Fig. 3.

Identification of T-ICs. (A) Detection of GFP^high and GFP^low subpopulations among brain tumor cells derived from NS-GFP-Tg mice. Tumors were dissociated with collagenase and GFP expression was analyzed in huKO⁺ tumor cells by flow cytometry. Tumors contained variable percentages of GFP^high and GFP^low cells. Three representative samples showing the GFP expression pattern in huKO⁺ tumor cells are shown. (B) GFP^high tumor cells exhibit immature cell properties. Cytospin smears of sorted GFP^high and GFP^low cells in A were fixed and immunostained to detect nestin (red). Blue, nuclear marker DAPI. Three representative samples of 5 independent experiments are shown. (Scale bars: 20 μm.) (C) GFP^high tumor cells can generate spheres. GFP^high and GFP^low cells from brain tumors in A were cultured for 14 days under sphere formation conditions. Data shown are the mean number ± SD. of spheres per indicated number of inoculated cells (n = 4 per group; *, P < 0.01). (D) Transplantation of GFP^high brain tumor cells curtails survival. The percentage survival of WT recipient mice injected with 10,000, 1,000, 100, or 10 GFP^high or GFP^low brain tumor cells is shown as indicated. (E) Maintenance of the GFP expression pattern between original and transplanted brain tumors. Analysis of histology (by H&E staining, Left) and GFP expression (by flow cytometry, Right) of brain tumors isolated from two WT recipient mice transplanted with 10 GFP^high brain tumor cells (as from D, Right). (Scale bars: 200 μm.) (F) A neurosphere derived from a single tumor cell by limiting dilution. (Left Upper) Bright field. (Left Lower) GFP fluorescence. (Scale bars: 200 μm.) Right, GFP fluorescence as determined by flow cytometry. (Upper) Neurospheres from control mice (P3). (Lower) Single cell-derived spheres from NS-GFP tumor. Representative data are shown. (G) Maintenance of GFP expression pattern between original tumors and tumors arising from transplantation of single-cell-derived neurospheres. Neurospheres derived in culture from single cells of an original tumor were transplanted into WT recipient mice. Tumors arising in these recipients were dissociated and the expression of their component cells analyzed. Data representative of 4 independent experiments are shown.