ES Cell-derived Neuroepithelial Cell Cultures

Step 1

1. Plate mitomycin-C ggrowth-inhibited (10 µg/ml for 2.5 hours) MS5 cells at a density of 70,000/cm2 on gelatin-coated (0.01 % for 30 minutes) 6 well plates in α-MEM media.
2. When cells are attached and have formed a monolayer (over night growth), switch to SRM.
3. Manually isolate ES cell colonies from the ES cell cultures using a syringe with a 27 ½ G needle.
4. Tritrurate the colonies carefully with a 1 ml blue tip and plate at low density on the MS5 cells (usually 2-3 colonies per 6 well plate).

Note: The ES cells can also be taken from enzymatic propagation using collagenase or trypsin.

Step 2

1. Grow the ES cells on the MS5 feeders for 14-21 days until rosette-containing colonies form.

Note: Change media often. The rosettes are usually at the outer edges of the colonies and their formation can be greatly enhanced if 300-500 ng/ml Noggin is added to the SRM (3). In some differentiation protocols, SRM can be exchanged with N2-A media and supplemented with growth or other factors to promote cell specification (2-5).

Step 3

1. Isolate and pick the rosettes with a 27 ½ G needle under a microscope.

Note: Avoid cutting and picking the MS5 stromal cells. If colonies are packed with rosettes, one can also isolate the entire colony.

Step 4

1. Aspirate the clusters of rosettes, tritrurate them carefully with a 1 ml blue tip, and plate them on poly-L-ornithine (0.001%) and fibronectin (1 µg/ml) or laminin (1 µg/ml) -coated 6 wells in N2-A media usually supplemented with bFGF (20 ng/ml). The rosettes will reform and expand.

Note: To enhance cell survival, one can plate the small clusters in droplets to increase cell density. This step can also be modified by supplementing the N2-A media with additional growth or other factors to promote cell specification (2-5).

This protocol demonstrates the different steps in generating and isolating neuroepithelial cells from human ES cells using SDIA. The application of this method is manifold and has been used in many protocols to produce specified neurons (e.g. 1, 2, 5-9). The rosettes are thought to resemble neural tube cells with an anterior phenotype (2, 5, 10) and also contain neural crest progenitors (11, 12). In addition, they retain a certain level of plasticity, since they can be patterned by specific factors in defined culture conditions. Thus, the SDIA-derived neural progenitors can give rise to many cell types from the central and peripheral nerve system making them a useful tool for the derivation of different neural cell populations in ES cell differentiation paradigms.