The NIH Center for Regenerative Medicine

Neural Stem Cell (NSC)

Differentiating EBs (Rosette Formation) and Midbrain Specification

View PDF version

Introduction:

Neural rosettes formed 2 days after replating (c) and magnified image of neural rosettes (d)¹

Protocol:

Neural stem cells (NSCs) will proliferate as progenitors a few times even after the complete growth medium is replaced with the appropriate differentiation medium. If the cells reach 90% confluency, it might be necessary to split the cells at a 1:2 ratio. However, do not split the cells once they reach day 9-10 of differentiation when they can get damaged during the passaging process.

1. After culturing the EBs in EB medium for 4 days, transfer the EBs from one T‑ 75 flask into a 50‑ mL centrifuge tube and centrifuge for 3 minutes at 200 × g.
2. Aspirate the EB medium and resuspend the EBs in 10 mL of pre‑ warmed neural induction medium.
3. Centrifuge the EBs for 3 minutes at 200 × g.
4. Aspirate the supernatant and resuspend the EBs in 40 mL of pre‑ warmed neural induction medium. Transfer the EBs into a fresh T‑ 75 flask and incubate the EBs in neural induction medium for 2 days in a 37 C incubator with a humidified atmosphere of 5% CO2. After the EBs float in the neural induction medium for 2 days, they are ready to be differentiated.
   Note: If the EB attach to the flask, use a 5‑ mL pipette to blow the attached EBs off the bottom of the flask.
5. Dilute laminin in D‑ PBS to 20 ug/mL and coat ten 100‑ mm culture dishes using 2.5-3 mL of laminin for each dish. Incubate the laminin‑ coated culture dishes in a 37 C incubator for several hours.
6. After incubation, aspirate the laminin and add 10 mL of pre‑ warmed neural induction medium into each 100 mm dish.
7. Transfer the EBs from the T‑ 75 flask into a 50‑ mL tube and centrifuge for 3 minutes at 200 × g.
8. Aspirate the supernatant and resuspend the EBs in 10 mL of pre‑ warmed neural induction medium.
9. Gently shake the 50‑ mL tube containing EBs to distribute the EBs evenly and add 1 mL of EB suspension into each laminin‑ coated culture dish.
10. Move the culture dishes in several quick back‑ and‑ forth and side‑ to‑ side motions to disperse the EBs across the surface of the dishes. Place the dishes gently in a 37 C incubator with a humidified atmosphere of 5% CO2.
11. Feed the EBs every other day with fresh pre‑ warmed neural induction medium until early rosettes form (approximately 2-3 days).
12. To direct the neural precursors to the midbrain fate, feed the differentiating EBs every other day with neural induction medium containing 100 ng/mL FGF‑ 8b and 200 ng/mL sonic hedgehog (SHH) for 5-6 days.
    Note: Plate the EBs at a density of 200-250 per one 100‑ mm dish. Generally, all EBs from hESCs cultured in one 100‑mm dish can be plated into eight to ten 100‑ mm dishes. The variation is from the confluence of hESCs and efficacy of EB formation

Materials:

Troubleshooting:

References:

1. Myung-Soo Cho, Dong-Youn Hwang & Dong-Wook Kim Efficient derivation of functional dopaminergic neurons from human embryonic stem cells on a large scale. Nature Protocols 3, 1888 - 1894 (2008).