Effect Of All-trans Retinoic Acid On Proliferation And Differentiation Of Brain Tumor Stem Cells In Vitro

Objective To isolate and culture brain tumor stem cells (BTSCs) from human glioblastoma (GBM),purify and identify them to obtain available BTSCs strain. To investigate the effect of all-trans retinoic acid (ATRA) on proliferation and differentiation of BTSCs in vitro.Methods (1) BTSCs were isolated from 3 cases of fresh-resected GBM and cultured in simplified serum-free medium (SFM). They were purified by limit dilution analysis and monoclonal formation assasy. Their proliferation and differentiation were observed. Immunohistochemistry detection was conducted to identify their characteristic of stem cells. (2) The BTSCs obtained were cultured in SFM and divided into control group, ATRA group, growth factor group, and ATRA/growth factor group with corresponding treatments. The proliferation of the treated BTSCs was evaluated using methyl thiazolyl tetrazolium (MTT) assay. (3) The BTSCs were induced in serum-containing medium (SCM) and treated with ATRA or diluted solvent, and the expression of CD133 and glial fibrillary acidic protein (GFAP) in the cells were detected by immunofluorescence on day 10 of induction. (4) The differentiated BTSCs were cultured in SFM, and the percentage of and time needed for cell sphere formation were observed.Results (1) The BTSCs had been successfully isolated from human GBM. They could not only survive and grow by means of suspension in SFM but proliferate into brain tumor sphere (BTS). They had strong ability of self-renewal and proliferation, and expressed specific marker CD133. The BTSCs attached to poly-L-lysine-coated coverslips and differentiated when SCM was added. The differentiated BTSCs expressed specific antigens neurone specific enolase (NSE) and GFAP of neuron and astrocyte. (2) The proliferation of BTSCs in ATRA group was faster than that in the control group and slower than that in growth factor group and ATRA/growth factor group, and the size of BTS in ATRA group was smaller than that in the latter two groups. (3) The percentage of CD133- and GFAP-positive differentiated BTSCs were (2.29±0.27)% and (75.60±4.03)% in ATRA group, and (7.05±0.49)% and (12.51±0.77)% in the control group, respectively. The differentiation rate of BTSCs was significantly higer in ATRA group than in the control group (P<0.05), and some of the differentiated BTSCs expressed CD133. (4) The differentiated BTSCs could form BTS in serum-free medium, and in ATRA group, the percentage of BTS formation was significantly lower and time needed for BTS formation was significantly longer than those in the control group [(4.84±0.32)% vs (17.71±0.78)%, P<0.05; 10.07±1.03 vs 4.08±0.35 days, P<0.05].Conclusion There were a small amount of BTSCs in human GBM which had ability of self-renewal, continuous proliferation and multi-potent differentiation. they could be isolated, cultured and purified in vitro. ATRA can promote the proliferation and induce the differentiation of BTSCs, but the differentiated BTSCs can not achieve terminal differentiation, and tend to form BTS again.