Identification of U251 glioma stem cells and their heterogeneous stem-like phenotypes

Abstract

Glioblastoma, the most common and lethal type of intracranial tumor, is characterized by extensive heterogeneity at the cellular and molecular levels. The discovery of glioma stem cells (GSCs) lends support to a new paradigm in tumor biology. In the present study, we aimed to clarify the validity of using U251 glioma cells as a source of GSC culture and critically evaluate the heterogeneous stem-like phenotypes of these cells when grown under various culture conditions. The findings suggested that U251 cells (U251-Adh, U251-SC-Sph and U251-SC-Adh) showed distinctive growth patterns and self-renewal capacity. The U251 glioma cell line is endowed with certain GSC phenotypes that may be moderately enriched in vitro when transferred into stem cell culture conditions, although this is not sustainable and reproducible in vivo. Notably, glioma cells are plastic in response to their environment. The reversible adaptive plasticity contributes to the GSC heterogeneity, which may lead to the heterogeneity of glioblastoma and the differing responses to current therapies. Therefore, an improved understanding of GSC heterogeneity is urgently required for designing more effective therapies against this highly malignant brain tumor.

Keywords: U251; glioma stem cells; heterogeneity; phenotype.

Figure 1

Differing growth patterns and proliferation rates of U251 glioma cells cultured under three growth conditions. (A) U251-Adh cells showed adherent growth in conventional 10% serum medium. (B) U251-SC-Sph cells showed non-adherent sphere growth in serum-free medium and (C) U251-SC-Adh cells exhibited adherent growth on laminin-coated flasks in serum-free medium. Scale bar, 50 μm. (D) Growth curves of U251 glioma cells under respective culture conditions. The horizontal axis represents the number of cell passages and the vertical axis represents the ln values, which were derived from ln_{(numbers of cells/106)}. (E–G) Cell cycle analysis: Flow cytometry analysis of propidium iodide-stained U251 cells under respective culture conditions. (H) Fraction of cell population in S and G₂-M phase. (I) Capability of tumor sphere formation from gradient cell titers at 10, 20, 50, 100, 200, 500 and 1,000 cells per 96-well plate, respectively. (J) Capability of neurosphere formation from a single cell per 96-well plate. ^*P<0.05 and ^**P<0.01. Adh, adherent growth; Sph, spherical growth; SC, stem cell (serum-free) medium.

Figure 2

Discriminatory cell marker expression of U251 cells under three culture conditions. (A–C) Immunofluorescence analysis of CD133, nestin and GFAP expression. Although a lack of immunoreactivity was observed for CD133, a higher expression of nestin was found in U251-SC-Sph and U251-SC-Adh cells. While a higer expression of GFAP was detected only in U251-Adh. The lower rows showed Nuclei counterstained with DAPI. (D) Western blot analysis of CD133, nestin and GFAP expression. (E) RT-PCR analysis of CD133, nestin, GFAP, Notch1 MMP2 and MMP9 expression. Adh, adherent growth; Sph, spherical growth; SC, stem cell (serum-free) medium; GFAP, glial fibrillary acidic protein; MMP, matrix metalloproteinase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Figure 3

SP analysis. (A, C and E) Flow cytometry analysis of SP cells under respective culture conditions. (B, D and F) The cells were treated with 50 μM verapamil during the labeling procedure. SP, side population; Adh, adherent growth; Sph, spherical growth; SC, stem cell (serum-free) medium.

Figure 4

Differences in tumorigenicity. (A) Kaplan-Meier blot showing survival of nude mice and (B) mean survival time of mice after being injected with U251 cells under respective culture conditions. Adh, adherent growth; Sph, spherical growth; SC, stem cell (serum-free) medium.