Mtesr1 media

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).

hPSCs (H9 hESC (WA09) line from WiCell), iSOX17 H9 line and knockout SOX17 H9 line were maintained and passaged on Matrigel in mTeSR1 media (WiCell). The cell lines were differentiated on collagen IV (ColIV)-coated plate (Uenishi et al., 2014 (link)). Cell lines were plated at a density of 5,000 cells/cm² onto 6 well plates with E8 media containing 10 μM Rock inhibitor (Y-27632, Cayman Chemicals). The following day, the media was changed 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 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 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 cultured in normoxia (5% CO₂, 20% O₂). DOX (Sigma) was added to cultures on day 2 of differentiation at concentration of 2 μg/ml.

H9 (WA09, WiCell) and H1 OCT4-EGFP (WiCell) human embryonic stem cells (hESC) were cultured in mTESR1 media (cat. # 85850, StemCell Technologies) on six-well plates pre-coated with Matrigel (cat. # 354277, Corning). hESCs were passaged every 4–6 days at a split ratio of 1:6 to 1:12 using ReLeSR (cat. # 05872, StemCell Technologies) and mTESR1 was supplemented with 10 μM ROCK inhibitor Y-27632 (cat. # 1254, Tocris) on the first day of passage. Media was replenished daily. H9-derived neural stem cells (NSC-H9, WiCell) were cultured in StemPro NSC SFM complete medium (cat. # A1050901, ThermoFisher) on six-well plates pre-coated with Geltrex (cat. # A1569601, ThermoFisher). NSCs were passaged with StemPro Accutase using a ratio of 1:3 to 1:6. Cranial neural crest cells (CNCCs) were generated from H9-hESCs as previously described^{26 (link)}. CNCCs were harvested at passages 4–5 for all subsequent downstream experiments. For each biological replicate, different passages were used for hESC and NSCs whereas different differentiations were used for CNCCs. All stem cells were cultured at 37°C in 20% O₂ and 5% CO₂. hESCs work was authorised by the Steering Committee for the UK Stem Cell Bank and for Use of Stem Cells (MRC).