Junying Lei

Much current knowledge of oligodendrocyte biology, the myelin-forming cells in the central nervous system, comes from cell culture studies mainly from postnatal rat tissue but mouse cells have been much more difficult to produce in large quantities. We have developed a high yield protocol for production of oligodendrocyte precursor cells from mouse embryonic neural progenitors grown as neurospheres. Neurospheres can be maintained and expanded for long periods in culture in the presence of epidermal growth factor (EGF). When floating neurospheres were plated on substrate-coated dishes in media supplemented with platelet derived growth factor (PDGF) and basic fibroblast growth factor (bFGF), the spheres attached and generated migrating cells that were predominantly oligodendrocyte-lineage cells. Furthermore, cells in spheres could be shifted to the oligodendrocyte phenotype prior to plating on substrate, by incubation in suspension with PDGF/bFGF. Single cell suspensions plated after dissociation of either EGF-treated neurospheres or PDGF/bFGF-treated oligospheres had the bipolar, elongated morphology characteristic of oligodendrocyte precursor cells. mRNA and protein expression analysis of the cells generated by this method confirmed their oligodendrocyte lineage. Oligodendrocyte precursors generated by this method matured in response to ciliary neurotrophic factor treatment, producing cells with multiple processes and myelin-like membranes. The most important aspect of this protocol is the ability to generate very high numbers of relatively pure mouse oligodendrocyte progenitor cells, which can be easily transfected. These studies open up many kinds of investigations on transgenic and mutant mouse oligodendrocytes, thereby providing a valuable tool to study oligodendrocyte biology and development.

CONTEXT: Thyroid cancer is a major component of Cowden syndrome (CS). CS patients with an underlying PTEN mutation (PTEN(mut+)) have a 70-fold increased risk of developing epithelial thyroid cancer. In contrast, less than 1% of sporadic epithelial thyroid cancer patients carry a germline PTEN mutation. Cost-efficient markers capable of shortlisting thyroid cancers for CS genetic testing would be clinically useful. OBJECTIVE: Our objective was to analyze the utility of patient blood phosphate and tensin homolog deleted on chromosome 10 (PTEN) protein levels in predicting germline PTEN mutations. DESIGN, SETTING, AND PATIENTS: We conducted a 5-yr, multicenter prospective study of 2792 CS and CS-like patients, all of whom had comprehensive PTEN analysis. Analysis of PTEN and downstream proteins by immunoblotting was performed on total protein lysates from patient-derived lymphoblast lines. We compared blood PTEN protein levels between PTEN(mut+) patients and those with variants of unknown significance or wild-type PTEN (PTEN(wt/vus)). MAIN OUTCOME MEASURES: We assessed the utility of PTEN protein levels in predicting germline PTEN mutations. RESULTS: Of 2792 CS/CS-like patients, 721 patients had thyroid cancer; 582 of them (81%) had blood PTEN protein analyzed. PTEN germline pathogenic mutations were present in 27 of 582 patients (4.6%). Ninety-six percent (26 of 27) of PTEN(mut+) patients had blood PTEN protein levels in the lowest quartile as compared with 25% (139 of 555) of PTEN(wt/vus) patients (P < 0.001). Low blood PTEN levels predicted for PTEN(mut+) cases with a 99.76% negative predictive value (95% confidence interval = 98.67-99.96) and a positive test likelihood ratio of 3.84 (95% confidence interval = 3.27-4.52). CONCLUSIONS: Our study shows that low blood PTEN protein expression could serve as a screening molecular correlate to predict for germline PTEN mutation in CS and CS-like presentations of thyroid cancer.

Cystic fibrosis-related diabetes (CFRD) is one the most common comorbidities in cystic fibrosis (CF). Pancreatic oxidative stress has been postulated in the pathogenesis of CFRD, but no studies have been done to show an association. The main obstacle is the lack of suitable animal models and no immediate availability of pancreas tissue in humans. In the CF porcine model, we found increased pancreatic total glutathione (GSH), glutathione disulfide (GSSG), 3-nitrotyrosine- and 4-hydroxynonenal-modified proteins, and decreased copper zinc superoxide dismutase (CuZnSOD) activity, all indicative of oxidative stress. CF pig pancreas demonstrated increased DHE oxidation (as a surrogate marker of superoxide) in situ compared to non-CF and this was inhibited by a SOD-mimetic (GC4401). Catalase and glutathione peroxidase activities were not different between CF and non-CF pancreas. Isolated CF pig islets had significantly increased DHE oxidation, peroxide production, reduced insulin secretion in response to high glucose and diminished secretory index compared to non-CF islets. Acute treatment with apocynin or an SOD mimetic failed to restore insulin secretion. These results are consistent with the hypothesis that CF pig pancreas is under significant oxidative stress as a result of increased O2●− and peroxides combined with reduced antioxidant defenses against reactive oxygen species (ROS). We speculate that insulin secretory defects in CF may be due to oxidative stress.