Abstract
ObjectiveTo investigate the effect of all-trans retinoic acid(ATRA) on the proliferation and differentiation of brain tumor stem cells(BTSCs) in vitro.MethodsLimiting dilution and clonogenic assay were used to isolate and screen BTSCs from the fresh specimen of human brain glioblastoma. The obtained BTSCs, which were cultured in serum-free medium, were classified into four groups in accordance with the composition of the different treatments. The proliferation of the BTSCs was evaluated by MTT assay. The BTSCs were induced to differentiate in serum-containing medium, and classified into the ATRA group and control group. On the 10th day of induction, the expressions of CD133 and glial fibrillary acidic protein (GFAP) in the differentiated BTSCs were detected by immunofluorescence. The differentiated BTSCs were cultured in serum-free medium, the percentage and the time required for formation of brain tumor spheres (BTS) were observed.ResultsBTSCs obtained by limiting dilution were all identified as CD133-positive by immunofluorescence. In serum-free medium, the proliferation of BTSCs in the ATRA group was observed significantly faster than that in the control group, but slower than that in the growth factor group and ATRA/growth factor group, and the size of the BTS in the ATRA group was smaller than that in the latter two groups(P < 0.01). In serum-containing medium, the expression percentages of CD133 and GFAP in the differentiated BTSCs were (2.29% ± 0.27%) and (75.60% ± 4.03%) respectively in the ATRA group, and (7.05% ± 0.49%) and (12.51% ± 0.77%) respectively in the control group. The differentiation rate of BTSCs in the ATRA group was significantly higher than that in the control group (P < 0.05), but there was still CD133 expressed in the ATRA group. The differentiated BTSCs could re-form BTSs in serum-free medium. The percentage of BTS formation in the ATRA group was(4.84% ± 0.32%), significantly lower than that in the control group (17.71% ± 0.78%) (P < 0.05), and the time required for BTS formation in the ATRA group was (10.07 ± 1.03)d, significantly longer than that in the control group (4.08 ± 0.35)d (P < 0.05).ConclusionATRA can promote the proliferation and induce the differentiation of BTSCs, but the differentiation is incomplete, terminal differentiation cannot be achieved and BTSs can be formed again.
Highlights
All-trans retinoic acid (ATRA) is one of the strongest and most thoroughly studied differentiation inducers
(5) Immunofluorescent detection of the differentiated brain tumor stem cells (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, incubated with antiCD133 or anti-glial fibrillary acidic protein (GFAP) antibody overnight at 4°C, and 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
Through MTT experiment, we have found that ATRA alone can promote the proliferation of BTSCs, but the promoting effect is weaker than Epidermal growth factor (EGF) +basic fibroblast growth factor (bFGF), and there is no obvious synergistic or antagonistic effect between ATRA and EGF+bFGF
Summary
Limiting dilution and clonogenic assay were used to isolate and screen BTSCs from the fresh specimen of human brain glioblastoma. The obtained BTSCs, which were cultured in serum-free medium, were classified into four groups in accordance with the composition of the different treatments. The proliferation of the BTSCs was evaluated by MTT assay. The BTSCs were induced to differentiate in serum-containing medium, and classified into the ATRA group and control group. On the 10th day of induction, the expressions of CD133 and glial fibrillary acidic protein (GFAP) in the differentiated BTSCs were detected by immunofluorescence. The differentiated BTSCs were cultured in serum-free medium, the percentage and the time required for formation of brain tumor spheres (BTS) were observed
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