L glutamin

Drosophila S2 cells (Thermo Fisher R69007) were cultured at 25 °C in serum free medium (Express Five SFM, Gibco), supplemented with 20 mM l-Glutamin (GlutaMAX, Gibco). Cells were passaged with 10% of conditioned medium each passage. Cells were maintained at a density of 1–12million ml⁻¹.
Mouse embryonic stem cells were cultured in a humidified incubator at 37 °C and 5% CO₂. Cells were grown on Attachment Factor (Thermo Fisher) in 2i medium (KnockOut^TM Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) supplemented with 15% KnockOut^TM Serum Replacement (Gibco), 0.1 mM MEAA (Gibco, non-essential aminoacid), 1 nM Na-Pyruvate (Gibco), 0.1 mM 2-mercaptoethanol (Gibco), 4 mM l-Glutamin (GlutaMAX, Gibco), 5 μg ml⁻¹ Insulin (Sigma), 50 U ml⁻¹ Pen-Strep, 200 U ml⁻¹ LIF (ESGRO), 1 μM PD0325901 (StemGent), 3 μM CHIR99021 (StemGent).
Human fibroblast cell lines were established from skin biopsies of confirmed Koolen de-Vries syndrome patients and healthy controls. Cells were maintained in in a humidified incubator at 37 °C and 5% CO2 in DMEM (GlutaMAX supplement, Life Technologies), 100 U ml⁻¹ penicillin, 100 μg ml⁻¹ streptomycin and 10% foetal calf serum (FCS). All fibroblast lines tested negative for Hepatitis B, Hepatitis C, HIV and human Herpes virus 4 and 8. Fibroblasts were passaged at ~90% confluency.

Human endothelial cells were generated as previously described^{7 (link)}. Maintenance and formation of EBs was performed as described in the paragraph cardiac differentiation. For induction of differentiation of mesodermal progenitor cells, EBs were cultivated in Pluronic F-127 (Sigma-Aldrich, P2443) -coated flasks in StemPro^®-34 (Gibco 10639-011) supplemented with 4 mg/ml PVA; 400 µM 1-thioglycerol, Sigma-Aldrich M6145; 2 mM L-glutamin, Gibco 25030; 5 mg/L transferrin, Sigma-Aldrich T8158; 5 µg/L selenium, Sigma-Aldrich S5261; 0.5% Penicillin/Streptomycin, Gibco 15140; 10 µM Y-27632, biorbyt orb60104 and 10 ng/ml BMP-4, R&D Systems 314-BP; 6 ng/ml Activin-A, R&D systems 338-AC; 5 ng/ml basic FGF, R&D systems 233-FB for 3 days at 37 °C, 90% humidity, 5% CO₂, 5% O₂ with daily medium change. To differentiate endothelial cells, EBs were then cultured on Geltrex^® in StemPro^®-34, containing 400 µM 1-thioglycerol, Sigma-Aldrich M6145; 2 mM L-glutamin, Gibco 25030; 1 mM magnesium ascorbyl phosphate; 5 mg/L transferrin, Sigma-Aldrich T8158; 5 µg/L selenium, Sigma-Aldrich S52610.5% Penicillin/Streptomycin, Gibco 15140; 1 µM Y-27632, biorbyt orb60104; 100 ng/ml VEGF, R&D Systems 293-VE and 10 ng/ml bFGF, R&D Systems 233-FB. For 3 days, EBs were cultured in a hypoxic environment (5% CO₂, 5% O₂), followed by 9 days under normoxic conditions. Medium was changed every other day.

Human melanoma cell line M230 was provided by Pr A. Ribas and was cultured in RPMI 1640 (Invitrogen, Cergy Pontoise, France) containing 10% (v/v) fetal calf serum (FCS; Perbio, Bredières, France), L-glutamin (2 mM; Gibco, Cergy pontoise, France), antibiotics (100 U/mL penicillin and 1000 μg/mL streptomycin; Gibco). Human melanoma cell lines HBL and LND1 were provided by Pr G E Gahnem and were cultured in F10 (Invitrogen) containing 10% (v/v) fetal calf serum (FCS; Perbio), L-glutamin (2 mM; Gibco), antibiotics (100 U/mL penicillin and 1000 μg/mL streptomycin; Gibco). Imatinib, nilotinib, sorafenib, dasatinib, pexidartinib, and trametinib were from Selleck Chemicals, dissolved in DMSO and used at 1 µM final concentration for KIT inhibitors and 0.2 µM for trametinib. FGF2 was from PeproTech and used at 20 ng/mL final concentration.