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

Nonsense mutations are present in 10{\%} of patients with CF,produce a premature termination codon in CFTR mRNA causing early termination of translation,and lead to lack of CFTR function. There are no currently available animal models which contain a nonsense mutation in the endogenous Cftr locus that can be utilized to test nonsense mutation therapies. In this study,we create a CF mouse model carrying the G542X nonsense mutation in Cftr using CRISPR/Cas9 gene editing. The G542X mouse model has reduced Cftr mRNA levels,demonstrates absence of CFTR function,and displays characteristic manifestations of CF mice such as reduced growth and intestinal obstruction. Importantly,CFTR restoration is observed in G542X intestinal organoids treated with G418,an aminoglycoside with translational readthrough capabilities. The G542X mouse model provides an invaluable resource for the identification of potential therapies of CF nonsense mutations as well as the assessment of in vivo effectiveness of these potential therapies targeting nonsense mutations. View Publication

Cellular plasticity contributes to the regenerative capacity of plants,invertebrates,teleost fishes and amphibians. In vertebrates,differentiated cells are known to revert into replicating progenitors,but these cells do not persist as stable stem cells. Here we present evidence that differentiated airway epithelial cells can revert into stable and functional stem cells in vivo. After the ablation of airway stem cells,we observed a surprising increase in the proliferation of committed secretory cells. Subsequent lineage tracing demonstrated that the luminal secretory cells had dedifferentiated into basal stem cells. Dedifferentiated cells were morphologically indistinguishable from stem cells and they functioned as well as their endogenous counterparts in repairing epithelial injury. Single secretory cells clonally dedifferentiated into multipotent stem cells when they were cultured ex vivo without basal stem cells. By contrast,direct contact with a single basal stem cell was sufficient to prevent secretory cell dedifferentiation. In analogy to classical descriptions of amphibian nuclear reprogramming,the propensity of committed cells to dedifferentiate is inversely correlated to their state of maturity. This capacity of committed cells to dedifferentiate into stem cells may have a more general role in the regeneration of many tissues and in multiple disease states,notably cancer. View Publication

Epithelial-mesenchymal transition (EMT) in cancer cells plays a pivotal role in determining metastatic prowess,but knowledge of EMT regulation remains incomplete. In this study,we defined a critical functional role for the Forkhead transcription factor FOXQ1 in regulating EMT in breast cancer cells. FOXQ1 expression was correlated with high-grade basal-like breast cancers and was associated with poor clinical outcomes. RNAi-mediated suppression of FOXQ1 expression in highly invasive human breast cancer cells reversed EMT,reduced invasive ability,and alleviated other aggressive cancer phenotypes manifested in 3-dimensional Matrigel (BD Biosciences) culture. Conversely,enforced expression of FOXQ1 in differentiated human mammary epithelial cells (HMLER) or epithelial cancer cell lines provoked an epithelial to mesenchymal morphologic change,gain of stem cell-like properties,and acquisition of resistance to chemotherapy-induced apoptosis. Mechanistic investigations revealed that FOXQ1-induced EMT was associated with transcriptional inactivation of the epithelial regulator E-cadherin (CDH1). Our findings define a key role for FOXQ1 in regulating EMT and aggressiveness in human cancer. View Publication

When inhaled,ozone (O3) interacts with cholesterols of airway epithelial cell membranes or the lung lining fluid,generating chemically reactive oxysterols. The mechanism by which O3-derived oxysterols affect molecular function is unknown. Our data show that in vitro exposure of human bronchial epithelial cells to O3 results in the formation of oxysterols,epoxycholesterol-α and β (α-EpCh,β-EpCh) and Secosterol A and B (Seco A,SecoB),in cell lysates and apical washes. Similarly,bronchoalveolar lavage fluid obtained from human volunteers exposed to O3 contained elevated levels of these oxysterol species. As expected,O3-derived oxysterols have a pro-inflammatory effect and increase NF-κB activity. Interestingly,expression of the cholesterol efflux pump ATP-binding cassette transporter 1 (ABCA1),which is regulated by activation of the liver X receptor (LXR),was suppressed in epithelial cells exposed to O3. Additionally,exposure of LXR knockout mice to O3 enhanced pro-inflammatory cytokine production in the lung,suggesting LXR inhibits O3-induced inflammation. Using alkynyl surrogates of O3-derived oxysterols,our data demonstrate adduction of LXR with Seco A. Similarly,supplementation of epithelial cells with alkynyl-tagged cholesterol followed by O3 exposure causes observable lipid-LXR adduct formation. Experiments using Seco A and the LXR agonist T0901317 (T09) showed reduced expression of ABCA1 as compared to stimulation with T09 alone,indicating that Seco A-LXR protein adduct formation inhibits LXR activation by traditional agonists. Overall,these data demonstrate that O3-derived oxysterols have pro-inflammatory functions and form lipid-protein adducts with LXR,thus leading to suppressed cholesterol regulatory gene expression and providing a biochemical mechanism mediating O3-derived formation of oxidized lipids in the airways and subsequent adverse health effects. View Publication

Human breast tumors contain a breast cancer stem cell (BCSC) population with properties reminiscent of normal stem cells. We found 37 microRNAs that were differentially expressed between human BCSCs and nontumorigenic cancer cells. Three clusters,miR-200c-141,miR-200b-200a-429,and miR-183-96-182 were downregulated in human BCSCs,normal human and murine mammary stem/progenitor cells,and embryonal carcinoma cells. Expression of BMI1,a known regulator of stem cell self-renewal,was modulated by miR-200c. miR-200c inhibited the clonal expansion of breast cancer cells and suppressed the growth of embryonal carcinoma cells in vitro. Most importantly,miR-200c strongly suppressed the ability of normal mammary stem cells to form mammary ducts and tumor formation driven by human BCSCs in vivo. The coordinated downregulation of three microRNA clusters and the similar functional regulation of clonal expansion by miR-200c provide a molecular link that connects BCSCs with normal stem cells. View Publication

The sympathetic (SNS) and parasympathetic (PNS) branches of the autonomic nervous system have been implicated in the modulation of the renewal of many tissues,including the intestinal epithelium. However,it is not known whether these mechanisms are direct,requiring an interaction between autonomic neurotransmitters and receptors on proliferating epithelial cells. To evaluate the existence of a molecular framework for a direct effect of the SNS or PNS on intestinal epithelial renewal,we measured gene expression for the main autonomic neurotransmitter receptors in this tissue. We separately evaluated intestinal epithelial regions comprised of the stem,progenitor,and mature cells,which allowed us to investigate the distinct contributions of each cell population to this proposed autonomic effect. Notably,we found that the stem cells expressed the receptors for the SNS-associated alpha2A adrenoreceptor and the PNS-associated muscarinic acetylcholine receptors (M1 and M3). In a separate experiment,we found that the application of norepinephrine or acetylcholine decreases the expression of cyclin D1,a gene necessary for cell cycle progression,in intestinal epithelial organoids compared with controls (P {\textless} 0.05). Together,these results provide evidence of a direct mechanism for the autonomic nervous system influence on intestinal epithelial stem cell proliferation. View Publication