1 Major reagents and instruments
(1) Major reagents: DMEM/F12 and B27 were purchased from Gibco(U.S.A). Epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) were purchased from PeproTech (U.S.A.). ATRA,3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT), fetal bovine serum (FBS), trypsin, Cy3-labeled sheep anti-rabbit IgG and diamidino-phenyl-indole (DAPI) were all purchased from Sigma (U.S.A). Rabbit anti-human CD133 antibody was purchased from Abcam (U.S.A). Rabbit anti-glial fibrillary acidic protein (GFAP) antibody and FITC-labeled goat anti-rabbit IgG were purchased from Boster (Wuhan, China).
(2) Major instruments: BB16 CO2 incubator and HF-safe-1200 purifying worktable (Heraeus and Lishen company, Germany). CKX41 inverted phase contrast microscope, BX51 fluorescence microscope and imaging system (Olympus, Japan). ELISA Reader 2010 (Anthos, Austria).
2 Experimental methods
(1) Isolation, culture and purification of BTSCs: The tissue samples were obtained from 3 surgical patients in Department of Neurosurgery, Anhui Provincial Hospital Affiliated to Anhui Medical University who had been diagnosed with glioblastoma during February-May, 2009. Fresh glioblastoma tissues without cystic degeneration, necrosis, calcification and electric coagulation were resected from the margin of tumor. By method in Ref[
2], fresh glioblastoma tissues without cystic degeneration, necrosis, calcification and electric coagulation were resected from the margin of tumor, put in simplified serum-free medium (DMEM/F12, containing 2% B27, 20 g/L EGF and 20 g/L bFGF), and trimmed off necrotic tissues and residual blood vessels. After rinsing with serum-free medium for three times, the tissue masses were cut into pieces, disaggregated into single cell suspensions by using a Pasteur pipette (blowing repeatedly), and filtered with a sieve with the aperture of 74 m. The filtered single cell suspensions were stained with Trypan Blue. The living cells were counted, and primary culture was completed within 2 h, followed by inoculation in simplified serum-free medium (DMEM/F12, containing 2% B27, 20 μg/L EGF and 20 μg/L bFGF), and then culture at 37°C in 5% CO
2 saturated humidity incubator. The medium was changed every 3~4 days. The cells were passaged by 1:2 subculture every 7 days and observed under the inverted phase contrast microscope. The cells were passaged three times. After the cell spheres became regularly shaped, they were dissociated into single cells with 0.25% trypsin + mechanical method, and inoculated into a 96-well plate at 1 living cell/well, with each well added with 100 μL simplified serum-free medium. The wells containing only one cell were labeled under the inverted microscope, and supplemented with 100 μL simplified serum-free medium for further culture. The formation of single cell colonies was recorded by dynamic observation. The cells were observed under the inverted microscope after culture for about one week, and the proliferated cells were collected and transferred into a culture flask for further culture and proliferation. The purified BTSCs after colony screening were used in the following experiments.
(2) Immunofluorescent identification of BTSCs: On the 5th day of passage, BTSs that grew well were re-suspended in culture medium containing a small amount of serum (DMEM/F12 containing 10%FBS), and dropped onto a poly-L-lysine-coated coverslip. After standing still for about 4 h until the solution adhered to the coverslip, the coverslip was fixed in 4% paraformaldehyde for 30 min, blocked with normal goat serum for 20 min, incubated with rabbit anti-human CD133 antibody overnight at 4°C, and then incubated with Cy3-labeled sheep anti-rabbit IgG at 37°C for 60 min, followed by DAPI counterstaining of the nuclei and coverslipping with buffered glycerol. Following each step, the coverslip was rinsed with 0.01 mol/L PBS three times, each for 5 minutes. The coverslip was observed after mounting and pictures were taken.
(3) Assessment of the effect of ATRA on proliferation of BTSCs: The BTSCs were collected and divided into groups as described below, put into the corresponding culture medium, disaggregated into single cell suspensions by mechanical dissociation, and inoculated into a 96-well plate at the density of 1000 living cells/well, with 100 Ml in each well. According to the different treatments, the BTSCs were divided into: (1) control group: basic medium (DMEM/F12 with 2% B27) containing the same amount of anhydrous ethanol as in the ATRA group (the final concentration < 0.1%); (2) ATRA group: containing 1 μmol/L ATRA; (3) ATRA/growth factor group: containing 1 μmol/L ATRA, and 20 μg/L EGF and 20 μg/L bFGF; (4) growth factor group: containing 20 μg/L EGF and 20 μg/L bFGF. The cells in the plate were then cultured at 37°C in 5% CO2 saturated humidity incubator, each well was added with the corresponding medium 20 μL every other day, and the cell proliferation was observed under the inverted microscope every day. MTT assay was performed to evaluate the proliferation consecutively from the 1st to the 9th day of culture. Each well was added with 20 μL MTT solution (5 g/L), and the cells were cultured for 4 h, followed by 10 min centrifugation at 1000r/min. The supernatant in the wells was absorbed carefully and discarded. Each well was added with 150 μL DMSO. After shaking for 10 min to achieve dissolution and crystallization, the optical density value of each well was measured by ELISA at the wavelength of 570 nm. Six duplicate wells were set up for each group. The experiments were repeated 3 times, and the averages were obtained.
(4) Assessment of the effect of ATRA on differentiation of BTSCs: The collected BTSCs were adjusted to 2 × 105 living cells/mL using serum-containing medium (DMEM/F12 containing 10%FBS), and inoculated into a 6-well plate with PLL-coated coverslips, with 2 mL in each well. The cells were divided into two groups: (1) ATRA group: serum-containing medium added with ATRA with the final concentration of 1 μmol/L; (2) control group: serum-containing medium containing the same amount of anhydrous ethanol as in the ATRA group (the final concentration < 0.1%). The cells were cultured at 37°C in 5% CO2 saturated humidity incubator. The culture medium was changed every 3 days. The growth and differentiation of BTSCs were observed dynamically.
(5) Immunofluorescent detection of the differentiated BTSCs: The coverslips were taken out on the 10th day of induction, fixed in 40 g/L paraformaldehyde for 30 min, blocked with normal goat serum for 20 min (those for GFAP staining were treated with 0.3%Triton X-100 for 20 min before serum blocking), incubated with anti-CD133 or anti-GFAP antibody overnight at 4°C, and then incubated at 37°C for 60 min with Cy3-labeled and FITC-labeled secondary antibodies respectively, followed by DAPI counterstaining of the nuclei and mounting with buffered glycerol. Following every step, the coverslips were rinsed with 0.01 mol/L PBS three times, each for 5 minutes. Randomly, 20 microscopic fields were selected on each coverslip and investigated under the fluorescence microscope to calculate the percentages of CD133 and GFAP positive cells among adherent cells. The calculation formula is: percentage of CD133 (or GFAP) positive cells = (CD133 (or GFAP) positive cells)/(DAPI positive cells)× 100%.
(6) Proliferation of the differentiated BTSCs: The adherent cells of the above two groups after 10 days of induction were digested with 0.25% trypsin, added with simplified serum-free medium, and inoculated into a 96-well plate at 5 living cells/well (density adjusted by limited dilution), with each well added with 100 μL simplified serum-free medium. The cells were then cultured at 37°C in 5% CO2 saturated humidity incubator. Each well was added with 20 μL simplified serum-free medium every other day, and the BTS formation was observed. The sphere formation and growth rate were observed at specified times every day, and the emergence of regularly-shaped BTSs (containing over 10 cells) was considered as positive result. The time required for BTS formation and the number of BTSs were recorded and used to calculate the percentage of BTS and the time for colony formation. The formed BTSs were dropped on PLL-coated coverslips to be dried for CD133 immunofluorescence staining as described previously.