Generating Midbrain Dopaminergic Neurons from hPSCs

Table of Contents

• Introduction
• Materials
• Media & Buffers
• Protocol
• References

Introduction

Welcome! If you are looking to generate midbrain dopaminergic (mDA) neurons from human embryonic stem cells (hESCs), 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. Because mDA neurons hold such incredible promise for translational research and regenerative medicine—particularly for modeling and treating Parkinson's disease—optimizing this workflow is crucial. Let's get started on successfully deriving your mDA neurons!

Materials

Human embryonic stem cells (e.g., WA09) Basement membrane extract (BME) Neurobasal medium N2 supplement B27 supplement L-glutamine SB 431542 (HB3555) LDN 193189 DAPT

CHIR 99021 (HB1261) Y-27632 (HB2297) Recombinant SHH Recombinant BDNF (HB3485) Recombinant GDNF (HB5735) L-Ascorbic acid (HB1238) Recombinant TGF-β3 Dibutyryl cAMP

Media & Buffers

Days 1-3 Medium

Component	Concentration
Neurobasal medium supplemented with N2, B27, and L-Glutamine	Base
LDN 193189	250 nM
SB 431542 (HB3555)	10 μM
CHIR 99021 (HB1261)	0.7 μM
Y-27632 (HB2297)	10 μM (Day 1 only)
SHH	500 ng/ml

Days 4-6 Medium

Component	Concentration
Neurobasal medium supplemented with N2, B27, and L-Glutamine	Base
LDN 193189	250 nM
SB 431542 (HB3555)	10 μM
CHIR 99021 (HB1261)	7.5 μM
SHH	500 ng/ml

Days 7-9 Medium

Component	Concentration
Neurobasal medium supplemented with N2, B27, and L-Glutamine	Base
CHIR 99021 (HB1261)	7.5 μM

mDA Differentiation Medium (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)	200 μM
Dibutyryl cAMP	500 μM
Recombinant TGF-β3	1 ng/ml
CHIR 99021 (HB1261)	3 μM

mDA Differentiation Medium (Days 12-16)

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)	200 μM
Dibutyryl cAMP	500 μM
Recombinant TGF-β3	1 ng/ml
DAPT	10 μM

Protocol

1. At Day 0, plate hESCs (e.g., WA09) onto culture vessels pre-coated with basement membrane extract (BME).
2. Maintain the cells in Neurobasal medium containing N2, B27, and L-glutamine. Supplement this base media with SB 431542, LDN 193189, CHIR 99021, SHH, and Y-27632 according to the Days 1-3 Medium specifications. Execute full media changes daily.
3. Withdraw the Y-27632 supplement entirely beginning on Day 1.
4. On Day 4, adjust the CHIR 99021 concentration to 7.5 μM to appropriately optimize midbrain lineage specification (refer to the Days 4-6 Medium).
5. Discontinue the addition of LDN 193189, SB 431542, and SHH from the culture medium starting on Day 7, shifting exclusively to the Days 7-9 Medium formulation.
6. On Day 10, transition the cultures into the primary mDA Differentiation Medium, consisting of a Neurobasal base supplemented with B27, L-glutamine, BDNF, Ascorbic Acid, GDNF, TGF-β3, dibutyryl cAMP, and CHIR 99021.
7. Dissociate the cells on Day 11 and subsequently re-seed them into fresh mDA Differentiation Medium.
8. Introduce DAPT into the mDA Differentiation Medium starting on Day 12 to finalize the neural maturation phase.
9. Upon reaching Day 16, dissociate the generated mDA neurons and re-plate them for immediate in vitro studies, or prepare the cellular suspension for cryopreservation protocols.

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