Apo transferrin

Unless otherwise stated, all reagents were purchased from Sigma-Aldrich. HOS and MG63 cell lines and bone-marrow MSC were purchased from the American Type Culture Collection (ATCC) and cultured, in IMDM (Life Technologies) for tumor cells or alpha-modified Minimum Essential Medium (αMEM) for MSC plus penicillin (20U/mL), streptomycin (100 mg/mL), and 10% heat-inactivated fetal bovine serum (complete medium). Cells were maintained at 37°C in humidified atmosphere with 5% CO_2.Sphere-forming cells were obtained as previously described [3 (link)]. Briefly, tumor cells were cultured in anchorage-independent conditions in CSC complete medium [DMEM:F12 medium with progesterone (20 nM), putrescine (10 mg/mL), sodium selenite (30 nM), apo-transferrin (100 mg/mL), and insulin (25 mg/mL), human epidermal growth factor (20 ng/mL) and basic fibroblast growth factor (10 ng/mL, PeproTech)], in low-attachment flasks (Nunc). MSC cells that had been cultured for more than 6 passages were never used.

Neurospheres (NS) were generated from the striata of 1‐day‐old Sprague–Dawley (SD) rat using a method modified by Reynolds and Weiss (1996) and differentiated into astrocytes as described in Sorensen, Moffat, Thomson, and Barnett (2008). Briefly, the tissue was enzymatically dissociated and plated in NS medium (NSM) containing, Dulbecco's Modified Eagle Medium/F12 (DMEM/F12, 1:1, DMEM containing 4,500 mg/L glucose, Life Technologies, Carlsbad, CA), enriched with 0.105% NaHCO₃, l‐glutamine, 5,000 IU/ml penicillin, 5 μg/ml streptomycin, 5.0 mM HEPES (all from Invitrogen, UK), 100 μg/ml apotransferrin, 25 μg/ml insulin, 60 μM putrescine, 20 μM progesterone, and 30 μM sodium selenite, supplemented with 20 ng/ml of epithelial growth factor (EGF; all from Peprotech, UK). The cell suspension was maintained until NS were formed. To generate astrocytes, the NS were triturated to produce smaller cell sphere suspensions, transferred to 13 mm poly‐l‐lysine (PLL; 13 μg/ml, Sigma) coated coverslips in a 24‐well plate (Corning, UK) and incubated for a further 5–7 days in vitro (DIV) at 37°C in an atmosphere of 7% CO₂/93% air until a confluent monolayer formed. NS‐derived astrocytes were maintained in DMEM‐1 g/ml glucose (Life Technologies) with 10% fetal bovine serum (Sigma, Poole, Dorset, UK) and 2 mM l‐glutamine (Sigma).

pd‐GBSCs were cultivated under stem cell conditions following the procedure described in [18 (link)] in order to retain as many of the original tumour features as possible. Cell expansion and subsequent experiments were conducted in Dulbecco's Modified Eagle Medium (DMEM/F12, Cat. No: 1331020, Gibco, Waltham, MA, USA) supplemented with N2 (100×: 25 μg·mL⁻¹ insulin (Cat. No: I6634, Sigma, Darmstadt, Germany)), 100 μg·mL⁻¹ apo‐transferrin (Cat. No: 11108016, Gibco), 50 μg·mL⁻¹ bovine serum albumin fraction V 7.5% (Cat. No: 15260037, Gibco), 0.02 μg·mL⁻¹ progesterone (Cat. No: P8783, Sigma), 16 μg·mL⁻¹ putrescine (Cat. No: P5780, Sigma) and 5.18 ng·mL⁻¹ sodium selenite (Cat. No: S5261, Sigma), 1× B‐27 (Cat. No: 17504044, Gibco), 1 mm sodium pyruvate (Cat. No: S8636, Sigma), 500 units·mL⁻¹ penicillin/streptomycin (Cat. No: P4333, Sigma) and supplemented daily with 10 ng·mL⁻¹ EGF (Cat. No: 78006.1, Stem Cell Tech., Vancouver, BC, Canada), and 10 ng·mL⁻¹ FGF‐2 (Cat. No: 78003, Stem Cell Tech). Cells were cultivated in flasks and plates previously coated with 10 μg·mL⁻¹ laminin (Cat No: L2020, Sigma) for 4 h at 37 °C. Cell cultures were incubated at 37 °C in a humidified atmosphere with 5% CO₂ and were routinely checked to ensure they were mycoplasma‐free (Mycoplasma Gel Detection Kit, Cat. No: 90.022‐4544, Biotools, Madrid, Spain).