Differentiation of Spinal Cord Neural Progenitor Cells into Motor Neurons

1. Thawing Neural Progenitor Cells (NPC) cultures

1. Coat 10 cm plates with polyornithine (PLO) and laminin the day prior to thawing the cells.
   1. Add 5 mL of PLO diluted to 100 µg/mL in phosphate buffered saline (PBS) or distilled water (dH₂O) to plates. Incubate at 37 °C for a minimum of 1 h up to overnight.
   2. Aspirate the PLO solution and rinse the plates three times with PBS. (PLO is toxic if not properly washed.)
   3. Add laminin diluted in PBS to a concentration of 10 µg/mL to the PLO-coated plates. Incubate at 37 °C for a minimum of 1 h, preferably overnight. After incubation, aspirate the laminin solution without rinsing to enhance cell adhesion.
      NOTE: The plates can be coated with PLO ahead of time, washed three times with water, dried in a sterile hood, and stored at 4 °C.
2. Thaw one NPC vial containing 6 x 10⁶ cells/vial for each 10 cm cell culture plate.
3. Remove the NPC cryotube from liquid nitrogen and place it immediately in a 37 °C water bath for up to 2 min. Quickly thaw by shaking the vial until there is a small ice piece surrounded by liquid.
4. Transfer cell aggregates into a 15 mL conical tube using a 1000 µL pipette and add up to 15 mL of pre-warmed Neural differentiation medium (NDM) or Dulbecco's Modified Eagles Medium (DMEM) / Ham's F21 F12 supplement to dilute the dimethyl sulfoxide (DMSO).
5. Centrifuge at 300 x g for 5 min.
6. Aspirate the supernatant, resuspend the cell pellet with motor neuron (MN) differentiation medium (Table 1) + ROCK-I (20 µM), and use a 1000 µL pipette to triturate up and down until a single cell suspension is obtained (up to 5 times).
7. Transfer the single-cell suspension to a PLO-Laminin coated 10 cm plate.
8. Transfer the plate to an incubator and leave it undisturbed overnight to allow for proper cell adhesion

2. Differentiating spinal cord NPC into motor neurons

1. Perform initial steps as mentioned in steps 1.1-1.8, with the final medium being MN differentiation medium + ROCK-I (20 μM).
2. The day after plating, perform a complete exchange of the medium with MN differentiation medium without ROCK-I, and then change the media every other day. Over the weekend, feed cells on Friday and then again on Monday. Rinse the cells with PBS when needed to remove cell debris.
3. Change the medium with MN differentiation medium + cytosine arabinoside (ARA-C) (0.02 µM) and incubate for 48 h when glial committed progenitors emerge as single proliferating flat cells under post-mitotic MN progenitors aggregated in cell clusters.
   NOTE: Usually, this occurs within the first 7 days after initial plating, although there may be variability depending on the cell line.
4. After 48 h, aspirate the medium containing ARA-C, rinse cultures gently with PBS three times, and change the medium to fresh MN medium without ARA-C.
5. After treatment with ARA-C, perform medium exchanges every other day (or Monday, Wednesday, and Friday) by removing the old medium with a manual pipette rather than a vacuum aspirator to prevent premature detachment of neuronal clusters. In addition, enrich the MN medium with Laminin 1 µg/mL once a week to further enhance neuronal attachment to the culture plates.

Table 1: Culture Media