growth protocol: Normal, non-neoplastic cells were derived from patient tissue specimens of neurosurgical resection in accordance with a Cleveland Clinic Institutional Review Board-approved protocol. Informed consent was obtained by the tissue bank, which provided de-identified excess tissue to the laboratory immediately following surgical resection. Specimens used for cell culture were dissociated with a Papain dissociation kit (Worthington). Cells were cultured adherently in media containing 50% Neurobasal medium (Gibco) and 50% Dulbecco's modified Eagle medium (DMEM) with B27 (without vitamin A, Invitrogen, basic fibroblast growth factor (10 ng/ml), epidermal growth factor (10 ng/ml), sodium pyruvate and L-glutamine, and 5% FBS. All cultured cells were used within five passages of dissociation.
cell characterization: Single cells were sorted using anti-A2B5 MicroBeads (130-093-388, Miltenyi Biotec) according to manufacturer’s protocol. In brief, live cells were incubated first with FcR Blocking Reagent (Miltenyi) for 10 mins then A2B5 antibody for 15 min at 4oC (2.5 μg A2B5 antibody per million cells). Cells positive for A2B5 were double enriched by passing through magnetic field (MACS Separator) twice. Both A2B5 positive and negative fractions were collected, lysed with TRIzol (Invitrogen) for RNA isolation. A population of A2B5+ cells were plated and allowed to attach overnight before fixing and staining with standard immunofluorescence techniques. A2B5+ cells stained positive for GFAP (DAKO, 1:5000) and Nestin (DSHB, 1:200).
growth protocol: Human OPCs were differentiated from the human embryonic stem cell line H7 over a 154 day protocol (NIH Human Embryonic Stem Cell Registry WA07; NIH Approval Number: NIHhESC-10-0061) as previously demonstrated (Hu et al., 2009; Wang et al., 2013).
cell characterization: hESC-derived OPCs were characterized by co-staining of Sox10 (R&D Systems,AF2864; 1:100) and Olig2 (Millipore,AB9610 1:500).
growth protocol: Human OPCs were differentiated from the human embryonic stem cell line H7 over a 154 day protocol (NIH Human Embryonic Stem Cell Registry WA07; NIH Approval Number: NIHhESC-10-0061) as previously demonstrated (Hu et al., 2009; Wang et al., 2013).
cell characterization: hESC-derived OPCs were characterized by co-staining of Sox10 (R&D Systems,AF2864; 1:100) and Olig2 (Millipore,AB9610 1:500).
growth protocol: IN528 cells from a primary patient derived GBM tumor model were injected subcutaneously into the flank of a immunocomprimised athymic nude mouse and allowed to grow into a tumor.
cell characterization: Cells were dissociated from the tumor and sorted on CD133 to collect the cancer stem cell population.
growth protocol: IN528 cells from a primary patient derived GBM tumor model were injected subcutaneously into the flank of a immunocomprimised athymic nude mouse and allowed to grow into a tumor.
cell characterization: Cells were dissociated from the tumor and sorted on CD133 to collect the cancer stem cell population.