Basic fgf

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.

Mycoplasma‐free patient‐derived glioma stem cells (h543 and h676) were cultured in neurosphere medium from NeuroCult (Stem Cell Technologies Inc, Vancouver, Canada) supplemented with 20 ng/ml basic‐FGF (RD Systems) and 10 ng/ml EGF (Gibco).
Mycoplasma‐free Trf1^lox/loxp53^−/− MEFs and K‐Ras^Δ/LG12Vgeop53^−/− tumor‐derived CHA‐9‐3 cell line were cultured in DMEM supplemented with 10% fetal bovine serum (FBS). All cell lines and primary cultures were regularly tested mycoplasma‐free.
For retroviral infection, supernatants were produced in 293T cells transfected with the ecotropic packaging plasmid pCL‐Eco, the envelope plasmid pCMV‐VSV‐G and either pBabe‐Cre and/or EGFP‐TRF1 pWzl‐Hygro (a gift from T. de Lange, Addgene plasmid #19834) using Fugene transfection reagent. Two days later, retroviral supernatants were collected and Trf1^lox/lox p53^−/− MEFs were infected with the corresponding retroviral supernatant at 12‐h intervals.
For lentiviral infection, supernatants were produced in 293T cells transfected with the envelope plasmid pCMV‐VSV‐G, the ecotropic packaging plasmid psPAX2 and either pLKO.1 empty and/or pLKO.1 vectors expressing short hairpin RNA for either ERK1 or ERK2 (SIGMA) using Fugene transfection reagent. Two days later, lentiviral supernatants were collected and p53^−/− MEFs were infected with the corresponding lentiviral supernatant at 12‐h intervals.