“…Briefly, after three passages, hiPSC were seeded on Matrigel-coated 12-well plates and cultured in mTeSR until 80% confluent. Then a series of chemical compounds were added sequentially (500 nM LDN193189 (Sigma, SML0559) days 0-3, 10 µM SB431542 (Sigma, S4317) days 0-4, 3 µM CHIR99021 + 10 µM DAPT (Sigma, D5942) + 0.2 µM PD173074 (Sigma, P2499) days 2-7, 60 ng ml −1 Shh C25II (Gibco, PMC8034) + 1 µM PMP (Sigma, SML0868) days 3-12, 10 ng ml −1 BMP4 (R&D Systems, 314-BP-010) days [10][11][12][13][14] to the culture medium to modify pathways involved in the induction of neuromesodermal progenitors and neural crest cells. Culture medium mTeSR was gradually replaced by N2 medium (Neurobasal Plus medium (Gibco, A3582901) supplemented with 2 mM L-glutamine, B-27 Plus (Gibco, A3582801), N-2 supplements (Gibco, 11520536), 0.2 mM ascorbic acid (Sigma, A8960), 0.2 nM dbcAMP (Sigma, D0260), 10 ng ml −1 NGF (Bio-Techne, 256-GF-100), 10 ng ml −1 BDNF (R&D Systems, 248-BDB-010), 10 ng ml −1 GDNF (R&D Systems, 212-GD-010)) to promote sympathetic neuron development from day 4 (days 4-5: 75% mTeSR + 25% N2 medium; days 6-7: 50% mTeSR + 50% N2 medium; days 8-9: 25% mTeSR + 75% N2 medium; days 10-12: N2 medium).…”