Neurosphere Culture. Tumors and neurosphere cultures were performed as according to Svendsen et al. (1) with some modifications. Briefly, tissue was washed, minced, and digested with trypsin, and then 100- 500-mm³ pieces of tumor or brain ventricular tissue were washed in chilled sterile PBS (pH 7.4) with 0.6% glucose. The tissues were minced with scissors and incubated in 0.1% trypsin (GIBCO-Invitrogen) and 0.04% DNase (Sigma type II) in Hanks’ balanced salt solution (HBSS) for 45 min at 37°C. Tissues were washed three times in 0.04% DNase in HBSS and then triturated in the same solution using first a 5-ml pipette (Falcon) then with a 1,000-m l Pipetteman (Rainin), and then a fire-polished Pasteur pipette. Cells were passed first through a 100-m m strainer and then through a 70-m m strainer (Falcon). Cells were seeded at a concentration of 100,000 per ml into tissue culture flasks and grown in proliferation medium containing DMEM-F12 (GIBCO-Invitrogen), penicillin G, streptomycin sulfate, amphotericin B (1:100; GIBCO-Invitrogen), B-27 (1:50; GIBCO-Invitrogen), recombinant human fibroblast growth factor (FGF-2, 20 ng/ml; Peprotech), recombinant human epidermal growth factor (EGF, 20 ng/ml; R&D Systems), leukemia inhibitory factor (LIF, 20 ng/ml; Chemicon), and heparin (5 m g/ml; Sigma). Fresh FGF-2 and EGF were added twice each week. Spheres were passaged by trituration through a fire-polished Pasteur pipette and reseeding into fresh proliferative medium. To create clonal density spheres, spheres originally cultured at 100,000 cells per ml were triturated with a fire-polished pipette, and the cells were passed through a 40-m m cell strainer to create a single-cell suspension (2). Cells were reseeded in 2:3 filtered mouse-neurosphere conditioned medium diluted in proliferation medium at a density of 1,000 cells per ml, a density previously shown to be clonal for primary neurospheres (3).

Semiquantitative RT-PCR. Total RNA was isolated from brain and tumor samples, neurosphere cultures, and neurospheres differentiated for 7days by TRIzol (GIBCO BRL), and 1 m g of RNA from each sample was converted to cDNA by reverse transcriptase, following the supplier’s protocol (Superscript II, Life Technologies). Glyceraldehyde-3-phosphate dehydrogenase(GAPDH) expression served as an internal control. After the amount of cDNA was adjusted, it was subjected to PCR analysis using primers specific to genes, listed below. The protocol for the thermal cycler was as follows: denaturation at 94°C for 5 min, followed by 36 cycles of 94°C (30 sec), 60°C (1 min), and 72°C (1 min), with the reaction terminated by a final 10-min incubation at 72°C. Controls experiments run without reverse transcriptase or template cDNA revealed no nonspecific hybridization. Expression levels were analyzed on ethidium bromide-stained 2% agarose gels. The intensities of signal were scored as –, ±, +, ++, +++ by an observer who did not know their identities.

S, sense; AS, antisense.

Statistical Analysis. Total cell counts of clonal neurospheres were performed by counting the number of DAPI-stained nuclei. Cell counts are reported as a percentage of total cells expressing nestin, TuJ1, and/or GFAP among several clonal neurospheres plated across two to three coverslips. Data were analyzed by using the paired t test and presented as the mean ± standard error of the mean. Controls consisted of the identical cultures incubated in the absence of primary antibodies.

Transplantation of Neurosphere Cells into Neonatal Rat Brain. Transplantation of 50,000 cells into the neostriata of neonatal rats was performed according to the methods of Uchida et al. (4) with modifications. Animals were treated in accordance with protocols approved by the University of California Los Angeles Institutional Review Board. Spheres were dissociated by trituration through a fire-polished pipette, and the number of live cells was determined by trypan blue exclusion. Postnatal day 1 rats were cryoanesthetized, placed on a stereotactic device, and injected with 1 m l containing 50,000 cells into the neostriatum and returned to their dams after recovery.

Immunohistochemical Analysis of Transplanted Rat Brain. Four weeks after transplantation, rats were perfused with 4% paraformaldehyde. Brains were removed, postfixed for 2 h in 4% paraformaldehyde, cryoprotected in 20% sucrose overnight, and sectioned at 20 m m on a cryostat. Sections were incubated in primary and secondary antibodies as described above. To detect human cells in the transplanted brains, sections were stained with a mouse monoclonal antibody against human nuclei (1:100; Chemicon).

Cellular Transplantation. Human cells were found seeding the subventricular zone of the lateral ventricle (Fig. 5A), the corpus callosum (Fig. 5B), and at the injection site (Fig. 5C) by 4 weeks after injection. Those human cells localized to the corpus callosum had elongated nuclei, consistent with the possibility that transplanted cells were migrating (Fig. 5B) (5). Some human cells in all locations expressed the neuronal marker Hu (Fig. 5 A and B), whereas others expressed GFAP (data not shown). Approximately 50% of transplanted cells expressed Ki-67, a marker of cycling cells (6), indicating that at least half of the tumor-derived progenitors and their derivatives continued to proliferate 4 weeks after transplantation into rat brains (Fig. 5C).

1. Svendsen, C. N., ter Borg, M. G., Armstrong, R. J., Rosser, A. E., Chandran, S., Ostenfeld, T. & Caldwell, M. A. (1998) J. Neurosci. Methods 85, 141-152.

2. Geschwind, D. H., Ou, J., Easterday, M. C., Dougherty, J. D., Jackson, R. L., Chen, Z., Antoine, H., Terskikh, A., Weissman, I. L., Nelson, S. F. & Kornblum, H. I. (2001) Neuron 29, 325-339.

3. Groszer, M., Erickson, R., Scripture-Adams, D. D., Lesche, R., Trumpp, A., Zack, J. A., Kornblum, H. I., Liu, X. & Wu, H. (2001) Science 294, 2186-2189.

4. Uchida, N., Buck, D. W., He, D., Reitsma, M. J., Masek, M., Phan, T. V., Tsukamoto, A. S., Gage, F. H. & Weissman, I. L. (2000) Proc. Natl. Acad. Sci. USA 97, 14720-14725.

5. Yang, H., Mujtaba, T., Venkatraman, G., Wu, Y. Y., Rao, M. S. & Luskin, M. B. (2000) Proc. Natl. Acad. Sci. USA 97, 13366-13371.

6. Gerdes, J., Schwab, U., Lemke, H. & Stein, H. (1983) Int. J. Cancer 31, 13-20.