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Immunosuppressants are powerful drugs,capable of triggering severe adverse effects. Hence,there is tremendous interest in replacing them with less-toxic agents. Adoptive therapy with CD25(+)CD4(+) T regulatory cells (Tregs) holds promise as an alternative to immunosuppressants. Tregs have been described as the most potent immunosuppressive cells in the human body. In a number of experimental models,they have been found to quench autoimmune diseases,maintain allogeneic transplants,and prevent allergic diseases. A major stumbling block in their clinical application is related to Treg phenotype and the very limited number of these cells in the periphery,not exceeding 1-5% of total CD4(+) T cells. Recent progress in multicolor flow cytometry and cell sorting as well as cellular immunology has found ways of overcoming these obstacles,and has opened the doors to the clinical application of Tregs. In the review,we describe Treg sorting and expansion techniques that have been developed in recent years. In the experience of our laboratory,as well as some published reports,Treg adoptive therapy is a promising tool in immunosuppressive therapy,and should be considered for clinical trials. View Publication

During anemia erythropoiesis is bolstered by several factors including KIT ligand,oncostatin-M,glucocorticoids,and erythropoietin. Less is understood concerning factors that limit this process. Experiments performed using dual-specificity tyrosine-regulated kinase-3 (DYRK3) knock-out and transgenic mice reveal that erythropoiesis is attenuated selectively during anemia. DYRK3 is restricted to erythroid progenitor cells and testes. DYRK3-/- mice exhibited essentially normal hematological profiles at steady state and reproduced normally. In response to hemolytic anemia,however,reticulocyte production increased severalfold due to DYRK3 deficiency. During 5-fluorouracil-induced anemia,both reticulocyte and red cell formation in DYRK3-/- mice were elevated. In short term transplant experiments,DYRK3-/- progenitors also supported enhanced erythroblast formation,and erythropoietic advantages due to DYRK3-deficiency also were observed in 5-fluorouracil-treated mice expressing a compromised erythropoietin receptor EPOR-HM allele. As analyzed ex vivo,DYRK3-/- erythroblasts exhibited enhanced CD71posTer119pos cell formation and 3HdT incorporation. Transgenic pA2gata1-DYRK3 mice,in contrast,produced fewer reticulocytes during hemolytic anemia,and pA2gata1-DYRK3 progenitors were compromised in late pro-erythroblast formation ex vivo. Finally,as studied in erythroid K562 cells,DYRK3 proved to effectively inhibit NFAT (nuclear factor of activated T cells) transcriptional response pathways and to co-immunoprecipitate with NFATc3. Findings indicate that DYRK3 attenuates (and possibly apportions) red cell production selectively during anemia. View Publication

Erythroid Krüppel-like factor (EKLF) is a Krüppel-like transcription factor identified as a transcriptional activator and chromatin modifier in erythroid cells. EKLF-deficient (Eklf(-/-)) mice die at day 14.5 of gestation from severe anemia. In this study,we demonstrate that early progenitor cells fail to undergo terminal erythroid differentiation in Eklf(-/-) embryos. To discover potential EKLF target genes responsible for the failure of erythropoiesis,transcriptional profiling was performed with RNA from wild-type and Eklf(-/-) early erythroid progenitor cells. These analyses identified significant perturbation of a network of genes involved in cell cycle regulation,with the critical regulator of the cell cycle,E2f2,at a hub. E2f2 mRNA and protein levels were markedly decreased in Eklf(-/-) early erythroid progenitor cells,which showed a delay in the G(1)-to-S-phase transition. Chromatin immunoprecipitation analysis demonstrated EKLF occupancy at the proximal E2f2 promoter in vivo. Consistent with the role of EKLF as a chromatin modifier,EKLF binding sites in the E2f2 promoter were located in a region of EKLF-dependent DNase I sensitivity in early erythroid progenitor cells. We propose a model in which EKLF-dependent activation and modification of the E2f2 locus is required for cell cycle progression preceding terminal erythroid differentiation. View Publication

The aryl hydrocarbon receptor (AhR) mediates the carcinogenicity of a family of environmental contaminants,the most potent being 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Increased incidence of lymphoma and leukemia in humans is associated with TCDD exposure. Although AhR activation by TCDD has profound effects on the immune system,precise cellular and molecular mechanisms have yet to be determined. These studies tested the hypothesis that alteration of marrow populations following treatment of mice with TCDD is due to an effect on hematopoietic stem cells (HSCs). Treatment with TCDD resulted in an increased number and proliferation of bone marrow (BM) populations enriched for HSCs. There was a time-dependent decrease in B-lineage cells with a concomitant increase in myeloid populations. The decrease in the B-cell lineage colony-forming unit-preB progenitors along with a transient increase in myeloid progenitors were consistent with a skewing of lineage development from lymphoid to myeloid populations. However,HSCs from TCDD-treated mice exhibited diminished capacity to reconstitute and home to marrow of irradiated recipients. AhR messenger RNA was expressed in progenitor subsets but is downregulated during HSC proliferation. This result was consistent with the lack of response following the exposure of 5-fluorouracil-treated mice to TCDD. The direct exposure of cultured BM cells to TCDD inhibited the growth of immature hematopoietic progenitor cells,but not more mature lineage-restricted progenitors. Overall,these data are consistent with the hypothesis that TCDD,through AhR activation,alters the ability of HSCs to respond appropriately to signals within the marrow microenvironment. View Publication

An attractive target for therapeutic intervention is constitutively activated,mutant FLT3,which is expressed in a subpopulation of patients with acute myelocyic leukemia (AML) and is generally a poor prognostic indicator in patients under the age of 65 years. PKC412 is one of several mutant FLT3 inhibitors that is undergoing clinical testing,and which is currently in late-stage clinical trials. However,the discovery of drug-resistant leukemic blast cells in PKC412-treated patients with AML has prompted the search for novel,structurally diverse FLT3 inhibitors that could be alternatively used to override drug resistance. Here,we report the potent and selective antiproliferative effects of the novel mutant FLT3 inhibitor NVP-AST487 on primary patient cells and cell lines expressing FLT3-ITD or FLT3 kinase domain point mutants. NVP-AST487,which selectively targets mutant FLT3 protein kinase activity,is also shown to override PKC412 resistance in vitro,and has significant antileukemic activity in an in vivo model of FLT3-ITD(+) leukemia. Finally,the combination of NVP-AST487 with standard chemotherapeutic agents leads to enhanced inhibition of proliferation of mutant FLT3-expressing cells. Thus,we present a novel class of FLT3 inhibitors that displays high selectivity and potency toward FLT3 as a molecular target,and which could potentially be used to override drug resistance in AML. View Publication

The c-myb proto-oncogene encodes an obligate hematopoietic cell transcription factor important for lineage commitment,proliferation,and differentiation. Given its critical functions,c-Myb regulatory factors are of great interest but remain incompletely defined. Herein we show that c-Myb expression is subject to posttranscriptional regulation by microRNA (miRNA)-15a. Using a luciferase reporter assay,we found that miR-15a directly binds the 3'-UTR of c-myb mRNA. By transfecting K562 myeloid leukemia cells with a miR-15a mimic,functionality of binding was shown. The mimic decreased c-Myb expression,and blocked the cells in the G(1) phase of cell cycle. Exogenous expression of c-myb mRNA lacking the 3'-UTR partially rescued the miR-15a induced cell-cycle block. Of interest,the miR-15a promoter contained several potential c-Myb protein binding sites. Occupancy of one canonical c-Myb binding site was demonstrated by chromatin immunoprecipitation analysis and shown to be required for miR-15a expression in K562 cells. Finally,in studies using normal human CD34(+) cells,we showed that c-Myb and miR-15a expression were inversely correlated in cells undergoing erythroid differentiation,and that overexpression of miR-15a blocked both erythroid and myeloid colony formation in vitro. In aggregate,these findings suggest the presence of a c-Myb-miR-15a autoregulatory feedback loop of potential importance in human hematopoiesis. View Publication

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Hematopoiesis during development is a dynamic process,with many factors involved in the emergence and regulation of hematopoietic stem cells (HSCs) and progenitor cells. Whereas previous studies have focused on developmental signaling and transcription factors in embryonic hematopoiesis,the role of well-known adult hematopoietic cytokines in the embryonic hematopoietic system has been largely unexplored. The cytokine interleukin-1 (IL-1),best known for its proinflammatory properties,has radioprotective effects on adult bone marrow HSCs,induces HSC mobilization,and increases HSC proliferation and/or differentiation. Here we examine IL-1 and its possible role in regulating hematopoiesis in the midgestation mouse embryo. We show that IL-1,IL-1 receptors (IL-1Rs),and signaling mediators are expressed in the aorta-gonad-mesonephros (AGM) region during the time when HSCs emerge in this site. IL-1 signaling is functional in the AGM,and the IL-1RI is expressed ventrally in the aortic subregion by some hematopoietic,endothelial,and mesenchymal cells. In vivo analyses of IL-1RI-deficient embryos show an increased myeloid differentiation,concomitant with a slight decrease in AGM HSC activity. Our results suggest that IL-1 is an important homeostatic regulator at the earliest time of HSC development,acting to limit the differentiation of some HSCs along the myeloid lineage. View Publication

Endoglin is an accessory receptor for TGF-beta signaling and is required for normal hemangioblast,early hematopoietic,and vascular development. We have previously shown that an upstream enhancer,Eng -8,together with the promoter region,mediates robust endothelial expression yet is inactive in blood. To identify hematopoietic regulatory elements,we used array-based methods to determine chromatin accessibility across the entire locus. Subsequent transgenic analysis of candidate elements showed that an endothelial enhancer at Eng +9 when combined with an element at Eng +7 functions as a strong hemato-endothelial enhancer. Chromatin immunoprecipitation (ChIP)-chip analysis demonstrated specific binding of Ets factors to the promoter as well as to the -8,+7+9 enhancers in both blood and endothelial cells. By contrast Pu.1,an Ets factor specific to the blood lineage,and Gata2 binding was only detected in blood. Gata2 was bound only at +7 and GATA motifs were required for hematopoietic activity. This modular assembly of regulators gives blood and endothelial cells the regulatory freedom to independently fine-tune gene expression and emphasizes the role of regulatory divergence in driving functional divergence. View Publication

The growth factors that drive the division and differentiation of stem cells during early human embryogenesis are unknown. The secretion of endorphins,progesterone (P(4)),human chorionic gonadotropin,17beta-estradiol,and gonadotropin-releasing hormone by trophoblasts that lie adjacent to the embryoblast in the blastocyst suggests that these pregnancy-associated factors may directly signal the growth and development of the embryoblast. To test this hypothesis,we treated embryoblast-derived human embryonic stem cells (hESCs) with ICI 174,864,a delta-opioid receptor antagonist,and RU-486 (mifepristone),a P(4) receptor competitive antagonist. Both antagonists potently inhibited the differentiation of hESC into embryoid bodies,an in vitro structure akin to the blastocyst containing all three germ layers. Furthermore,these agents prevented the differentiation of hESC aggregates into columnar neuroectodermal cells and their organization into neural tube-like rosettes as determined morphologically. Immunoblot analyses confirmed the obligatory role of these hormones; both antagonists inhibited nestin expression,an early marker of neural precursor cells normally detected during rosette formation. Conversely,addition of P(4) to hESC aggregates induced nestin expression and the formation of neuroectodermal rosettes. These results demonstrate that trophoblast-associated hormones induce blastulation and neurulation during early human embryogenesis. View Publication

Human embryonic stem (ES) cells can be maintained in an undifferentiated state if the culture medium is first conditioned on a layer of mouse embryonic fibroblast (MEF) feeder cells. Here we show that human ES cell proliferation is coordinated by MEF-secreted heparan sulfate proteoglycans (HSPG) in conditioned medium (CM). These HSPG and other heparinoids can stabilize basic fibroblast growth factor (FGF2) in unconditioned medium at levels comparable to those observed in CM. They also directly mediate binding of FGF2 to the human ES cell surface,and their removal from CM impairs proliferation. Finally,we have developed a purification scheme for MEF-secreted HSPG in CM. Using column chromatography,immunoblotting,and mass spectrometry-based proteomic analysis,we have identified multiple HSPG species in CM. The results demonstrate that HSPG are key signaling cofactors in CM-based human ES cell culture. View Publication

Expression of Mpl is restricted to hematopoietic cells in the megakaryocyte lineage and to undifferentiated progenitors,where it initiates critical cell survival and proliferation signals after stimulation by its ligand,thrombopoietin (TPO). As a result,a deficiency in Mpl function in patients with congenital amegakaryocytic thrombocytopenia (CAMT) and in mpl(-/-) mice produces profound thrombocytopenia and a severe stem cell-repopulating defect. Gene therapy has the potential to correct the hematopoietic defects of CAMT by ectopic gene expression that restores normal Mpl receptor activity. We rescued the mpl(-/-) mouse with a transgenic vector expressing mpl from the promoter elements of the 2-kb region of DNA just proximal to the natural gene start site. Transgene rescued mice exhibit thrombocytosis but only partial correction of the stem cell defect. Furthermore,they show very low-level expression of Mpl on platelets and megakaryocytes,and the transgene-rescued megakaryocytes exhibit diminished TPO-dependent kinase phosphorylation and reduced platelet production in bone marrow chimeras. Thrombocytosis is an unexpected consequence of reduced Mpl expression and activity. However,impaired TPO homeostasis in the transgene-rescued mice produces elevated plasma TPO levels,which serves as an unchecked stimulus to drive the observed excessive megakaryocytopoiesis. View Publication