Oct 15, 2025
Ventral midbrain dopaminergic (mDA) Neuron Differentiation Protocol
- Regine Tipon1,
- Niraj Sawarkar1,
- Gist Croft1
- 1New York Stem Cell Foundation
Protocol Citation: Regine Tipon, Niraj Sawarkar, Gist Croft 2025. Ventral midbrain dopaminergic (mDA) Neuron Differentiation Protocol . protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvmebdog3p/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: August 28, 2024
Last Modified: February 21, 2026
Protocol Integer ID: 106536
Keywords: ASAPCRN, iPSC, midbrain dopaminergic neurons (mDA), Parkinson's disease, Differentiation, ASAPCRN, ventral midbrain dopaminergic neuron, including caudal ventral midbrain dopaminergic neuron, development of midbrain cell type, ventral midbrain dopaminergic, caudal ventral midbrain dopaminergic neuron, midbrain cell type, pluripotent stem cell, induced pluripotent stem cell, selective enrichment of da neuron, human induced pluripotent stem cell, stem cell, other resident neural cell type, neuron differentiation protocol, da neuron, vulnerable in parkinson, other regional cell type, parkinson, variability between different cell line, enabled cell replacement therapy, dopaminergic neuron, neuron, midbrain
Funders Acknowledgements:
ASAP
Grant ID: ASAP-000472
Abstract
Generating authentic functional ventral midbrain dopaminergic neurons, those most vulnerable in Parkinson's disease, from human induced pluripotent stem cells (iPSCs) has enabled cell replacement therapy clinical trials and increasingly sophisticated in vitro disease models. This protocol features further optimizations on a recently updated developmental patterning approach (Kim et al., 2021), focusing on optimizing key stages of t including cell maintenance, induction, cryopreservation, reassembly, and maturation. This comprehensive protocol details the conditions required for the development of midbrain cell types including caudal ventral midbrain dopaminergic neurons and other resident neural cell types, including morphogens, media, substrates, timing, and enables selective enrichment of DA neurons as well as other regional cell types, and integrations with organoid and assembloid cultures. Importantly, variability between different cell line genetic backgrounds, and isogenic pairs, is reduced by by reducing the impact of non-disease-related varitation in iPSC line-intrisic phenotypic variablkes like plating and repassaging efifciency at multiple bottlenecks,and leverages strategies for further inter-line normalization.
Guidelines
- Day -X: PSC expansion
- iPSC are cultured following Croft lab Protocols. It should have perfectly undifferentiated appearance and high viability. Almost all other substrates and media are fine as well, with some caveats (Eb, see discussion) .https://docs.google.com/document/d/1mgfOIq4uSawHrjZtxbICilmuu64zIxRlk6SAWFPGm3Y/edit
Day -2/-1 Form standardized pluripotent 3D aggregates (embryoid bodies); see Aggrewell Plates protocol
Seed in Stemfleex media + RI or CEPT, 2000 cells/microwell (2.4M cells per well of 400/800 aggrewell 24wp wells)
Note: smooth spherical EBs should have formed the next day.
Note: EBs can be formed 1 or 2 days before differentiation. Increase cells/microwell if using day -1. Day 2 is standard
Transfer EBs to ULA plates
- ULA 6wp, 10cm non-tc-treated dishes can be used based on user preference
- All culture plates are kept on a shaker at 80 rpm (after release for 2 days )
CHIR Dose:
CHIR99021 concentration may have to be altered to obtain the highest mDA neuron differentiation efficiency for different iPSC / ESC cell lines due to line-intrinsic differences in Wnt signaling tone and interacting signalling pathways,
Day 1 and Day 3 = CHIR99021 range of 0.7 μM to 2 μM; Standard is 1 μM
Day 4, Day 6, and Day 9 = CHIR99021 range of 5-6 μM, Standard is 6 μM
Materials
| A | B | C | |
| Neurobasal | Life Technologies | 21103049 | |
| N2 | Life Technologies | 17502-048 | |
| B27 without RA | Life Technologies | 12587-010 | |
| SB431542 | Selleck Chemicals | S1067 | |
| LDN191389 | Reprocell | 27,120.00 | |
| Puromorphamine | Selleck Chemicals | S3042 | |
| SAG | EMD Millipore | 566660-1MG | |
| NEAA | Life Technologies | 11,140,050.00 | |
| Albumax | Thermo Scientific | 11,020,021.00 | |
| CHIR | R&D Systems | 4423/50 | |
| BDNF | R&D Systems | 248-BDB-050 | |
| GDNF | R&D Systems | 212-GD-050 | |
| AA | Sigma-Aldrich | A4403-100MG | |
| TGFb3 | R&D Systems | 7754-BH-025 | |
| dbCAMP | Sigma-Aldrich | D0627-1G | |
| GLUTAMAX | Thermo Scientific | 31,980,030.00 | |
| DAPT | Tocris | 2,634.00 | |
| Neurobrew521 | Miltenyi Biotec | 130-093-566 | |
| Reconstitution buffer | R&D Systems | RB02 | |
| DMSO | Sigma-Aldrich | D2650 |
Troubleshooting
Safety warnings
CHIR99021 concentrations above 7.5 μM results in dose-dependent cell death ( Kim et al., 2021)
Notes: Before starting the protocol
PSC cultures should be completely undifferentiated
Embryoid bodies (EB) formation
Form EB aggregates (Day -2): iPSC are first nucleated and generated into embryoid bodies. Aggrewells, iPSC medium + CEPT (or Y27632) using the following protocol EB protocols.io
Neural Induction and patterning
Neural induction and patterning (Day 0-11), CHIR boost 4-10
Static or shaking suspension culture (80 RPM) in 6-well plates or 10 cm plate
. in Neurobasal media.
| Components | Concentration | |
| Neurobasal | ||
| N2 | 10.0% | |
| B27 without retinoic acid (RA) | 20.0% | |
| SB-431542 | 10 μM | |
| LDN193189 | 100 nM | |
| Purmorphamine | 1 μM | |
| SAG | 1 μM | |
| NEAA | 10% | |
| Albumax | 1% | |
| CHIR-99021 | 1 μM |
Media Day 4-6 in Neurobasal media.
Followed by CHIR BOOST from Day 4-6
| Component | Concentration | |
| Neurobasal | ||
| N2 | 10% | |
| B27 without retinoic acid (RA) | 20% | |
| SB-431542 | 10 μM | |
| LDN193189 | 100 nM | |
| Purmorphamine | 1 μM | |
| SAG | 1 μM | |
| NEAA | 10% | |
| Albumax | 1% | |
| CHIR99021 | 6 μM |
Media day 4-6
Remove SB/LDN+SAG/PURO on Day 6
| Component | Concentration | |
| Neurobasal | ||
| N2 | 10% | |
| B27 without retinoic acid (RA) | 20% | |
| CHIR99021 | 3 μM | |
| Albumax | 1% |
Media day 7-9
Add trophic factors from Day 10
| Component | Concentration | |
| Neurobasal | ||
| N2 | 10% | |
| B27 without retinoic acid (RA) | 20% | |
| CHIR99021 | 3 μM | |
| BDNF | 20 ng/mL | |
| GDNF | 20 ng/mL | |
| Ascorbic Acid | 0.2 mM | |
| TGFb3 | 1 ng/mL | |
| dbCAMP | 0.2 mM | |
| Albumax | 1% |
Media 10-11
Remove CHIR followed by Expansion and trophic support (Day 12-33)
| Components | Concentration | |
| Small Molecules | Conc. (ng/mL) | |
| Neurobasal | ||
| N2 | ||
| B27without RA | ||
| BDNF | 20ng/ml | |
| GDNF | 20ng/ml | |
| AA | 0.2mM | |
| TGFb3 | 1ng/ml | |
| dbCAMP | 0.2mM |
Media Day 12-35
Cultures can be harvested and cryopreserved or reconstituted at (Day 12-33)
see Neural dissociation protocol .io version, See Worthington protocol.io version, See crypreservation protocol.io version,
Further maturation and extended Culture can be performed in the following ways
- Reseed as Mono-, co- or tri-culture (see survival assay protocol.io versions)
- Organoids / Assembloids (See Assembloid protocol)
Protocol references
Kim TW, Piao J, Koo SY, Kriks S, Chung SY, Betel D, Socci ND, Choi SJ, Zabierowski S, Dubose BN, Hill EJ, Mosharov EV, Irion S, Tomishima MJ, Tabar V, Studer L. Biphasic Activation of WNT Signaling Facilitates the Derivation of Midbrain Dopamine Neurons from hESCs for Translational Use. Cell Stem Cell. 2021 Feb 4;28(2):343-355.e5. doi: 10.1016/j.stem.2021.01.005. PMID: 33545081; PMCID: PMC8006469