Jul 24, 2025
Generation of Dopaminergic (DA) neurons from human ES cells
- Arun Thiruvalluvan1,2,
- Agnete Kirkeby1,2
- 1Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815, USA.;
- 2Novo Nordisk Foundation Center for Stem Cell Medicine (renew), University of Copenhagen, 2200 Copenhagen, Denmark
Protocol Citation: Arun Thiruvalluvan, Agnete Kirkeby 2025. Generation of Dopaminergic (DA) neurons from human ES cells. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7nbd1lwz/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: July 16, 2025
Last Modified: July 24, 2025
Protocol Integer ID: 222631
Keywords: ASAPCRN, human ventral midbrain dopaminergic progenitor, generation of dopaminergic, mature dopaminergic neuron, neurons from human es cell, human es cell, dopaminergic, neuron, generation
Funders Acknowledgements:
Aligning Science Across Parkinson’s through the Michael J. Fox Foundation for Parkinson’s Research
Grant ID: ASAP-000520
Aligning Science Across Parkinson’s through the Michael J. Fox Foundation for Parkinson’s Research
Grant ID: ASAP-024296
Aligning Science Across Parkinson’s through the Michael J. Fox Foundation for Parkinson’s Research
Grant ID: ASAP-025170
Abstract
This protocol details how to generate mature dopaminergic neurons from human ventral midbrain dopaminergic progenitors.
Troubleshooting
Generation of high-purity human ventral midbrain dopaminergic progenitors
Embryonic stem cells were maintained and differentiated towards ventral midbrain (vMB) fate based on a
previously published protocol (dx.doi.org/10.17504/protocols.io.kxygx4eqol8j/v1)
Terminal differentiation to DA neurons
Post differentiation towards ventral midbrain specification, day 16 progenitors were plated for terminal differentiation on Lam-111 (i.e., 2 μg/cm2; Biolaminin: LN111-0501) plates in Neurobasal medium containing:
| Reagent | Dilution/Concentration | Provider | Cat# Number | |
| NB-21 supplement without vitamin A | 1:500 | Miltenyi Biotec | 130-093-566 | |
| Penicillin/streptomycin | 1:1000 | Thermo Fisher Scientific | 15140122 | |
| L-glutamine | 1:1000 | Thermo Fisher Scientific | 25030081 | |
| NEAA | 1:1000 | Thermo Fisher Scientific | 11140050 | |
| BDNF | 20 ng/ml | Miltenyi Biotec | 130-096-286 | |
| Ascorbic acid | 0.2 mM | Sigma-Aldrich | A4403-100MG | |
| GDNF | 10 ng/ml | Miltenyi Biotec | 130-129-548 | |
| db-cAMP | 500 μM | Sigma-Aldrich | D0627-1G |
Media composition for terminal differentiation of DA neurons
Day 16 ventral midbrain progenitors were plated onto Lam-111-coated plates (2 μg/cm2) at a density of 155,000 cells/cm2.
Every 2–3 days, remove old medium and add new medium to the cells: B27 medium + BDNF (20 ng/ml) + AA (0.2 mM) + GDNF (10 ng/ml) + db-cAMP (500 μM) + DAPT (1 μM). Add 300–500 μl of medium per cm2, depending on cell confluency.
Mature DA neurons phenotypes start to appear from around day 42 and onward, and the neurons can be characterised by immunocytochemistry for mature DA markers such as TH, LMX1, EN1 and MAP2.