Generating Neural Progenitors from Embryonic Stem Cells in Serum-Free Monolayer Cultures

1. Media Preparation

NOTE: The protocol relies on using a mix of two separate media: DMEM/F12 supplemented with modified N2 supplement and Neurobasal supplemented with B27 supplement, typically in a 1:1 ratio.

1. Prepare the modified N2 supplement by mixing the components in a 15 ml tube. Do not vortex or filter this; mix by inverting the tube until the solution is clear.
   1. Start by pipetting 7.2 ml of DMEM/F12, then add 1 ml of 25 mg/ml insulin (made up in 0.01 M HCl) and mix well by inverting the tube. It takes a couple of minutes until the solution is clear.
   2. Add 1 ml of 100 mg/ml apo-transferrin (made up in water), 33 µl of 0.6 mg/ml progesternone (made up in ethanol), 100 µl of 160 mg/ml (1 M) putrescine (made up in water), 10 µl of 3 mM sodium selenite (made up in water) and 666 µl of 7.5% bovine serum albumin and mix the tube well. Aliquot into 2.5 ml and store at -20 °C for up to 2 months.
2. Prepare the media.
   1. To 250 ml of DMEM/F12 add 2.5 ml of N2. Mix well but do not shake or filter.
   2. To 250 ml of Neurobasal media, add 5 ml of B27 supplement. Mix well but do not shake or filter.
   3. Mix the media from steps 1.2.1 and 1.2.2 in a 1:1 ratio. In some cases a 1:3 mix may be required (supplemented as the 1:1 mix in step 1.2.4).
   4. To the mix add 0.5 ml of L-glutamine (final concentration 0.2 mM) and 3.5 µl of 2-mercaptoethanol (final concentration 0.1 mM) and mix well without shaking. Store the media at 4 °C for up to 3 weeks and avoid exposing to light. This final mix is called N2B27.
3. Prepare the gelatin solution.
   1. Prepare a 1% gelatin solution in ultrapure water and autoclave at 121 °C, 15 psi, for 15 min. It is important that the bottle used for this is completely clean from detergents or disinfectants, so it is recommended that a new bottle is used for this and is only ever rinsed in ultrapure water between uses. Aliquot the 1% solution into 50 ml aliquots and keep at 4 °C for months.
   2. Warm an aliquot of 1% gelatin in a 37 °C water bath until dissolved and add to 500 ml of warm phosphate-buffered saline (PBS). Mix well and pipet enough to cover the bottom of each well or plate. Allow the gelatin to coat the surface for at least 30 min.

2. Plating the Cells

NOTE: This protocol applies equally to mouse ESC grown in 10% serum with leukemia inhibitory factor (LIF), serum replacement with LIF or serum-free media with LIF and bone morphogenetic protein 4 (BMP4) or MEK and GSK3 inhibitors (2i media) with or without LIF. However, the timing and efficiency of the differentiation may vary depending on the media and cells. For the experiments shown here we used the 46C mouse ESC line (with an EGFP reporter knocked into one of the endogenous Sox1 alleles), grown in GMEM with 10% serum and LIF. For optimal results, it is important that cells are dissociated and replated in the N2B27 media; simply changing of media from GMEM/serum/LIF to N2B27 always results in a reduced differentiation efficiency compared to replating the cells.

1. Grow the cells in GMEM with 10% serum, 1 mM sodium pyruvate, 1x non-essential amino acids, 0.1 M 2-mercaptoethanol, and 100 units/ml LIF13. To get a subconfluent culture of mouse ESC, plate 1 x 10⁶ cells into a T25 culture flask which will take 2 – 3 days.
2. Observe cells under a bright-field microscope. When the cells are subconfluent and ready to passage, rinse them twice in PBS. Add 1 ml of cell dissociation reagent and return to the incubator for 2 – 5 min. Although trypsin and other enzymes can also be used for cell dissociation, they can negatively impact cell attachment, so the plating densities will need to be adjusted.
3. Once the cells begin to lift from the plate, tap the vessel to dislodge them into a single cell suspension. Collect the cells into 10 ml of media and cell dissociation reagent and pipet into a 15 ml centrifuge tube.
4. Spin the cells at 300 x g for 3 min at RT.
5. Carefully aspirate the supernatant and resuspend the pellet in 10 ml of pre-warmed N2B27 by pipetting up and down 3 – 5 times. When pipetting the suspension down, pipet against the side of the tube to avoid creating bubbles. Count the cells with an automated cell counter or a hemocytometer and record the concentration.
6. Prepare a cell suspension containing the desired number of cells per unit of volume to be plated per well in pre-warmed N2B27. A density of 10,500 cells per cm² in a 6-well dish (i.e., 1 x 10⁵ cells per well of a 6-well dish) is optimal. See Table 1 for the suggested number of cells per cm² and plating media volumes for various cell culture vessels.
7. Aspirate the gelatin from the culture vessel and plate the cell suspension according to the suggested density and volume in Table 1. Do not swirl the plates, as this will concentrate the cells to the center. Place the cultures in a humid incubator at 37 °C, 5% CO₂.
8. Change media every 1 – 2 days by replacing all the media with fresh N2B27. Pipette gently as flushing the cells might affect the density and lessen differentiation efficiency in the following days. Where initial viability after plating is poor, plate the cells in a 1:3 mix of the N2 and B27-supplemented media and change this to N2B27 after the first two days. Cells will begin to differentiate and should show Sox1 expression (visible by green fluorescence of the reporter in the 46C cell line used here) within 4 – 6 days.

Table 1. Suggested plating densities and media volumes for different vessel sizes. The plating densities in this table were determined using the 46C cell line. For optimal results, the plating density of each individual line may have to be adjusted.