搜索结果: 'gmp t cell activator'

Human B cells can be cultured ex vivo for a few weeks,following stimulation of the CD40 cell surface molecule in the presence of recombinant cytokines such as interleukin-4 (IL-4). However,attempts to produce polyclonal antigen-specific human antibodies by in vitro culture of human B cells obtained from immunized donors have not been successful. It has been shown in mice that lipopolysaccharide (LPS) is a potent mitogen for B cells and plays an important role in the generation of antigen-specific antibody responses. Although it has long been believed that LPS has no direct effect on human B cells,recent data indicating that IL-4-activated human B cells are induced to express Toll-like receptor-4,the main LPS receptor,prompted us to study the effects of LPS on the proliferation and antibody secretion of human B cells. Our results showed that LPS caused a reduction in the expansion of CD40-activated human B cells,accompanied by an increase in antigen-specific antibody secretion. This result suggested that some,but not all,B cells were able to differentiate into antibody-secreting cells in response to LPS. This increased differentiation could be explained by the observation that LPS-stimulated human B cells were induced to secrete higher amounts of IL-6,a pleiotropic cytokine well-known for its B-cell differentiation activity. In vivo,the effect of LPS on cytokine secretion by B cells may not only enhance B-cell differentiation but also help to sustain a local ongoing immune response to invading Gram-negative bacteria,until all pathogens have been cleared from the organism. 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

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

Recently it has been demonstrated that CD30 expression was rather specific for transformed than for normal human ES cells and therefore CD30 maybe suggested as a potential marker for human ES cells bearing chromosomal abnormalities. Using immunohistochemistry and RT-PCR analysis we examined �?¡D30 expression in 10 hESCs lines with normal and abberant karyotypes. All hESC lines expressed CD30 antigen and RNA in undifferentiated state whether cell line beared chromosomal abnormalities or not. In contrast to previous notions our data demonstrate that CD30 could be considered as marker of undifferentiated hESCs without respect to karyotype changes. View Publication

Previous studies have demonstrated that normal mouse mammary tissue contains a rare subset of mammary stem cells. We now describe a method for detecting an analogous subpopulation in normal human mammary tissue. Dissociated cells are suspended with fibroblasts in collagen gels,which are then implanted under the kidney capsule of hormone-treated immunodeficient mice. After 2-8 weeks,the gels contain bilayered mammary epithelial structures,including luminal and myoepithelial cells,their in vitro clonogenic progenitors and cells that produce similar structures in secondary transplants. The regenerated clonogenic progenitors provide an objective indicator of input mammary stem cell activity and allow the frequency and phenotype of these human mammary stem cells to be determined by limiting-dilution analysis. This new assay procedure sets the stage for investigations of mechanisms regulating normal human mammary stem cells (and possibly stem cells in other tissues) and their relationship to human cancer stem cell populations. View Publication

Defects in apoptosis contribute to poor outcome in pediatric acute lymphoblastic leukemia (ALL),calling for novel strategies that counter apoptosis resistance. Here,we demonstrate for the first time that small molecule inhibitors of the antiapoptotic protein XIAP cooperate with TRAIL to induce apoptosis in childhood acute leukemia cells. XIAP inhibitors at subtoxic concentrations,but not a structurally related control compound,synergize with TRAIL to trigger apoptosis and to inhibit clonogenic survival of acute leukemia cells,whereas they do not affect viability of normal peripheral blood lymphocytes,suggesting some tumor selectivity. Analysis of signaling pathways reveals that XIAP inhibitors enhance TRAIL-induced activation of caspases,loss of mitochondrial membrane potential,and cytochrome c release in a caspase-dependent manner,indicating that they promote a caspase-dependent feedback mitochondrial amplification loop. Of note,XIAP inhibitors even overcome Bcl-2-mediated resistance to TRAIL by enhancing Bcl-2 cleavage and Bak conformational change. Importantly,XIAP inhibitors kill leukemic blasts from children with ALL ex vivo and cooperate with TRAIL to induce apoptosis. In vivo,they significantly reduce leukemic burden in a mouse model of pediatric ALL engrafted in non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice. Thus,XIAP inhibitors present a promising novel approach for apoptosis-based therapy of childhood ALL. View Publication

The basic helix-loop-helix transcription factor stem cell leukemia gene (Scl) is a master regulator for hematopoiesis essential for hematopoietic specification and proper differentiation of the erythroid and megakaryocyte lineages. However,the critical downstream targets of Scl remain undefined. Here,we identified a novel Scl target gene,transcription factor myocyte enhancer factor 2 C (Mef2C) from Scl(fl/fl) fetal liver progenitor cell lines. Analysis of Mef2C(-/-) embryos showed that Mef2C,in contrast to Scl,is not essential for specification into primitive or definitive hematopoietic lineages. However,adult VavCre(+)Mef2C(fl/fl) mice exhibited platelet defects similar to those observed in Scl-deficient mice. The platelet counts were reduced,whereas platelet size was increased and the platelet shape and granularity were altered. Furthermore,megakaryopoiesis was severely impaired in vitro. Chromatin immunoprecipitation microarray hybridization analysis revealed that Mef2C is directly regulated by Scl in megakaryocytic cells,but not in erythroid cells. In addition,an Scl-independent requirement for Mef2C in B-lymphoid homeostasis was observed in Mef2C-deficient mice,characterized as severe age-dependent reduction of specific B-cell progenitor populations reminiscent of premature aging. In summary,this work identifies Mef2C as an integral member of hematopoietic transcription factors with distinct upstream regulatory mechanisms and functional requirements in megakaryocyte and B-lymphoid lineages. View Publication

Ectopic delivery of HOXB4 elicits the expansion of engrafting hematopoietic stem cells (HSCs). We hypothesized that inhibition of tumor necrosis factor-alpha (TNF-alpha) signaling may be central to the self-renewal signature of HOXB4. Because HSCs derived from Fanconi anemia (FA) knockout mice are hypersensitive to TNF-alpha,we studied Fancc(-/-) HSCs to determine the physiologic effects of HOXB4 on TNF-alpha sensitivity and the relationship of these effects to the engraftment defect of FA HSCs. Overexpression of HOXB4 reversed the in vitro hypersensitivity to TNF-alpha of Fancc(-/-) HSCs and progenitors (P) and partially rescued the engraftment defect of these cells. Coexpression of HOXB4 and the correcting FA-C protein resulted in full correction compared with wild-type (WT) HSCs. Ectopic expression of HOXB4 resulted in a reduction in both apoptosis and reactive oxygen species in Fancc(-/-) but not WT HSC/P. HOXB4 overexpression was also associated with a significant reduction in surface expression of TNF-alpha receptors on Fancc(-/-) HSC/P. Finally,enhanced engraftment was seen even when HOXB4 was expressed in a time-limited fashion during in vivo reconstitution. Thus,the HOXB4 engraftment signature may be related to its effects on TNF-alpha signaling,and this pathway may be a molecular target for timed pharmacologic manipulation of HSC during reconstitution. View Publication

The development of cell therapies to treat peripheral vascular disease has proven difficult because of the contribution of multiple cell types that coordinate revascularization. We characterized the vascular regenerative potential of transplanted human bone marrow (BM) cells purified by high aldehyde dehydrogenase (ALDH(hi)) activity,a progenitor cell function conserved between several lineages. BM ALDH(hi) cells were enriched for myelo-erythroid progenitors that produced multipotent hematopoietic reconstitution after transplantation and contained nonhematopoietic precursors that established colonies in mesenchymal-stromal and endothelial culture conditions. The regenerative capacity of human ALDH(hi) cells was assessed by intravenous transplantation into immune-deficient mice with limb ischemia induced by femoral artery ligation/transection. Compared with recipients injected with unpurified nucleated cells containing the equivalent of 2- to 4-fold more ALDH(hi) cells,mice transplanted with purified ALDH(hi) cells showed augmented recovery of perfusion and increased blood vessel density in ischemic limbs. ALDH(hi) cells transiently recruited to ischemic regions but did not significantly integrate into ischemic tissue,suggesting that transient ALDH(hi) cell engraftment stimulated endogenous revascularization. Thus,human BM ALDH(hi) cells represent a progenitor-enriched population of several cell lineages that improves perfusion in ischemic limbs after transplantation. These clinically relevant cells may prove useful in the treatment of critical ischemia in humans. View Publication

Correction of murine models of beta-thalassemia has been achieved through high-level globin lentiviral vector gene transfer into mouse hematopoietic stem cells (HSCs). However,transduction of human HSCs is less robust and may be inadequate to achieve therapeutic levels of genetically modified erythroid cells. We therefore developed a double gene lentiviral vector encoding both human gamma-globin under the transcriptional control of erythroid regulatory elements and methylguanine methyltransferase (MGMT),driven by a constitutive cellular promoter. MGMT expression provides cellular resistance to alkylator drugs,which can be administered to kill residual untransduced,diseased HSCs,whereas transduced cells are protected. Mice transplanted with beta-thalassemic HSCs transduced with a gamma-globin/MGMT vector initially had subtherapeutic levels of red cells expressing gamma-globin. To enrich gamma-globin-expressing cells,transplanted mice were treated with the alkylator agent 1,3-bis-chloroethyl-1-nitrosourea. This resulted in significant increases in the number of gamma-globin-expressing red cells and the amount of fetal hemoglobin,leading to resolution of anemia. Selection of transduced HSCs was also obtained when cells were drug-treated before transplantation. Mice that received these cells demonstrated reconstitution with therapeutic levels of gamma-globin-expressing cells. These data suggest that MGMT-based drug selection holds promise as a modality to improve gene therapy for beta-thalassemia. View Publication

Effects of angiotensin (Ang)-(1-7),an AngII metabolite,on bone marrow-derived hematopoietic cells were studied. We identified Ang-(1-7) to stimulate proliferation of human CD34(+) and mononuclear cells in vitro. Under in vivo conditions,we monitored proliferation and differentiation of human cord blood mononuclear cells in NOD/SCID mice. Ang-(1-7) stimulated differentially human cells in bone marrow and accumulated them in the spleen. The number of HLA-I(+) and CD34(+) cells in the bone marrow was increased 42-fold and 600-fold,respectively. These results indicate a decisive impact of Ang-(1-7) on hematopoiesis and its promising therapeutic potential in diseases requiring progenitor stimulation. View Publication

Objective: Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) constitute unique sources of pluripotent cells,although the molecular mechanisms involved in their differentiation into specific lineages are just beginning to be defined. Here we evaluated the ability of MEDII (medium conditioned by HepG2 cells,a human hepatocarcinoma cell line) to selectively enhance generation of mesodermal derivatives,including hematopoietic cells,from hESCs and hiPSCs. Materials and Methods: Test cells were exposed to MEDII prior to being placed in conditions that promote embryoid body (EB) formation. Hematopoietic activity was measured by clonogenic assays,flow cytometry,quantitative real-time polymerase chain reaction of specific transcript complementary DNAs and the ability of cells to repopulate sublethally irradiated nonobese diabetic/severe combined immunodeficient interleukin-2 receptor ??-chain-null mice for almost 1 year. Results: Exposure of both hESCs and hiPSCs to MEDII induced a rapid and preferential differentiation of hESCs into mesodermal elements. Subsequently produced EBs showed a further enhanced expression of transcripts characteristic of multiple mesodermal lineages,and a concurrent decrease in endodermal and ectodermal cell transcripts. Frequency of all types of clonogenic hematopoietic progenitors in subsequently derived EBs was also increased. In vivo assays of MEDII-treated hESC-derived EBs also showed they contained cells able to undertake low-level but longterm multilineage repopulation of primary and secondary nonobese diabetic/severe combined immunodeficient interleukin-2 receptor ??-chain-null mice. Conclusions: MEDII treatment of hESCs and hiPSCs alike selectively enhances their differentiation into mesodermal cells and allows subsequent generation of detectable levels of hematopoietic progenitors with in vitro and in vivo differentiating activity. ?? 2009 ISEH - Society for Hematology and Stem Cells. View Publication