Fibroblast growth factor 2 (fgf2)

hPSCs were maintained and passaged on Matrigel in mTeSR1 media (WiCell). Hematopoietic differentiation was performed on collagen IV (ColIV)-coated plates in chemically defined serum-free medium as described.¹⁴ (link) The iSOX18 H1 line from hPSCs (H1 hESC line from WiCell) was maintained and passaged on Matrigel in mTeSR1 media (WiCell). The cell lines were differentiated on a collagen IV (ColIV)-coated plate.¹⁴ (link) To initiate differentiation, cells were plated at 5,000 cells/cm² onto 6 well plates with E8 media and 10 μM Rock inhibitor (Y-27632, Cayman Chemicals). This media was changed the following day to IF9S media with 50 ng/mL FGF2 (PeproTech), 50 ng/mL BMP4 (PeproTech), 15 ng/mL Activin A (PeproTech), and 2 mM LiCl (Sigma), and cells were cultured in hypoxia (5% CO₂, 5% O₂). On day 2, the media was changed to IF9S media with 50 ng/mL FGF2, 50 ng/mL VEGF (PeproTech), and 2.5 μM TGF-β inhibitor (SB-431542, Cayman), and cells were cultured in hypoxia (5% CO₂, 5% O₂). On days 4 and 6, the media was changed to IF9S media with 50 ng/mL FGF2, 50 ng/mL VEGF, 50 ng/mL TPO (PeproTech), 50 ng/mL IL-6 (PeproTech), 20 ng/mL SCF (PeproTech), and 10 ng/mL IL-3 (PeproTech), and cells were cultured in normoxia (20% CO₂, 5% O₂).

Pluripotent cells were differentiated as discussed previously
[14 (link),18 ]. Briefly, pluripotent cells cultured on E-cadherin-IgG Fc were harvested using Accutase (Millipore) and plated onto 6-well tissue culture-treated plates pre-coated with 2 mg/ml Matrigel (Geltrex; Invitrogen). Typically, one 100 mm dish of cells cultured on E-cadherin-IgG Fc provided enough cells for 2 wells of a 6-well plate. Approximately 24 hours after seeding the cells onto Matrigel, when the cells were 85-95% confluent, differentiation was initiated by culture for 5 days with 50 ng/ml Activin A (R&D Systems) in RPMI/B27 (without Insulin) supplement (Invitrogen) under ambient oxygen/5%CO₂. In addition, we included 10 ng/ml BMP4 (Peprotech) and 20 ng/ml FGF-2 (Invitrogen) for the first 2 days. This resulted in reproducible differentiation into definitive endoderm at efficiencies of greater than 80%. Cells were cultured for 5 days with 20 ng/ml BMP4 (Peprotech)/10 ng/ml FGF-2 (Invitrogen) in RPMI/B27 (containing Insulin) under 4%O₂/5%CO₂, then 5 days with 20 ng/ml HGF (Peprotech) in RPMI/B27 (containing Insulin) under 4%O₂/5%CO₂, and finally for 5 days with 20 ng/ml Oncostatin-M (R&D Systems) in Hepatocyte Culture Media (Lonza) supplemented with SingleQuots (without EGF) in ambient oxygen/5% CO₂.

Cortical organoids were generated as previously described (Trujillo et al, 2019). Briefly, PSCs cultured for approximately seven days were dissociated with 1:1 Accutase (Life Technologies):PBS, and cells were plated into a 6‐well plate (4 × 10⁶ cells/well) in mTeSR1 supplemented with 10 µM SB (Stemgent), 1 μM Dorso (R&D Systems), and 5 µM Y‐27632 (EMD‐Millipore, Burlington, MA, USA) and cultured hereafter in shaker suspension (95 rpm at 37°C). Formed spheres were fed mTeSR1 (with 10 μM SB and 1 μM Dorso) for three days followed by Media1 [Neurobasal (Life Technologies), 1× Glutamax (Life Technologies), 2% Gem21‐NeuroPlex (Gemini Bio‐Products, Sacramento, CA, USA), 1% N2‐NeuroPlex (Gemini Bio‐Products), 1% non‐essential amino acids (NEAA; Life Technologies), 1% P/S (Life Technologies), 10 μM SB, and 1 μM Dorso] for six days, every other day; Media2 (Neurobasal, 1× Glutamax, 2% Gem21, 1% NEAA, and 1% P/S) with 20 ng/ml FGF‐2 (Life Technologies) for seven days, daily; Media2 with 20 ng/ml each of FGF‐2 and EGF (PeproTech, Rocky Hill, NJ, USA) for 6 days, every other day; and Media2 with 10 ng/ml each of BDNF, GDNF, and NT‐3 (all PeproTech), 200 μM L‐ascorbic acid (Sigma‐Aldrich, St. Louis, MO, USA), and 1 mM dibutyryl‐cAMP (Sigma‐Aldrich) for 6 days, every other day. Cortical organoids were subsequently maintained indefinitely in Media2 without supplementation.