Isolation and Characterization of Glioma Stem Cells

Human brain microvessel endothelial cells (hBMECs) were purchased from ScienCell Research Laboratories (San Diego, CA, USA). The hBMECs were maintained in endothelial cell medium (ECM; ScienCell Research Laboratories). Six patient-derived primary glioma stem cells from WHO grade II to IV (grade II: GSC205 and GSC207; grade III: GSC306 and GSC307; grade IV: GSC406 and GSC408) were isolated, and neurosphere cultures were obtained as previously described [23 (link)]. The detailed clinicopathological information is presented in Table S2. Briefly, freshly resected glioma samples were dissociated into single cells and grown in serum-free DMEM/F12 with 2% B27, 20 ng/mL rh-bFGF, and rh-EGF (Gibco, Gaithersburg, MD, USA). The stem cell markers of GSCs were detected by immunofluorescence using anti-CD133 (Abcam Technology, Cambridge, UK) and nestin antibodies (Abcam). The immunofluorescence staining of glial fibrillary acidic protein (GFAP; Abcam) and β-III tubulin (Abcam) was used to evaluate the multi-lineage differentiation capacity of GSCs.