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  • A protocol for the differentiation of human embryonic stem cells into midbrain dopaminergic neurons.

A protocol for the differentiation of human embryonic stem cells into midbrain dopaminergic neurons.

STAR protocols (2023-06-13)
Kaneyasu Nishimura, Emilía Sif Ásgrímsdóttir, Shanzheng Yang, Ernest Arenas
ABSTRACT

Here, we present a protocol for the generation of functional midbrain dopaminergic (mDA) neurons from human embryonic stem cells (hESCs), which mimics the development of the human ventral midbrain. We describe steps for hESC proliferation, induction of mDA progenitors, freezing stocks of mDA progenitors as an intermediate starting point to reduce the time to make mDA neurons, and maturation of mDA neurons. The entire protocol is feeder-free and uses chemically defined materials. For complete details on the use and execution of this protocol, please refer to Nishimura et al. (2023).1.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Anti-Tyrosine Hydroxylase Antibody, Chemicon®, from rabbit
Sigma-Aldrich
SU-5402, ≥98% (HPLC)
Sigma-Aldrich
PD 0325901, ≥98% (HPLC)
Sigma-Aldrich
DAPT, ≥98% (HPLC), solid
Sigma-Aldrich
Anti-LMX-1 Antibody, serum, from rabbit
Sigma-Aldrich
Anti-Sox2 Antibody, Chemicon®, from rabbit
Sigma-Aldrich
CHIR99021, ≥98% (HPLC)
Sigma-Aldrich
N6,2′-O-Dibutyryladenosine 3′,5′-cyclic monophosphate sodium salt, ≥96% (HPLC), powder
Sigma-Aldrich
GW3965 hydrochloride, ≥98% (HPLC), powder