• hESC medium
To prepare 500 ml of hESC medium, 400 ml of DMEM/F12, 100 ml of KOSR, 15 ml of FBS, 5 ml of GlutaMAX, 5 ml of MEM-NEAA, and 3.5µl of 2-ME were mixed together and sterile-filtered with a 22µm filter bottle. FGF2/bFGF and/or RI were added freshly just before usage. Medium can be stored at 4 °C for up to 2 weeks after preparation.
• Neural Induction (NI) medium
To prepare 500 ml of NI medium, 500 ml of DMEM/F12, 5 ml of N2 supplement, 5 ml of GlutaMAX, 5 ml of MEM-NEAA, and 500 µl of Heparin solution were mixed and sterile-filtered using a 22 µm filter bottle. Medium can be stored at 4 °C for up to 2 weeks after preparation.
• Differentiation Medium
To prepare 500 ml of differentiation medium, 250 ml of DMEM/F12, 250 ml of Neurobasal, 2.5 ml of N2 supplement, 5 ml of B27 (with or without vitamin A supplement), 125 µl of Insulin, 175 µl of a 1:100 solution of 2-ME (in DMEM/F12), 5 ml of GlutaMAX, 2.5 ml of MEM-NEAA, and 5 ml of P/S solution were mixed and sterile-filtered using a 22 µm filter bottle. Medium can be stored at 4 °C for up to 2 weeks after preparation.
• FGF2/bFGF stock solution
To prepare FGF2/bFGF stock solution (10 µg/ml), 50 µg of FGF2/bFGF was reconstituted in 5 ml PBS +0.1% BSA, and aliquoted into 50 or 100 µl aliquots. Aliquots can be stored at -20 °C for up to 1 year.
• Heparin stock solution:
Heparin stock solution (1 mg/ml) were prepared in PBS, and stored at -20oC for up to 1 year.
• Rock Inhibitor (RI) stock solution
To prepare RI stock solution, 5 mg of RI was reconstituted in 2.96 ml of H2O, and aliquoted into 0.5-1 ml aliquots. Aliquots can be stored at -20 °C.
• Gelatin solution for coating
Gelatin solution was prepared as 0.1% wt/vol in H2O. For 500 ml solution, 0.5 g of Gelatin were reconstituted in H2O at 50 °C, and sterile-filtered with a 22µm filter bottle. Gelatin solution can be stored at 4 °C for up to 1 year.
• Collagenase IV solution
Collagenase IV solution was prepared as 1 mg/ml in DMEM/F-12 medium, and sterile-filtered with a 22µm filter. Aliquots can be stored at -20 °C for up to 6 months.
• Dispase solution
Dispase solution was prepared as 0.5 mg/ml in DMEM/F-12 medium, and sterile-filtered with a 22µm filter. Aliquots can be stored at -20 °C for up to 6 months.
A) Cell maintenance
• FF hESCs
FF hESCs were cultured in a feeder-free manner on Matrigel-coated plate with mTeSR medium in a 5% CO2 incubator at 37 °C. For coating, low-growth-factor Matrigel (0.5 mg per 6-well plate) was dissolved in ice-cold DMEM/F12. FF hESCs were routinely splitted using 0.5 mM EDTA in PBS.
• FD hESCs
FD hESCs were maintained with hESC medium containing 20 ng/ml FGF2/bFGF in a 5% CO2 incubator at 37 °C on the gelatin- and MEF (1.87×105 cells/well)-coated 6-well cell culture plates. FD hESCs were routinely passaged using collagenase IV solution (0.1% wt/vol in H2O).
B) Generation of Cerebral organoids
Day 0, Embryoid body (EB) formation
- Single cell suspension was prepared as described previously using hESCs cultured in either feeder-independent or feeder-dependent manner (Lancaster et al., 2013).
Cell density was counted using automated cell counter. Nine thousand live cells/well in 150 µl hESC medium containing RI (1:100) and low FGF2/bFGF (1:2500, 4 ng/ml) were seeded in a 96-well low-attachment U-bottom cell culture plate.
Day 3, Exchanging medium
The medium was exchanged with fresh hESC medium without RI and FGF2/bFGF.
Day 5 or 6, Neural induction (NI)
hESC medium was replaced by NI medium to induce neural lineage differentiation when the sizes of EBs are more than 500 µm.
Day 5 or 6 to Day 11 or 12, Neuroectoderm expansion
NI medium was exchanged every second days with fresh NI medium for 6 days.
\! Attention: EBs with expanded radialized neuroepithelial structure were selected for the further procedure.
Day 11 or 12, Nucleofection of plasmid cocktails to introduce gene mutations/amplifications
- Nucleofector solution is prepared according to manufacturer’s protocol by mixing 82 µl of solution 1 and 18 µl of supplement 1 for one reaction. Maximum 5 µg of plasmid cocktail, including 500 ng of transposase expression vector pCAG-SB100X, 1 µg of transposon vector for pCAG-eGFP, and 1 µg of each transposon vectors to express oncogene and/or CRISPR-Cas9 vectors were added into nucleofector solution.
About 15 EBs were collected, washed with PBS, resuspended with nucleofection solution with plasmid cocktails, and transferred into nucleofection cuvettes.
! Attention: a) Using widened pipette tips to transfer EBs. b) Gentle operation is required for entire procedure to avoid the damage of EBs. c) More than 15 EBs performed with nucleofection using NucleofectorTM 2b (Lonza) will significantly reduce the nucleofection efficiency.
- EBs in the nucleofection cuvettes were nucleofected under program A-023 using NucleofectorTM 2b.
EBs in the nucleofection cuvettes were gently added 1 ml of NI medium, and poured out into 6-cm dishes containing NI medium, and incubated in a 5% CO2 incubator at 37 °C for overnight.
Day 12 or 13, EB embedding
- EBs were embedded into matrigel based on previous described procedure. Briefly, EBs were transferred onto parafilm in 6- or 10-cm petri dish. The excess medium was carefully removed.
- EBs were embedded into a droplet of matrigel, and adjusted into the center of the droplet before the matrigel droplet solidifies. Embedded EBs then were incubated into 37 °C incubator for 20 min to solidify the matrigel.
- Differentiation medium (-A) was added onto EBs to wash embedded EBs off the parafilm. The parafilm was discarded from the petri dish.
Embedded EBs were cultured in a 5% CO2 incubator at 37 °C without shaking.
Day 16 or 17, Differentiation of cerebral organoids
- Differentiation medium (-A) were replaced by differentiation medium (+A).
The dish was transferred onto an orbital shaker in a 5% CO2 incubator at 37 °C.
Day 16 or 17 to Day 40, Initiation of neoplastic cerebral organoids
Medium was changed twice a week with differentiation medium (+A).
Day 40 and later, Neoplastic cerebral organoids selection for further analysis
- Neoplastic cerebral organoids with overgrowth of GFP-positive mutated cells will be collected for further analysis.
- Differentiation medium (+A) was exchanged twice per week for neoplastic cerebral organoid culture to the desired age of collection for subsequent analyses such as immunofluorescence staining, RNA-seq, renal implantation, drug testing, and viral infection.