搜索结果: 'methocult media formulations for mouse hematopoietic cells serum containing'

Murine embryonic stem cells can differentiate in vitro to form cystic embryoid bodies (CEB) that contain different structures and cell types. The blood islands are one such structure that consist of immature hematopoietic cells surrounded by endothelial cells,the first identifiable vascular cells. CEBs differentiated in vitro developed blood islands initially,and subsequently these blood islands matured to form vascular channels containing hematopoietic cells. Phase contrast microscopy demonstrated the presence of channels in mature CEBs grown in suspension culture,and high resolution light and electron microscopy showed that the cells lining these channels were endothelial cells. The channels appeared less organized than the vasculature of the mature yolk sac. The hematopoietic cells were occasionally seen 'flowing' through the CEB channels,although their numbers were reduced relative to the yolk sac. Analysis of primary CEB cultures showed the presence of cells with two characteristics of endothelial cells: approximately 30% of the cells labelled with fluorescent acetylated low density lipoprotein and a small number of cells were positive for von Willebrand's factor by immunostaining. Thus we conclude that a primitive vasculature forms in CEBs differentiated in vitro,and that not only primary differentiation of endothelial cells but also some aspects of vascular maturation are intrinsic to this cell culture system. CEBs are therefore a useful model for the study of developmental blood vessel formation. View Publication

Menin is the product of the tumor suppressor gene Men1 that is mutated in the inherited tumor syndrome multiple endocrine neoplasia type 1 (MEN1). Menin has been shown to interact with SET-1 domain-containing histone 3 lysine 4 (H3K4) methyltransferases including mixed lineage leukemia proteins to regulate homeobox (Hox) gene expression in vitro. Using conditional Men1 knockout mice,we have investigated the requirement for menin in hematopoiesis and myeloid transformation. Men1 excision causes reduction of Hoxa9 expression,colony formation by hematopoietic progenitors,and the peripheral white blood cell count. Menin directly activates Hoxa9 expression,at least in part,by binding to the Hoxa9 locus,facilitating methylation of H3K4,and recruiting the methylated H3K4 binding protein chd1 to the locus. Consistent with signaling downstream of menin,ectopic expression of both Hoxa9 and Meis1 rescues colony formation defects in Men1-excised bone marrow. Moreover,Men1 excision also suppresses proliferation of leukemogenic mixed lineage leukemia-AF9 fusion-protein-transformed myeloid cells and Hoxa9 expression. These studies uncover an important role for menin in both normal hematopoiesis and myeloid transformation and provide a mechanistic understanding of menin's function in these processes that may be used for therapy. View Publication

Growth of human embryonic stem (hES) cells as a pluripotent population requires a balance between survival,proliferation and self-renewal signals. Here we demonstrate that hES cells express receptors of the tropomyosin-related kinase (TRK) family,which mediate antiapoptotic signals. We show that three TRK ligands,brain-derived neurotrophic factor,neurotrophin 3 and neurotrophin 4,are survival factors for hES cells. Addition of neurotrophins to hES cell cultures effects a 36-fold improvement in their clonal survival. hES cell cultures maintained in medium containing neurotrophins remain diploid and retain full developmental potency. In the presence of neurotrophins,TRK receptors in hES cells are phosphorylated; TRK receptor inhibition leads to hES cell apoptosis. The survival activity of neurotrophins in hES cells is mediated by the phosphatidylinositol-3-kinase pathway but not the mitogen-activated protein kinase pathway. Neurotrophins improve hES cell survival and may facilitate their manipulation and the development of high-throughput screens to identify factors responsible for hES cell differentiation. View Publication

Combined deletion of chromosomes 1p and 19q is associated with improved prognosis and responsiveness to therapy in patients with anaplastic oligodendroglioma. The deletions usually involve whole chromosome arms,suggesting a t(1;19)(q10;p10). Using stem cell medium,we cultured a few tumors. Paraffin-embedded tissue was obtained from 21 Mayo Clinic patients and 98 patients enrolled in 2 North Central Cancer Treatment Group (NCCTG) low-grade glioma trials. Interphase fusion of CEP1 and 19p12 probes detected the t(1;19). 1p/19q deletions were evaluated by fluorescence in situ hybridization. Upon culture,one oligodendroglioma contained an unbalanced 45,XX,t(1;19)(q10;p10). CEP1/19p12 fusion was observed in all metaphases and 74% of interphase nuclei. Among Mayo Clinic oligodendrogliomas,the prevalence of fusion was 81%. Among NCCTG patients,CEP1/19p12 fusion prevalence was 55%,47%,and 0% among the oligodendrogliomas,mixed oligoastrocytomas,and astrocytomas,respectively. Ninety-one percent of NCCTG gliomas with 1p/19q deletion and 12% without 1p/19q deletion had CEP1/19p12 fusion (P textless 0.001,chi(2) test). The median overall survival (OS) for all patients was 8.1 years without fusion and 11.9 years with fusion (P = 0.003). The median OS for patients with low-grade oligodendroglioma was 9.1 years without fusion and 13.0 years with fusion (P = 0.01). Similar significant median OS differences were observed for patients with combined 1p/19q deletions. The absence of alterations was associated with a significantly shorter OS for patients who received higher doses of radiotherapy. Our results strongly suggest that a t(1;19)(q10;p10) mediates the combined 1p/19q deletion in human gliomas. Like combined 1p/19q deletion,the 1;19 translocation is associated with superior OS and progression-free survival in low-grade glioma patients. View Publication

T cell activation is predicated on the interaction between the T cell receptor and peptide-major histocompatibility (pMHC) ligands. The factors that determine the stimulatory potency of a pMHC molecule remain unclear. We describe results showing that a peptide exhibiting many hallmarks of a weak agonist stimulates T cells to proliferate more than the wild-type agonist ligand. An in silico approach suggested that the inability to form the central supramolecular activation cluster (cSMAC) could underlie the increased proliferation. This conclusion was supported by experiments that showed that enhancing cSMAC formation reduced stimulatory capacity of the weak peptide. Our studies highlight the fact that a complex interplay of factors determines the quality of a T cell antigen. View Publication

The present study reports for the first time a dual antiglioma effect of the well-known antidiabetic drug metformin. In low-density cultures of the C6 rat glioma cell line,metformin blocked the cell cycle progression in G(0)/G(1) phase without inducing significant cell death. In confluent C6 cultures,on the other hand,metformin caused massive induction of caspase-dependent apoptosis associated with c-Jun N-terminal kinase (JNK) activation,mitochondrial depolarization and oxidative stress. Metformin-triggered apoptosis was completely prevented by agents that block mitochondrial permeability transition (cyclosporin A) and oxygen radical production (N-acetylcisteine),while the inhibitors of JNK activation (SP600125) or glycolysis (sodium fluoride,iodoacetate) provided partial protection. The antiglioma effect of metformin was reduced by compound C,an inhibitor of AMP-activated protein kinase (AMPK),and was mimicked by the AMPK agonist AICAR. Similar effects were observed in the human glioma cell line U251,while rat primary astrocytes were completely resistant to the antiproliferative and proapoptotic action of metformin. View Publication

The present study explored the sensitivity of leukaemic blasts derived from 30 acute myeloid leukaemia (AML) patients to Bortezomib. Bortezomib induced apoptosis of primary AML blasts: 18/30 AMLs were clearly sensitive to the proapoptotic effects of Bortezomib,while the remaining cases were moderately sensitive to this molecule. The addition of tumour necrosis factor-related-apoptosis-inducing ligand,when used alone,did not induce apoptosis of AML blasts and further potentiated the cytotoxic effects of Bortezomib. The majority of AMLs sensitive to Bortezomib showed immunophenotypic features of the M4 and M5 French-American-British classification subtypes and displayed myelomonocytic features. All AMLs with mutated FLT3 were in the Bortezomib-sensitive group. Biochemical studies showed that: (i) Bortezomib activated caspase-8 and caspase-3 and decreased cellular FLICE [Fas-associated death domain (FADD)-like interleukin-1beta-converting enzyme]-inhibitory protein (c-FLIP) levels in AML blasts; (ii) high c-FLIP levels in AML blasts were associated with low Bortezomib sensitivity. Finally,analysis of the effects of Bortezomib on leukaemic cells displaying high aldehyde dehydrogenase activity suggested that this drug induced in vitro killing of leukaemic stem cells. The findings of the present study,further support the development of Bortezomib as an anti-leukaemic drug and provide simple tools to predict the sensitivity of AML cells to this drug. View Publication

BACKGROUND Human embryonic stem cells (hESC) should enable novel insights into early human development and provide a renewable source of cells for regenerative medicine. However,because the three-dimensional hESC aggregates [embryoid bodies (hEB)] typically employed to reveal hESC developmental potential are heterogeneous and exhibit disorganized differentiation,progress in hESC technology development has been hindered. METHODOLOGY/PRINCIPAL FINDINGS Using a centrifugal forced-aggregation strategy in combination with a novel centrifugal-extraction approach as a foundation,we demonstrated that hESC input composition and inductive environment could be manipulated to form large numbers of well-defined aggregates exhibiting multi-lineage differentiation and substantially improved self-organization from single-cell suspensions. These aggregates exhibited coordinated bi-domain structures including contiguous regions of extraembryonic endoderm- and epiblast-like tissue. A silicon wafer-based microfabrication technology was used to generate surfaces that permit the production of hundreds to thousands of hEB per cm(2). CONCLUSIONS/SIGNIFICANCE The mechanisms of early human embryogenesis are poorly understood. We report an ultra high throughput (UHTP) approach for generating spatially and temporally synchronised hEB. Aggregates generated in this manner exhibited aspects of peri-implantation tissue-level morphogenesis. These results should advance fundamental studies into early human developmental processes,enable high-throughput screening strategies to identify conditions that specify hESC-derived cells and tissues,and accelerate the pre-clinical evaluation of hESC-derived cells. View Publication

Decay-accelerating factor (DAF) is a cell surface regulator that accelerates the dissociation of C3/C5 convertases and thereby prevents the amplification of complement activation on self cells. In the context of transplantation,DAF has been thought to primarily regulate antibody-mediated allograft injury,which is in part serum complement-dependent. Based on our previously delineated link between DAF and CD4 T cell responses,we evaluated the effects of donor Daf1 (the murine homolog of human DAF) deficiency on CD8 T cell-mediated cardiac allograft rejection. MHC-disparate Daf1(-/-) allografts were rejected with accelerated kinetics compared with wild-type grafts. The accelerated rejection predominantly tracked with DAF's absence on bone marrow-derived cells in the graft and required allograft production of C3. Transplantation of Daf1(-/-) hearts into wild-type allogeneic hosts augmented the strength of the anti-donor (direct pathway) T cell response,in part through complement-dependent proliferative and pro-survival effects on alloreactive CD8 T cells. The accelerated allograft rejection of Daf1(-/-) hearts occurred in recipients lacking anti-donor Abs. The results reveal that donor DAF expression,by controlling local complement activation on interacting T cell APC partners,regulates the strength of the direct alloreactive CD8(+) T cell response. The findings provide new insights into links between innate and adaptive immunity that could be exploited to limit T cell-mediated injury to an allograft following transplantation. View Publication

BACKGROUND Interleukin (IL-6) and transforming growth factor (TGF)-beta have been shown to play a role in skin development and maintenance. OBJECTIVES A link between these two cytokines has yet to be identified and therefore in this study we investigated the modulation of TGF-beta1 and TGF-beta type 2 receptor (TGF-betaR2) by IL-6 in skin. METHODS An IL-6 knockout (IL-6KO) fibroblast-populated lattice model and intradermal injections of IL-6 into unwounded IL-6KO mice were used to investigate the direct effects of IL-6 treatment on TGF-beta and TGF-betaR2 expression and to determine the signalling mechanism. In addition,IL-6KO and C57BL/6 control mice were wounded by a 4-mm punch biopsy to monitor expression of TGF-beta1 and TGF-betaR2 within a wound over time. The expression of TGF-beta1 and TGF-betaR2 was assessed by real-time quantitative polymerase chain reaction,enzyme-linked immunosorbent assay and immunohistology. RESULTS Recombinant IL-6 treatment of IL-6KO lattices and intradermal injections of IL-6 showed a significant induction of TGF-beta1 mRNA and protein,with TGF-beta1 expression localized in the dermis,while TGF-betaR2 expression was primarily in the epidermis in IL-6KO mice. During healing,the expression of TGF-beta1 and TGF-betaR2 mRNA was significantly greater in unwounded and 7-day-old wounds from wild-type mice; however,protein expression did not differ. Treatment with signal transduction inhibitors indicated that IL-6 modulates TGF-beta through a mitogen-activated protein kinase/extracellular signal-regulated kinase (Mapk/Erk)-dependent mechanism. CONCLUSION These studies indicate that IL-6 has the ability to modulate the expression of TGF-beta and TGF-betaR2 to varying degrees in the skin,which may provide a possible mechanism for defining the role of IL-6 in skin maintenance and a new association of IL-6 with TGF-beta in pathologies associated with fibrosis. View Publication

The presentation of proteins on surfaces is fundamental to numerous cell culture and tissue engineering applications. While a number of physisorption and cross-linking methods exist to facilitate this process,few avoid denaturation of proteins or allow control over protein orientation,both of which are critical to the functionality of many signal proteins and ligands. Often recombinant protein sequences include a poly-histidine tag to facilitate purification. We utilize this sequence to anchor proteins to biosurfaces via a peptide bonded to the surface which conjugates with the poly-histidine tag in the presence of zinc rather than nickel,which is more traditionally used to conjugate poly-histidine tags to surfaces. We demonstrate that this strategy enables the display of proteins on 2D and 3D surfaces without compromising protein function through direct cross-linking or physisorption. View Publication

OBJECTIVES: Hematopoietic stroma promotes resistance to immune control by APO2L/TRAIL in multiple myeloma (MM) cells in part by increasing synthesis of the anti-apoptotic protein c-FLIP. Here,we tested whether bortezomib can reverse the APO2L/TRAIL environmental mediated-immune resistance (EM-IR). MATERIAL AND METHODS: MM cell lines (RPMI 8226 and U266) and CD138+ patient's MM cells were directly adhered to HS5 stroma exposed to HS5 or bone marrow stroma of patients with MM released soluble factors in a transwell system. Cells were treated with either APO2L/TRAIL (10 ng/mL),bortezomib (10 nm) or both. RESULTS: Pretreatment with bortezomib effectively overcomes APO2L/TRAIL apoptosis resistance in myeloma cell lines and in CD138+ cells while directly adhered or in transwell assay. Bortezomib was not cytotoxic to HS5 stroma cells and only altered monocyte chemotactic protein-2-3 and IL-10 levels in the stroma-myeloma milieu. Factors released by HS5 stroma increased expression of c-FLIP,induced STAT-3 and ERK phosphorylation and reduced DR4 receptor expression in MM cells. HS5 stroma-released factor(s) induced NF-kappaB activation after 20 h exposure in association with an enhanced c-FLIP transcription. Bortezomib effectively reduced c-FLIP protein expression without affecting other proteins. Bortezomib also increased DR4 and DR5 expression in the presence of stroma. CONCLUSIONS: These findings provide the rationale to combine bortezomib and APO2L/TRAIL to disrupt the influence of the stroma microenvironment on MM cells. View Publication