Aug 01, 2025
Version 1
Dopaminergic neuron differentiation V.1
- Khaja Mohieddin Syed1,2,
- Oriol Busquets3,2,
- Frank Soldner3,2,
- Dirk Hockemeyer1,2
- 1University of California, Berkeley, Berkeley, CA 94720, USA;
- 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 2081;
- 3Albert Einstein College of Medicine, 1301 Morris Park Ave., Bronx, NY 10461, USA.
Protocol Citation: Khaja Mohieddin Syed, Oriol Busquets, Frank Soldner, Dirk Hockemeyer 2025. Dopaminergic neuron differentiation. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4q8yovo5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 09, 2024
Last Modified: August 01, 2025
Protocol Integer ID: 93189
Keywords: ASAPCRN, dopaminergic neuron differentiation this protocol, dopaminergic neuron differentiation, dopaminergic neurons from hpsc, dopaminergic neuron, feeder free culture system
Funders Acknowledgements:
Aligning Science Across Parkinson's (ASAP)
Grant ID: ASAP-000486
Aligning Science Across Parkinson's (ASAP)
Grant ID: ASAP-024409
Abstract
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
Attachments
Materials
| Item | Vendor | Catalog number | |
| DMEM/F12 | Gibco | 11320033 | |
| mTESR-plus Medium | StemCell Tech | 100-0276 | |
| Neurobasal medium | Gibco | 21103049 | |
| N2 supplement | Gibco | 17502048 | |
| B27 supplement without vitamin A | Gibco | 12587-010 | |
| L-Glutamine | Sigma | G8450 | |
| Penicillin-Streptomycin | Gibco | 15140122 | |
| Y-27632 Dihydrochloride Rock inhibitor | ToCris | CD0141 | |
| DPBS (No calcium, No magnesium) | Cytiva | SH30028 | |
| Accutase | Innovative Cell Tech | AT104 | |
| Matrigel | Corning | 354230 | |
| Laminin | R&D Systems | 3400-010-02 | |
| Poly-L-Ornithine | Sigma | P3655 | |
| LDN | Stemgent | 04-0074 | |
| SHH C25II | R&D Systems | 1845-SH-100 | |
| SB431542 | SelleckChem | S1067 | |
| CHIR99021 | ToCris | 4423 | |
| GDNF | PeProtech | 450-10 | |
| BDNF | PeProtech | 450-02 | |
| Dibutyryl-cAMP (Bucladesine) | SelleckChem | S7858 | |
| Sodium L-Ascorbate | Sigma | A40-34 | |
| TGFꞵ3 | R&D Systems | 8420-B3-005 | |
| DAPT | Tocris | 2634 |
Troubleshooting
Plate preparation
Plate preparation:
Note
This protocol has been used to differentiate dopaminergic neurons from hPSC adapted to feeder free culture systems. Our version of the protocol has been adapted from Kim et al 2021; Cell Stem Cell 28, 343–355 e5, February 4, 2021 and Piao et al. 2021; Cell Stem Cell 28, 217-229 e7, February 4, 2021.
Note
We have used two alternative protocol variants with minor modifications which will be outlined at the respective steps as SP (Soldner Protocol) and HP (Hockemeyer Protocol).
Note
It is important to start the differentiation from pristine, undifferentiated feeder free cultures. For more details consult: https://doi.org/10.17504/protocols.io.b4mcqu2w
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 00:30:00 to 1 hour.
30m
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 Overnight at 37 °C .
1h
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 06:00:00 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 Overnight at 37 °C . Do not let the wells dry.
12h
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 06:00:00 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 Overnight at 37 °C . Do not let the wells dry.
12h
Media Preparation
Media recipes:
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).
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).
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).
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).
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).
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).
Dopaminergic neuron differentiation
1h 35m
Day 0: Dissociate hPSCs using accutase (00:10:00 - 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 00:05:00 . Remove the supernatant, resuspend the cells in mTeSR-plus media + 10 µM Y-27632 and count them.
15m
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).
HP: Plate the cells at 400-600k/well in a 6-well plate in mTeSR-plus + 10 µM Y-27632 onto matrigel coated plates.
Day 1-3: Change the media - media A (change daily or every other day as necessary).
Day 4 and day 6: Change the media- media B (change daily or every other day as necessary).
Day 7 and Day 9: Change the media - media C (change daily or every other day as necessary).
Day 10: Change the media - media D.
Day 11: Passage #1. Dissociate cells using accutase (approximately 00:10:00 - 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 - 00:05:00 . Remove the supernatant, resuspend the cell in media D + 10 µM Y-27632.
15m
SP: Count the cells and plate them at 800k/cm2 in media D + 10 µM Y-27632 onto PLO+F+L coated plates.
HP: Split 1:2 in precursor splitting media + 10 µM Y-27632 onto laminin-coated plates.
Day 12-15: Change the media - maturation media (change daily or every other day as necessary).
Day 16: Passage #2. Dissociate cells using accutase (approximately 00:10:00 - 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 - 00:05:00 . Remove the supernatant, resuspend the cell in precursor splitting media + 10 µM Y-27632 and count them.
15m
SP: Plate the cells at 800k/cm2 in precursor splitting media + 10 µM Y-27632 onto PLO+F+L coated plates.
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.
Day 17-24: Change the media - maturation media (change daily or every other day as necessary).
Day 25: Passage #3.
SP: Dissociate cells using papain solution (00:30:00 - 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 - 00:05:00 . 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.
35m
HP: Dissociate cells using accutase (approximately 00:10:00 - 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 - 00:05:00 . 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.
15m
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.