Ldn 193189

Except where specified, reset cells were ʻre-primed' before initiating differentiation. Cells were plated on Geltrex in t2iLGö and after 48 h the medium was changed to E8. Cultures were maintained in E8, passaging at confluence. Lineage-specific differentiation was initiated between 25 and 44 days.
Definitive endoderm was induced according to Loh et al. (2014) (link). Cells were cultured in CDM2 medium (in-house according to Loh et al., 2014) supplemented with 100 ng/ml activin A (produced in-house), 100 nM PI-103 (Bio-Techne, 2930), 3 µM CHIR99021, 10 ng/ml FGF2, 3 ng/ml BMP4 (Peprotech) for 1 day. For the next 2 days the following supplements were applied: 100 ng/ml activin A, 100 nM PI-103, 20 ng/ml FGF2, 250 nM LDN193189.
For lateral mesoderm induction (Loh et al., 2016 (link)), cells were treated with CDM2 supplemented with 30 ng/ml activin A, 40 ng/ml BMP4 (Miltenyi Biotech, 130-098-788), 6 µM CHIR99021, 20 ng/ml FGF2, 100 nM PI-103 for 1 day, then with 1 µM A8301, 30 ng/ml BMP4 and 10 µM XAV939 (Sigma-Aldrich).
For neural differentiation via dual SMAD inhibition (Chambers et al., 2009 (link)), cells were treated with N2B27 medium supplemented with 500 nM LDN193189 (Axon, 1509) and 1 μM A 83-01 (Bio-Techne, 2939) for 10 days, then passaged to plates coated with poly-L-ornithine and laminin and further cultured in N2B27 without supplements.

The H9 hESCs (WA09, purchased from the WiCell research Institute, female, RRID: CVCL_9773) colonies were maintained on matrigel‐coated 6‐well plates (Corning) in E8 flex medium [E8 basal medium (Gibco) supplemented with E8 supplement Flex and 5 U ml⁻¹ Penicillin−Streptomycin]. The colonies were passaged twice a week (in 1:3 split ratio) using 0.5 mM EDTA (Gibco). To induce NPC, the hESCs were harvested into single‐cell suspensions by accutase (Sigma) and seeded at 2.5 million cells per well of 6‐well plate in the neural induction medium (NIM) consisting of neural maintenance medium (NMM) supplemented with the dual‐SMAD inhibitors SB431542 (10 µM, Tocris) and LDN193189 (1 µM, Miltenyi)^[34 (link)
^]. (For NMM preparation, see Supporting Information: Extended Description of Methods.) At day 10, the neuroepithelial cells were passaged three times with Dispase II (Sigma) in the NMM up to 34 days to further purify the NPC. The generated DIV (Day In Vitro) 34 NPC were stored in liquid nitrogen cell bank for subsequent experiments. To enable cell viability tracking, a genome‐engineered H9 hESCs line bearing the constitutive tdTomato reporter expression cassette in the Adeno‐Associated Virus Integration Site 1 (AAVS1) locus were used to generate NPC as described above. The use of hESCs was approved by the Human Ethics Committee at the University Hospital, Gasthuisberg, KU Leuven, Belgium.