stains at intermediate levels (ALDH(int)). The CD48(-)EPCR(+)ALDH(dim) cells are virtually all in G(0) and yield high levels of engraftment via both intravenous and intrabone routes. In contrast the CD48(-)EPCR(+)ALDH(int) cells are virtually all in G(1),have little intravenous engraftment potential,and yet can engraft long-term after intrabone transplantation. These data demonstrate that Aldefluor staining of unfractionated murine marrow does not identify known HSCs or progenitors. However,varying levels of Aldefluor staining when combined with CD48 and EPCR detection can identify novel populations in murine marrow including a highly enriched population of resting HSCs and a previously unknown HSC population in G(1) with an intravenous engraftment defect. View Publication

Transcriptional profiling of differentiating human embryonic stem cells (hESCs) revealed that MIXL1-positive mesodermal precursors were enriched for transcripts encoding the G-protein-coupled APELIN receptor (APLNR). APLNR-positive cells,identified by binding of the fluoresceinated peptide ligand,APELIN (APLN),or an anti-APLNR mAb,were found in both posterior mesoderm and anterior mesendoderm populations and were enriched in hemangioblast colony-forming cells (Bl-CFC). The addition of APLN peptide to the media enhanced the growth of embryoid bodies (EBs),increased the expression of hematoendothelial genes in differentiating hESCs,and increased the frequency of Bl-CFCs by up to 10-fold. Furthermore,APLN peptide also synergized with VEGF to promote the growth of hESC-derived endothelial cells. These studies identified APLN as a novel growth factor for hESC-derived hematopoietic and endothelial cells. View Publication

BACKGROUND Cryopreservation protocols have remained relatively unchanged since the first umbilical cord blood banking program was established. This study evaluated the preservation efficacy of a novel intracellular-like cryopreservation solution (CryoStor,BioLife Solutions,Inc.),the rate of addition of two cryopreservation solutions to cord blood units (CBUs),and reduced final dimethyl sulfoxide (DMSO) concentration of 5%. STUDY DESIGN AND METHODS Split-sample CBUs were cryopreserved with either an in-house 20% DMSO-based cryopreservation solution or CryoStor CS10 at a rate of 1 mL/min (n = 10; i.e.,slow addition) or as a bolus injection (n = 6; i.e.,fast addition). Infrared images of exothermic effects of the cryopreservation solutions were monitored relative to the rate of addition. Prefreeze and postthaw colony-forming unit assays,total nucleated cells,and CD34+ cell counts were compared. RESULTS Maximum temperature excursions observed were less than 6°C,regardless of the rate of solution addition. Fast addition resulted in peak excursions approximately twice that of slow addition but the magnitude and duration were minimal and transient. Slow addition of CryoStor CS10 (i.e.,final concentration % 5% DMSO) resulted in significantly better postthaw CD34+ cell recoveries; no other metrics were significantly different. Fast addition of CryoStor resulted in similar postthaw metrics compared to slow addition of the in-house solution. CONCLUSION Slow and fast addition of cryopreservation solutions result in mean temperature changes of approximately 3.3 to 4.45°C. Postthaw recoveries with CryoStor were equivalent to or slightly better than with the in-house cryopreservation solution. CryoStor also provides several advantages including reduced processing time,formulation consistency,and reduced DMSO in the frozen product (% 5%). View Publication

Lipid droplets,also called lipid bodies (LB) in inflammatory cells,are important cytoplasmic organelles. However,little is known about the molecular characteristics and functions of LBs in human mast cells (MC). Here,we have analyzed the genesis and components of LBs during differentiation of human peripheral blood-derived CD34(+) progenitors into connective tissue-type MCs. In our serum-free culture system,the maturing MCs,derived from 18 different donors,invariably developed triacylglycerol (TG)-rich LBs. Not known heretofore,the MCs transcribe the genes for perilipins (PLIN)1-4,but not PLIN5,and PLIN2 and PLIN3 display different degrees of LB association. Upon MC activation and ensuing degranulation,the LBs were not cosecreted with the cytoplasmic secretory granules. Exogenous arachidonic acid (AA) enhanced LB genesis in Triacsin C-sensitive fashion,and it was found to be preferentially incorporated into the TGs of LBs. The large TG-associated pool of AA in LBs likely is a major precursor for eicosanoid production by MCs. In summary,we demonstrate that cultured human MCs derived from CD34(+) progenitors in peripheral blood provide a new tool to study regulatory mechanisms involving LB functions,with particular emphasis on AA metabolism,eicosanoid biosynthesis,and subsequent release of proinflammatory lipid mediators from these cells. View Publication

BACKGROUND: Anaemia and microcytosis are common post kidney transplantation. The aim of this study was to evaluate the potential role of mammalian target of rapamycin (mTOR) inhibition in the development of anaemia and microcytosis in healthy animals and in human erythroid cultures in vitro. METHODS: Rats with normal kidney function were treated with sirolimus (n = 7) or vehicle (n = 8) for 15 weeks. Hemograms were determined thereafter. In the sirolimus withdrawal part of the study,rats received sirolimus (SRL) for 67 days (n = 4) 1 mg/kg three times per week or for 30 days (n = 4) and were observed until Day 120. Hemograms were performed regularly. Peripheral blood mononuclear cells from healthy controls (HC; n = 8),kidney transplant patients with sirolimus treatment with (SRL + MC; n = 8) or without microcytosis (SRL - MC; n = 8) were isolated and cultured in the absence or presence of SRL (5 ng/mL). RESULTS: SRL-treated animals had a reduced mean corpuscular volume (MCV) and elevated erythrocyte count compared with control animals after 15 weeks of treatment. This effect was evident as early as 4 weeks (MCV: 61.5 ± 1.8 versus 57 ± 1.7 fL; P = 0.0156; Red blood count 7.4 ± 0.3 × 10(9)/L versus 8.6 ± 0.5 × 10(9)/L; P = 0.0156) and was reversible 90 days after SRL withdrawal. SRL in the culture medium of erythroid cultures led to fewer colonies in cultures from HC as well as from kidney transplant patients (without SRL: 34.2 ± 11.4 versus with SRL: 27.5 ± 9.9 BFU-E-derived colonies P = 0.03),regardless if the cultures were derived from recipients with normocytic or with microcytic erythrocytes. The presence of tacrolimus in the culture medium had no influence on the number and size of colonies. CONCLUSION: mTOR inhibition induces microcytosis and polyglobulia,but not anaemia in healthy rats. This might be caused by growth inhibition of erythroid precursor cells. View Publication