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

Receptor activator of nuclear factor kappa-B ligand (RANKL) is a critical osteoclastogenic factor expressed in bone marrow stromal/osteoblast lineage cells. Tumor necrosis factor (TNF) related apoptosis-inducing ligand (TRAIL) levels are elevated in pathologic conditions such as multiple myeloma and inflammatory arthritis,and have been positively correlated with osteolytic markers. Osteoprotegerin (OPG) which inhibits osteoclastogenesis is a decoy receptor for RANKL and also known to interact with TRAIL. Herein,we show that TRAIL increases DR5 and DcR1 receptors but no change in the levels of DR4 and DcR2 expression in human bone marrow derived stromal/preosteoblast (SAKA-T) cell line. We further demonstrated that TRAIL treatment significantly decreased OPG mRNA expression. Interestingly,TRAIL treatment induced RANKL mRNA expression in these cells. In addition,TRAIL significantly increased NF-kB and c-Jun N-terminal kinase (JNK) activity. Human transcription factor array screening by real-time RT-PCR identified TRAIL up-regulation of the signal transducers and activators of the transcription (STAT)-6 expression in SAKA-T cells. TRAIL stimulation induced p-STAT-6 expression in human bone marrow derived primary stromal/preosteoblast cells. Confocal microscopy analysis further revealed p-STAT-6 nuclear localization in SAKA-T cells. Chromatin immunoprecipitation (ChIP) assay confirmed p-STAT-6 binding to the hRANKL gene distal promoter region. In addition,siRNA suppression of STAT-6 expression inhibits TRAIL increased hRANKL gene promoter activity. Thus,our results suggest that TRAIL induces RANKL expression through a STAT-6 dependent transcriptional regulatory mechanism in bone marrow stromal/preosteoblast cells. View Publication

Meis1 and Hoxa9 expression is upregulated by retroviral integration in murine myeloid leukemias and in human leukemias carrying MLL translocations. Both genes also cooperate to induce leukemia in a mouse leukemia acceleration assay,which can be explained,in part,by their physical interaction with each other as well as the PBX family of homeodomain proteins. Here we show that Meis1-deficient embryos have partially duplicated retinas and smaller lenses than normal. They also fail to produce megakaryocytes,display extensive hemorrhaging,and die by embryonic day 14.5. In addition,Meis1-deficient embryos lack well-formed capillaries,although larger blood vessels are normal. Definitive myeloerythroid lineages are present in the mutant embryos,but the total numbers of colony-forming cells are dramatically reduced. Mutant fetal liver cells also fail to radioprotect lethally irradiated animals and they compete poorly in repopulation assays even though they can repopulate all hematopoietic lineages. These and other studies showing that Meis1 is expressed at high levels in hematopoietic stem cells (HSCs) suggest that Meis1 may also be required for the proliferation/self-renewal of the HSC. View Publication

Frontotemporal dementia (FTD) and other tauopathies characterized by focal brain neurodegeneration and pathological accumulation of proteins are commonly associated with tau mutations. However,the mechanism of neuronal loss is not fully understood. To identify molecular events associated with tauopathy,we studied induced pluripotent stem cell (iPSC)-derived neurons from individuals carrying the tau-A152T variant. We highlight the potential of in-depth phenotyping of human neuronal cell models for pre-clinical studies and identification of modulators of endogenous tau toxicity. Through a panel of biochemical and cellular assays,A152T neurons showed accumulation,redistribution,and decreased solubility of tau. Upregulation of tau was coupled to enhanced stress-inducible markers and cell vulnerability to proteotoxic,excitotoxic,and mitochondrial stressors,which was rescued upon CRISPR/Cas9-mediated targeting of tau or by pharmacological activation of autophagy. Our findings unmask tau-mediated perturbations of specific pathways associated with neuronal vulnerability,revealing potential early disease biomarkers and therapeutic targets for FTD and other tauopathies. View Publication

It has become apparent that chromatin modification plays a critical role in the regulation of cell-type-specific gene expression. Here,we show that an inhibitor of histone deacetylase,valproic acid (VPA),induced neuronal differentiation of adult hippocampal neural progenitors. In addition,VPA inhibited astrocyte and oligodendrocyte differentiation,even in conditions that favored lineage-specific differentiation. Among the VPA-up-regulated,neuron-specific genes,a neurogenic basic helix-loop-helix transcription factor,NeuroD,was identified. Overexpression of NeuroD resulted in the induction and suppression of neuronal and glial differentiation,respectively. These results suggest that VPA promotes neuronal fate and inhibits glial fate simultaneously through the induction of neurogenic transcription factors including NeuroD. View Publication

The potential for directed differentiation of human-induced pluripotent stem (iPS) cells to functional postmitotic neuronal phenotypes is unknown. Following methods shown to be effective at generating motor neurons from human embryonic stem cells (hESCs),we found that once specified to a neural lineage,human iPS cells could be differentiated to form motor neurons with a similar efficiency as hESCs. Human iPS-derived cells appeared to follow a normal developmental progression associated with motor neuron formation and possessed prototypical electrophysiological properties. This is the first demonstration that human iPS-derived cells are able to generate electrically active motor neurons. These findings demonstrate the feasibility of using iPS-derived motor neuron progenitors and motor neurons in regenerative medicine applications and in vitro modeling of motor neuron diseases. View Publication

Glioblastoma multiforme (GBM) is an aggressive,Grade IV astrocytoma with a poor survival rate,primarily due to the GBM tumor cells migrating away from the primary tumor site along the nanotopography of white matter tracts and blood vessels. It is unclear whether this nanotopography influences the biomechanical properties (i.e. cytoskeletal stiffness) of GBM tumor cells. Although GBM tumor cells have an innate propensity to migrate,we believe this capability is enhanced due to the influence of nanotopography on the tumor cells' biomechanical properties. In this study,we used an aligned nanofiber film that mimics the nanotopography in the tumor microenvironment to investigate the mechanical properties of GBM tumor cells in vitro. The data demonstrate that the cytoskeletal stiffness,cell traction stress,and focal adhesion area were significantly lower in the GBM tumor cells compared to healthy astrocytes. Moreover,the cytoskeletal stiffness was significantly reduced when cultured on aligned nanofiber films compared to smooth and randomly aligned nanofiber films. Gene expression analysis showed that tumor cells cultured on the aligned nanotopography upregulated key migratory genes and downregulated key proliferative genes. Therefore,our data suggest that the migratory potential is elevated when GBM tumor cells are migrating along aligned nanotopographical substrates. View Publication

CRISPR genome engineering has become a powerful tool to functionally investigate the complex mechanisms of immune system regulation. While decades of work have aimed to genetically reprogram innate immunity,the utility of current approaches is restricted by poor knockout efficiencies or limited specificity for mature cell lineages in vivo. Here,we describe an optimized strategy for non-viral CRISPR-Cas9 ribonucleoprotein (cRNP) genomic editing of mature primary mouse innate lymphocyte cells (ILCs) and myeloid lineage cells that results in an almost complete loss of single or double target gene expression from a single electroporation. Furthermore,we describe in vivo adoptive transfer mouse models that can be utilized to screen for gene function during viral infection using cRNP-edited naive natural killer (NK) cells and bone-marrow-derived conventional dendritic cell precursors (cDCPs). This resource will enhance target gene discovery and offer a specific and simplified approach to gene editing in the mouse innate immune system. View Publication

Lentiviral-mediated integration of a CFTR transgene cassette into airway basal cells is a strategy being considered for cystic fibrosis (CF) cell-based therapies. However,CFTR expression is highly regulated in differentiated airway cell types and a subset of intermediate basal cells destined to differentiate. Since basal stem cells typically do not express CFTR,suppressing the CFTR expression from the lentiviral vector in airway basal cells may be beneficial for maintaining their proliferative capacity and multipotency. We identified miR-106b as highly expressed in proliferating airway basal cells and extinguished in differentiated columnar cells. Herein,we developed lentiviral vectors with the miR-106b-target sequence (miRT) to both study miR-106b regulation during basal cell differentiation and detarget CFTR expression in basal cells. Given that miR-106b is expressed in the 293T cells used for viral production,obstacles of viral genome integrity and titers were overcome by creating a 293T-B2 cell line that inducibly expresses the RNAi suppressor B2 protein from flock house virus. While miR-106b vectors effectively detargeted reporter gene expression in proliferating basal cells and following differentiation in the air-liquid interface and organoid cultures,the CFTR-miRT vector produced significantly less CFTR-mediated current than the non-miR-targeted CFTR vector following transduction and differentiation of CF basal cells. These findings suggest that miR-106b is expressed in certain airway cell types that contribute to the majority of CFTR anion transport in airway epithelium. View Publication

Monitoring genome-wide,cell-specific responses to human disease,although challenging,holds great promise for the future of medicine. Patients with injuries severe enough to develop multiple organ dysfunction syndrome have multiple immune derangements,including T cell apoptosis and anergy combined with depressed monocyte antigen presentation. Genome-wide expression analysis of highly enriched circulating leukocyte subpopulations,combined with cell-specific pathway analyses,offers an opportunity to discover leukocyte regulatory networks in critically injured patients. Severe injury induced significant changes in T cell (5,693 genes),monocyte (2,801 genes),and total leukocyte (3,437 genes) transcriptomes,with only 911 of these genes common to all three cell populations (12%). T cell-specific pathway analyses identified increased gene expression of several inhibitory receptors (PD-1,CD152,NRP-1,and Lag3) and concomitant decreases in stimulatory receptors (CD28,CD4,and IL-2Ralpha). Functional analysis of T cells and monocytes confirmed reduced T cell proliferation and increased cell surface expression of negative signaling receptors paired with decreased monocyte costimulation ligands. Thus,genome-wide expression from highly enriched cell populations combined with knowledge-based pathway analyses leads to the identification of regulatory networks differentially expressed in injured patients. Importantly,application of cell separation,genome-wide expression,and cell-specific pathway analyses can be used to discover pathway alterations in human disease. View Publication

Human CD56(bright) natural killer (NK) cells possess little or no killer immunoglobulin-like receptors (KIRs),high interferon-gamma (IFN-gamma) production,but little cytotoxicity. CD56(dim) NK cells have high KIR expression,produce little IFN-gamma,yet display high cytotoxicity. We hypothesized that,if human NK maturation progresses from a CD56(bright) to a CD56(dim) phenotype,an intermediary NK cell must exist,which demonstrates more functional overlap than these 2 subsets,and we used CD94 expression to test our hypothesis. CD94(high)CD56(dim) NK cells express CD62L,CD2,and KIR at levels between CD56(bright) and CD94(low)CD56(dim) NK cells. CD94(high)CD56(dim) NK cells produce less monokine-induced IFN-gamma than CD56(bright) NK cells but much more than CD94(low)CD56(dim) NK cells because of differential interleukin-12-mediated STAT4 phosphorylation. CD94(high)CD56(dim) NK cells possess a higher level of granzyme B and perforin expression and CD94-mediated redirected killing than CD56(bright) NK cells but lower than CD94(low)CD56(dim) NK cells. Collectively,our data suggest that the density of CD94 surface expression on CD56(dim) NK cells identifies a functional and likely developmental intermediary between CD56(bright) and CD94(low)CD56(dim) NK cells. This supports the notion that,in vivo,human CD56(bright) NK cells progress through a continuum of differentiation that ends with a CD94(low)CD56(dim) phenotype. View Publication