Generation of hESC/iPSC Derived Midbrain Dopamine Neurons

Briefly, culture hESCs (1 day after passaging) on MEF feeder layer in the neural induction medium (NIM) (DMEM/F-12, 1X NEAA, 1X N2 supplement) supplemented with 10micromolar (µM) and 2micromolar (µM).

To pattern the differentiating cells to the midbrain FP progenitors, add 500nanogram per milliliter (ng/mL) and 0.4micromolar (µM) to the cultures from day 1 till day 7.

On day 7, gently blow off individual colonies of neuroepithelial cells with a pipette and replate on mouse embryonic fibroblast feeder in the NIM containing 1micromolar (µM) and 100nanogram per milliliter (ng/mL) and 0.4micromolar (µM) for additional 6 days (D7-12).

On day 12, remove CHIR99021, reduce the following: 20nanogram per milliliter (ng/mL), 0.5micromolar (µM) and 100nanogram per milliliter (ng/mL) and add to the culture to expand the progenitors in suspension till day 19.

Then, keep 20nanogram per milliliter (ng/mL) and 20nanogram per milliliter (ng/mL) in the neural induction medium till transplantation/in vitro analysis at day 32.

For in vitro analysis, dissociate neurospheres by incubation in Accutase at 37°C for 0h 3m 0s-0h 5m 0s on day 32 and plate onto glass coverslips that were previously coated with Matrigel.

Feed cells with Neural differentiation medium (Neurobasal^TM Medium, 1 X N2 supplement, 1 X B27 supplement) (NDM) supplemented with brain-derived neurotrophic factor (10nanogram per milliliter (ng/mL)), glial cell line derived neurotrophic factor (10nanogram per milliliter (ng/mL)), ascorbic acid (200micromolar (µM)), 1micromolar (µM), transforming growth factor β3 (1nanogram per milliliter (ng/mL)) and Compound E (0.1micromolar (µM)).