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

The PI3K/AKT/mTOR pathway is commonly over activated in glioblastoma (GBM),and Rictor was shown to be an important regulator downstream of this pathway. EGFR overexpression is also frequently found in GBM tumors,and both EGFR and Rictor are associated with increased proliferation,invasion,metastasis and poor prognosis. This research evaluated in vitro and in vivo whether the combined silencing of EGFR and Rictor would result in therapeutic benefits. The therapeutic potential of targeting these proteins in combination with conventional agents with proven activity in GBM patients was also assessed. In vitro validation studies were carried out using siRNA-based gene silencing methods in a panel of three commercially available human GBM cell lines,including two PTEN mutant lines (U251MG and U118MG) and one PTEN-wild type line (LN229). The impact of EGFR and/or Rictor silencing on cell migration and sensitivity to chemotherapeutic drugs in vitro was determined. In vivo validation of these studies was focused on EGFR and/or Rictor silencing achieved using doxycycline-inducible shRNA-expressing U251MG cells implanted orthotopically in Rag2M mice brains. Target silencing,tumor size and tumor cell proliferation were assessed by quantification of immunohistofluorescence-stained markers. siRNA-mediated silencing of EGFR and Rictor reduced U251MG cell migration and increased sensitivity of the cells to irinotecan,temozolomide and vincristine. In LN229,co-silencing of EGFR and Rictor resulted in reduced cell migration,and increased sensitivity to vincristine and temozolomide. In U118MG,silencing of Rictor alone was sufficient to increase this line's sensitivity to vincristine and temozolomide. In vivo,while the silencing of EGFR or Rictor alone had no significant effect on U251MG tumor growth,silencing of EGFR and Rictor together resulted in a complete eradication of tumors. These data suggest that the combined silencing of EGFR and Rictor should be an effective means of treating GBM. View Publication

The polarization and migration of neural progenitor cells (NPCs) are critical for embryonic brain development and neurogenesis after brain injury. Although stromal-derived factor-1α (SDF-1α,CXCL12) and its receptor CXCR4 are well-known to mediate the migration of NPCs in the developing brain,the dynamic cellular processes and structure-related molecular events remain elusive. Transwell and microfluidic-based assays are classical assays to effectively study cellular migration. However,both of them have limitations in the analysis of a single cell. In this study,we modified the stripe assay and extended its applications in the study of NPC polarization and intracellular molecular events associated with CXCL12-mediated migration. In response to localized CXCL12,NPCs formed lamellipodia in the stripe assay. Furthermore,CXCR4 and Rac1 quickly re-distributed to the area of lamellipodia,indicating their roles in NPC polarization upon CXCL12 stimulation. Although the chemokine stripes in the assay provided concentration gradients that can be best used to study cellular polarization and migration through immunocytochemistry,they can also generate live imaging data with comparable quality. In conclusion,stripe assay is a visual,dynamic and economical tool to study cellular mobility and its related molecule mechanisms. View Publication

NMDA receptors play a central role in shaping the strength of synaptic connections throughout development and in mediating synaptic plasticity mechanisms that underlie some forms of learning and memory formation in the CNS. In the hippocampus and the neocortex,GluN1 is combined primarily with GluN2A and GluN2B,which are differentially expressed during development and confer distinct molecular and physiological properties to NMDA receptors. The contribution of each subunit to the synaptic traffic of NMDA receptors and therefore to their role during development and in synaptic plasticity is still controversial. We report a critical role for the GluN2B subunit in regulating NMDA receptor synaptic targeting. In the absence of GluN2B,the synaptic levels of AMPA receptors are increased and accompanied by decreased constitutive endocytosis of GluA1-AMPA receptor. We used quantitative proteomic analysis to identify changes in the composition of postsynaptic densities from GluN2B(-/-) mouse primary neuronal cultures and found altered levels of several ubiquitin proteasome system components,in particular decreased levels of proteasome subunits. Enhancing the proteasome activity with a novel proteasome activator restored the synaptic levels of AMPA receptors in GluN2B(-/-) neurons and their endocytosis,revealing that GluN2B-mediated anchoring of the synaptic proteasome is responsible for fine tuning AMPA receptor synaptic levels under basal conditions. View Publication

Cellular damage by reactive oxygen species and altered neurogenesis are implicated in the etiology of AD and the pathogenic actions of amyloid β-peptide (Aβ); the underlying mechanisms and the early oxidative intracellular events triggered by Aβ are not established. In the present study,we found that mouse embryonic cortical neural progenitor cells exhibit intermittent spontaneous mitochondrial superoxide (SO) flashes that require transient opening of mitochondrial permeability transition pores (mPTPs). The incidence of mitochondria SO flash activity in neural progenitor cells (NPCs) increased during the first 6-24 hours of exposure to aggregating amyloid β-peptide (Aβ1-42),indicating an increase in transient mPTP opening. Subsequently,the SO flash frequency progressively decreased and ceased between 48 and 72 hours of exposure to Aβ1-42,during which time global cellular reactive oxygen species increased,mitochondrial membrane potential decreased,cytochrome C was released from mitochondria and the cells degenerated. Inhibition of mPTPs and selective reduction in mitochondrial SO flashes significantly ameliorated the negative effects of Aβ1-42 on NPC proliferation and survival. Our findings suggest that mPTP-mediated bursts of mitochondrial SO production is a relatively early and pivotal event in the adverse effects of Aβ1-42 on NPCs. If Aβ inhibits NPC proliferation in the brains of AD patients by a similar mechanism,then interventions that inhibit mPTP-mediated superoxide flashes would be expected to protect NPCs against the adverse effects of Aβ. View Publication

BACKGROUND & AIMS The etiology of abdominal pain in postinfectious,diarrhea-predominant irritable bowel syndrome (PI-IBS-D) is unknown,and few treatment options exist. Catechol-O-methyltransferase (COMT),an enzyme that inactivates and degrades biologically active catecholamines,plays an important role in numerous physiologic processes,including modulation of pain perception. Our objective was to determine the mechanism(s) of how decreased colonic COMT in PI-IBS-D patients contributes to the chronic abdominal pain phenotype after enteric infections. METHODS Colon neurons,epithelial cells,and macrophages were procured with laser capture microdissection from PI-IBS-D patients to evaluate cell-specific colonic COMT,microRNA-155 (miR-155),and tumor necrosis factor (TNF) ? expression levels compared to recovered patients (infection cleared: did not develop PI-IBS-D) and control individuals. COMT-/-,colon-specific COMT-/-,and miR-155-/- mice and human colonoids were used to model phenotypic expression of COMT in PI-IBS-D patients and to investigate signaling pathways linking abdominal pain. Citrobacter rodentium and trinitrobenzene sulfonic acid animal models were used to model postinflammatory changes seen in PI-IBS-D patients. RESULTS Colonic COMT levels were significantly decreased and correlated with increased visual analog scale abdominal pain ratings in PI-IBS-D patients compared to recovered patients and control individuals. Colonic miR-155 and TNF-? were increased in PI-IBS-D patients with diminished colonic COMT. COMT-/- mice had significantly increased expression of miR-155 and TNF-? in both colon tissues and dorsal root ganglia. Introduction of cV1q antibody (anti-TNF-?) into mice reversed visceral hypersensitivity after C rodentium and trinitrobenzene sulfonic acid. CONCLUSIONS Decreased colonic COMT in PI-IBS-D patients drives abdominal pain phenotypes via the COMT/miR-155/TNF-? axis. These important findings will allow new treatment paradigms and more targeted and personalized medicine approaches for gastrointestinal disorders after enteric infections. View Publication

Na{\{i}}ve T cell activation in secondary lymphoid organs such as lymph nodes (LNs) occurs upon recognition of cognate antigen presented by antigen presenting cells (APCs). T cell activation requires cytoskeleton rearrangement and sustained interactions with APCs. Enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) proteins are a family of cytoskeletal effector proteins responsible for actin polymerization and are frequently found at the leading edge of motile cells. Ena/VASP proteins have been implicated in motility and adhesion in various cell types but their role in primary T cell interstitial motility and activation has not been explored. Our goal was to determine the contribution of Ena/VASP proteins to T cell-APC interactions T cell activation and T cell expansion in vivo. Our results showed that na{\"{i}}ve T cells from Ena/VASP-deficient mice have a significant reduction in antigen-specific T cell accumulation following Listeria monocytogenes infection. The kinetics of T cell expansion impairment were further confirmed in Ena/VASP-deficient T cells stimulated via dendritic cell immunization. To investigate the cause of this T cell expansion defect we analyzed T cell-APC interactions in vivo by two-photon microscopy and observed fewer Ena/VASP-deficient na{\"{i}}ve T cells interacting with APCs in LNs during priming. We also determined that Ena/VASP-deficient T cells formed conjugates with significantly less actin polymerization at the T cell-APC synapse and that these conjugates were less stable than their WT counterparts. Finally we found that Ena/VASP-deficient T cells have less LFA-1 polarized to the T cell-APC synapse. Thus we conclude that Ena/VASP proteins contribute to T cell actin remodeling during T cell-APC interactions which promotes the initiation of stable T cell conjugates during APC scanning. Therefore Ena/VASP proteins are required for efficient activation and expansion of T cells in vivo." View Publication

BACKGROUND In kidney transplantation,early allograft inflammation impairs long-term allograft function. However,precise mediators of early kidney allograft inflammation are unclear,making it challenging to design therapeutic interventions. METHODS We used an allogeneic murine kidney transplant model in which CD45.2 BALB/c kidneys were transplanted to CD45.1 C57BL/6 recipients. RESULTS Donor kidney resident macrophages within the allograft expanded rapidly in the first 3 days. During this period,they were also induced to express a high level of Ccl8,which,in turn,promoted recipient monocyte graft infiltration,their differentiation to resident macrophages,and subsequent expression of Ccl8. Enhanced graft infiltration of recipient CCR8+ T cells followed,including CD4,CD8,and ?? T cells. Consequently,blocking CCL8-CCR8 or depleting donor kidney resident macrophages significantly inhibits early allograft immune cell infiltration and promotes superior short-term allograft function. CONCLUSIONS Targeting the CCL8-CCR8 axis is a promising measure to reduce early kidney allograft inflammation. View Publication

Despite advances in therapy,glioblastoma remains an incurable disease with a dismal prognosis. Recent studies have implicated cancer stem cells within glioblastoma (glioma stem cells,GSCs) as mediators of therapeutic resistance and tumor progression. In this study,we investigated the role of the transforming growth factor-$\beta$ (TGF-$\beta$) superfamily,which has been found to play an integral role in the maintenance of stem cell homeostasis within multiple stem cell systems,as a mediator of stem-like cells in glioblastoma. We find that BMP and TGF-$\beta$ signaling define divergent molecular and functional identities in glioblastoma,and mark relatively quiescent and proliferative GSCs,respectively. Treatment of GSCs with BMP inhibits cell proliferation,but does not abrogate their stem-ness,as measured by self-renewal and tumorigencity. Further,BMP pathway activation confers relative resistance to radiation and temozolomide chemotherapy. Our findings define a quiescent cancer stem cell population in glioblastoma that may be a cellular reservoir for tumor recurrence following cytotoxic therapy. View Publication

Progressive resistance exercise training (PRT) is the most effective known intervention for combating aging skeletal muscle atrophy. However,the hypertrophic response to PRT is variable,and this may be due to muscle inflammation susceptibility. Metformin reduces inflammation,so we hypothesized that metformin would augment the muscle response to PRT in healthy women and men aged 65 and older. In a randomized,double-blind trial,participants received 1,700 mg/day metformin (N = 46) or placebo (N = 48) throughout the study,and all subjects performed 14 weeks of supervised PRT. Although responses to PRT varied,placebo gained more lean body mass (p = .003) and thigh muscle mass (p {\textless} .001) than metformin. CT scan showed that increases in thigh muscle area (p = .005) and density (p = .020) were greater in placebo versus metformin. There was a trend for blunted strength gains in metformin that did not reach statistical significance. Analyses of vastus lateralis muscle biopsies showed that metformin did not affect fiber hypertrophy,or increases in satellite cell or macrophage abundance with PRT. However,placebo had decreased type I fiber percentage while metformin did not (p = .007). Metformin led to an increase in AMPK signaling,and a trend for blunted increases in mTORC1 signaling in response to PRT. These results underscore the benefits of PRT in older adults,but metformin negatively impacts the hypertrophic response to resistance training in healthy older individuals. ClinicalTrials.gov Identifier: NCT02308228. View Publication

The vascular wall is a source of progenitor cells that are able to induce skeletal repair,primarily by paracrine mechanisms. Here,the paracrine role of extracellular vesicles (EVs) in bone healing was investigated. First,purified human perivascular stem cells (PSCs) were observed to induce mitogenic,pro-migratory,and pro-osteogenic effects on osteoprogenitor cells while in non-contact co-culture via elaboration of EVs. PSC-derived EVs shared mitogenic,pro-migratory,and pro-osteogenic properties of their parent cell. PSC-EV effects were dependent on surface-associated tetraspanins,as demonstrated by EV trypsinization,or neutralizing antibodies for CD9 or CD81. Moreover,shRNA knockdown in recipient cells demonstrated requirement for the CD9/CD81 binding partners IGSF8 and PTGFRN for EV bioactivity. Finally,PSC-EVs stimulated bone repair,and did so via stimulation of skeletal cell proliferation,migration,and osteodifferentiation. In sum,PSC-EVs mediate the same tissue repair effects of perivascular stem cells,and represent an 'off-the-shelf' alternative for bone tissue regeneration. View Publication

Human cortical neural progenitor cell transplantation holds significant potential in cortical stroke treatment by replacing lost cortical neurons and repairing damaged brain circuits. However,commonly utilized human cortical neural progenitors are limited in yield a substantial proportion of diverse cortical neurons and require an extended period to achieve functional maturation and synaptic integration,thereby potentially diminishing the optimal therapeutic benefits of cell transplantation for cortical stroke. Here,we generated forkhead box G1 (FOXG1)-positive forebrain progenitors from human inducible pluripotent stem cells,which can differentiate into diverse and balanced cortical neurons including upper- and deep-layer excitatory and inhibitory neurons,achieving early functional maturation simultaneously in vitro. Furthermore,these FOXG1 forebrain progenitor cells demonstrate robust cortical neuronal differentiation,rapid functional maturation and efficient synaptic integration after transplantation into the sensory cortex of stroke-injured adult rats. Notably,we have successfully utilized the non-invasive 18F-SynVesT-1 PET imaging technique to assess alterations in synapse count before and after transplantation therapy of FOXG1 progenitors in vivo. Moreover,the transplanted FOXG1 progenitors improve sensory and motor function recovery following stroke. These findings provide systematic and compelling evidence for the suitability of these FOXG1 progenitors for neuronal replacement in ischemic cortical stroke. Human NPCs show promise for stroke treatment,but challenges remain in neuron diversity and maturation time. Here,the authors describe the generation of FOXG1 progenitors from iPSCs that quickly mature into functional cortical neurons,enhancing stroke recovery in rats. View Publication

IntroductionCRISPR/Cas9-edited induced pluripotent stem cells (iPSCs) are valuable research models for mechanistic studies. However,gene conversion between a gene-pseudogene pair that share high sequence identity and form direct repeats in proximity on the same chromosome can interfere with the precision of gene editing. Mutations in the human beta-glucocerebrosidase gene (GBA1) are associated with Gaucher disease,Parkinson’s disease,and Lewy body dementia. During the creation of a GBA1 KO iPSC line,we detected about 70% gene conversion from its pseudogene GBAP1. These events maintained the reading frame and resulted from GBA1-specific cleavage by CRISPR/Cas9,without disrupting the GBA1 gene.MethodTo increase the percentage of alleles with out-of-frame indels for triggering nonsense-mediated decay of the GBA1 mRNA,we supplied the cells with two single-stranded oligodeoxynucleotide (ssODN) donors as homology-directed repair (HDR) templates.ResultsWe demonstrate that HDR using the ssODN templates effectively competes with gene conversion and enabled biallelic KO clone isolation,whereas the nonallelic homologous recombination (NAHR)-based deletion rate remained the same.DiscussionHere,we report a generalizable method to direct cellular DNA repair of double strand breaks at a target gene towards the HDR pathway using exogenous ssODN templates,allowing specific editing of one gene in a gene-pseudogene pair without disturbing the other. View Publication