TITLE: Human iPSC culture and directed differentiation to Midbrain Dopaminergic neurons.
AUTHORS: gurvir.virdi

[DESCRIPTION]
Generation of hiPSC derived midbrain dopaminergic neurons.

[STEPS]
SECTION: iPSC culture and maintenance
1. Human induced pluripotent stem cells (hiPSCs) were maintained in feeder-free monolayers on Geltrex (ThermoFisherScientific) and fed daily with Essential 8 medium (Life Technologies). 
Grow cells on 6-well tissue culture plates.
When confluent, hiPSCs were passaged using 0.5 μM EDTA (Life Technologies). All cells were maintained at 37°C and 5% carbon dioxide.
SECTION: Midbrain Dopaminergic Neuron protocol
2. Media for neuronal induction is Neurobasal and DMEM-F12 supplemented as follows:
Neurobasal (B27): 
Neurobasal phenol red free (500ml): thermofisher cat number: 12348017
Add the following supplements into 1x 500ml bottle:
B27 Supplement (10ml): thermofisher cat number: 17504044
Glutamax (5ml): thermofisher gibco cat number: 35050-038
Pen/Strep (2.5ml): thermofisher gibco cat number: 15140122 10,000U/ml
DMEM-F12 (N2): 
DMEM-F12 (500ml): thermofisher cat number: 10565018
Add the following supplements into 1x 500ml bottle:
N2 Supplement (5ml) : thermofisher cat number: 17502048
MEM Non-essential amino acids (5ml): thermofisher gibco cat number: 11140-050
Pen/Strep (2.5ml): thermofisher gibco cat number: 15140122 10,000U/ml
Beta-mercaptoethanol (910ul): thermofisher gibco cat number: 21985-023
Insulin solution human (250ul): sigma cat number: I9278
Media we use is a 1:1 mix of the supplemented Neurobasal (B27) and supplemented DMEM-F12 (N2), termed “N2B27”.

SECTION: Midbrain Dopaminergic Neuron protocol
2.1. Before starting an induction, make the media above. Then make “Media A” which consists of:
50ml N2B27
2uM Dorsomorphin (Tocris 3093/10) 
5uM SB 431542 (Tocris 1614/10)
1uM CHIR99021 (Miltenyi Biotec 130-104-172)
2. 
2.2. 
2.1. 
SECTION: Midbrain Dopaminergic Neuron protocol
2.2. On Day 0, check iPSCs to see if they are ~100% confluent. Aspirate iPSC media and wash cells one with 2ml PBS (ThermoFisher Gibco) per well. Then add 5ml of "Media A" to each 6-well plate to initiate differentiation. 
SECTION: Midbrain Dopaminergic Neuron protocol
2.5. On Day 3, aspirate media and replace with fresh 5ml of "Media B".
SECTION: Midbrain Dopaminergic Neuron protocol
2.4. On Day 2, prepare "Media B" which consists of:
50ml N2B27
2uM Dorsomorphin (Tocris 3093/10) 
5uM SB 431542 (Tocris 1614/10)
1uM CHIR99021 (Miltenyi Biotec 130-104-172)
1uM Purmorphamine (Merck Millipore SML0868)

Aspirate media and replace with 5ml of "Media B".
SECTION: Midbrain Dopaminergic Neuron protocol
2.3. On Day 1, aspirate media and replace with fresh 5ml of "Media A".
SECTION: Midbrain Dopaminergic Neuron protocol
2.8. On Day 8, prepare "Media C" which consists of:
50ml N2B27
2uM Dorsomorphin (Tocris 3093/10) 
1uM Purmorphamine (Merck Millipore SML0868)

Aspirate media and replace with 5ml of "Media C".
SECTION: Midbrain Dopaminergic Neuron protocol
2.7. On Day 5 till and including day 7 aspirate the media and replace with fresh 5ml of "Media B". Daily media change.
SECTION: Midbrain Dopaminergic Neuron protocol
2.6. On Day 4, split the cells in a 1:2 dilution following the "Dispase protocol" below, and plate the split cells into new Geltrex pre-coated 6-well plates in 2ml per well of "Media B" + 10μM Y-27632 dihydrochloride (Tocris). 
SECTION: Midbrain Dopaminergic Neuron protocol
2.12. On Day 14, split the cells in a 2:3 dilution following the "Dispase protocol" below, and plate the split cells into new Geltrex pre-coated 6-well plates in 2ml per well of just N2B27 + 10μM Y-27632 dihydrochloride (Tocris). At this point, NPCs can also be cryopreserved
SECTION: Midbrain Dopaminergic Neuron protocol
2.11. On Day 11 till and including day 13 aspirate the media and replace with fresh 5ml of "Media C". Daily media change.
SECTION: Midbrain Dopaminergic Neuron protocol
2.10. On Day 10, split the cells in a 1:2 dilution following the "Dispase protocol" below, and plate the split cells into new Geltrex pre-coated 6-well plates in 2ml per well of "Media C" + 10μM Y-27632 dihydrochloride (Tocris).
SECTION: Midbrain Dopaminergic Neuron protocol
2.9. On Day 9, aspirate media and replace with fresh 5ml of "Media C".
SECTION: Midbrain Dopaminergic Neuron protocol
2.13. On day 15, aspirate the media and replace with 5ml of N2B27. The cells are now midbrain NPCs, and media can be changed every 2 days until final plating.
SECTION: Midbrain Dopaminergic Neuron protocol
2.14. On day 19 prior to final plating, coat plates, ibidi chambers, 96-well plates, coverslips with Geltrex for final plating of NPCs for terminal differentiation into neurons. At this point, NPCs can also be cryopreserved
Using Accutase (ThermoFisher Gibco), get cells into a single cell suspension and count the cells.
Make up N2B27 + 10μM Y-27632 dihydrochloride to plate cells in.
Prepare cell stocks to obtain a final plating density of 500,000 cells per ml.
Plate the cells into final chambers: 
100,000 cells per well of a µ-Slide 8 Well (Ibidi chamber).
50,000 cells per well of a 96-well plate.
500,000 cells of a 12-well plate.

SECTION: Midbrain Dopaminergic Neuron protocol
2.15. On day 20, prepare Terminal Differentiation media:
50ml N2B27
10μM Y-27632 dihydrochloride
0.1μM Compound E (Enzo Life Sciences).

Aspirate media and replace with "Terminal Differentiation media". 

Cells need to be changed with Terminal Differentiation media twice weekly until desired differentiation time point (3-5 weeks).
SECTION: Dispase Protocol
3. On the days indicated above, cells need to be enzymatically dissociated using Dispase. Before beginning coat 6-well plates with Geltrex. On day 4, and 10 cells are split 1:2 (1 well of a 6-well plate is split into 2 wells). 
On day 14, cells are split 2:3 (2 wells of a 6-well plate are split into 3 wells).
Prepare dispase: Dissolve dispase powder in sterile PBS (ThermoFisher Gibco) to obtain a concentration of 1mg/ml. Make sure dispase is completely dissolved. Can increase speed of dissolving by shaking the falcon tube.
Filter the dissolved dispase in a sterile environment through a 0.22um filter.
Add a 1:10 dilution to each well of the 6-well plate containing the cells (500ul of dispase to each well containing 5ml of media).
Leave in the incubator for ~20 minutes checking the plate every 10 minutes.
Whilst cells are in the dispase, add 10ml of sterile PBS to a 15ml falcon, 1 falcon for each iPSC line.
Add DNase I (Roche) to each falcon containing PBS to achieve a final concentration of 0.1mg/ml DNase I (stock is made up to be 10mg/ml). Add 100ul per falcon containing 10ml of PBS.
Once cells have lifted (they should lift as a continuous sheet), add the cells to its respective 15ml falcon containing PBS and DNase I. Invert the falcon 2-3 times to break up, and wash the cells. 
Allow the cells to settle to the bottom of the falcon with gravity flow.
Once they have settled, aspirate the PBS and 10ml of fresh sterile PBS to each falcon. Invert the falcon again 2-3 times to break up cells into smaller clumps.
Allow the cells to settle to the bottom of the falcon with gravity flow.
In the meantime prepare the required media with the extra addition of 10μM Y-27632 dihydrochloride.
When cells have all settled to the bottom, aspirate the PBS and add the required amount of media to each falcon to achieve a final volume of 2ml per well of a 6-well plate (for eg. splitting 1 well into 2, the total will be 4ml in the falcon. 2ml will then be added to each well).
Pipette up and down using a 5ml stripette to break up clumps of cells.
Add the cell suspension to the 6-well plate achieving the desired cell split. Move the cells in several quick side-to-side and back-and-forth motions to disperse the cells uniformly across the well. Place them in the incubator.