Enrichment and Transcriptional Characterization of Stem Cells Isolated from Human Glioblastoma Cell Lines.

Glioblastomas (GBM) are the most frequent and aggressive brain tumors due to their recurrence and resistance to current therapies. These characteristics are associated with the presence of glioma stem cells (GSCs), mainly identified by the detection of the membrane antigens CD133 and CD15. The main source of GSCs has been biopsies of tumors. However, alternatives are sought from cell lines because more homogeneous populations can be obtained with high yields. This chapter describes a method for the enrichment and characterization of GSCs from cell lines derived from human GBM by selective culture with serum-free neural stem cell medium and growth factors. The technique offers alternatives for the enrichment and characterization of GSCs, that could contribute to a better understanding of the biology of GBMs.

Estradiol Induces Epithelial to Mesenchymal Transition of Human Glioblastoma Cells.

The mesenchymal phenotype of glioblastoma multiforme (GBM), the most frequent and malignant brain tumor, is associated with the worst prognosis. The epithelial-mesenchymal transition (EMT) is a cell plasticity mechanism involved in GBM malignancy. In this study, we determined 17ß-estradiol (E2)-induced EMT by changes in cell morphology, expression of EMT markers, and cell migration and invasion assays in human GBM-derived cell lines. E2 (10 nM) modified the shape and size of GBM cells due to a reorganization of actin filaments. We evaluated EMT markers expression by RT-qPCR, Western blot, and immunofluorescence.We found that E2 upregulated the expression of the mesenchymal markers, vimentin, and N-cadherin. Scratch and transwell assays showed that E2 increased migration and invasion of GBM cells. The estrogen receptor-α (ER-α)-selective agonist 4,4',4''-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT, 10 nM) affected similarly to E2 in terms of the expression of EMT markers and cell migration, and the treatment with the ER-α antagonist methyl-piperidino-pyrazole (MPP, 1 µM) blocked E2 and PPT effects. ER-ß-selective agonist diarylpropionitrile (DNP, 10 nM) and antagonist 4-[2-phenyl-5,7-bis(trifluoromethyl)pyrazole[1,5-a]pyrimidin-3-yl]phenol (PHTPP, 1 µM) showed no effects on EMT marker expression. These data suggest that E2 induces EMT activation through ER-α in human GBM-derived cells.

Effects of progesterone on the cell number of gliomaspheres derived from human glioblastoma cell lines.

AIMS: The malignancy of the Glioblastomas (GBM), the most frequent and aggressive brain tumors, have been associated with the presence of glioma stem cells (GSCs) which can form gliomaspheres (GS) in vitro. Progesterone (P) increases the proliferation, migration, and invasion of GBM cell lines through the interaction with its intracellular receptor (PR). However, it is unknown if the PR is expressed and the possible effects of P in the formation/differentiation of GS. MAIN METHODS: GS were grown from U251 and U87 cell lines by selective culture with serum-free neural stem cell medium. GSCs were identified by the detection of Sox2, Ki67, Nestin, CD133, and CD15 by immunofluorescence. Additionally, the relative expression of PROM1, NES, SOX2, OLIG2, EZH2, BMI1 and PR genes was evaluated by RT-qPCR. The GS were treated with P, and the number of cells was quantified. By RT-PCR the ßIII-TUB and GFAP differentiation genes were evaluated. KEY FINDINGS: GS were maintained until passage four. The expression of all GSCs markers was significantly higher in GS as compared with the basal culture of U251 and U87 cells. We demonstrated for the first time that PR is expressed in GS and this expression was higher as compared with the U251 and U87 cells in basal conditions. Also, we observed that P increased the number of cells derived primary gliomaspheres (GS1) from the U251 line, as well as the expression of the neuronal differentiation marker ßIII-TUB. SIGNIFICANCE: These results suggest the participation of P in the growth of GSCs.