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

The cancer stem cell (CSC) model suggests that a subpopulation of cells within the tumor,the CSCs,is responsible for cancer relapse and metastasis formation. CSCs hold unique characteristics,such as self-renewal,differentiation abilities,and resistance to chemotherapy,raising the need for discovering drugs that target CSCs. Previously we have found that the antihypertensive drug spironolactone impairs DNA damage response in cancer cells. Here we show that spironolactone,apart from inhibiting cancerous cell growth,is also highly toxic to CSCs. Notably,we demonstrate that CSCs have high basal levels of DNA double-strand breaks (DSBs). Mechanistically,we reveal that spironolactone does not damage the DNA but impairs DSB repair and induces apoptosis in cancer cells and CSCs while sparing healthy cells. In vivo,spironolactone treatment reduced the size and CSC content of tumors. Overall,we suggest spironolactone as an anticancer reagent,toxic to both cancer cells and,particularly to,CSCs. View Publication

Fatty-acid-binding proteins (FABPs) are key intracellular molecules involved in the uptake,transportation and storage of fatty acids and in the mediation of signal transduction and gene transcription. However,little is known regarding their expression and function in the oligodendrocyte lineage. We evaluate the in vivo and in vitro expression of FABP5 and FABP7 in oligodendrocyte lineage cells in the cortex and corpus callosum of adult mice,mixed cortical culture and oligosphere culture by immunofluorescent counter-staining with major oligodendrocyte lineage markers. In all settings,FABP7 expression was detected in NG2(+)/PDGFRα(+) oligodendrocyte progenitor cells (OPCs) that did not express FABP5. FABP5 was detected in mature CC1(+)/MBP(+) oligodendrocytes that did not express FABP7. Analysis of cultured OPCs showed a significant decrease in the population of FABP7-knockout (KO) OPCs and their BrdU uptake compared with wild-type (WT) OPCs. Upon incubation of OPCs in oligodendrocyte differentiation medium,a significantly lower percentage of FABP7-KO OPCs differentiated into O4(+) oligodendrocytes. The percentage of mature MBP(+) oligodendrocytes relative to whole O4(+)/MBP(+) oligodendrocytes was significantly lower in FABP7-KO and FABP5-KO than in WT cell populations. The percentage of terminally mature oligodendrocytes with membrane sheet morphology was significantly lower in FABP5-KO compared with WT cell populations. Thus,FABP7 and FABP5 are differentially expressed in oligodendrocyte lineage cells and regulate their proliferation and/or differentiation. Our findings suggest the involvement of FABP7 and FABP5 in the pathophysiology of demyelinating disorders,neuropsychiatric disorder and glioma,conditions in which OPCs/oligodendrocytes play central roles. View Publication

Hypoxia is a near-universal feature of cancer,promoting glycolysis,cellular proliferation,and angiogenesis. The molecular mechanisms of hypoxic signaling have been intensively studied,but the impact of changes in oxygen partial pressure (pO2) on the state of signaling networks is less clear. In a glioblastoma multiforme (GBM) cancer cell model,we examined the response of signaling networks to targeted pathway inhibition between 21% and 1% pO2. We used a microchip technology that facilitates quantification of a panel of functional proteins from statistical numbers of single cells. We find that near 1.5% pO2,the signaling network associated with mammalian target of rapamycin (mTOR) complex 1 (mTORC1)--a critical component of hypoxic signaling and a compelling cancer drug target--is deregulated in a manner such that it will be unresponsive to mTOR kinase inhibitors near 1.5% pO2,but will respond at higher or lower pO2 values. These predictions were validated through experiments on bulk GBM cell line cultures and on neurosphere cultures of a human-origin GBM xenograft tumor. We attempt to understand this behavior through the use of a quantitative version of Le Chatelier's principle,as well as through a steady-state kinetic model of protein interactions,both of which indicate that hypoxia can influence mTORC1 signaling as a switch. The Le Chatelier approach also indicates that this switch may be thought of as a type of phase transition. Our analysis indicates that certain biologically complex cell behaviors may be understood using fundamental,thermodynamics-motivated principles. View Publication

Understanding the molecular mechanisms that underlie neurodegenerative disorders has been hampered by a lack of readily available model systems that replicate the complexity of the human disease. Recent advances in stem cell technology have facilitated the derivation of patient-specific stem cells from a variety of differentiated cell types. These induced pluripotent stem cells (iPSCs) are attractive disease models since they can be grown and differentiated to produce large numbers of disease-relevant cell types. However,most iPSC lines are derived in advance of,and without the benefit of,neuropathological confirmation of the donor - the gold standard for many disease classifications and measurement of disease severity. While others have reported the generation of autopsy-confirmed iPSC lines from patient explants,these methods require outgrowth of cadaver tissue,which require additional time and is often only successul 50% of the time. Here we report the rapid generation of autopsy-confirmed iPSC lines from peripheral blood mononuclear cells (PBMCs) drawn postmortem. Since this approach doesn't require the propagation of previously frozen cadaver tissue,iPSC can be rapidly and efficiently produced from patients with autopsy-confirmed pathology. These matched iPSC-derived patient-specific neurons and postmortem brain tissue will support studies of specific mechanisms that drive the pathogenesis of neurodegenerative diseases. View Publication

BACKGROUND AKI is a common sequela of infection with SARS-CoV-2 and contributes to the severity and mortality from COVID-19. Here,we tested the hypothesis that kidney alterations induced by COVID-19-associated AKI could be detected in cells collected from urine. METHODS We performed single-cell RNA sequencing (scRNAseq) on cells recovered from the urine of eight hospitalized patients with COVID-19 with (n=5) or without AKI (n=3) as well as four patients with non-COVID-19 AKI (n=4) to assess differences in cellular composition and gene expression during AKI. RESULTS Analysis of 30,076 cells revealed a diverse array of cell types,most of which were kidney,urothelial,and immune cells. Pathway analysis of tubular cells from patients with AKI showed enrichment of transcripts associated with damage-related pathways compared with those without AKI. ACE2 and TMPRSS2 expression was highest in urothelial cells among cell types recovered. Notably,in one patient,we detected SARS-CoV-2 viral RNA in urothelial cells. These same cells were enriched for transcripts associated with antiviral and anti-inflammatory pathways. CONCLUSIONS We successfully performed scRNAseq on urinary sediment from hospitalized patients with COVID-19 to noninvasively study cellular alterations associated with AKI and established a dataset that includes both injured and uninjured kidney cells. Additionally,we provide preliminary evidence of direct infection of urinary bladder cells by SARS-CoV-2. The urinary sediment contains a wealth of information and is a useful resource for studying the pathophysiology and cellular alterations that occur in kidney diseases. View Publication

Mutations causing loss of the NF-$\kappa$B regulator I$\kappa$BNS,result in impaired development of innate-like B cells and defective plasma cell (PC) differentiation. Since productive PC differentiation requires B cell metabolic reprogramming,we sought to investigate processes important for this transition using the bumble mouse strain,deficient for I$\kappa$BNS. We report that LPS-activated bumble B cells exhibited elevated mTOR activation levels,mitochondrial accumulation,increased OXPHOS and mROS production,along with a reduced capacity for autophagy,compared to wildtype B cells. Overall,our results demonstrate that PC differentiation in the absence of I$\kappa$BNS is characterized by excessive activation during early rounds of B cell division,increased mitochondrial metabolism and decreased autophagic capacity,thus improving our understanding of the role of I$\kappa$BNS in PC differentiation. View Publication

α-Lipoic acid (LA) is a thiol with antioxidant properties that protects against oxidative stress-induced apoptosis. LA is absorbed from the diet,taken up by cells and tissues,and subsequently reduced to dihydrolipoic acid (DHLA). Recently,DHLA has been used as the hydrophilic nanomaterial preparations,and therefore,determination of its bio-safety profile is essential. In this article,we show that DHLA (50-100 μM) induces apoptotic processes in mouse embryonic stem cells (ESC-B5),but exerts no injury effects at treatment dosages below 50 μM. Higher concentrations of DHLA (50-100 μM) directly increased the reactive oxygen species (ROS) content in ESC-B5 cells,along with a significant increase in cytoplasmic free calcium and nitric oxide (NO) levels,loss of mitochondrial membrane potential (MMP),activation of caspases-9 and -3,and cell death. Pretreatment with NO scavengers suppressed the apoptotic biochemical changes induced by 100 μM DHLA and promoted the gene expression levels of p53 and p21 involved in apoptotic signaling. Our results collectively indicate that DHLA at concentrations of 50-100 μM triggers apoptosis of ESC-B5 cells,which involves both ROS and NO. Importantly,at doses of less than 50 μM (0-25 μM),DHLA does not exert hazardous effects on ESC-B5 cell properties,including viability,development and differentiation. These results provide important information in terms of dosage safety and biocompatibility of DHLA to facilitate its further use as a precursor for biomaterial preparation. View Publication

Notch signaling has been demonstrated to have a central role in glioblastoma (GBM) cancer stem cells (CSCs) and we have demonstrated recently that Notch pathway blockade by γ-secretase inhibitor (GSI) depletes GBM CSCs and prevents tumor propagation both in vitro and in vivo. In order to understand the proteome alterations involved in this transformation,a dose-dependent quantitative mass spectrometry (MS)-based proteomic study has been performed based on the global proteome profiling and a target verification phase where both Immunoassay and a multiple reaction monitoring (MRM) assay are employed. The selection of putative protein candidates for confirmation poses a challenge due to the large number of identifications from the discovery phase. A multilevel filtering strategy together with literature mining is adopted to transmit the most confident candidates along the pipeline. Our results indicate that treating GBM CSCs with GSI induces a phenotype transformation towards non-tumorigenic cells with decreased proliferation and increased differentiation,as well as elevated apoptosis. Suppressed glucose metabolism and attenuated NFR2-mediated oxidative stress response are also suggested from our data,possibly due to their crosstalk with Notch Signaling. Overall,this quantitative proteomic-based dose-dependent work complements our current understanding of the altered signaling events occurring upon the treatment of GSI in GBM CSCs. View Publication

The sub-population of tumor cells termed 'cancer stem cells' (CSCs) possess the capability to generate tumors,undergo epithelial-mesenchymal transition (EMT) and are implicated in metastasis,making treatments to specifically target CSCs an attractive therapeutic strategy. Tumor hypoxia plays a key role in regulating EMT and cancer stem cell function. Carbonic anhydrase IX (CAIX) is a hypoxia-inducible protein that regulates cellular pH to promote cancer cell survival and invasion in hypoxic microenvironments and is a biomarker of poor prognosis for breast cancer metastasis and survival. Here,we demonstrate that inhibition of CAIX expression or activity with novel small-molecule inhibitors in breast cancer cell lines,or in primary metastatic breast cancer cells,results in the inhibition of breast CSC expansion in hypoxia. We identify the mTORC1 axis as a critical pathway downstream of CAIX in the regulation of cancer stem cell function. CAIX is also required for expression of EMT markers and regulators,as well as drivers of 'stemness',such as Notch1 and Jagged1 in isolated CSCs. In addition,treatment of mice bearing orthotopic breast tumors with CAIX-specific small-molecule inhibitors results in significant depletion of CSCs within these tumors. Furthermore,combination treatment with paclitaxel results in enhanced tumor growth delay and eradication of lung metastases. These data demonstrate that CAIX is a critical mediator of the expansion of breast CSCs in hypoxic niches by sustaining the mesenchymal and 'stemness' phenotypes of these cells,making CAIX an important therapeutic target for selectively depleting breast CSCs. View Publication

Prominin-1 (CD133) is commonly used to isolate stem and progenitor cells from the developing and adult nervous system and to identify cancer stem cells in brain tumors. However,despite extensive characterization of Prominin-1(+) precursor cells from the adult subventricular zone,no information about the expression of Prominin-1 by precursor cells in the subgranular zone (SGZ) of the adult hippocampus has been available. We show here that Prominin-1 is expressed by a significant number of cells in the SGZ of adult mice in vivo and ex vivo,including postmitotic astrocytes. A small subset of Prominin-1(+) cells coexpressed the nonspecific precursor cell marker Nestin as well as GFAP and Sox2. Upon fluorescence-activated cell sorting,only Prominin-1/Nestin double-positive cells fulfilled the defining stem cell criteria of proliferation,self-renewal,and multipotentiality as assessed by a neurosphere assay. In addition,isolated primary Prominin-1(+) cells preferentially migrated to the neurogenic niche in the SGZ upon transplantation in vivo. Finally,despite its expression by various stem and progenitor cells,Prominin-1 turned out to be dispensable for precursor cell proliferation in vitro and in vivo. Nevertheless,a net decrease in hippocampal neurogenesis,by ∼30% was found in Prominin-1 knock-out mice,suggesting other roles in controlling adult hippocampal neurogenesis. Remarkably,an upregulation of Prominin-2 was detected in Prominin-1-deficient mice highlighting a potential compensatory mechanism,which might explain the lack of severe symptoms in individuals carrying mutations in the Prom1 gene. View Publication