The NIH Center for Regenerative Medicine

Neural Stem Cell (NSC)

Isolating Dopaminergic Progenitors

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Introduction:

Protocol:

1. Label all differentiating colonies containing rosettes using a microscope marker.
2. Using a 200-uL pipette tip pointing to the center of each marked colony, blow off the cells in rosettes.
3. Use a 10-mL pipette to transfer the detached cell clumps into a 50-mL centrifuge tube.
Note: You can combine the cell clumps from five 100-mm dishes into one 50-mL tube.
4. Centrifuge the cells for 3 minutes at 200 × g.
5. Aspirate the supernatant and resuspend the cell clumps in 40 mL of neural expansion medium containing 100 ng/mL FGF-8b and 200 ng/mL SHH.
6. Transfer the cell clumps to a T-75 flask and place the flask in a 37 C incubator with a humidified atmosphere of 5% CO2. The rosettes will roll up to form neurospheres after about 1 day in the incubator.
7. Replace half of the neural expansion medium containing 100 ng/mL FGF-8b and 200 ng/mL SHH with fresh medium every other day.
   Note: Contaminating non-neural cells tend to attach to the flask. When changing the medium, set the flask down at a tilted angle to allow the neurospheres to settle in one corner of the flask. Aspirate half of the neural expansion medium and use a 10-mL pipette to transfer the neurospheres with the rest of the spent neural expansion medium to a fresh T-75 flask. Add 20 mL of pre-warmed fresh neural expansion medium to the flask and incubate in a 37 C incubator with a humidified atmosphere of 5% CO2.

Materials:

Neural expansion medium
FGF-8b
SHH
Neural Expansion Medium

Troubleshooting:

References:

1. Houbo Jiang, Yong Ren, Eunice Y. Yuen, et al. Parkin controls dopamine utilization in human midbrain dopaminergic neurons derived from induced pluripotent stem cells. Nature Communications 3, Article number: 668 (2011).