Evaluation of the behavior of murine and human embryonic stem cells in in vitro migration and invasion assays.

Cell migration and invasion are essential processes in a variety of physiological events in the body, but also in several patho-physiological events. In this paper, the behavior of murine and human embryonic stem cells is examined in in vitro migration and invasion models. mESC and hESC were applied as spheroids, also known as patches, and as single cells, to mimic possible cell therapy application strategies. Two known in vitro migration assays, the ECM (extracellular matrix) assay and the Boyden chamber migration assay were selected. These assays revealed that mESC are statistically significantly more infiltrative than hESC. Application as spheroid results in a slightly higher infiltrative capacity compared single cells. The PHF (precultured chick heart fragment) assay was selected as an invasion assay. In the PHF assay a more 3D examination of the infiltrative nature of the ESC can be observed. The mESC showed infiltrative behavior, as spheroids and as single cells. The hESC were infiltrative as single cells but not as spheroids. The results of these assays are mostly complementary and prove the applicability of these assays, which were originally applied in tumor biology, in migratory behavior studies regarding stem cells and their progeny in basic and other conditions.

Expansion of human embryonic stem cells: a comparative study.

OBJECTIVES: Human embryonic stem cells (hESC) are promising for tissue engineering (TE) purposes due to their unique properties. However, current standard mechanical passaging techniques limit rates of possible TE experiments, as it is difficult to obtain high enough numbers of the cells for experimentation. In this study, several dissociative solutions and application methods are tested for their applicability to, and influence on, hESC culture and expansion. MATERIALS AND METHODS: Expansion of two hESC lines, H1 and VUB01, subjected to different passaging techniques, was evaluated. Four dissociative solutions - TrypLE™ Express, Trypsin-EDTA, Cell Dissociation Solution and Accutase™- were combined with two application protocols. As reference conditions, manual and bead-based passaging techniques were used. RESULTS: Results showed that use of Cell Dissociation Solution in combination with a slow adaptation protocol, generated the best expansion profile for both cell lines. The hESC single cell lines remained pluripotent, had good expansion profiles and were capable of differentiation into representatives of all three germ layers. Reproducibility of the results was confirmed by adaptation for three other hESC lines. CONCLUSION: Use of Cell Dissociation Solution, combined with slow adaptation protocol, allows a fast switch from the mechanical passaging technique to a single-cell split technique, generating stable and robust hESC cell lines, which allow for large scale expansion of hESC for TE purposes.