Differentiating Mouse Embryonic Stem Cells into a Networked Neuron Population

1. Neuronal Differentiation

NOTE: Conduct steps 1.1 – 1.5 before noon and step 1.7 after noon. An overview of the differentiation procedures is presented in Figure 1A.

1. Transfer 350 µl (3.5 x 10⁶) of ESCs to a 10 cm low-attachment dish containing 25 ml differentiation medium. Place on an orbital shaker set to 30 – 45 rpm inside a tissue culture incubator at 37 °C with 5% CO₂. This is the first day of differentiation, termed day in vitro (DIV) -8.
   NOTE: The use of an ultra-low attachment dish increases the cost of the method but produces slightly larger yields than bacterial Petri dishes since aggregates can occasionally adhere to Petri dishes. If different dish sizes are preferred, medium volumes and cell numbers can be scaled accordingly.
2. After 48 hr (DIV -6), use a 25 ml pipet to transfer the differentiating cell aggregates to a 50 ml conical tube. Immediately add a fresh 25 ml of differentiation medium to the Petri dish.
3. Allow aggregates to settle over 2 – 5 min, producing a visible pellet that is 1 – 2 mm deep. Ignore single cells or small aggregates remaining in suspension. Carefully aspirate the medium and transfer the cell pellet back to the petri dish using a P1000. Place on rotary shaker in a tissue culture incubator.
4. At DIV -4, repeat step 1.2. The pellet will be 2 – 4 mm deep. Replace 30 ml differentiation medium supplemented with 6 µM all-trans retinoic acid (RA) to the petri dish. Return to the rotary shaker in the tissue culture incubator for an additional 48 hr.
5. At DIV -2, repeat step 1.3. The pellet will be 4 – 8 mm deep at this point.
6. At DIV -1, prepare plating surfaces as in Section 2.
7. At DIV 0, thaw 5 ml of pre-aliquoted and frozen NPC trypsinization medium at 37 °C for 5 – 10 min and place in a tissue culture hood. Using a 25 ml pipet, transfer differentiating aggregates to a 50 ml conical tube. Allow aggregates to settle and carefully aspirate the medium. Wash the pellet twice with 10 ml PBS, allowing the aggregates to settle between washes.
8. After the second PBS wash, add 5 ml of NPC trypsinization medium to the pellet and incubate at 37 °C for 5 min. Gently flick the tube after 2.5 min.
9. Add 5 ml of 0.1% soybean trypsin inhibitor (STI) to inactivate trypsin and mix by inverting. Gently triturate 10 – 15 times with a 10 ml serological pipet until a relatively homogenous cell suspension is produced.
10. Slowly transfer the cell suspension to a 40 µm or 70 µm cell strainer placed on the top of a 50 ml conical tube. Once all the suspension has been filtered, add 1 ml of N2 medium to wash the remaining cells through the filter and pellet the dissociated cell suspension for 6 min at 200 x g.
11. Aspirate medium without disturbing the pellet. Wash cells twice with 10 ml N2 medium, pelleting cells for 5 min at 200 x g and triturating between washes with a P1000. Prior to the second wash, count cells using a hemocytometer.
12. Resuspend cells in N2 medium at 1 x 10⁷ cells per ml and plate ESNs at a cell density of 150,000 – 200,000 cells/cm².
13. Transfer newly plated ESNs to a humidified tissue culture incubator at 37 °C and 5% CO₂ and maintain as in Section 3.

2. Preparing Culture Surfaces for Plating Neural Precursors at DIV 0

1. Prepare tissue culture-treated dishes at least 1 day prior to plating. Add sufficient polyethyleneimine (PEI; 25 µg/ml in sterile H₂O) or poly-D-lysine (PDL; 100 µg/ml in sterile H₂O) to cover tissue-culture-treated plastic dishes and incubate O/N at 37 °C.
2. On the morning of plating, wash dishes twice with double-distilled H₂O and once with PBS. After the final wash, add sufficient N2 medium to cover the dish (e.g., 1 ml per well of a 12-well dish or 4 ml per 6 cm dish).
3. Prepare 18 mm glass coverslips at least one day prior to neuron plating. Clean the coverslips by plasma-cleaning for 4 min.
4. Immediately transfer cleaned coverslips to an ethanol-washed parafilm in the bottom of a large sterile dish and add 400 µl of PEI or PDL solution, prepared as in step 2.1. Incubate O/N at 37 °C in a tissue culture incubator.
5. In the morning, wash coverslips three times with water and add 5 µg/ml laminin in PBS for 1 – 3 hr at 37 °C. Prior to dissociating neurons, aspirate the laminin and immediately transfer the coverslip to a well of a 12-well dish containing 1 ml of NPC medium, being sure to keep the treated side facing up.
6. Store dishes and coverslips at 37 °C until NPCs are ready to plate.

3. Maintenance of Neurons

1. At DIV 1, aspirate medium and replace with N2 culture medium.
2. At DIV 2 and 4, aspirate medium and replace with B27 culture medium.
3. At DIV 8, aspirate medium and replace with B27 culture medium containing mitotic inhibitors to eliminate contaminating non-neuronal cells.
4. At DIV 12, replace with B27 culture medium.
5. Do not remove DIV 12⁺ ESNs from 5% CO₂ until ready to use.