Generating Neural Progenitors from Mouse Embryonic Stem Cells by the Hanging Drop Method

1. mESC culture

1. Coat a 10 cm tissue-culture-treated plate with 0.1% gelatin and allow the gelatin to set for at least 15–30 min before aspirating it out.
2. Seed γ-irradiated mouse embryonic fibroblasts (MEFs) one day before culturing the mESCs in the pre-warmed mESC medium (Dulbecco's modified Eagle medium (DMEM) with 15% fetal bovine serum (FBS), non-essential amino acids, β-mercaptoethanol, L-glutamine, penicillin/streptomycin, sodium pyruvate, Leukemia inhibitory factor (LIF), PD0325901 (PD), and CHIR99021 (CH)).
3. Allow the γ-irradiated MEFs to settle and attach to the plate surface before culturing E14 cells.
4. Thaw E14 ESCs in 37 °C water bath and quickly transfer the cells in a 15 cm conical tube with warm mESC medium. Pellet the cells at 200 x g for 3 min and remove the supernatant.
5. Resuspend the cells in 10 mL of mESC medium and plate the cells on the culture plate containing the γ-irradiated MEFs seeded earlier. Incubate the cell culture in a 37 °C incubator under 5% CO₂.
6. For culture passaging, aspirate the medium and wash the plate with sterile 1x Phosphate-buffered saline (PBS). Add enough 0.05% trypsin to cover the plate surface and incubate at 37 °C for 3 min.
7. Neutralize the trypsin with mESC medium and pipette to generate single-cell suspension. Centrifuge the cells at 200 x g for 3 min and remove the supernatant.
8. Count the cells with a hemocytometer or cell counter and seed about 5.0 x 10⁵ cells in a 10 cm culture plate.
9. Resuspend the cells in 10 mL of mESC medium, plate the cells on the gelatin-coated tissue culture plate and incubate cultures as described earlier.
   NOTE: It is recommended that mESCs be passaged every 2 days to prevent the cells from differentiating in their colonies. Phenol red in the medium functions solely as a pH indicator and depending on the cellular density, it can turn yellowish (more acidic), sooner than 2 days. Hence, it may be necessary to change the medium every day. The γ-irradiated MEFs will eventually die off after a couple of passages.

2. EB, NPC, and neuron differentiation

1. Perform culture passaging protocol and count the cells.
2. Hanging drop method (day 0)
   1. For a 10 cm cell culture plate, count roughly 2.5 x 10⁴ cells where 5.0 x 10² cells will be suspended in 20 µL differentiation medium (DMEM with 15% FBS, non-essential amino acids, β-mercaptoethanol, L-glutamine, penicillin/streptomycin, and sodium pyruvate). Roughly fifty 20 µL droplets containing the cells can be plated on one 10 cm plate.
   2. Aliquot the appropriate number of cells, and then centrifuge the cells at 200 x g for 3 min and remove the supernatant.
   3. Resuspend the cells in the appropriate volume of differentiation medium for a cell density of 5.0 x 10² cells per 20 µL (e.g., 2.5 x 10⁴ cells in 1 mL of differentiation medium).
   4. Using a micropipette or a repeater pipette, place 20 µL droplets of the cell suspension onto the lid of the tissue culture plate. Make sure that the droplets are not too close to one another to prevent them from merging.
      NOTE: The droplets can be plated on either a tissue-culture-treated attachment plate or suspension plate as they will be placed on the lid and not the plate itself. For a more feasible and sterile approach, plate the droplets on an attachment tissue-culture plate and transfer them to a suspension plate as described below.
   5. Fill up the plate with 5–10 mL of 1x PBS and carefully put the lid back on the plate. Incubate the culture in the 37 °C incubator.
      NOTE: PBS is added to the culture plate to prevent the droplets from drying up.
3. On day 2, use a micropipette to collect the droplets from the lid and place them in a 10 cm cell culture suspension plate filled with 10 mL of differentiation medium. Incubate the culture on an orbital shaker shaking at low speed in the incubator.
4. On day 4, to harvest the EBs, collect the cells, centrifuge at 200 x g for 3 min, and remove the supernatant.
   NOTE: The EBs can also be washed with 1x PBS as per the requirements of subsequent experiments.
5. To continue the procedure and induce the EB differentiation into neural progenitor cells (NPCs), prepare the differentiation medium with 5 µM retinoic acid (RA).
6. Remove the old medium by pelleting the EBs at 100 x g for 3 min or allow the EBs to settle before aspirating out the old medium. Add 10 mL of the differentiation medium containing 5 µM RA to the culture plate.
7. On day 6, replace at least half of the medium with fresh medium containing 5 µM RA by tilting the plate and pipetting out the medium as described above.
   NOTE: It is recommended that at least half of the medium be replaced with fresh RA-containing medium on days 5 and 7. Take note of the phenol red indicator; if it turns yellowish, it is best to replace all of the medium.
8. On day 8, harvest the NPCs by collecting the cells, centrifuging at 200 x g for 3 min, and removing the supernatant.
   NOTE: The NPCs can also be washed with 1x PBS according to the needs of subsequent experiments. If needed, NPCs can be frozen down and thawed again for later culture and analysis. If the NPCs are to be cultured, accutase can also be used as an alternative to trypsin.