Welcome! If you are aiming to successfully cultivate midbrain dopaminergic (mDA) neurons derived from human embryonic stem cells (hESCs), you are in the right place. This protocol outlines a highly reproducible and scalable methodology for generating these specialized neurons, which hold significant potential for applications in regenerative medicine and Parkinson's disease research. By carefully regulating signaling pathways across specific developmental windows, you can achieve robust mDA specification, eventually yielding cells ready for in vitro electrophysiology, cryopreservation, or in vivo transplantation.
Days 1-3 Medium (Neurobasal medium + N2/B27/L-Glutamine)
Component
Concentration
LDN 193189
250 nM
SB 431542
10 μM
CHIR 99021
0.7 μM
Y-27632
10 μM (Day 1 only)
SHH25II
500 ng/ml
Days 4-6 Medium (Neurobasal medium + N2/B27/L-Glutamine)
Component
Concentration
LDN 193189
250 nM
SB 431542
10 μM
CHIR 99021
7.5 μM
SHH25II
500 ng/ml
Days 7-9 Medium (Neurobasal medium + N2/B27/L-Glutamine)
Component
Concentration
CHIR 99021
7.5 μM
Days 10-12 mDA Differentiation Medium (Neurobasal medium + B27/L-Glutamine)
Component
Concentration
BDNF
20 ng/ml
GDNF
20 ng/ml
Ascorbic Acid
200 μM
Dibutyryl-cAMP
500 μM
TGF-β3
1 ng/ml
CHIR 99021
3 μM
Days 12-16 mDA Differentiation Medium
Component
Concentration
BDNF
20 ng/ml
GDNF
20 ng/ml
Ascorbic Acid
200 μM
Dibutyryl-cAMP
500 μM
TGF-β3
1 ng/ml
DAPT
10 μM
Protocol
On day 0, prepare your culture surfaces by coating them with basement membrane extract (BME). Plate your hESCs (such as the WA09 line) onto the coated surface.
Maintain the cells in a basal formulation of Neurobasal medium containing L-glutamine, supplemented with N2 and B27.
Introduce the specific day 0 small molecules and growth factors to the medium: 10 μM SB 431542, 250 nM LDN 193189, 0.7 μM CHIR 99021, 500 ng/ml SHH, and 10 μM of the ROCK inhibitor Y-27632. Replace the medium completely every day.
Beginning on day 1, discontinue the use of Y-27632 while continuing daily replacements of the other components.
On day 4, increase the concentration of CHIR 99021 in the medium to 7.5 μM. This step is critical to thoroughly drive the specification of midbrain cellular identities.
By day 7, remove LDN 193189, SB 431542, and SHH from your daily media formulation. Maintain CHIR 99021.
On day 10, completely shift the culture into the mDA differentiation medium. This consists of Neurobasal medium, L-glutamine, and B27, newly supplemented with 20 ng/ml BDNF, 20 ng/ml GDNF, 200 μM Ascorbic Acid, 1 ng/ml TGF-β3, 500 μM dibutyryl cAMP, and 3 μM CHIR 99021.
On day 11, enzymatically or mechanically detach the developing cells and replate them into fresh mDA differentiation medium.
Starting on day 12, introduce 10 μM DAPT to the mDA medium and discontinue the addition of CHIR 99021.
On day 16, dissociate the cultured cells once more. The resulting mDA neurons are now ready to be replated for further in vitro analysis (e.g., electrophysiology) or they can be cryopreserved for future 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
