TITLE: Dopaminergic neuron differentiation
AUTHORS: Khaja Mohieddin Syed, Oriol Busquets, Frank Soldner, Dirk Hockemeyer

[DESCRIPTION]
This protocol has been used to differentiate dopaminergic neurons from hPSC adapted to feeder free culture systems.

Protocol overview

A. Plate preparation
B. Media recipes
C. Dopaminergic neuron differentiation

[STEPS]
SECTION: Plate preparation
1. Plate preparation:

 

 

 

SECTION: Plate preparation
1.1. Matrigel/Geltrex coating: Prepare matrigel/geltrex (1:30) in cold DMEM/F12 or DPBS in a 15 ml tube as described by the manufacturer. Coat each well with 1-1.5 ml (6 well plate size) or 0.5 ml (12w plate size)  of solution and incubate at 37 °C incubator for at least 30 min to 1 hour.
SECTION: Plate preparation
1.2. Laminin coating: Prepare laminin (2 µg/ml) in cold DPBS and coat each well of a 6-well plate with 1.5ml laminin solution (2 µg/ml) and incubate 60 minat 37 °C.
SECTION: Plate preparation
1.3. Poly-L-Ornithine (PLO) + Fibronectin (F) + Laminin (L) coating: Prepare PLO (15 µg/ml) in sterile water and coat each well with 0.5-1 ml making sure it covers the entire surface. Incubate at 37 °C for 360 min to overnight. The day after, wash each well with sterile water 4 times and add a new solution with fibronectin (1 µg/ml) and laminin (2 µg/ml) (in water) and incubate 360 minat 37 °C. Do not let the wells dry.
SECTION: Plate preparation
1.4. Poly-L-Ornithine (PLO) + Laminin (L) coating: Prepare PLO (15 µg/ml) in sterile water and coat each well of a 12-well plate with 0.5-1 ml making sure it covers the entire surface. Incubate at 37 °C for 360 min to overnight. The day after, wash each well with sterile water 2 times and add a new solution with laminin (2 µg/ml) (in cold DPBS) and incubate 360 min at 37 °C. Do not let the wells dry.
SECTION: Media Preparation
2. Media recipes:
SECTION: Media Preparation
2.1. Media A: Neurobasal media + N2 supplement (1% vol/vol) + B27 supplement without vitamin A (2% vol/vol) + L-Glutamine (2 mM) + Penicillin-Streptomycin (100U/ml) + SHH (200 ng/ml) + CHIR99021 (0.7 µM) + LDN (250 nM) + SB431542 (10 µM).
SECTION: Media Preparation
2.2. Media B: Neurobasal media + N2 supplement (1% vol/vol) + B27 supplement without vitamin A (2% vol/vol) + L-Glutamine (2 mM) + Penicillin-Streptomycin (100U/ml) + SHH (200 ng/ml) + CHIR99021 (7.5 µM) + LDN (250 nM) + SB431542 (10 µM).
SECTION: Media Preparation
2.3. Media C: Neurobasal media + N2 supplement (1% vol/vol) + B27 supplement without vitamin A (2% vol/vol) + L-Glutamine (2 mM) + Penicillin-Streptomycin (100U/ml) + CHIR99021 (7.5 µM).
SECTION: Media Preparation
2.4. Media D: Neurobasal media + B27 supplement without vitamin A (2% vol/vol) + L-Glutamine (2 mM) + Penicillin-Streptomycin (100U/ml) + BDNF (20 ng/ml) + GDNF (20 ng/ml) + Ascorbic acid (200 µM) + Dibutyryl-cAMP (0.5 mM) + TGFꞵ3 (1ng/ml) + CHIR99021 (3 µM).
SECTION: Media Preparation
2.5. Precursor splitting media: Neurobasal media + B27 supplement without vitamin A (2% vol/vol) + L-Glutamine (2 mM) + Penicillin-Streptomycin (100U/ml) + BDNF (20 ng/ml) + GDNF (20 ng/ml) + Ascorbic acid (200 µM) + Dibutyryl-cAMP (0.5 mM) + TGFꞵ3 (1ng/ml).
SECTION: Media Preparation
2.6. Maturation media: Neurobasal media + B27 supplement without vitamin A (2% vol/vol) + L-Glutamine (2 mM) + Penicillin-Streptomycin (100U/ml) + BDNF (20 ng/ml) + GDNF (20 ng/ml) + Ascorbic acid (200 µM) + Dibutyryl-cAMP (0.5 mM) + TGFꞵ3 (1ng/ml) + DAPT (10 µM).
SECTION: Dopaminergic neuron differentiation
3. Day 0: Dissociate hPSCs using accutase (10 min - 37 °C). Quench dissociation by diluting the accutase solution with mTeSR-plus media + 10 µM Y-27632 and collect the cells into a 15ml conical tube. Spin the cells down at 105 rcf for 5 min. Remove the supernatant, resuspend the cells in mTeSR-plus media + 10 µM Y-27632 and count them. 
SECTION: Dopaminergic neuron differentiation
3.1. SP: Plate the cells at 400-600k/cm2 in media A + 10 µM Y-27632 onto geltrex coated plates (adjust cell number to the plate size being used).
SECTION: Dopaminergic neuron differentiation
3.2. HP: Plate the cells at 400-600k/well in a 6-well plate in mTeSR-plus + 10 µM Y-27632 onto matrigel coated plates.
SECTION: Dopaminergic neuron differentiation
4. Day 1-3: Change the media - media A (change daily or every other day as necessary).
SECTION: Dopaminergic neuron differentiation
5. Day 4 and day 6: Change the media- media B (change daily or every other day as necessary).
SECTION: Dopaminergic neuron differentiation
6. Day 7 and Day 9: Change the media - media C (change daily or every other day as necessary).
SECTION: Dopaminergic neuron differentiation
7. Day 10: Change the media -  media D.
SECTION: Dopaminergic neuron differentiation
8. Day 11: Passage #1. Dissociate cells using accutase (approximately 10 min - 37 °C  but longer incubations may be necessary to properly detach the cells). Quench dissociation by diluting the accutase solution with 1 ml/well media D + 10 µM Y-27632. Collect cells in 15ml conical tubes and spin them down at 300 rcf - 5 min. Remove the supernatant, resuspend the cell in media D + 10 µM Y-27632.

SECTION: Dopaminergic neuron differentiation
8.1. SP: Count the cells and plate them at 800k/cm2 in media D + 10 µM Y-27632 onto PLO+F+L coated plates.
SECTION: Dopaminergic neuron differentiation
8.2. HP: Split 1:2 in precursor splitting media + 10 µM Y-27632 onto laminin-coated plates.
SECTION: Dopaminergic neuron differentiation
9. Day 12-15: Change the media - maturation media (change daily or every other day as necessary).
SECTION: Dopaminergic neuron differentiation
10. Day 16: Passage #2. Dissociate cells using accutase (approximately 10 min - 37 °C but longer incubations may be necessary to properly dissociate collect the cells). Quench dissociation by diluting accutase with 1 ml/well precursor splitting media + 10 µM Y-27632. Collect the cells in 15 ml conical tubes and spin them down at 300 rcf - 5 min. Remove the supernatant, resuspend the cell in precursor splitting media + 10 µM Y-27632 and count them. 

SECTION: Dopaminergic neuron differentiation
10.1. SP: Plate the cells at 800k/cm2 in precursor splitting media + 10 µM Y-27632 onto PLO+F+L coated plates.
SECTION: Dopaminergic neuron differentiation
10.2. HP: Plate the cells at >1 million cells per well of 12-well plate in maturation media + 10 µM Y-27632 onto PLO+L coated plates.
SECTION: Dopaminergic neuron differentiation
11. Day 17-24: Change the media - maturation media (change daily or every other day as necessary).
SECTION: Dopaminergic neuron differentiation
12. Day 25: Passage #3.
SECTION: Dopaminergic neuron differentiation
12.1. SP: Dissociate cells using papain solution (30 min - 37 °C). Inactivate papain solution using ovomucoid trypsin inhibitor (10mg/ml). Collect your cells in 15 ml conical tubes and spin them down at 300 rcf - 5 min. Remove the supernatant, resuspend the cells in maturation media + 10 µM Y-27632 and count them. Plate the cells at 200-300k/cm2 in maturation media + 10 µM Y-27632 onto PLO+L+F coated plates.

SECTION: Dopaminergic neuron differentiation
12.2. HP: Dissociate cells using accutase (approximately 10 min - 37 °C is the usual standard but longer incubations may be necessary to properly detach the cells). Quench accutase by adding maturation media + 10 µM Y-27632. Collect your cells in 15 ml conical tubes and spin them down at 300 rcf - 5 min. Remove the supernatant, resuspend the cells in maturation media + 10 µM Y-27632 and count them. Plate the cells at >1 million cells per well of 12-well plate in maturation media + 10 µM Y-27632 onto PLO+L coated plates.
SECTION: Dopaminergic neuron differentiation
13. Day >26: Change media to maturation media (perform media changes every 2-3 days). Cells can be  maintained in this media for several months or until experiment.