Roland A. Fleck

Characterization of human embryonic stem cell lines by the International Stem Cell Initiative

The International Stem Cell Initiative characterized 59 human embryonic stem cell lines from 17 laboratories worldwide. Despite diverse genotypes and different techniques used for derivation and maintenance, all lines exhibited similar expression patterns for several markers of human embryonic stem cells. They expressed the glycolipid antigens SSEA3 and SSEA4, the keratan sulfate antigens TRA-1-60, TRA-1-81, GCTM2 and GCT343, and the protein antigens CD9, Thy1 (also known as CD90), tissue-nonspecific alkaline phosphatase and class 1 HLA, as well as the strongly developmentally regulated genes NANOG, POU5F1 (formerly known as OCT4), TDGF1, DNMT3B, GABRB3 and GDF3. Nevertheless, the lines were not identical: differences in expression of several lineage markers were evident, and several imprinted genes showed generally similar allele-specific expression patterns, but some gene-dependent variation was observed. Also, some female lines expressed readily detectable levels of XIST whereas others did not. No significant contamination of the lines with mycoplasma, bacteria or cytopathic viruses was detected.

DNA binding polarity, dimerization, and ATPase ring remodeling in the CMG helicase of the eukaryotic replisome

The Cdc45/Mcm2-7/GINS (CMG) helicase separates DNA strands during replication in eukaryotes. How the CMG is assembled and engages DNA substrates remains unclear. Using electron microscopy, we have determined the structure of the CMG in the presence of ATPγS and a DNA duplex bearing a 3′ single-stranded tail. The structure shows that the MCM subunits of the CMG bind preferentially to single-stranded DNA, establishes the polarity by which DNA enters into the Mcm2-7 pore, and explains how Cdc45 helps prevent DNA from dissociating from the helicase. The Mcm2-7 subcomplex forms a cracked-ring, right-handed spiral when DNA and nucleotide are bound, revealing unexpected congruencies between the CMG and both bacterial DnaB helicases and the AAA+ motor of the eukaryotic proteasome. The existence of a subpopulation of dimeric CMGs establishes the subunit register of Mcm2-7 double hexamers and together with the spiral form highlights how Mcm2-7 transitions through different conformational and assembly states as it matures into a functional helicase. DOI: http://dx.doi.org/10.7554/eLife.03273.001

OPTIMIZATION OF NB-4 AND HL-60 DIFFERENTIATION FOR USE IN OPSONOPHAGOCYTOSIS ASSAYS

SummaryProduction of effective vaccine formulations is dependent on the availability of assays for the measurement of protective immune responses. The development and standardization of in vitro human cell-based assays for functional opsonophagocytic antibodies require critical evaluation and optimization of the preparation of cells for the assay. We report evaluation of a number of protocols with two continuous cell lines (NB-4 and HL-60) for the provision of differentiated cells for use in functional assays. Flow cytometric analysis of CD11b antigen expression, as a marker of differentiation, indicated that all-trans-retinoic acid (ATRA) gave improved differentiation (>80% of cells differentiated at 96 h) when compared with dimethylformamide (DMF) (<60% of cells differentiated at 96 h). Morphological changes during differentiation toward a neutrophil-like phenotype were assessed by scanning electron microscopy. HL-60 and NB-4 cells treated with ATRA showed more spreading and flattening than cells treated with DMF, further evidence that they may have achieved a more differentiated phenotype. The number of cell divisions in culture appeared to be critical because cell lines maintained in exponential growth for >40 passages failed to express CD11b antigen or show morphological changes associated with differentiation after exposure to either differentiation-inducing reagent. Late-passage cells also demonstrated increased tolerance to DMF. Our results indicated that ATRA supplemented with vitamin D3 and granulocyte colony-stimulating factor affords robust, rapid, and reproducible differentiation of both cell types.

Confocal microscopy study of pertussis toxin and toxoids on CHO-cells

Pertussis toxin in its detoxified form is a major component of all current acellular pertussis vaccines. Here we report the membrane translocation and internalization activities of pertussis toxin and various pertussis toxoids using Chinese hamster ovary cells and confocal microscopy based on indirect immunofluorescence labeling. Chemically detoxified pertussis toxoids were able to translocate/internalize into cells at the concentration about 1,000 times higher than the native toxin. Pertussis toxoids detoxified with different procedures (glutaraldehyde, glutaraldehyde plus formaldehyde, hydrogen peroxide or genetic mutation) showed differences in fluorescence intensity under the same condition, indicating toxoids from different detoxification methods may have different translocation/internalization activities on cells.