Generation of Dopaminergic Neuronal Organoids from Human Embryonic Stem Cells

Abstract

Source: Cosset, E. et al. Human Neural Organoids for Studying Brain Cancer and Neurodegenerative Diseases. J. Vis. Exp. (2019)

This video demonstrates the differentiation of human embryonic stem cells (hESCs) into dopaminergic neurons, to provide a model for studying Parkinson's disease. The hESCs are cultured in a neural induction medium in a microwell plate to form 3D neurospheres. These are then cultured in a neural induction medium to promote the differentiation into dopaminergic progenitor neurons. The final maturation of the neurons occurs in a culture insert under air-liquid interface conditions, resulting in 3D organoids consisting of mature dopaminergic neurons.

Protocol

1. hESC-derived dopaminergic organoids for Parkinson's Disease (PD) studies

1. Day 0: Amplify hESCs in 2D culture up to 60% confluency (day 0), then replace stem cell media used to maintain pluripotency features of hESCs with a serum-free medium. Start neural induction by supplementing culture medium with 0.5 µM bone morphogenetic protein (BMP) inhibitor and 10 µM transforming growth factor beta (TGFβ)/Activin/Nodal inhibitor (dual-SMAD inhibition cocktail), then add 10 µM Rho-associated protein kinase (ROCK) inhibitor for 24 h to increase the survival rate of cells during passage.
2. Day 1: Prepare the microwell plate with 2.5 mL per well of serum-free medium supplemented with 0.5 µM BMP inhibitor, 10 µM TGFβ/Activin/Nodal inhibitor, and 10 µM ROCK inhibitor. To specify cells towards the ventral pattern of the neural tube, add 100 ng/mL sonic hedgehog (SHH), 100 ng/mL fibroblast growth factor 8 (FGF8), and 2 µM smoothened agonist. Centrifuge the plate (only with medium and without cells) at 1200 x g for 5 min to remove air bubbles from the microwells.
   1. After 1 day of neural induction in 2D, remove the medium and quickly wash with phosphate-buffered saline (PBS) without Ca²⁺/MgCl²⁺. Dissociate the colonies in single cells suspension by adding 7.5 mL of recombinant enzymatic solution in a T75 cm² flask. Incubate for 2 min at 37 °C then complete with 7.5 mL of Dulbecco's modified Eagle medium/nutrient mixture F-12 (DMEM-F12).
   2. Collect the cell suspension and centrifuge at 300 x g for 5 min. Remove the supernatant and count the cells in the same medium used to prepare the microwell plate.
   3. Adjust the medium volume to obtain a cell suspension allowing to form neurospheres containing 1000 cells per microwell (for example, the microwell plate used here contains 4,700 microwells per well). So, prepare 4.7 million cells in 2.5 mL of medium and add it to the previous 2.5 mL of medium already placed in the plate.
   4. To correctly distribute the cells in each microwell, gently shake the plate, and centrifuge the microwell plate 300 x g for 5 min. Incubate the plate at 37 °C for 24 h to generate spheres.
3. Day 2: Gently flush the microwells with the medium and collect then transfer the spheres in tissue-treated six-well plate. Replace medium with Neurobasal medium supplemented with 1% B27, 1x non-essential amino acids (NEAA), 2 mM L-glutamine, and 1% of penicillin/ streptomycin. Additionally, add regionalization factors SHH, FGF8, smoothened agonist, and dual-SMAD inhibition small molecules.
   1. Place spheres in rotation at 37 °C (60 rpm, orbital shaker) and change half-medium freshly supplemented every 2-3 days.
4. Day 3: To enhance neural induction and convert to neural progenitors with a midbrain identity, supplement the medium with 3 µM Glycogen synthase kinase-3 beta (GSK-3β) inhibitor, which activates the Wnt/β-catenin pathway. Maintain GSK-3β inhibitor in the medium up to day 13. Split into two new tissue-treated 6 well plates to reduce both sphere density per well and avoid sphere aggregation.
   NOTE: At Day 8, most of the cells should be positive for Nestin.
5. Day 8: Start the neural maturation: replace regionalization factors SHH, FGF8, smoothened agonist, and dual-SMAD inhibition cocktail with 0.5 mM dibutyryl cyclic adenosine monophosphate (cAMP) (to favor maturation), 20 nM inhibitor of histone deacetylase (for cell cycle exit), 1 µM γ-secretase inhibitor and growth factors, 10 ng/mL glial cell line-derived neurotrophic factor (GDNF), 10 ng/mL brain-derived neurotrophic factor (BDNF), 1 ng/mL transforming growth factor β3 (TGFβ3), and 5 ng/mL fibroblast growth factor 20 (FGF20) (both favor dopaminergic [DA] progenitor survival). Change the medium every 2-3 days.
6. Day 21: Generate the neural organoid: seed around 100 neurospheres under air-liquid interface conditions on polytetrafluoroethylene (PTFE membrane, 6 mm diameter). Transfer the membrane to a culture plate insert (0.4 mm) and add 1.2 mL of neural maturation medium used for neurosphere differentiation as previously described.
   1. Stop any rotation from this step. Change the medium every 2-3 days until the required differentiation time point is achieved.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
6-well plate (6-well plate)	Falcon / Corning	07-201-588
Aggrewell 400 (Microwell culture plates )	StemCell Technologies	34421
B27 supplements (B27)	Life Technologies / Invitrogen	1238	For both protocol, stock solution 100x, final solution 1x
Brain-derived neurotrophic factor (BDNF)	Cell Guidance	GFH1-2	For both protocol,  stock solution 100 µg/mL in pure H2O, final solution 20 ng/mL
CHIR-99021 (GSK-3β inhibitor )	Axon Medchem	ct99021	For Dopaminergic protocol,  stock solution 7.5 mM in DMSO, final solution 3 µM
Compound E a γ–secretase inhibitor (γ–secretase inhibitor)	Calbiochem	CAS 209986-17-4	For both protocol (gamma- secretase inhibitor XXI),  stock solution 5 mM in DMSO, final solution 1 µM
Dibutyryl cyclic-AMP (Dibutyryl cAMP)	Sigma	D0627	For Dopaminergic protocol,  stock solution 0.5 M in DMSO, final solution 0.5 mM
Dulbecco's Modified Eagle Medium Mixture F-12 (DMEM-F12)	Gibco	11320033	For cell culture, ready to use
EDTA 0.1 mM (EDTA)	Life Technologies	AM9912	For cell culture, ready to use
Fibroblast Growth Factor 20 (FGF20)	Peprotech	100-41	For Dopaminergic protocol,  stock solution 100 µg/mL in pure H2O, final solution 5 ng/mL
fibroblast growth factor 8  (FGF8)	Peprotech	GFH176-5	For Dopaminergic protocol,  stock solution 100 µg/mL in pure H2O, final solution 100 ng/mL
Glial cell-derived neurotrophic factor (GDNF)	Cell Guidance	GFH2-2	For both protocol,  stock solution 100 µg/mL in pure H2O, final solution 20 ng/mL
Hydrophilic polytetrafluoroethylene membrane (PTFE membrane)	BioCell-Interface	Discontinued
LDN-193189 (BMP inhibitor)	Axon Medchem /Stemgen	04-0072-02 /1509	Dual/Smad,  stock solution 5 mM in DMSO, final solution 0.5 µM
L-glutamine (L-glutamine)	Gibco	25030081	L-Glutamine (200 mM), stock solution 200 mM, final solution 2 mM
Millicell-CM Culture plate insert (0.4 µm) (Culture plate insert)	Millipore	PICM03050
MS Orbital Shaker, MS-NOR-30 (Orbital shaker)	Major Science	MS-NRC-30
N2 supplements (N2)	Invitrogen	17502-048	For GIC culture, stock solution 100x, final solution 1x
Neurobasal (Neurobasal)	Life Technologies / Gibco	21103049	Maintenance and maturation embryonic neuronal cell populations , ready to use
Non-Essential Amino Acids (NEAA)	Gibco	11140	Non-essential Amino Acids 100X, stock solution 100x, final solution 1x
Nutristem  (hESC medium )	Biological Industries	05-100-1A	Stem cell media, ready to use
Penicilin / Streptomycin  (Penicilin / Streptomycin )	Life Technologies / Gibco	15140122	For cell culture, stock solution 5 mg/mL, final solution 50 µg/mL
Phosphate Buffered Saline without Ca2+/Mg2+  (PBS without Ca2+/ Mg2+ )	Life Technologies	14190250	For cell culture, ready to use
Purmorphamin (smoothened agonist)	Calbiochem	SML0868	For Dopaminergic protocol,  stock solution 10 mM in DMSO, final solution 2 µM
Rho-associated Kinase Y-27632 (ROCK)	Abcam Biochemicals	ab120129-1	Rock Inhibitor,  stock solution 50 mM in DMSO, final solution 10 µM
SB-431542 (TGFβ/Activin/Nodal inhibitor )	Ascent	Asc- 163	Dual-Smad,  stock solution 50 mM in DMSO, final solution 10 µM
Sonic Hedgehog (SHH)	Cell Guidance	GFH168-5	For Dopaminergic protocol,  stock solution 100 µg/mL in pure H2O, final solution 100 ng/mL
StemPro Accutase (hESC enzymatic solution)	Gibco	A11105-01	hESC enzymatic solution, ready to use
T150 flask (T150 flask)	Falcon	08-772-1F
Transforming Growth Factors beta 3 (TGFβ3)	Cell Guidance	GFH109-2	For Dopaminergic protocol, stock solution 100 µg/mL in pure ethanol , final solution 1 ng/mL
Trichostatine A (inhibitor of histone deacetylase )	Sigma	T8552	For Dopaminergic protocol,  stock solution 100 µM in DMSO, final solution 20 nM
TrypLE (recombinant enzymatic solution)	Invitrogen	12604021	recombinant enzymatic solution, ready to use
Trypsin 0.25% (enzymatic solution)	Life Technologies	15050065	enzymatic solution, ready to use
X-vivo (serum free medium)	Lonza	BE04-743Q	serum free medium, ready to use