Essential 8 medium

Human GBM GSCs and NSGCs were isolated from GBM tissue from surgical specimens and from established U-1242/luc-GFP GBM cells. GBM tissue samples were dissociated and digested samples were filtered with 70 μm nylon cell strainer (BD) and resuspended in stem cell medium comprised of DMEM/F-12 50:50 containing K27 supplements, glutamine 2 μmol/L (Invitrogen), basic fibroblast and epidermal growth factors (PeproTech, 20 ng/mL each) for continuous culturing [59 (link)]. All primary cells were cultured as suspended spheres in ultra-low attachment T25 or T75 culture disks (Corning) in Essential 8 medium (Invitrogen) and analyzed prior to 5 passages to form GSC enriched neurospheres. Neurospheres were dissociated and then labeled with CD44 and CD133 antibody (Miltenyi Biotech) to isolate both pro-neural and mesenchymal GSC subtypes. Stained cells were sorted through a BD Aria II sorting station. Antibody negative and positive cell populations were counted and collected for further culturing. GSC and NSGC populations were counted and collected for further culturing. Xenografted human GSCs were isolated from mice and analyzed for cell surface and intracellular proteins by FACS. The GSCs were cultured in ultra-low attachment plates and flasks with Essential 8 medium (Invitrogen), unless indicated. Isolated NSGCs were cultured in monolayer with complete DMEM medium.

5 × 10⁵ PBMCs were resuspended in 24-well plates with PBMCs medium (StemPro™-34 + SCF 100 ng/mL, FLT-3 100 ng/mL, IL-3 20 ng/mL, IL-6 20 ng/mL). Viruses provided by CytoTune^®-iPS 2.0 Sendai Reprogramming Kit (Invitrogen, USA) were incubated for 24 h. The medium was replaced with fresh complete PBMCs medium to remove the CytoTune™ 2.0 Sendai reprogramming vectors. After 2 days, transduced cells were plated on vitronectin-coated culture dishes in complete StemPro™-34 medium (Invitrogen, USA) and the consumed medium was replaced every day for 4 days. After 7 days from transduction, cells were grown in Essential 8 Medium (Thermo Fisher Scientific, Waltham, MA USA). Consumed medium was replaced every two days for 3 weeks. Undifferentiated colonies were manually picked and plated on vitronectin-coated well. Colonies were split using 0.5 mM EDTA for 3–5 passages. Finally, cells were collected using 0.5 mM EDTA (Thermo Fisher Scientific) and cryopreserved in liquid nitrogen in Essential 8 Medium plus 10% DMSO (Figure 1).