| Embryonic stem (ES) cell differentiation is a complex process, with many interactive effects impacting upon the development of functional cells. This complexity motivates our use of experimental design strategies to make empirical investigations more efficient, and to reveal unexpected interactions from conventional dose-response analysis. Evaluation of a large number of test conditions in parallel requires development of a quantitative high throughput screening (HTS) technology. We have developed such a technology using automated fluorescence microscopy, employing endoderm differentiation as a model system. Starting with a previously described differentiation protocol (Lumelsky et al. 2001), combinations and concentrations of factors were tested to identify conditions that impacted endoderm-specific differentiation, with RT-PCR used to confirm HTS output. Retinoic acid was found to have negative effects on endoderm formation, while low glucose proved beneficial. Our HTS technology was demonstrated to be a powerful tool to study ES cell differentiation. |
