Correction: Isolation of cancer stem cells from three human glioblastoma cell lines: Characterization of two selected clones

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 forthe: expression of stem cell markers (CD133, Nestin, Musashi-1 and Sox2); proliferation; differentiation capability(determined by the expression of GalC,bIII-Tubulin and GFAP); Ca2+signaling and tumorigenicity in nude mice. Both D2 andF11 clones expressed higher levels of all stem cell markers with respect to the parental cell line. Clones grew more slowlythan LI cells with a two-fold increase in duplication time. Markers of differentiation (bIII-Tubulin and GFAP) were expressedat high levels in both LI cells and in neurospheres. The expression of Nestin, Sox2, andbIII-Tubulin was down-regulated inD2 and F11 when cultured in serum-containing medium, whereas Musashi-1 was increased. In this condition, duplicationtime of D2 and F11 increased without reaching that of LI cells. D2, F11 and parental cells did not express voltage-dependentCa2+-channels but they exhibited increased intracellular Ca2+levels in response to ATP. These Ca2+signals were larger in LIcells and in spheres cultured in serum-containing medium, while they were smaller in serum-free medium. The ATPtreatment did not affect cell proliferation. Both D2 and F11 induced the appearance of tumors when ortotopically injectedin athymic nude mice at a density 50-fold lower than that of LI cells. All these data indicate that both clones havecharacteristics of CSC and share the same stemness properties. The findings regarding the expression of differentiationmarkers and Ca2+-channels show that both clones are unable to reach the terminal differentiation. Both D2 and F11 mightrepresent a good model to improve the knowledge on CSC in glioblastoma and to identify new therapeutic approaches