Differentiation of Spinal Cord Neural Progenitor Cells into Motor Neurons

Abstract

Source: Taga, A. et al., Establishment of an Electrophysiological Platform for Modeling ALS with Regionally-Specific Human Pluripotent Stem Cell-Derived Astrocytes and Neurons. J. Vis. Exp. (2021).

This video showcases a method for differentiating spinal cord neural progenitor cells (NPCs) into motor neurons. It details the culturing process of the NPCs on a coated culture plate using specialized media enriched with specific inhibitors aimed at facilitating NPC differentiation into mature motor neurons.

Protocol

1. Thawing Neural Progenitor Cells (NPC) cultures Coat 10 cm plates with polyornithine (PLO) and laminin the day prior to thawing the cells. 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. Aspirate the PLO solution and rinse the plates three times with PBS. (PLO is toxic if not properly washed.) Add laminin diluted in PBS t…

Materials

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