Proteins were transferred onto polyvinylidene difluoride membranes at 4C for 1 h, which were then blocked in TBS-T (Tris-buffered saline with 0.05% Tween-20) with 5% nonfat dry milk for 1 h at room temperature. molecular pathways responsible for these effects, however, are not fully understood. Here, we describe the establishment of glioma stem-like cells from multiple different human oligodendrogliomas. We further demonstrate that BMP signaling is usually intact in these cells, and potently induces their astrocytic differentiation. Finally, we reveal cytoplasmic sequestration of oligodendrocyte lineage transcription factors (OLIG) 1 and 2 by BMP-induced ID proteins as a putative mechanism underlying this effect. Our findings have important implications for the development of therapies targeting the stem-like cell compartment of oligodendrogliomas. Materials and Methods Oligodendroglioma propagating cell isolation and culture OligPCs were isolated from primary surgical specimens from patients with known or suspected oligodendroglioma in keeping with protocols approved by the Northwestern University Institutional Review Board and produced as spheres as previously described (2). In brief, specimens were rinsed in 1x phosphate-buffered saline (PBS), mechanically dissociated with a scalpel and enzymatically dissociated using DNaseI (Roche) and Dispase (GIBCO) in DMEM/F12 media (Invitrogen) at 37C for 45 min. Red blood cells were lysed using ACK buffer (Gibco), and a single cell suspension was achieved using a 100 m strainer. Cells were IL8 plated in non-adherent flasks in DMEM/F12 made up of 1% penicillin/streptomycin, supplements N2 and B27 (Gibco), and the following growth factors: 20 ng/ml human recombinant EGF (Millipore), 20 ng/ml bFGF (Millipore) and 10 ng/ml LIF (Chemicon). Once spheres were visible, cell cultures were centrifuged at 100 x for 5 minutes and the supernatant was aspirated to remove lifeless cells and cellular debris as needed. Such centrifugation was often performed multiple occasions before the spheres were passaged. The final diagnosis for each tumor, including lineage-specific immunohistochemical stains and fluorescent in situ hybridization confirming the characteristic 1p19q chromosomal deletion, was obtained before cells were used in subsequent experiments. OligPC 40 was derived from a primary WHO grade III oligodendroglioma with 1p19q chromosomal deletion and polysomy for chromosome 10. Areas of focal anaplasia with increased proliferative index were apparent, and no astrocytic features were observed. OligPC 49 was derived from a recurrent WHO grade III oligodendroglioma Entasobulin also with 1p19q chromosomal deletion. This tumor exhibited frequent mitoses and microvascular proliferation, as well as marked cellular atypia, with some cells resembling common oligodendroglial cells and other with enlarged nuclei or multiple nuclei. However, no astrocytic component was apparent upon immunohistochemistry for GFAP. Mutations in IDH1 and IDH2 were not assessed in these tumors, as the pathological analyses were performed prior to the identification of these mutations in oligodendroglial tumors (20). OligPC spheres were passaged every 7C10 days by mechanical chopping. Cells Entasobulin were used at passage 10 or less for all experiments. For sphere-forming assays, cells were plated Entasobulin in 96-well plates at a density of 10 cells/well in 100l GSC media. After 10 days, each well was inspected for sphere formation, and the number of spheres per well were counted. Clonogenic frequency was estimated as the average number of spheres formed per 100 cells plated. For differentiation assays, cells were dissociated to a single cell suspension using Accutase (Sigma) and plated on glass coverslips coated with poly-D-lysine/laminin (BD Biosciences) and produced in GSC media without growth factor supplementation. For cultures with BMP Entasobulin treatment, human recombinant BMP4 (R&D Systems) was added to a final concentration of 100 ng/ml. Immunocytochemistry Cells were fixed in 4% paraformaldehyde (Sigma) in 1x PBS for 20 min, washed 3 times in PBS, and incubated Entasobulin with primary antibodies overnight at 4C in 1x PBS made up of 1% bovine serum albumin and 0.25% Triton X-100. After 3 more PBS washes, cells were incubated with the appropriate secondary antibody (Molecular Probes, Invitrogen) at 1:500 in 1x PBS for 1 h at room temperature. Nuclei were counterstained with Hoechst dye (1:5000 in 1x PBS), coverslips were mounted using Prolong Gold antifade reagent (Invitrogen) and imaged on a Zeiss UV-LSM 510 META or Leica SP-5 confocal microscope. National Institutes of Health Image J software was used to quantify images. The following primary antibodies were used: MAP2 (Abcam, mouse IgG1, 1:1000), GFAP (DakoCytomaton, rabbit polyclonal, 1:1000), O4 (Millipore, mouse IgM, 1:100 in 1x PBS; incubated with cells at room heat for 30 min prior to fixation), Sox2 (Millipore, rabbit polyclonal, 1:500), Nestin (BD biosciences, mouse IgG1, 1:500), Ki67 (Chemicon, mouse IgG1, 1:500), phospho-Smad1/5/8 (Cell Signaling, rabbit polyclonal, 1:500), ID2 (Santa Cruz, rabbit polyclonal, 1:100), ID4 (Santa Cruz, rabbit polyclonal, 1:50), OLIG1 (Millipore, mouse IgG2b, 1:250), OLIG2 (Millipore, mouse IgG2a, 1:250). Cell viability and death were decided using a LIVE/DEAD Viability/Cytotoxicity kit, which labels live.