1. Maintenance of hESCs
· Human embryonic stem cells (hESCs) are cultured on Matrigel coated tissue culture plates.
· hESC colonies are passaged every 6 to 7 days when they achieve around 80% confluence.
· All cultures are checked regularly for mycoplasma and chromosomal abnormalities.
2. Generation of hESCs expressing inducible ETV2
1. To generate hESCs expressing inducible ETV2, doxycycline-inducible (BC4) dCAS9-mCherry and rTTA (addgene plasmid # 73497) cassette was introduced into the AAVS1 locus of HES-3 NKX2-1GFP/w.
a. 2 million HES-3 NKX2-1GFP/w hESCs were electroporated with 8 µg donor plasmid, 1 µg AAVS1 TALEN-L and 1 µg AAVS1 TALEN-R by using Amaxa nucleofector device (AAB-1001, Lonza, program A-023).
b. Cells were seeded in mTeSR1 plus ROCK inhibitor Y-27632 (10 µM).
c. After 3 days, G-418 (Thermo Fisher Scientific) was applied for 7 days (400 µg/ml for the first 3 days and 300 µg/ml for the next 4 days) to obtain stable colonies.
d. A single isogenic colony was picked and expanded. This hESC line was named BC4-hESCs.
2. After generating the tetracycline-inducible lentivirus for ETV2 (FUW-tetO-ETV2 6), BC4-hESCs were infected with the virus at multiplicity of infection (MOI) ~ 4.
3. 24 hours later, infected hESCs were washed with 1X PBS three times. Then, fresh mTeSR1 media (Stem Cell Technologies) was added and cells were cultured for 5 more days.
3. Formation of embryoid bodies (EBs) and induction of neural differentiation and ETV2 expression
Cortical organoids (hCOs) with vasculature were generated by mixing ETV2-infected BC4 and non-infected parental HES3 hESCs (differentiation scheme is shown in Figure 1).
1. Aspirating the media and washing cells once with 1 ml DMEM-F12 medium was followed by the addition of 1 ml of 1x Accutase.
2. After 10 minutes of incubation at 37 °C, single cell suspension was confirmed under a microscope and transferred into a 15ml-falcon tube containing 5 ml DMEM-F12.
3. Cells were collected by centrifugation for 3 min at 1100 rpm.
4. After aspirating the supernatant, cells were resuspended in 1 ml neural induction medium (DMEM-F12, 15% (v/v) KSR, 1% (v/v) Glutamax, 1% (v/v) MEM-NEAA, 100 µM β-Mercaptoethanol, 10 µM SB-431542, 100 nM LDN-193189, 2 µM XAV-939).
5. Live cells were counted using a hematocytometer.
6. Cells were diluted with neural induction media to a final concentration of 9000 cells/150 ml (7200 parental HES-3 hESC cells and 1800 ETV2 infected hESC cells were combined for each 150 ml). Then, 50 µM ROCK inhibitor Y27632 and 5% (v/v) heat-inactivated FBS (Life Technologies) were added.
7. 150 ml of single cell suspension was added into each well of a U-bottom-ultra-low attachment 96-well plate (Day 0 of differentiation).
8. Basal activation of ETV2 was started on day 2 by adding 0.5 µM dox, whereas FBS and Y27632 were removed on days 2 and 4, respectively.
9. On days 4, 6, and 8, the neural induction media was replenished via removing 125 ml media and adding 150 ml fresh media.
4. Patterning of cortical organoids with functional vascular-like network
1. At day 10, organoids were transferred to ultra-low-attachment 6-well plates in hCO patterning media with minus vitamin A (1:1 mixture of DMEM-F12 and Neurobasal media, 0.5% (v/v) N2 supplement, 1% (v/v) B27 supplement without vitamin A, 0.5% (v/v) MEM-NEAA, 1% (v/v) Glutamax, 50 µM β-Mercaptoethanol, 1% (v/v) Penicillin/Streptomycin and 0.025% Insulin) for spinning culture and the media was changed every other day. Basal activation of ETV2 was preserved by adding 0.5 µM dox.
2. On day 10, 12, 14, and 16, 2.4 ml medium was removed from each well and 2.5 ml hCO patterning media with 0.5 µM dox was added.
3. The spinning culture was started by placing the plate on an orbital shaker at 80 rpm inside the incubator.
4. Maturation of vascularized cortical organoids
1. After day 18, the media was changed to hCO maturation media (1:1 mixture of DMEM-F12 and Neurobasal media, 0.5% (v/v) N2 supplement, 1% (v/v) B27 supplement with vitamin A, 0.5% (v/v) MEM-NEAA, 1% (v/v) Glutamax, 50 µM β-Mercaptoethanol, 1% (v/v) Penicillin/Streptomycin, 0.025% Insulin, 20 ng/ml BDNF and 200 µM ascorbic acid).
2. Final activation of ETV2 expression was performed from day 18 by adding 2 µM dox continuously in the media.
3. Media was changed every 4 days after day 18.