A Technique for Generating Neural Progenitor Cells from Mouse Embryonic Stem Cells

1. Mouse embryonic stem cell culture

1. Prepare 0.1% gelatin coated cell culture dishes or plates.
   1. Add 2 mL of sterilized 0.1% gelatin (0.1% w/v in water) to 60 mm cell culture dishes. Rock gently to ensure even coating of the cell culture dishes.
   2. Put the dishes into a 5% CO₂ incubator at 37 °C and allow coating for 1 h.
   3. Remove the 0.1% gelatin solution before seeding the cells.
      NOTE: After removing the gelatin, there is no need to dry or wash the coated dishes.
2. Mouse embryonic stem cells (A2lox and 129) culture
   1. Incubate mESCs (A2lox and 129) cells in the 0.1% gelatin coated 60 mm cell culture dishes in mESC growth medium at 37 °C in a 5% CO₂ incubator, respectively. The mESC growth medium consists of 85% knock-out Dulbecco's modified Eagle medium/nutrient mixture F-12 (DMEM/F12), 15% Knock-out serum replacement (KSR), 0.1 mM β-mercaptoethanol (2ME), 2 mM GlutaMAX, 1% non-essential amino acid (NEAA), 1% penicillin/streptomycin (P/S), 1000 U/mL leukemia inhibitory factor (LIF), 10 nM CHIR-99021 (GSK-3 inhibitor) and 0.33 nM PD0325901 (MEK inhibitor).
      CAUTION: β-mercaptoethanol is flammable and has inhalation toxicity. Keep away from fire sources and wear a mask to avoid inhalation when use.
   2. Change the mESC growth medium daily for better growth of A2lox and 129.
   3. When the cells reach 80% confluence, remove the medium and add 1 mL of 0.1% trypsin to the dish. Gently rock for 30 s to ensure even cover of trypsin on all cells.
   4. Leave the cells for about 1 min to trypsinize and then remove the trypsin using a 1 mL pipette.
   5. Add 2 mL of mESC growth medium to the dish, pipette up and down several times to make a single cell suspension.
   6. Count the density of the cells in the suspension as accurately as possible using a hemocytometer.
   7. Divide the cells into 7 groups and induce differentiation using different protocols shown in Table 1.

2. Differentiation from mESCs to NPCs

1. Prepare 0.1% gelatin coated cell culture plates or coverslips.
   1. Before use, prepare 0.1% gelatin coated 6-well plates or coverslips as in step 1.1.
2. Differentiation (Table 1)
   1. Add 1.5 x 10⁶ mESCs into a non-adhesive bacterial dish in 10 mL of basal differentiation medium I to allow for embryoid body formation at 37 °C in a 5% CO₂ incubator.
   2. After 2 days, transfer cell aggregates into 15 mL centrifuge tubes and let them settle by gravity.
   3. Remove the supernatant and add 10 mL of fresh basal differentiation medium I to resuspend the embryoid bodies. Replant them into a new non-adhesive bacterial dish and allow differentiation for another 2 days.
   4. Check the formation of embryoid bodies under the microscope (Figure 1A).
   5. Collect embryoid bodies as described in steps 2.2.2-2.2.3. Seed about 50 embryoid bodies in 2 mL of basal differentiation medium I per well onto 0.1% gelatin-coated 6-well plates.
   6. Prepare 1 mM all-trans retinoic acid (RA) stock (in DMSO) and store away from light in a -80 °C freezer after sub-packaging.
      NOTE: RA is unstable, and attention should be paid to keeping it away from light and reducing air contact during preparation of RA stock.
   7. For RA induction, add 2 µL of RA stock into each well to make a final concentration of 1 µM.
   8. Place the plate into the 5% CO₂ incubator at 37 °C and differentiate for another 4 days.
   9. Change the entire 2 mL of basal differentiation medium I (with 1 µM RA) every 2 days.

Table 1: Details of the protocol used in differentiation.