Figure 1.
Representative phase contrast microscopy analysis of cultured cells.
(A): top, parental cell lines (LI, U87 and U373 cells) cultured in their standard medium added of 10% serum. Middle, formation of neurospheres on top of the monolayers of U373 cell line. Images were taken after 1, 2 and 3 days in neurosphere medium. Bottom, images of primary NS generated by LI, U87 and U373 cells by day 8. (B) Clones derived from LI (D2 and F11) and U87 (C7 and E8) primary NS. Original magnification 200× or 400×. Scale bar = 100 µm.
Figure 2.
Parental cell lines and NS derived from a single mother cell were stained by stemness marker Nestin.
The inserts refer to negative controls. Original magnification 400×. Scale bar = 100 µm.
Figure 3.
A: Flow cytometry analysis to measure CD133 expression of cells derived from LI monolayer cultures and D2 and F11 clones. CaCo2 cells are positive control. CD133 expression was evaluated in both clones at 5th passage (5thP) and 16th passage (16thP). B: Data from a representative experiment of immunocytochemical staining for Msi-1 and Sox2 expression in LI, D2 and F11 cells. The inserts refer to negative controls. Original magnification 400×. Scale bar = 100 µm.
Figure 4.
A: Immunocytochemical staining for Nestin, Msi-1, Sox2 and βIII-Tubulin expression in D2 and F11 cells cultured in neurosphere medium (without serum) or in differentiation medium (presence of serum) for 5–45 days. Original magnification 400×. Scale bar = 100 µm. B: Some βIII-Tubulin positive cells exhibiting a typical neuronal-like morphology. Original magnification 630×. Scale bar = 50 µm. C: Western blot analysis of Nestin, Msi-1 and βIII-Tubulin expression in F11 cells cultured for different times in differentiation medium. Proteins levels were analyzed by immunoblot analysis and were quantified by densitometry and standardized against the levels β-actin as a loading control. Values are the mean ± SD of tree independent experiments. Similar results were obtained from D2 cells. Immunoblottings from representative experiments are shown. *p<0.05 Student’s t-test vs. parental cell lines, *p<0.01 vs. F11 cells.
Figure 5.
(A) Bar graph representing the mean amplitude at the peak of ATP-induced Ca2+ signals (expressed in terms of the ΔF/F ratio) at different points of permanence of CSC in differentiation medium. Extracellular application of 50 µM ATP induced intracellular Ca2+ transients in CSC whose fluorescence increased with the time of differentiation reaching the maximum in cells cultured for 5 days. Since 5th day fluorescence remained constant. (B) Bar graph representing the percentage of cells responsive to ATP (50 µM) with an increase in intracellular Ca2+. (C) Comparisons of the intracellular Ca2+ transients induced by 50 µM ATP in cells at 5th day differentiation in the presence (white) and in the absence (gray) of extracellular Ca2+. (D) Bar graph quantifying the mean amplitude at the peak of intracellular Ca2+ transients in the presence (white) and in the absence (gray) of extracellular Ca2+. *p<0.001.
Table 1.
Doubling time of D2 and F11 clones cultured in NSCM (serum-free; presence of growth factors) or in medium with 10% FBS added.
Table 2.
Tumorigenicity of LI cells and CSC clones (D2 and F11).
Figure 6.
Immunostaining of brain sections from mice implanted with 500,000 LI cells, 10,000 D2 cells and 10,000 F11 cells.
Original magnification 630×. Scale bar = 50 µm.