We describe a procedure for efficient and reproducible differentiation of neuroectodermal cells from human embryonic stem (ES) cells using an adherent colony culture. ES cell colonies are detached intact from the fibroblast feeder layer using dispase or collagenase. The ES cell aggregates, after 4-6 d in suspension culture, are adhered to the culture surface and form colonies of monolayer in a chemically defined medium. Under this culture condition, columnar neuroectodermal cells appear in the center of each colony and organize into neural tube-like rosettes after 14 d of differentiation culture. These neuroectodermal cells in the rosettes can be effectively isolated through differential enzymatic and adhesion treatment and the neural population accounts for at least 70% of the total differentiated progenies. Thus, our system not only provides a source of synchronized neuroectodermal cells, but also offers a paradigm to dissect mechanisms of neural induction and cell lineage specification during early human development.
