1. Dispase should be incubated in a 37°C water bath for 10-15 min to dissolve completely before filter. Make fresh dispase solution before use.

2. Under most circumstances, swirling the plate will dislodge most ES cell colonies. Use gentle force to blow the remaining attached ES cell colonies to avoid the detachment of fibroblasts. After removal of ES cell colonies, the plate will be left with a layer of mouse fibroblasts in which there are empty "holes" where ES cell colonies used to sit.

3. The amount of the medium depends on the number of ES cell colonies. In general, 10-12 mL of medium is appropriate for ES cells from two wells of a six-well plate.

4. The aggregates of ES cells described here is to initiate a differentiation process. The aggregates cultured for 4 d do not display structures of the three germ layers and form cysts inside; hence, they do not resemble an embryo. These aggregates, after plating onto the mouse fibroblast feeder layer, can regrow as ES cells. We therefore recommend the name of "ES cell aggregates" instead of embryoid bodies, a more widely used name.

5. ES cell aggregates usually do not attach under this culture condition. They may attach if there are a lot of contaminating fibroblasts, which attach and reform a "feeder." In that case, transfer the aggregates right after the fibroblasts attach to the flask. This can be repeated to remove the contaminating fibroblasts.

6. ES cell aggregates, after switching from ES cell growth medium to FGF-containing neural induction medium for 2 d, become brighter and healthier looking. They are now ready to attach to the plastic substrate for neural differentiation. If the aggregates do not attach, they may be plated onto the culture vessels that are precoated with fibronectin (20 |g/mL) or laminin (20 |g/mL). Alternatively, addition of 10% fetal bovine serum into the culture overnight will promote the attachment of the aggregates. It should be noted that fetal bovine serum has an adverse role in neural induction and thus it should be avoided or used as short time as possible.

7. This protocol mimics in vivo neuroectodermal development in terms of timing and morphology. It takes about 12-14 d for the human ES cells to differentiate into columnar neuroectodermal cells that organize into neural tube-like rosettes. Considering ES cells are equivalent to a d 5-6 embryo, development of the neuroectoderm in vitro takes about 18-20 d, the time window when the neural tube forms in a human embryo (8,20).

8. There is a temporal change of cell morphology in each colony. Such temporal pattern may change if the ES cells are already partially differentiated or if the ES cell aggregates do not attach according to the schedule described.

9. This method works well when neuroepithelial cells in the neural tube-like rosettes accumulate in the colony center whereas the peripheral cells remain a monolayer of flat cells, creating a clear boundary between rosettes and the peripheral cells. It happens usually around 10 d after plating the ES cell aggregates. Other techniques may be developed if a set of neuroectodermal cell-specific surface markers are available.

10. Because the differential response of rosette cells vs surrounding cells to dispase is very narrow, it is extremely important to watch the culture closely to obtain best separation.

11. These rosette clusters can be maintained or expanded in the suspension culture for several weeks (see Fig. 1E). These neuroectodermal cell clusters are different from the neurospheres that are formed from the fetal or adult tissues. The ES cell-generated neuroectodermal spheres contain rosettes inside. The rosettes gradually disappear after several weeks of expansion, which then resemble brain-derived neurospheres. The morphogens to be used depend on the objective of the study.

12. The human ES cell-derived neuroectodermal spheres do not normally attach to general plastic culture surface. If they attach easily, it usually suggests contamination of non-neural lineage cells. The neuroectodermal spheres can be detached by gently tapping the flask. Alternatively, they can be transferred to a bacterial-grade Petri dish, or poly-HEME coated flask to prevent cell attachment.

13. Neuroepithelial cells isolated using this protocol should consist of at least 95% of the cells that are stained positively for nestin and musahi-1, the commonly used neuroepithelial markers. These ES-generated neuroepithelial cells can be expanded as neurospheres or can be differentiated into neurons and glia cells under appropriate culture conditions (see Fig. 1F).


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