Nov 21, 2022
Generation of hESC/iPSC Derived Midbrain Dopamine Neurons
- Su-Chun Zhang1
- 1University of Wisconsin-Madison
Protocol Citation: Su-Chun Zhang 2022. Generation of hESC/iPSC Derived Midbrain Dopamine Neurons. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygxpzjzl8j/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 07, 2020
Last Modified: May 31, 2024
Protocol Integer ID: 44308
Keywords: generation, hESC, iPSC, midbrain, dopamine, neurons, differentiation, ASAPCRN
Abstract
This protocol details methods for generating Midbrain Dopamine Neuronds from hESC or iPSC sources.
Guidelines
References
CITATION
CITATION
Safety warnings
Please refer to the Safety Data Sheets (SDS) for health and environmental hazards.
Before start
Induction of the midbrain dopaminergic progenitors was carried out based on protocol described previously (Xi et al., 2012) with modification.
Pre-differentiation and Differentiation (Day 1-6)
Pre-differentiation and Differentiation (Day 1-6)
Briefly, culture hESCs (1 day after passaging) on MEF feeder layer in the neural induction medium (NIM) (DMEM/F-12, 1X NEAA, 1X N2 supplement) supplemented with 10 micromolar (µM) SB431542 and 2 micromolar (µM) DMH-1 .
To pattern the differentiating cells to the midbrain FP progenitors, add 500 nanogram per milliliter (ng/mL) SHH (C25II) and 0.4 micromolar (µM) CHIR99021 to the cultures from day 1 till day 7.
Differentiation (Day 7-11)
Differentiation (Day 7-11)
On day 7, gently blow off individual colonies of neuroepithelial cells with a pipette and replate on mouse embryonic fibroblast feeder in the NIM containing 1 micromolar (µM) SAG and 100 nanogram per milliliter (ng/mL) SHH and 0.4 micromolar (µM) CHIR99021 for additional 6 days (D7-12).
Differentiation (Day 12-19)
Differentiation (Day 12-19)
On day 12, remove CHIR99021, reduce the following: 20 nanogram per milliliter (ng/mL) SHH , 0.5 micromolar (µM) SAG and 100 nanogram per milliliter (ng/mL) FGF8b and add to the culture to expand the progenitors in suspension till day 19.
Differentiation (Day 19-31)
Differentiation (Day 19-31)
Then, keep 20 nanogram per milliliter (ng/mL) SHH and 20 nanogram per milliliter (ng/mL) FGF8b in the neural induction medium till transplantation/in vitro analysis at day 32.
In Vitro Analysis (Day 32+)
In Vitro Analysis (Day 32+)
8m
8m
For in vitro analysis, dissociate neurospheres by incubation in Accutase at 37 °C for 00:03:00 -00:05:00 on day 32 and plate onto glass coverslips that were previously coated with Matrigel.
8m
Feed cells with Neural differentiation medium (NeurobasalTM Medium, 1 X N2 supplement, 1 X B27 supplement) (NDM) supplemented with brain-derived neurotrophic factor (10 nanogram per milliliter (ng/mL) BDNF ), glial cell line derived neurotrophic factor (10 nanogram per milliliter (ng/mL) GDNF ), ascorbic acid (200 micromolar (µM) AA ), 1 micromolar (µM) cAMP , transforming growth factor β3 (1 nanogram per milliliter (ng/mL) ) and Compound E (0.1 micromolar (µM) ).
Citations
Xi J, Liu Y, Liu H, Chen H, Emborg ME, Zhang SC. Specification of midbrain dopamine neurons from primate pluripotent stem cells.
https://doi.org/10.1002/stem.1152Xiong M, Tao Y, Gao Q, Feng B, Yan W, Zhou Y, Kotsonis TA, Yuan T, You Z, Wu Z, Xi J, Haberman A, Graham J, Block J, Zhou W, Chen Y, Zhang SC. Human Stem Cell-Derived Neurons Repair Circuits and Restore Neural Function.
https://doi.org/pii:S1934-5909(20)30410-0.10.1016/j.stem.2020.08.014