Differentiation and Maturation of Human Embryonic Stem Cells into Neural Organoids

Abstract

Source: Cosset, E., et al. Human neural organoids for studying brain cancer and neurodegenerative diseases. J. Vis. Exp. (2019)

In this video, a stepwise protocol for Human embryonic stem cells into neural organoids is demonstrated. The method involves inducing neuronal rosette formation, followed by controlled maturation and final culturing on a hydrophobic membrane to achieve fully developed neural organoids.

Protocol

1. Maintenance and culture of undifferentiated human embryonic stem cells (hESCs)

1. Perform maintenance and expansion of hSECs on feeder-free conditions by pre-coating dishes with a specific extracellular matrix.
   1. Thaw 300 µL of extracellular matrix at 4 °C (typical range concentration 18-22 mg/mL, keep on ice) and gently mix with 15 mL of cold DMEM(Dulbecco's Modified Eagle Medium )medium to avoid premature gelation of the extracellular matrix. Add 7.5 mL of the extracellular matrix to both T150 flasks.
   2. Incubate the dishes coated with extracellular matrix at 37 °C for at least 1 h (maximum overnight).

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
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
Dimethyl Sulfoxide Pure (DMSO)	Sigma-Aldrich	C6164	Compounds solvent, ready to use
Dulbecco's Modified Eagle Medium (DMEM)	Life Technologies	12491-015	For cell culture, ready to use
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
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
Matrigel (extracellular matrix)	BD Biosciences	354277	hESC-qualified Matrix, stock solution 18-22 mg/mL, final solution 180-220 µg/mL
Millicell-CM Culture plate insert  (0.4 µm) (Culture plate insert)	Millipore	PICM03050
Monoclonal Anti-β-Tubulin III antibody	Sigma	T8660	1/1000 dilution
MS Orbital Shaker, MS-NOR-30 (Orbital shaker)	Major Science	MS-NRC-30
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
Nurr1 Antibody (M-196)	Santa Cruz	Sc-5568	1/100 dilution
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
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
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
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 u