Multi-well chip for forming a uniform embryoid body in a tiny droplet with mouse embryonic stem cells.

A multi-well chip (MWC) is described by which mouse embryonic carcinoma (EC) stem cells form a comparatively more rapid and uniform embryoid body (EB) over the conventional hanging drop (HD) method. The newly developed MWC consists of an array of extruded through-holes, each of which holds a droplet of the cell suspension. The study found that the small curvature radius of the droplet in the MWC improved the EB formation rate of a hanging drop from 70% to 98%. Furthermore, the EBs formed by the MWC were uniformly round in shape regardless of the number of suspended cells ranging from 0.5 x 10(3) to 20 x 10(3). The ratio of beating colonies from the MWC was over 2-fold larger than that from HD. The experiments demonstrate that the MWC will be a valuable experimental tool for robust and reproducible EB-based differentiation of a defined number of ES cells.

Coaction of DNA topoisomerase IIIalpha and a RecQ homologue during the germ-line mitosis in Caenorhabditis elegans.

BACKGROUND: Among the four RecQ homologues predicted from the Caenorhabditis elegans genomic DNA sequence, T04A11.6 is most similar to Bloom syndrome's protein in humans. To investigate a possible interaction of the protein with topoisomerase IIIalpha (TOP3alpha), as observed between TOP3 and RecQ homologues in yeast and human, the top3alpha gene expression was suppressed by RNA interference (RNAi) in the him-6(e1104) C. elegans strain which is mutated in T04A11.6 (F. Mueller & C. Wicky, personal communication). RESULTS: Germ cells in the gonads of the progeny him-6(e1104);top3alpha(RNAi) showed severe chromosomal abnormalities and were arrested during mitosis with a subsequent failure in meiotic entry. Most of the aberrant chromosomes were stained by the TUNEL assay but not by the SYTO12 dye, suggesting extensive DNA breaks not associated with apoptosis. The phenotypes in the germ cells of him-6(e1104);top3alpha(RNAi) were also observed in the progeny produced by double RNA interference of the top3alpha and him-6 gene expression, though at a reduced level. The over-expressed TOP3alpha and Him-6 proteins showed specific physical interaction in vitro, in agreement with the genetic interaction in C. elegans. CONCLUSION: In C. elegans, TOP3alpha and the RecQ homologue (T04A11.6) contribute to genome stability during germ-line mitosis, probably by acting in a complex.