Stock Solutions

1. l-glutamine solution (200 mM) (Sigma, St. Louis, MO; cat. no. G7513). Make aliquots of 2.5 mL and store at -20°C.

2. MEM nonessential amino acids solution (Gibco-BRL, Rockville, MD; cat. no. 11140-050).

3. Kockout serum replacer (Gibco-BRL; cat. no. 10828-028). Make aliquots of 50 mL and store at -20°C.

4. Dulbecco's modified Eagle's medium (DMEM): nutrient mixture F-12 1:1 (DMEM/F12) (Gibco-BRL; cat. no. 11330-032).

5. P-mercaptoethanol (14.3 M) (Sigma; cat. no. M7522).

6. Recombinant human FGF basic (R&D Systems, Minneapolis, MN; cat. no. 233-FB) is dissolved in sterilized PBS with 0.1% bovine serum albumin (Sigma; cat. no. A-7906) at a final concentration of 10 |g/mL. Aliquot 0.5 mL into sterilize tubes and store at -80°C.

7. Dispase solution (1 mg/mL): dissolve 10 mg dispase (Gibco-BRL; cat. no. 17105-041) in 10 mL F12/DMEM in a water bath for 15 min (see Note 1) and filter-sterilize the dispase solution with a 50-mL Steri-flip (Fisher Scientific; cat. no. SCGP00525).

8. Heparin (1 mg/mL); dissolve 10 mg heparin (Sigma; cat. no. H3149) in 10 mL DMEM medium. Aliquot 0.5 mL into sterilized tubes and store at -80°C.

10. F-12 nutrient mixture (HAM) (Gibco-BRL; cat. no. 11765-054).

11. N2 supplement (Gibco-BRL; cat. no. 17502-048).

12. Fibronectin from human plasma (Sigma; cat. no. F2006).

13. Fetal bovine serum (Gibco-BRL; cat. no. 16000-044).

14. Laminin from human placenta (Sigma; cat. no. L6274).

15. Poly-2-hydroxyethylmethacrylate (Poly-HEME) (Sigma; cat. no. P3932).

2.3. Media

1. ES cell growth medium. In a 500-mL filter unit (0.22-^m sterilizing low protein-binding membrane) (Corning Incorporated, Corning, NY; cat. no. 430513), combine 392.5 mL DMEM-F12, 100 mL knockout serum replacer, 5 mL MEM nonessential amino acids solution, 2.5 mL of 200 mM l-glutamine solution (final concentration of 1 mM), and 3.5 ^L 14.3 M P-mercaptoethanol (final concentration of 0.1 mM). Filter and store at 4°C for up to 7-10 d.

2. Neural induction medium (DMEM/F12/N2). Sterilely combine: 163 mL F-12, 326 mL DMEM, 5 mL N2 supplement, 5 mL MEM nonessential amino acids solution, and 1 mL of 1 mg/mL Heparin (see Subheading 2.2., item 8). Media be stored at 4°C for up to 2 wk.

3. Methods

3.1. Aggregation of ES Cells (Formation of Embryonic Bodies)

1. Aspirate medium from each well of the six-well plate containing the ES cells (see Fig. 1A).

2. Add to each well 1 mL of fresh ES cell growth medium (see Subheading 2.3., item 1) with dispase (see Subheading 2.2., item 7) at a final concentration of 0.5-1 mg/mL.

3. Incubate in a CO2 incubator for 5 min; observe the cells every 2 min under a phase-contrast microscope until the edges of ES cell colonies begin to curve up (see Fig. 1B). It usually takes about 5-10 min to reach this point.

4. Aspirate the dispase-containing medium from the six-well plate and add fresh, warm ES cell medium to each well (1.5 mL/well).

5. Swirl the six-well plate to dislodge the ES cell colonies from the mouse fibroblast feeder layer.

6. Use a 5-mL pipet to gently blow the ES cell colonies off the bottom of the well; if some ES cell colonies remain attached, pool all the cells to a 15-mL tube (see Note 2).

7. Centrifuge at 200g for 5 min at room temperature.

8. Aspirate the medium off the cell pellet.

9. Resuspend the ES cell colonies in 10-12 mL ES cell medium and culture them in a T25 flask (see Note 3).

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Fig. 1. Differentiation of neuroectodermal cells from human embryonic stem (ES) cells. (A) Phase contrast image showing an ES cell colony grown on the mouse embryonic fibroblasts feeder layer. (B) The edge of the ES cell colony, after treatment with dispase for 5 min, began to curve up (arrows). (C) ES cell aggregates suspended in the ES cell growth medium for 4 d. (D) ES cell aggregates, after 10 d of culture in the neural induction medium, developed multiple rosettes (arrows) in the colony center. (E) Rosettes cells, separated from peripheral flat cells and cultured in neural induction medium with FGF2 (10 ng/mL), formed spheres, which have rosettes inside (arrows). (F) After differentiating the neuroectodermal cells on the polyornithine and laminin substrate for 1 wk, numerous neurites extended out of the sphere. Bar = 50 |im.

10. The ES cell aggregates (see Note 4, Fig. 1C) should be fed every day and cultured for 4 d. If there are mouse embryonic fibroblasts attaching to the flask, you may get rid of them simply by transferring the floating ES cell aggregates to a new flask (see Note 5). To feed the ES cell aggregates, set the flask down at a titled angle so that the ES cell aggregates settle in one corner of the flask. Aspirate off about half of the medium and add fresh ES cell medium to replace the amount of aspirated.

3.2. Neuroepithelial Differentiation (Formation of Neural Tube-Like Rosettes)

1. After culturing for 4 d, the ES cell aggregates are ready to be differentiated.

2. Collect the ES cell aggregates and transfer them to a 15-mL centrifuge tube.

3. Centrifuge at 50g for 1-2 min at room temperature.

4. Wash with 5 mL neural induction medium (see Subheading 2.3., item 2).

5. Centrifuge at 50g for 1-2 min at room temperature.

6. Aspirate off medium and resuspend ES cell clusters in 8-10 mL neural induction medium supplemented with 10 ng/mL FGF2 (final concentration, see Subheading 2.2., item 6) and transfer to a T25 flask.

7. After 2 d in the neural induction medium, the ES cell aggregates attach to the plastic substrate (see Note 6). Once attached, feed the culture with neural induction medium with 10 ng/mL FGF2 every other day.

8. Observe the culture daily (see Note 7). There is a temporal change of cell morphology in each colony. At 1-2 d after attachment, cells in the colony center become elongated compared with those in the periphery. In another 2-3 d, these elongated cells in the colony center become compacted and start to form rosettes. With further culturing in the same medium for another 4-5 d, the area and thickness of rosettes increase and almost every colony contains multiple neural tube-like rosettes often with lumens (see Note 8, Fig. 1D).

3.3. Isolation of Neuroepithelial Cells in the Neural Tube-Like Rosettes (see Note 9)

1. Rinse the rosette culture with PBS once and add fresh neural induction medium with dispase (see Subheading 2.2, item 7) at a final concentration of 0.2-0.5 mg/mL.

2. Incubate the culture for 15-30 min in a CO2 incubator until the clump of rosettes retract and begin to come off the plate. At this stage, most of the peripheral flat cells remain attached (see Note 10).

3. Gently swirl the flask to release rosettes clumps. Let the flask stand and collect the rosette clumps into a 15-mL centrifuge tube. Triturate the clumps with a 5- or 10-mL serological pipet up and down twice, but not to break up the clumps.

4. Centrifuge at 50g for 2 min at room temperature.

5. Wash cells with 5 mL of neural induction medium and centrifuge again at 50g for 2 min at room temperature.

6. Aspirate the medium, resuspend the cells in 10-15 mL of neural induction medium +10 ng/mL FGF2, and transfer the cells to a T25 flask. Place in an incubator for about 2 h to allow the non-neural cells to differentially attach to the flask.

7. Transfer the floating cells (mostly aggregates of neural rosette cells) to a new flask. The rosette aggregates will roll up to form round clusters after about 1 d (see Note 11, Fig. 1E).

8. Feed the neuroectodermal aggregates every other day with neural induction medium +10 ng/mL FGF2 (see Notes 12 and 13).

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