Differentiate ES cells into glial cells and neurons

Day -1? Pass ES cells at normal density on gelatinized plate to free the culture of contamination fibroblast cells.

Day 1? Trypsinized the cells as for normal passaging until the colonies lift off. Try to keep the loosely connected clumps of cells together by gentle handling. Then directly plate the cells 1:3 into bacterial grade Petri dishes in LIF free medium containing 1 microM all-trans retinoic acid (RA).

Day 3? Collect cell aggregates and replate in tissue culture dishes (appr. 25 cell aggregates per 6 cm tissue culture dish) in ES cell culture medium without LIF and RA. Aspirate the medium carefully.

Day 8? Change half of the medium. From this day on at least 10% of the cells exhibits neuronal phenotype. They are specifically stained with Cresyl Violet and stongly positive for the N-CAM antigen.