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.
The authors have nothing to disclose.
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 |