High frequency of tumorigenic cells in a murine brain tumor. (A) Development of brain tumors. (i) Gross appearance of a recipient brain. A massive lesion can be seen in the cerebrum. (ii–iv) H&E staining of representative sections of brain tumors. Regions of increased cell density, nuclear pleomorphism, and prominent mitotic figures can be seen. Arrows, giant cells; arrowheads, areas of necrosis with pseudopalisading; asterisks, invading tumor cells adjacent to blood vessels. (Scale bars: 200 μm.) (B) Survival of recipient mice after transplantation of 1,000, 100, or 10 huKO⁺ tumor cells, as indicated.

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.

Migration potential of T-ICs. (A) High expression of c-Met in GFP^high cells. Total RNA was purified from GFP^high and GFP^low cells isolated from three independent original tumors and c-Met mRNA levels were evaluated by RT-PCR. β-actin, control. (B) c-Met is phosphorylated in T-ICs. Serial sections of one of the single cell-derived tumors were subjected to H&E (i and ii); anti-GFP (iii and iv); and anti-phospho-c-Met (v and vi) staining. Magnified views of the areas indicated by the squares in i, iii and v are shown in ii, iv, and vi, respectively. (Scale bars: i, iii, v, 1 mm; ii, iv, vi, 200 μm.) (C) Migration of T-ICs in a collagen gel. Dissociated cells from a tumor were inoculated into either a plain collagen gel (control) or a collagen gel containing HGF (10 ng/mL) and incubated for 4 days. Representative GFP⁺ tumor cells with and without HGF in a collagen gel are shown. (Scale bars: 50 μm.)

Correlation of in vivo differentiation status of NSC/NPC and GFP fluorescence intensity in the NS-GFP-Tg brain. (A) High GFP expression correlates with high nestin expression in cells of NS-GFP-Tg embryonic forebrains. Coronal sections of forebrains of E14.5 NS-GFP-Tg mice were subjected to immunohistochemical analysis using anti-GFP (green) plus anti-type III β-tubulin (TuJ1; red, Top), or anti-GFP plus anti-nestin (red, Middle and Bottom). Images of the ventricular zone (VZ) cells in the Middle are shown at higher magnification in Bottom. (Scale bars: Top and Middle, 100 μm; Bottom, 10 μm). (B) Fractionation of GFP-expressing cells from E14.5 NS-GFP-Tg brain. Total brain cells from E14.5 NS-GFP-Tg mice were dissociated and fractionated by flow cytometry into 4 subpopulations, GFP^−/+, GFP⁺⁺, GFP⁺⁺⁺ and GFP⁺⁺⁺⁺, based on GFP fluorescence intensity as indicated. Gray peak, WT C57BL/6 mice; black peak, NS-GFP-Tg mice. (C) Correlation of high GFP expression with immature cell properties. The fractionated brain cell subpopulations from B were fixed and analyzed by immunocytochemistry to detect expression of nestin (red, i–iv) or TuJ1 (red, v–viii) as indicated. Blue, nuclear marker TOTO-3. (Scale bars, 20 μm.) (D) Correlation of GFP expression with neurosphere-forming capacity in E14.5 NS-GFP-Tg brain tissues. Fractionated brain cell subpopulations from B were cultured under conditions allowing neurosphere formation. Data shown are the mean number ± SD. of neurospheres generated per 2,000 cells (n = 3 per group). (E) Fractionation of P3 GFP-expressing brain cells. Total brain cells from P3 NS-GFP-Tg mice were dissociated and fractionated by flow cytometry into 4 subpopulations, GFP^−/+, GFP⁺⁺, GFP⁺⁺⁺ and GFP⁺⁺⁺⁺, based on GFP fluorescence intensity as indicated. Gray peak, WT C57BL/6 mice; black peak, NS-GFP-Tg mice. (F) Correlation of GFP expression with neurosphere-forming capacity in P3 NS-GFP-Tg brain tissues. The fractionated brain cell subpopulations in E were cultured to allow neurosphere formation as for D. Data shown are the mean number ± SD. of neurospheres generated per 2,000 cells (n = 3 per group).

Localization of T-ICs. Serial sections of a single-cell-derived tumor were stained with H&E (A and F) or anti-GFP (B–E) to identify T-ICs. (A) H&E staining of a tumor invading normal brain tissue. (B) Anti-GFP staining of the tissue in A. (a and b) Low-power views of areas shown at high power in C and D, respectively. (E) High-power view of area c in D. (F) H&E staining of E. (Scale bars: A and B, 1 mm; C and D, 200 μm; E and F, 50 μm.)