Differentiation of Induced Pluripotent Stem Cells (iPSCs) into Dopaminergic Neurons
Differentiating iPSCs into midbrain dopaminergic neurons offers incredible potential for researching neurological conditions, particularly Parkinson's disease. Whether you are investigating dopamine signaling pathways or modeling neurodegenerative diseases in vitro, this protocol outlines a clear, step-by-step method to efficiently drive your stem cells toward a dopaminergic fate. You will find it straightforward to follow, helping you achieve high-quality, functional neuronal cultures for all your downstream assays!
Featured Key Products
Neuronal Induction
LDN193189 hydrochloride (LDN)
Potent, selective ALK2/ALK3 inhibitor which also promotes neural induction of hPSCs
View ProductNeuronal Maturation
Recombinant BDNF
Member of the neurotrophin growth factor family. Plays an important role in synaptic plasticity.
View ProductNeuronal Maturation
Recombinant human GDNF protein
GDNF is a key protein used to differentiate hPSC-derived neural progenitor cells into neurons
View ProductNeuronal Differentiation
DAPT
γ-secretase inhibitor and classical notch inhibitor. Induces neuronal differentiation.
View ProductMaterials
- DAPT (HB3345)
- Recombinant human FGF-8a
- BDNF (HB3485)
- GDNF (HB5735)
- TGF-ß3
- Primary antibodies against dopaminergic markers such as tyrosine hydroxylase
- Secondary antibodies (see our range of validated secondary antibodies)
Media Recipes
Aseptically combine components and filter the media before use.
Media A - Day 1
| Component | Concentration |
|---|---|
| Knockout DMEM | 85% |
| Knockout Serum replacement | 15% |
| GlutaMax | 1x |
| MEM Non-Essential Amino Acids | 1x |
| 2-Mercaptoethanol | 10 mM |
| LDN193189 (HB5624) | 100 nM |
| SB431542 (HB3555) | 10 µM |
Media B - Days 2 - 3
| Component | Concentration |
|---|---|
| Knockout DMEM | 85% |
| Knockout Serum replacement | 15% |
| GlutaMax | 1x |
| MEM Non-Essential Amino Acids | 1x |
| 2-Mercaptoethanol | 10 mM |
| LDN193189 (HB5624) | 100 nM |
| SB431542 (HB3555) | 10 µM |
| SAG (HB3107) | 100 nM |
| Purmorphamine (HB3412) | 2 µM |
| Human FGF-8a | 100 ng/ml |
Media C - Days 4 - 5
| Component | Concentration |
|---|---|
| Knockout DMEM | 85% |
| Knockout Serum replacement | 15% |
| GlutaMax | 1x |
| MEM Non-Essential Amino Acids | 1x |
| 2-Mercaptoethanol | 10 mM |
| LDN193189 (HB5624) | 100 nM |
| SB431542 (HB3555) | 10 µM |
| SAG (HB3107) | 100 nM |
| Purmorphamine (HB3412) | 2 µM |
| Human FGF-8a | 100 ng/ml |
| CHIR99021 (HB1261) | 3 µM |
Media D - Days 6 - 7
| Component | Concentration |
|---|---|
| Knockout DMEM | 63.2% |
| Knockout Serum replacement | 11.6% |
| Neurobasal Medium | 24.8% |
| N-2 supplement | 0.13% |
| B-27 supplement | 0.26% |
| GlutaMax | 1x |
| MEM Non-Essential Amino Acids | 1x |
| 2-Mercaptoethanol | 10 µM |
| LDN193189 (HB5624) | 100 nM |
| CHIR99021 (HB1261) | 3 µM |
| SAG (HB3107) | 100 nM |
| Purmorphamine (HB3412) | 2 µM |
| Human FGF-8a | 100 ng/ml |
Media E - Days 8 - 9
| Component | Concentration |
|---|---|
| Knockout DMEM | 42.3% |
| Knockout Serum replacement | 7.6% |
| Neurobasal Medium | 49.3% |
| N-2 supplement | 0.26% |
| B-27 supplement | 0.5% |
| GlutaMax | 1x |
| MEM Non-Essential Amino Acids | 1x |
| 2-Mercaptoethanol | 10 µM |
| LDN193189 (HB5624) | 100 nM |
| CHIR99021 (HB1261) | 3 µM |
Media F - Days 10 - 11
| Component | Concentration |
|---|---|
| Knockout DMEM | 21% |
| Knockout Serum replacement | 3.8% |
| Neurobasal Medium | 74% |
| N-2 supplement | 0.38% |
| B-27 supplement | 0.74% |
| GlutaMax | 1x |
| MEM Non-Essential Amino Acids | 1x |
| 2-Mercaptoethanol | 10 µM |
| LDN193189 (HB5624) | 100 nM |
| CHIR99021 (HB1261) | 3 µM |
Media G - Days 12 - 13
| Component | Concentration |
|---|---|
| Neurobasal Medium | 96% |
| B-27 supplement | 1x |
| GlutaMax | 1x |
| DAPT (HB3345) | 10 µM |
| Dibutyryl cyclic-AMP sodium salt | 500 µM |
| Ascorbic acid (HB1238) | 200 µM |
| CHIR99021 (HB1261) | 3 µM |
| BDNF (HB3485) | 20 ng/ml |
| GDNF (HB5735) | 20 ng/ml |
| TGF-β3 | 1 ng/ml |
Protocol
1
Cell culture and maintenance: Cultivate human iPSCs using E8 medium on 6-well plates coated with 5 µg/ml Vitronectin. Every 3 to 4 days, perform passaging with 0.5 µM EDTA (HB5135) and split the cells at a 1:6 ratio to ensure they remain high quality prior to differentiation.
2
Preparation for differentiation: Once the iPSCs have reached the required quality and confluence, detach them using Accutase. Seed the cells into a 24-well plate previously coated with basement membrane matrix at a density of 3.8x105 cells/ml. Incubate until a 90-100% confluent monolayer is established.
3
Neural induction (Day 1): Initiate differentiation toward neural progenitor cells (NPCs) by applying dual SMAD inhibition. Exchange media for Media A which encorporates SB431542 (HB3555) to suppress pluripotency maintenance pathways and guide the cells toward a neural lineage.
4
Neural Induction (Days 2 - 3): Exchange the culture medium with freshly prepared Media B
5
Neural Induction (Days 4 - 6): Exchange the culture medium with freshly prepared Media C
6
Neural Induction (Days 6 - 7): Exchange the culture medium with freshly prepared Media D
7
Neural Induction (Days 8 - 9): Exchange the culture medium with freshly prepared Media E
8
Neural Induction (Days 8 - 9): Exchange the culture medium with freshly prepared Media E
9
Neural Induction (Days 10 - 11): Exchange the culture medium with freshly prepared Media F
10
Neural Induction (Days 12 - 13): Exchange the culture medium with freshly prepared Media G
11
Neuronal Maturation (Days 14 - 19): Exchange the culture medium with freshly prepared Media H (Maturation Media)
12
Passaging of Neuronal Progenitors (Day 20): Passage cells using a mixture of Accutase and Papain then wash and filter cells in DMEM/F12 containing DNase I. Subseqeuntly wash and filter cells a second time in DMEM/F12 without Dnase I. Next seed cells at a density of 8.6x105 cells / ml into a Geltrex-coated 24 plate well containing Media I (Seeding Media).
13
Neuronal Maturation (Days 21 - 35): Exchange the culture medium with freshly prepared Media H (Maturation Media).
14
Phenotypic Analysis: Fix cells with 4% paraformaldehyde (PFA) then stain for dopaminergic markers such as tyrosine hydroxylase (TH). For a full guide to immunocytochemistry (ICC), please see our ICC protocol.
References
1
O Klein, M. et al. 2019. Dopamine: Functions, Signaling, and Association with Neurological Diseases. Cellular and Molecular Neurobiology. PMID: 30446950
2
Zhuang, Y. et al. 2021. Mechanism of dopamine binding and allosteric modulation of the human D1 dopamine receptor. Cell Research PMID: 33750903
3
Marcotto et al., 2026 Differentiation of human iPSCs into dopaminergic neurons: comparative analysis of 2D and 3D protocols for disease modeling and pharmacology.Neuroscience Applied PMID: 41756022