β fgf

Stem-like RN1, JK2, RKI1 and HW1 cells were derived from glioblastoma specimens [39 (link),40 (link)]. Cells were cultured in KnockOut DMEM/F-12 basal medium supplemented with StemPro NSC SFM supplement, 2 mM GlutaMAX-ICTS, 20 ng/mL EGF, 10 ng/mL FGF-β and Antibiotic–Antimycotic solution (all Life Technologies) as adherent cells on flasks coated with MatriGel Matrix (Corning Life Sciences, Cat# 354234, Corning, NY, USA). The protocols were approved by the Human Ethics Committee of the Royal Brisbane and Women’s Hospital (RBWH 2004/161). All cell cultures were routinely tested for mycoplasma contamination and the cumulative length of culturing did not exceed 15 passages.

A172 and U251 cells were obtained from the European Collection of Cell Cultures (EACC, UK) in 2014 and authenticated in 2020 by Cell Bank Australia using short tandem profiling. Cells were cultured in DMEM supplemented with 10% FBS and Antibiotic-Antimycotic solution (Life Technologies, Carlsbad, CA, USA) at 37 °C and 5% CO₂. Patient-derived HW1, RN1, MMK1 and RKI1 glioblastoma stem cells^{34 (link)} were cultured in KnockOut DMEM/F-12 supplemented with StemPro NSC SFM, 2 mM GlutaMAX-ICTS, 20 ng/mL EGF, 10 ng/mL FGF-β and Antibiotic-Antimycotic solution (all Life Technologies) as adherent cells on flasks coated with MatriGel (Corning, NY, USA). The protocols were approved by the Human Ethics Committee of the Royal Brisbane & Women’s Hospital (RBWH 2004/161). Cell cultures were routinely tested for mycoplasma infection and culturing did not exceed 15 passages.

The human NSCs used in this study were derived from NIH-approved H9 human embryonic stem cells (Thermo Fisher Scientific, MA, USA). The cells were handled following a kit’s protocol. The complete medium used to maintain the human NSCs consisted of 1 × KnockOut D-MEM/F12, 2 mM GlutaMAX-I supplement, 20 ng/ml FGF-β, 20 ng/ml EGF and 2% StemPro neural supplement (all from Thermo Fisher Scientific, MA, USA). Adherent human NSCs were thawed and subcultured in plates precoated with a matrix consisting of CELLStart substrate and D-PBS containing calcium and magnesium (both from Thermo Fisher Scientific, MA, USA). The cells were incubated at 37 °C with 5% CO_2, and the medium was replaced with new complete medium every 2 days. When the NSCs were approximately 90% confluent, they were ready to be passaged. The spent medium was removed from the cells, and the cellular surface was rinsed with 1 × D-PBS. To detach the cells, they were treated with Accutase and incubated at 37 °C in a 5% CO₂ incubator for 1–2 min, after which complete medium was added to stop the dissociation reaction. The cells were gently pipetted several times, transferred to a 15 ml tube, and centrifuged at 200 × g for 5 min. The supernatant was aspirated, and the cells were resuspended in complete medium and seeded in precoated plates for maintenance.