Chir99021

hiPSCs (414C2 line) were cultured for 12 days in adhesion cultures with mouse embryonic fibroblasts and then allowed to form embryonic bodies in floating culture for 30 days. Aggregated cells were differentiated into NS/PCs derived from hiPSC-NS/PCs using various factors during each day of the incubation period (Okada et al., 2008 (link)).
hiPSCs (201B7 line) were pretreated for 6 days with 3 μM SB431542 (Tocris, 301836-41-9) and 150 nM LDN193189 (StemRD, 1062368-24-4). The cells were then dissociated and seeded at a density of 1×10⁵ cells per milliliter in ultra-low-attachment culture dishes (Corning) in neuronal induction medium consisting of medium hormone mix (MHM) (Okada et al., 2008 (link)) supplemented with 2% B27 supplement without vitamin A (Thermo Fisher, 17504-044), 20 ng/mL FGF-2, 10 μM Y27632 (Nacalai Tesque, 08945-71), 1 μM retinoic acid (RA; Sigma, R2625-1G), 3 μM CHIR 99021 (Reprocell, 04-0004) and 10 μM SB431542 (Calbiochem, 301836-41-9) in a hypoxic and humidified atmosphere (4% O₂, 5% CO₂) for 6 days. The formed neurospheres were passaged by dissociation into single cells and then cultured in slightly modified neuronal induction medium, MHM supplemented with 2% B27 without vitamin A, 20 ng/mL FGF-2, 10 μM Y27632, and 1 μM RA for 6 days under 4% O₂ (hypoxic) conditions.

viEC were differentiated from iPSCs via previously established protocol (Atchinson et al., 2020). On day 0, iPSCs were harvested with Accutase and reseeded on Matrigel-coated six-well plate at a cell density of 47,000 cells/cm². On day 1, mTeSR1 media was replaced with N2B27 media, which contains neurobasal media (Invitrogen) and DMEM/F12 Glutamax (Invitrogen) in 1:1 ratio with N2 (100×) (Invitrogen) and B27 (-) Vitamin A (Invitrogen). The media was then supplemented with 8 µM CHIR-99021 (Reprocell, Inc., Beltsville, MD, USA), and 25 ng/mL hBMP4 (VWR International LLC, Radnor, PA, USA). Cells were cultured in N2B27 media without media changing for 3 days to achieve lateral mesoderm induction.
On Day 4, N2B27 media was replaced with endothelial induction media which contained StemPro-34 SFM media (Invitrogen) supplemented with Glutamax (Invitrogen) and Pen-Strep (Gibco Cell Culture) in 100:1:1 ratio, and supplemented with 2 µM forskolin (Abcam Inc., Cambridge, UK) and 200 ng/mL VEGF165 (Invitrogen). Media was changed daily from Day 4 to Day 6 and conditioned media was collected on Day 5 to Day 7. viEC were then dissociated on Day 7 with Accutase, and sorted to harvest CD144+ (VE-Cadherin) and CD31+ (PECAM-1) cells, as described below.

EGC was established as described previously^{42 (link)}. Briefly, sorted PGCs were cultured on Mitomycin C (Kyowa Kirin, Japan)-inactivated Sl/Sl4-m220 feeder cells^{43 (link)} in DMEM (D6429, Sigma-Aldrich) supplemented with 15% KSR (10828010, Thermo Fisher Scientific), 1% Glutamax (35050061, Thermo Fisher Scientific), 1% non-essential amino acids (11140050, Thermo Fisher Scientific), 0.1 mM β-mercaptoethanol (198-15781, FUJIFILM Wako, Japan), 1000 unit/mL mLIF (ESG1107, Merck Millipore), and 12 ng/mL bFGF (450-33, PeproTech, NJ) until day 3. On day 3, the media was replaced with fresh media containing 1 μM PD325901 (04-0006, ReproCELL), 1 μM CHIR99021 (04-0004, ReproCELL, Japan) and 250 nM A83-01(039-24111, FUJIFILM Wako, Japan).
Cells were cultured under 20% O₂ and 5% CO₂ at 37 °C and the media were refreshed after 1, 3, and 5 days of culture. The efficiency of colony formation was determined after 7 days in culture as the number of colonies per seeded cell in a culture well.

H1 human embryonic stem cells and E14 mouse embryonic stem cells were obtained from the 4D Nucleome Consortium and cultured according to 4D Nucleome Consortium-approved protocols (https://www.4dnucleome.org/). In brief, H1 cells were grown at 37 °C under 5% CO₂ on Matrigel (Corning, 354277)-coated dishes. Cells were maintained in complete mTeSR medium prepared from basal medium (Corning, 85851) with 5× supplement (Corning, 85852). Medium was replaced daily. Cell passage numbers were kept below P10. E14 cells were cultured on plates coated with 0.1% gelatin (EMD, SF008) in serum-free 2i/LIF medium; this medium was made from base medium (1:1 mixture of NeuroBasal medium (Gibco, 21103-049) and DMEM/F12 medium (Gibco, 11320-033) supplemented with 0.5× N2 supplement (Gibco, 17502-048), 0.5× B27 supplement (Gibco, 17504-044) and 0.05% bovine serum albumin (BSA) fraction V (Gibco, 15260-037)), supplemented with 1 µM PD0325901 (Reprocel, 04-0006-02C), 3 µM CHIR99021 (Reprocell, 04-0004-02C), 0.15 mM monothioglycerol (Sigma, M6145-25ML) and 1,000 U ml⁻¹ LIF (Cell Guidance Systems, GFM200). Medium was replaced daily, and cell passage number was kept below P10.