Differentiation of Midbrain Dopaminergic Neurons from Human Embryonic Stem Cells (hESCs)
Table of Contents
Introduction
If you are looking to generate midbrain dopaminergic (mDA) neurons from human embryonic stem cells (hPSCs), you've come to the right place. Based on the highly reproducible methodology developed by Kim and colleagues, this scalable protocol outlines the key steps to derive these specialized neurons. mDA neurons are vital for research into translational therapies and regenerative medicine with a particular relevance to the modeling and treatment of Parkinson's disease. Let's get started on successfully deriving your mDA neurons!
Materials
Media & Buffers
Media A - Day 1
| Component | Concentration |
|---|---|
| Neurobasal medium supplemented with N2, B27, and L-Glutamine | Base |
|
LDN 193189 (HB5624) |
250 nM |
| SB 431542 (HB3555) | 10 μM |
| CHIR 99021 (HB1261) | 0.7 μM |
| Y-27632 (HB2297) | 10 μM |
| SHH | 500 ng/ml |
Media B - Days 2-3
| Component | Concentration |
|---|---|
| Neurobasal medium supplemented with N2, B27, and L-Glutamine | Base |
|
LDN 193189 (HB5624) |
250 nM |
| SB 431542 (HB3555) | 10 μM |
| CHIR 99021 (HB1261) | 0.7 μM |
| SHH | 500 ng/ml |
Media C - Days 4-6
| Component | Concentration |
|---|---|
| Neurobasal medium supplemented with N2, B27, and L-Glutamine | Base |
| LDN 193189 (HB5624) | 250 nM |
| SB 431542 (HB3555) | 10 μM |
| CHIR 99021 (HB1261) | 7.5 μM |
| SHH | 500 ng/ml |
Media D - Days 7-9
| Component | Concentration |
|---|---|
| Neurobasal medium supplemented with N2, B27, and L-Glutamine | Base |
| CHIR 99021 (HB1261) | 7.5 μM |
Media E - Days 10-12
| Component | Concentration |
|---|---|
| Neurobasal medium supplemented with B27 and L-Glutamine | Base |
| Recombinant BDNF (HB3485) | 20 ng/ml |
| Recombinant GDNF (HB5735) | 20 ng/ml |
| L-Ascorbic acid (HB1238) | 0.2 μM |
| Dibutyryl cAMP | 500 μM |
| Recombinant TGF-β3 | 1 ng/ml |
| CHIR 99021 (HB1261) | 3 μM |
Protocol
- At Day 0, plate hESCs (e.g., WA09) onto culture vessels pre-coated with basement membrane extract (BME) at a density of 400,000 cells / cm2.
- Day 1: Culture the cells in Media A.
- Days 2-3: Culture the cells in Media B with daily media changes.
- Days 4-6: Culture the cells in Media C with daily media changes..
- Days 7-9: Culture the cells in Media D with daily media changes..
- Day 10: Culture the cells in Media E
- Day 11: Dissociate the cells and replate in Media E on polyornithine (15µg/ml), laminin (1µg/ml) and fibronectin (2µg/ml) coated dishes at a density of 800,000 cells / cm2.
- Days 12-15: Culture the cells in Media F with daily media changes.
- Day 16 onwards: Dissociate the cells and replate in Media G on polyornithine (15µg/ml), laminin (1µg/ml) and fibronetcin (2µg/ml) coated dishes at a density of 800,000 cells / cm2. Cells can be cryopreserved at this point if desired.
- Day 25: Dissociate the cells using accutase and replate at 200,000 - 300,000 cells / cm2 in Media G until needed for electrophysiology experiments.
References
- Kim et al. (2021) Biphasic activation of WNT signaling facilitates the derivation of midbrain dopamine neurons from hESCs for translational use. Cell Stem Cell 28 343. PMID: 33545081