Primary newborn astroglial cells were isolated and maintained as
previously described78 . For
gliomasphere preparation, astroglial cells were plated at 20-30% confluence in
6-well plates in 2 ml astrocyte medium (DMEM medium supplemented with 10% FBS,
glutamine and antibiotics). The next day cells were transduced with lentiviral
vectors coding for oncogenes, or with empty vector as negative controls. After
24 hours (day 3), transduced astrocytes were switched to NSC medium (DMEM/F12
supplemented with 100X N2, 20 ng/ml mEGF, 20 ng/ml mbFGF, glutamine, and
antibiotics). Spheres arising from the cell monolayer were evident after
approximately 3 weeks. Sphere passaging was performed as described
previously74 (link).
To evaluate self-renewal properties (Extended Data Fig. 7d and 9b ),
gliomaspheres were dissociated to single cells and replated in Ultra Low
Attachment 24-wells plates (Corning), at the concentration of 2,000 cells per
well; fully gliomaspheres were counted by visual inspection.
previously described78 . For
gliomasphere preparation, astroglial cells were plated at 20-30% confluence in
6-well plates in 2 ml astrocyte medium (DMEM medium supplemented with 10% FBS,
glutamine and antibiotics). The next day cells were transduced with lentiviral
vectors coding for oncogenes, or with empty vector as negative controls. After
24 hours (day 3), transduced astrocytes were switched to NSC medium (DMEM/F12
supplemented with 100X N2, 20 ng/ml mEGF, 20 ng/ml mbFGF, glutamine, and
antibiotics). Spheres arising from the cell monolayer were evident after
approximately 3 weeks. Sphere passaging was performed as described
previously74 (link).
To evaluate self-renewal properties (
gliomaspheres were dissociated to single cells and replated in Ultra Low
Attachment 24-wells plates (Corning), at the concentration of 2,000 cells per
well; fully gliomaspheres were counted by visual inspection.
