Isolation of Cancer Stem Cells from Three Human Glioblastoma Cell Lines: Characterization of Two Selected Clones

Abstract

Cancer stem cells (CSC) were isolated via a non-adherent neurosphere assay from three glioma cell lines: LI, U87, and U373. Using a clonal assay, two clones (D2 and F11) were selected from spheres derived from LI cells and were characterized for the: expression of stem cell markers (CD133, Nestin, Musashi-1 and Sox2); proliferation; differentiation capability (determined by the expression of GalC, βIII-Tubulin and GFAP); Ca2+ signaling and tumorigenicity in nude mice. Both D2 and F11 clones expressed higher levels of all stem cell markers with respect to the parental cell line. Clones grew more slowly than LI cells with a two-fold increase in duplication time. Markers of differentiation (βIII-Tubulin and GFAP) were expressed at high levels in both LI cells and in neurospheres. The expression of Nestin, Sox2, and βIII-Tubulin was down-regulated in D2 and F11 when cultured in serum-containing medium, whereas Musashi-1 was increased. In this condition, duplication time of D2 and F11 increased without reaching that of LI cells. D2, F11 and parental cells did not express voltage-dependent Ca2+-channels but they exhibited increased intracellular Ca2+ levels in response to ATP. These Ca2+ signals were larger in LI cells and in spheres cultured in serum-containing medium, while they were smaller in serum-free medium. The ATP treatment did not affect cell proliferation. Both D2 and F11 induced the appearance of tumors when ortotopically injected in athymic nude mice at a density 50-fold lower than that of LI cells. All these data indicate that both clones have characteristics of CSC and share the same stemness properties. The findings regarding the expression of differentiation markers and Ca2+-channels show that both clones are unable to reach the terminal differentiation. Both D2 and F11 might represent a good model to improve the knowledge on CSC in glioblastoma and to identify new therapeutic approaches.

Reduced gliotransmitter release from astrocytes mediates tau‐induced synaptic dysfunction in cultured hippocampal neurons

“…Confocal Ca 2+ imaging was performed as previously described (Iacopino et al, 2014; Santoro et al, 2014) with minor modifications. Briefly, cultured hippocampal astrocytes (either WT or APP KO) were incubated for 30 min at 37 °C with the Ca 2+ sensitive fluorescent dye Fluo-4 AM (2.5 μM, Thermo) in Tyrode's solution containing (in mM): 150 NaCl, 4KCl, 1MgCl 2 , 10 glucose, 10 HEPES and 2 CaCl 2 (pH adjusted to 7.4 with NaOH).…”

Suberoylanilide hydroxamic acid represses glioma stem-like cells

“…1a) and U373MG (Additional file 1: Figure S1A) GBM cells, since a spheroid environment can be employed to enrich CSCs [25, 26]. After 2–14 days of incubation, stem cell-related properties in the spheroids were further examined via stemness-related markers, such as CD133, Bmi1, Nanog, Oct3/4, and Sox2.…”

miR-340 inhibits glioblastoma cell proliferation by suppressing CDK6, cyclin-D1 and cyclin-D2

“…Our report shows that miR-340 overexpression in U87 and T98G significantly reduced the number of neurospheres that formed. Neurospheres are comprised of neural stem cells and progenitor cells, and several studies have shown distinct CSC populations in U87 and T98G cell lines [25,26]. Therefore, it is possible that endogenous miR-340 might be downregulated in the CSC population of glioblastoma cells and in the absence of the negative regulation of CDK and cyclins, glioblastoma CSCs might undergo continual renewal, proliferation, and differentiate into cancer cells.…”