Egf and fgf2

For neuronal differentiation, induction medium containing Neurobasal media (Gibco, USA), B27 supplement (Gibco, USA), EGF and FGF2 (10 ng/ml each) (PeproTech Asia), L-Glutamine (Gibco, USA) PenStrep (Gibco, USA) was used. The induction protocol was carried out for 12 days with media change on every 3^rd day. Another set of study group included induction of hMSCs using the above mentioned induction media cocktail for 8 days, followed by addition of BDNF (50 ng/ml) on 9^th day. After termination of induction period at 12 days in both the study groups, the cells were used for further experiments^{18 (link), 20 (link)}.

Human glioblastoma cell cultures were developed as follows: human GBM grade IV biopsies were obtained in accordance with the protocol approved by the Uppsala ethical review board (2007/353), and were graded by neuropathologist Irina Alafuzoff, Uppsala University Hospital, according to WHO guidelines. Tumor biopsies were minced (1 mm × 1 mm pieces) and digested by 1:1 ratio of Accutase (eBioscience, San Diego, CA)/TrypLE (Life technologies, Grand Island, NY) at 37°C for 15 min and triturated through 18g and 21g needles 5 times. Dissociated cells were resuspended in DMEM/F12 Glutamax (Life technologies, Grand Island, NY) and Neurobasal medium (Life technologies, Grand Island, NY) mixed 1:1 with addition of 1% B27 (Life technologies, Grand Island, NY), 0.5% N2 (Life technologies, Grand Island, NY), 1% penicillin/streptomycin (Sigma-Aldrich, St Louis, MO), 10 ng/ml EGF and FGF2 (Peprotech, Rocky Hill, NJ), and plated at 100,000 cells/ml. After primary sphere formation, spheres were seeded onto poly-ornithine/laminin-coated dishes and cultured as adherent cells as described in Pollard et al. [71 (link)].

NSCs were isolated from adult hippocampus of C57BL/6J mice as described in Babu et al (2011) (link). Unless otherwise stated, NSCs were maintained on poly-D-lysine (Sigma-Aldrich) and laminin (Invitrogen) coated plates in Neurobasal A medium (Invitrogen) with 1x B27 supplement without vitamin A (GIBCO), 1x GlutaMax (Invitrogen) and 20 ng/ml each of EGF and FGF2 (Peprotech), as per (Babu et al, 2011 (link)). No cells were used past passage 20. Two separate lines were used in all experiments, one from five pooled C57BL/6J male mice and one from five pooled C57BL/6J female mice. No differences between NSCs isolated from males and females were found.

Patient-derived GCGR cell lines were provided by the glioma cellular genetics resource: (www.gcgr.org.uk). All GSC lines were cultured adherently in serum-free GSC media (N2 (1/200), B27 (1/100) (Life Technologies), 1 mg/ml laminin (Sigma), 10 ng/mL EGF and FGF-2 (Peprotech), 1× MEM NEAA (Gibco), 0.1 mM betamercaptoethanol, 0.012% BSA (Gibco), 0.2 g/L glucose (Sigma), 1000 U/ml penicillin-streptomycin (Sigma))^{25 (link)}. Medium was changed every 3 d, and cells were dissociated using Accutase solution (Sigma). For induction of SOX10 expression, doxycycline was used at a concentration of 3 μg/ml. For experiments involving assessment of proliferation, cells were exposed to 10 μm EdU for 4 h prior to collection.

All 60 human GSC cultures used in this study have been established in our laboratory and are part of the HGCC biobank (hgcc.se). Handling of human tissues and data were performed in accordance with the protocol approved by Uppsala ethical review board (2007/353) and following informed written consent from all patients. All have been previously described and most have been authenticated^{22 (link),25 (link),26 (link)}. Cultures were maintained on poly-ornithine/laminin-coated dishes in DMEM/F12 Glutamax (Gibco) and Neurobasal medium (Gibco) mixed 1:1 with addition of 1% B27 (Invitrogen), 0.5% N2 (Invitrogen), 1% penicillin/streptomycin (Sigma), 10 ng/ml each of EGF and FGF2 (Peprotech). Human cells used in all experiments were below passage 20. All human cell cultures are listed in Supplementary Data 1.
All cell cultures, both mouse and human, have been regularly analyzed for mycoplasma infection using either a PCR-based method with the primers Myco1 (50-GGCGAATGGGTGAGTAACACG) and Myco2 (50-CGGATAACGCTTGCGACTATG) (Invitrogen), or the KAPA kit (Techtum, cat# 25-KK7352), and have at all times tested negative.

The cancer stem cells (CSCs) used in this study (PANC-1, PSN-1, SW620, HT29, WiDr, and SW480) were maintained as monolayer stem cell cultures [28 (link),29 (link)]. Briefly, the cells were cultured on collagen-I-coated dishes (IWAKI, Tokyo, Japan) in stem cell culture medium (DMEM/F-12 supplemented with 1% B27 (Thermo Fisher Scientific, Waltham, MA, USA), 20 ng/mL of EGF and FGF2 (Peprotech, Rocky Hill, NJ, USA), D-(+)-glucose (final concentration, 26.2 mM), L-glutamine (final concentration, 4.5 mM), 100 units/mL of penicillin, and 100 mg/mL of streptomycin). The stem cell culture medium was changed every 3 days, and EGF and FGF2 were added to the stem cell culture medium daily. To obtain isogenic non-CSC counterparts, the CSCs were induced to lose their stemness by culturing in DMEM/F-12 medium supplemented with 10% fetal bovine serum (Thermo Fisher Scientific), 100 units/mL of penicillin, and 100 mg/mL of streptomycin for 1 week. Then, the cells were used in the experiments in this study as non-CSCs.