Differentiation of Spinal Cord Neural Progenitor Cells into Motor Neurons

Protocollo

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

Medium name	Reagents	Concentrations
Neural differentiation medium (NDM)	Neurobasal L-Glutamine Non-Essential Amino Acids Supplement N - N2 Supplement Penicillin/streptomycin	1x 100x 100x 100x 100x
Motor neuron differentiation medium	Neural differentiation medium Ascorbic acid Puromorphamine Brain-derived neurotrophic factor Ciliary neurotrophic factor Insulin-like growth factor 1 Glial cell line derived neurotrophic factor Retinoic acid Compound E Supplement B - B27 Supplement	1x 0.4 µg/mL 1 µM 10 ng/mL 10 ng/mL 10 ng/mL 10 ng/mL 1 μM 125 nM 50x

Materiali

Name	Company	Catalog Number	Comments
10 cm sterile culture plates	Falcon	353003
Non-Essential Amino Acids (NEAA)	Thermofisher	11140050	Working concentration 100x
Supplement N - N2 Supplement	Thermofisher	17502048	Working concentration 100x
L-Glutamine	Thermofisher	25030	Working concentration 100x
Neurobasal	Thermofisher	21103049	Working concentration 1x
Retinoic acid (RA)	Sigma	R2625	Dissolve 100 mg into 3.3 mL of DMSO to get 100 mM stock solution. Aliquot the stock 100 µL/tube and freeze at -80 ºC. Take 200 µL of 100 mM stock and dilute 10x (add 1.8 mL of DMSO) to make 10 mM stock. Aliquot 50 µL/tube and store at -80 ºC. Dilute at 1:10000 for use. (working concentration 2 µM).
Polyornithine (PLO)	Sigma-Aldrich	P3655	Dissolve 100 mg in 1 mL of ddW to get 100 mg/mL stock solution. Aliquot the stock in 100 µL tubes. (working concentration 100 µg/mL)
Laminin	Thermofisher	23017-015	Stock solution 1 mg/mL, working concentration 10 µg/mL (coating), 1 µg/mL (cell media)
Ascorbic acid (ASAC)	Sigma	A4403	Dissolve 100 mg into 250 mL of dH2O to get 0.4mg/ml stock. Sterile filter through a 0.22 µm filter, aliquot and freeze at -80 ºC. Dilute at 1:1000 for use. (working concentration 0.4 µg/mL).
Ciliary neurotrophic factor (CNTF)	Peprotech	450-13	Dissolve 100 µg in 1mL of sterile PBS, and then add 9 mL of sterile 0.1% BSA-PBS to 10 µg/mL. Aliquot and freeze at -80 ºC. Dilute at 1: 1000 for use. (working concentration 10 ng/mL).
Compound E	Abcam	ab142164	Dissolve 250 µg in 2.0387 mL of DMSO to get a 250 µM stock. Aliquot and freeze at -80 ºC. Dilute 1:2000 for use. (working concentration 125 nM).
DMEM/F12	Thermofisher	113300	Working concentration 1x
Glial cell line-derived neurotrophic (GDNF)	Peprotech	450-10	Dissolve 100 µg in 1mL of PBS to 100 µg/mL, and then add 9 mL of sterile 0.1% BSA-PBS to 10 µg/mL. Aliquot and freeze at -80 ºC. Dilute at 1: 1000 for use. (working concentration 10 ng/mL).
ROCK-I nhibitor	Peprotech	1293823	Dissolve 5 mg in 1480 µL of dH2O to get 10 mM stock, aliquot and freeze at -80 ºC. Dilute at 1: 500 for use. (working concentration 20 µM).
Pencillin/Streptomycin	Thermofisher	15140122	Working concentration 100x
Purmorphamine (PMN)	Millipore-Sigma	540223	Dissolve 5 mg in 9.6 mL of DMSO to get 1 mM solution. Aliquot and freeze at -80 ºC. Dilute 1:1000 for use. (working concentration 1 µM).
Recombinant human-brain-derived neurotrophic factor (BDNF)	Peprotech	450-02	Centrifuge briefly before reconstitution. Dissolve 100 µg in 1 mL of PBS to 100 µg/mL, and then add 9 mL of sterile 0.1% BSA-PBS to 10 µg/mL. Aliquot and freeze at -80 ºC. Dilute at 1: 1000 for use. (working concentration 10 ng/mL).
Insulin-like growth factor 1 (IGF-1)	R&D systems	291-G1-200	Dissolve 200 µg in 2 mL of PBS to 100 µg/mL, then add 18 mL of 0.1%BSA-PBS to make 10 µg/mL stock and store at -80 ºC. Aliquot and freeze at -80 ºC. Dilute 10 µg/mL stock at 1: 1000 for use. (working concentration 10 ng/mL).
Glial cell line-derived neurotrophic (GDNF)	Peprotech	450-10	Dissolve 100 µg in 1mL of PBS to 100 µg/mL, and then add 9 mL of sterile 0.1% BSA-PBS to 10 µg/mL. Aliquot and freeze at -80 ºC. Dilute at 1: 1000 for use. (working concentration 10 ng/mL).
Retinoic acid (RA)	Sigma	R2625	Dissolve 100 mg into 3.3 mL of DMSO to get 100 mM stock solution. Aliquot the stock 100 µL/tube and freeze at -80 ºC. Take 200 µL of 100 mM stock and dilute 10x (add 1.8 mL of DMSO) to make 10 mM stock. Aliquot 50 µL/tube and store at -80 ºC. Dilute at 1:10000 for use. (working concentration 2 µM).
Compound E	Abcam	ab142164	Dissolve 250 µg in 2.0387 mL of DMSO to get a 250 µM stock. Aliquot and freeze at -80 ºC. Dilute 1:2000 for use. (working concentration 125 nM).
Supplement B - B27 Supplement	Thermofisher	21985023	Working concentration 50x
Sterile cell culture hoods	Baker Company	SG-600
Table top cell culture centrifuge	ThermoFisher	75004261	Sorvall Legend X1R
Waterbath	ThermoFisher	2332	Isotemp
Humidity controlled Cell culture incubator	ThermoFisher	370	set to 37 ºC, 5 % CO2