CD133+ glioblastoma (GBM) sphere culture derived from treated and recurrence GBM tumors express neural and mesenchymal stem cell-associated genes. A Under neural stem cell (NSC)-selective conditions, passaged and dissociated GBM spheres can generate single cells, small spheres, and large spheres (>50 cells), indicating tumor spheres consist of progeny with different proliferative potentials. Scale bar = 50 μm. B Propagated GBM sphere cultures contain ~7% to 10% of the CD133+ GBM cells determined by flow cytometry analysis. C The clonogenic efficiency of dissociated CD133+ GBM spheres assayed by the limiting dilutions relatively correlates to the % of the CD133+ cells determined in the GBM spheres. D CD133+ GBM spheres express neural and mesenchymal/chondrogenic-associated genes as indicated, determined by immunocytochemical analysis. Scale bar = 25 or 50 μm, as indicated

CD133+ GBM cells can reconstitute an infiltrating GBM tumor in mouse brain that displays hypervascularity and pseudopalisading necrosis-like features. A Passaged CD133+ GBM spheres (>20 passage) can radially migrate out of spheres extensively (a–f). Magnification, 40× (f), 100× (e), 200× (a, d), 400× (b, c). B The flow cytometry analysis indicated that the adherent GBM sphere cultures contain a higher percentage of CD133+ cells (20–70%) that coexpressed SOX2 and CD44 (a–d). Replating adherent CD133+ GBM sphere culture cells at clonal cell density can re-initiate spheres that contain ~10% CD133+ GBM cells (e, f). C Genomic abnormalities that are associated with glioblastoma were detected in CD133+ GBM cells. CD133+ GBM cells were evaluated for allelic imbalances and chromosomal copy number abnormalities by using a high-density single nucleotide polymorphism array analysis. X axis, length of chromosomes 17, 10, and 7; Y axis, score of the evidence of LOH or gain of gene copy. D Intracranial injection of purified CD133+, not CD133− GBM daughter cells, can lead to the development of infiltrating tumors. HE staining shows hypercellular zones surrounding necrotic foci and the formation of a clear space (a–i). The hypervascularity was displayed by the strong positivity of CD31/PECAM-1 (platelet endothelial cell adhesion molecule-1) as determined by immunostaining (k, l). CD133 immunoreactive cells were occasionally found in small clusters (m, n). The expression of nestin, SOX2, and YKL-40 in the infiltrating cells validates the origin of human malignant GBM tumor (o–q). No immunoreactivity was determined when the control antibody was applied (j). Magnification, 200× (a–g; j–q), 400× (h, i)

CD133+ GBM cells are sphere-reinitiating cells capable of undergoing clonal self-renewing and proliferative cell division in order to repopulate spheres. A Purification of CD133+ GBM cells from bulk CD133+ sphere cultures using specific anti-CD133 antibody and fluorescence-activated cell sorter. B A single CD133+ GBM cell can undergo proliferative cell division to generate daughter cells that are morphologically heterogeneous as revealed by cell size. Scale bar = 25 μm. C GBM spheres initiated by a single CD133+ GBM cell contain ~10% CD133+ GBM cells as determined by flow cytometry analysis. D RT-PCR analysis showed that purified CD133+ GBM cells overexpress CD133 transcripts compared with GBM spheres. Sorted CD133− GBM cells, serum-cultured GBM cell lines, and fibroblasts do not express CD133 transcripts. Normal neural stem cells served as positive control cells, show a strong signal for CD133. Beta-actin was served as an internal loading control. E The growth expansion assays indicated that GBM sphere cultures initiated by CD133+ GBM daughter cells, not CD133− GBM daughter cells, can be propagated for indefinite passages. (a) Cells were seeded in 6-well plates at a cell density of 10⁴ cells per well in triplicates. Cells were counted approximately every 2 weeks and reseeded at the same cell density. (b) Short-term proliferation assay performed in day 2 cultures indicated that freshly sorted CD133+ GBM cells exhibited less proliferative activity compared to the that of CD133− GBM cells sorted from the same sphere culture, as determined by MTS/PMS colorimetric assay. Bars represent the mean ± standard error of triplicate wells. (c) CD133+ GBM cells, not CD133− GBM cells, sorted from the same CD133+ GBM sphere cultures can repopulate GBM spheres for indefinite passages

Analyses of gene expression profiles of purified, tumorigenic CD133+ GBM cells sorted from the CD133+ GBM sphere cultures. A All plots show normalized gene expression values converted into a heat map. The log2 of the fold difference is indicated by the heat map scale at the bottom. Each column is an individual sample organized into cell types and culture conditions defined at the top. Each row is a single probe set measurement of transcript abundance for an individual gene. The genes are listed in the same order from top to bottom as the corresponding tables for each of the lists. (a) All genes were filtered to select transcripts with ≥3-fold expression in the tumorigenic CD133+ GBM cells (D431 and S496) sorted from the CD133+ sphere cultures (passage (p) 20, p29, and p40) compared with the non-tumorigenic, autologous CD133− GBM cells cultured in serum-containing media (p5, p10 and p15) with or without switching to a short-term NSC culture condition for 24 h, 48 h and 6 days. Sixty-four shared genes were identified from the intersection of the comparisons between CD133+ D431 GBM cells and CD133− D431 cells, and the comparison between CD133+ S496 GBM cells and CD133− S496 GBM cells. Functional categories of gene clusters upregulated in the CD133+ GBM cells were analyzed using a gene ontology annotation–based gene function enrichment analysis (d-chip software). (b, c) Gene changes in CD133− GBM daughter cells compared to CD133+ GBM daughter cells sorted from same CD133+ GBM sphere cultures. Genes that were upregulated or down-regulated with ≥1.5-fold expression in CD133− GBM daughter cells compared with CD133+ GBM daughter cells were collected. The CD133+ and CD133− GBM cells were sorted from the sphere cultures at p20, p29, and p40. Functional categories of gene clusters in GO terms were shown. B RT-PCR analysis showed that CD133+ GBM stem cell-associated transcripts are expressed in patient-derived GBM tumors