iNDI Transcription Factor-NGN2 differentiation of human iPSC into cortical neurons Version 1

Induction Medium:

For day 0 to day 3

A	B	C	D
Reagent	Stock	Final concentration	Amount for 50mL of medium
Knock out DMEM/F12	---------	-------	48.5 mL
N2 supplement	100X	1X	0.5 mL
Non-essential amino acids (NEAA )	100X	1X	0.5 mL
Glutamax	100X	1X	0.5 mL
Doxycycline	2mg/mL	2µg/mL	0.05 mL
Chroman I	50 µM	50 nM	0.05 mL

Neuronal Maturation Medium:

For day 4 and 7

A	B	C	D
Reagent	Stock	Final concentration	Amount for 50mL of medium
Knockout DMEM/F12	---------	-------	24mL
Brainphys	---------	---------	24mL
N21MAX	50X	1X	1mL
GDNF (in 0.1%BSA/PBS)	10 µg/mL	10 ng/mL	0.05 mL
BDNF (in 0.1%BSA/PBS)	10 µg/mL	10 ng/mL	0.05 mL
NT-3 ( in 0.1%BSA/PBS)	10 µg/mL	10 ng/mL	0.05 mL
Laminin	6 mg/mL	1 µg/mL	0.01 mL
Doxycycline	2mg/mL	2µg/mL	0.05 mL

Neuronal Maturation Medium:

For day 10 to day 28

A	B	C	D
Reagent	Stock	Final concentration	Amount for 50mL of medium
BrainPhys neuronal medium	------------	-------------	49 mL
N21MAX	50X	1X	1 mL
GDNF (in 0.1%BSA/PBS)	10 µg/mL	10 ng/mL	0.05 mL
BDNF (in 0.1%BSA/PBS)	10 µg/mL	10 ng/mL	0.05 mL
NT-3 (in 0.1% BSA/PBS)	10 µg/mL	10 ng/mL	0.05 mL
Laminin	6 mg/mL	1 µg/mL	0.01 mL
Doxycycline	2mg/mL	2µg/mL	0.05 mL

Day 0

The iPSCs with a stably integrated human NGN2 using PiggyBac system under a tetracycline-inducible promoter were exposed to doxycycline as follows:

Coat a well of 6 well plate or 10cm dish to be used for differentiation with 1mL or 4mL respectively of Matrigel solution, tilting to ensure coverage of entire surface area. Place in 37°C incubator for 0h 30m 0s to 1h 0m 0s .

Aspirate supernatant and resuspend cell pellet with Induction Medium.

Count cells, Gently transfer 0.5-1 x 10⁶iPSCs per one well of 6-well plate in 2-3 mL of Induction Medium or 4-6 × 10⁶ per 10-cm dish in 10-12 mL to be differentiated.

Gently rock plate to evenly distribute cells and place in 37°C incubator.

Prepare Induction Medium and place in 37°C water or bead bath to warm during dissociation.

Observe iPSCs under a phase contrast microscope to assess confluency and presence of cell debris. Dish should be dissociated at ~70% to 80% confluency.

Aspirate culture medium and wash with PBS 1X.

Aspirate PBS and add half of culture volume of Accutase

Transfer to 37°C incubator for0h 10m 0s

When Incubation is ready, tilt the plate and pipet the accutase solution two to three times up and down the culture surface to singularize as single cells.

Quench the Accutase adding half of the culture volume of PBS. Transfer to a new conical tube and rinse with more PBS the culture surface, combine with the cell solution in the tube.

Centrifuge 0h 5m 0s at 200 - 300 x g at Room temperature

Day 1

Check cells under the microscope, nascent neuritic extensions should begin to be evident after 24 h of doxyxycline exposure.

Prepare Induction Medium but without Chroman I and warm it.

Aspirate medium, wash once with PBS 1X and replace with warm induction medium.

Day 2

Check cells under the microscope, neuritic extensions should be more evident.

Repeat medium change with induction medium as on day 1.

Day 3

Check cells under a microscope. Neurites should be obvious by this time.

Repeat medium exchange with induction medium + Uridine and Fluorodeoxyuridine (FdU) both at Uridine and Fluorodeoxyuridine (FdU) both at 1micromolar (µM) .

Day 4

Check cells under a microscope. Pre-differentiated neurons are ready to be re-plated.

Coating dishes

Freshly prepared poly-L-ornithine (PLO), at final concentration at 0.1mg/mL :

• Using Sodium Borate Buffer pH 8.2, make a 1mg/mL stock PLO solution.
• To prepare working solution dilute to a 0.1mg/mL with cell culture water then filter through a 0.22µm sterile filter and it is ready to use.
• Add half of the culture volume of PLO working solution to dishes and Place in 37°C incubator for 1h 0m 0s to .
• Aspirate PLO working solution from the dishes.
• Wash dishes with cell culture water three times.
• Let dry completely in a culture hood.
• Dishes are ready to use.

Plating pre-differentiated neurons day 4

Once cells are confirmed to be healthy, they should be dissociated with Accutase to re-plated onto final dishes for neuronal maturation and experimental manipulation

Prepare fresh Neuronal Maturation Medium + Uridine and Fluorodeoxyuridine (FdU) both at 1 micromolar (µM)

After dissociating cells with Accutase as step 2.4 to 2.9 resuspend cell pellet with Neuronal Maturation Medium + Uridine and Fluorodeoxyuridine (FdU) both at 1 micromolar (µM) and count.

Plate 2 x 10⁶ pre-differentiated neurons onto a PLO-coated 6 well with 3-4mL of Neuronal Maturation Medium + Uridine and Fluorodeoxyuridine (FdU) both at 1 micromolar (µM) .

After day 4 do half of the medium change every 3-4 days with Neuronal Maturation Medium + Uridine and Fluorodeoxyuridine (FdU) both at 1 micromolar (µM) .