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).
   3. Remove the medium and seed hESCs to a density of 6.5 x 104 cell/cm².
2. Maintain H1 (hESC cell line) in hESC medium and 1% penicillin/streptomycin.
3. Pass the cells with enzymatic procedure: add 7.5 mL of enzymatic solution to a T75 cm2 flask over a period of 1-2 min at 37 °C. Once cells are completely detached, add 7.5 mL of DMEM-F12 then centrifuge for 5 min at 300 x g. To allow for better survival, re-plate cells at the desired density onto extracellular matrix-coated dishes, in the same medium containing Rho-associated protein kinase (ROCK) inhibitor (10 µM) for 24 h.

2. hESC-derived neural organoids

1. 24 h before starting the 3D culture, replace the hESC medium with a serum-free medium supplemented with 10 µM ROCK inhibitor (both components are necessary to support cell survival and spontaneous neurosphere formation during the aggregation phase in a microwell plate). The cells should be at 60% confluency. The next day (day 0), detach hESC colonies as single cells: remove the medium, rinse with PBS without Ca²⁺/Mg²⁺, add 5 mL of enzymatic dissolution solution, and incubate at 37 °C for 1-2 min.
2. Collect the cells in serum-free medium supplemented with 10 µM of ROCK inhibitor and centrifuge them at 300 x g for 5 min. Remove the supernatant and count the cells in 10 mL of serum-free medium supplemented with 10 µM of ROCK inhibitor.
3. In parallel, rinse the microwell plate with 2 mL of serum-free medium per well and centrifuge the plate at 1200 x g for 5 min to remove all bubbles, which can prevent neurosphere formation.
4. Prepare 28.2 x 10⁶ of cells in 12.5 mL of serum-free medium supplemented with 10 µM ROCK inhibitor. Dispense 1000 cells/microwell. Centrifuge the cells at 300 x g for 5 min and place the plate in the incubator at 37 °C overnight (maximum 36 h). For example, to obtain 30 human neural organoids, use one T150 flask at 70%-80% of confluence (about 30 million cells).
5. The next day (day 1), collect the spheres (with a P1000) and place them in a 6 well plate. In each well, add 2 mL of B27 medium and DMEM-F12 GlutaMAX and Neurobasal medium (mix at 1:1), supplemented with 1% B27 supplements and 1% non-essential amino acids (NEAA). To promote fast neural induction, supplement the medium with a dual-SMAD (Sma and Mad Related Protein) inhibition cocktail composed of 10 µM TGFβ(Transforming Growth Factor-beta)/Activin/Nodal inhibitor and 0.5 µM bone morphogenic protein (BMP) inhibitor. From this step forward, the spheres are cultured in rotation (60 rpm, orbital shaker). The rotation is critical to prevent the spheres from sticking together or to the plate.
6. Change the medium every 2-3 days: bend the plate and let the spheres fall down for 5 min, remove half of the medium (2 mL), and add 2 mL of fresh B27 medium supplemented with growth factors and inhibitors. Do not centrifuge the spheres.
7. Perform neural induction according to the following time course:
   1. From days 1-4, culture the spheres in B27 medium supplemented with dual-SMAD. The dual-SMAD inhibition cocktail (10 µM TGFβ/Activin/Nodal inhibitor and 0.5 µM BMP inhibitor) promotes neural induction.
   2. From days 4-11, promote proliferation of hESC-derived neural rosettes (into the spheres), by adding 10 ng/mL epidermal growth factor (EGF) and 10 ng/mL basic fibroblast factor (bFGF) to the B27 medium supplemented with dual-SMAD cocktail.
      NOTE: On day 11, most cells should be positive for Nestin.
   3. From days 11-13, culture the spheres in B27 medium supplemented with 0.5 µM BMP inhibitor.
   4. From days 13-21, culture the spheres in B27 medium supplemented with 10 ng/mL glial derived neurotrophic factor (GDNF), 10 ng/mL brain derived neurotrophic factor (BDNF) and 1 µM of γ-secretase inhibitor. GDNF and BDNF promote neuronal and glial differentiation. The γ-secretase inhibitor allows for greater neural maturation.
   5. On day 21, plate the spheres (about 1,000 spheres) on a hydrophilic polytetrafluoroethylene (PTFE) membrane (6 mm diameter, 0.4 µm) deposited on a culture plate insert designed for 6 well plates. Stop any rotation from this step. The presence of rosettes, observed with a bright-field microscope, indicates the initiation of neural differentiation. The neural rosettes can be observed 2-3 days after plating spheres on the PTFE membrane.
   6. Add 1 mL of B27 medium supplemented with growth factors and inhibitors (as followed) to each well underneath the membrane insert, every 2-3 days (usually on Monday, Wednesday and Friday), for a following 3 weeks of differentiation.
   7. From days 21-25, cultivate human neural organoids in the same neural maturation medium.
   8. From days 25-28, only complement B27 medium with 1 µM γ-secretase inhibitor.
   9. From days 28-39, stop adding the γ-secretase inhibitor and continue human neural organoid culture in the B27 medium only.
      NOTE: After 3 weeks, neural organoids are ready to use for GIC implantation. Along the neural maturation, a decrease of neural immature marker Nestin and increase of mature neural markers β3-tubulin and GFAP were observed.

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