Neuroectoderm Differentiation
ReagentVendorCatalog Number
Corning™ Matrigel™ hESC-Qualified Matrix Fisher Scientific 354277
Falcon™ Polystyrene Microplates Fisher Scientific Various
Corning™ Cell Culture PBS (1X) Thermo Fisher / Life Technologies MT21040CV
DMEM-F12 Thermo Fisher / Life Technologies MT10092CV
Essential 6 Medium Thermo Fisher / Life Technologies A1516401
mTesR Medium Stemcell Technologies 85850
Rock Inhibitor Stemcell Technologies 72302
Accutase Innovative Cell Technologies AT-104
LDN193189 TOCRIS 6053
SB431542 TOCRIS 1614
Required Equipment / Consumables:
  • Biosafety Cabinet
  • CO2, O2, humidified, water jacketed incubator
  • Sterile, glass, borosilicate pipets (Fisherbrand)
  • Reagent Setup:
    LDN193189 (500 μM Stock):
    • Dissolve 2mg of LDN193189 in 9.1 ml of DMSO.
    • Divide the solution into 200-μl aliquots in 1.5-ml tubes.
    • Store it at −80 °C for up to 6 months.
    SB431542 (10 mM Stock):
    • Dissolve 10mg of SB431542 in 2.4 ml of 200 proof ethanol.
    • Divide the solution into 500-μl aliquots in 1.5-ml tubes.
    • Store it at −80 °C for up to 6 months.
    Prepare Matrigel Plates:
    1. Aliquot Matrigel according to manufacturer’s instructions. Freeze aliquots at -80°C.
    2. To prepare tissue culture plates, take 1 aliquot of matrigel and thaw on ice or overnight at 4°C.
    3. Add the thawed matrigel in cold DMEM-F12 and plate in the appropriate sized cell culture vessel.
      • For a 6-well plate, we use 1mL of matrigel/DMEM-F12 per well.
      • For a 24-well plate, we use 250μl of matrigel/DMEM-F12 per well.
      • For a 96-well plate, we use 50μl of matrigel/DMEM-F12 per well
    4. Allow ECM to attach to plate by incubating at 37°C for at least an hour. Plates can be left overnight at 37°C.
    Procedure:
    Culture of hPSCs
    1. Prepare a matrigel-coated 6-well plate.
    2. Quickly thaw 1 vial of hPSCs from liquid N2 in a 37°C water bath.
    3. Transfer the cells to a sterile 15mL conical tube.
    4. Add a few drops of mTeSR1 and gently swirl the conical tube.
    5. Add 2mL of mTeSR1 to the conical tube.
    6. Centrifuge the cells at 120g for 3 min at room temperature. Aspirate and discard the supernatant.
    7. Aspirate the liquid from the matrigel-coated plates.
    8. Resuspend the cell pellet in 2mL of mTeSR1 + Rock inhibitor. Add the cell suspension to 1 well of the matrigel-coated plate.
    9. Gently shake the plate back and forth and front to back to evenly distribute the cells—avoid circular motions to prevent concentrating cells in the middle of the well.
    10. Incubate a 37°C incubator overnight.
    11. On the next day, aspirate the medium in each well. Add 2mL of fresh mTeSR1 media. Repeat daily with increasing volumes of mTeSR1 until cells are ready to be passaged (usually about 3-4 days).
    Neuroectoderm Induction
    1. Prepare a matrigel-coated 24-well plate.
    2. When cells are 80-90% confluent, aspirate the old medium and add 1mL of Accutase to each well to be passaged.
    3. Incubate for 6-10 minutes at 37°C until cells detach.
    4. Once incubation is complete, add 3mL of mTeSR1 to neutralize the Accutase, and collect cells to be passaged using fresh media using a 5mL pipet. 

    5. Spin the cells at 120g for 3 minutes.
    6. Aspirate the supernatant, leaving a trace amount to prevent the pellet from drying.
    7. Prepare the combined medium: Add 5 μl of 500 μm LDN (final concentration is 500 nm) and 5 μl of 10 mM SB (final concentration is 10 μm) to 50mL of E6.
    8. Resuspend the cell pellet in 1mL of the combined medium.
    9. Aspirate the liquid from the matrigel-coated plates and add 500 μl of the combined medium.
    10. Take 20 μl of the cell suspension and count the cells.
    11. Add the appropriate number of cells to the medium-containing wells.
      • For a 24-well plate, seed the wells with 400,000 cells.
    12. Gently shake the plate back and forth and front to back to evenly distribute the cells—avoid circular motions to prevent concentrating cells in the middle of the well.
    13. Place culture vessel into incubator overnight.
    14. On the next day, aspirate the medium in each well. Add 1mL of the combined media. Repeat daily until day 7.