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Objective: To investigate the target cells and molecules with sodium valproate induced differentiation of human glioma cells., Methods: Nude mice bearing human glioma xenogenic graft subcutaneously were treated with sodium valproate. The expressions of HDAC1 and Tob genes of xenografts were analyzed with semiquantitative RT-PCR. The CD133+ cells (BTSCs) were isolated from glioma specimens by immunomagnetic sorting, and cultured in the medium containing FCS or in the serum-free medium supplemented with growth factors, respectively, followed by treatment with sodium valproate in vitro for 21 days. The cell surface markers were detected with flow cytometry and confocal microscopy., Results: Sodium valproate inhibited the growth of subcutaneous xenografs bearing on nude mice (P<0.05), and up-regulated the HDAC1 expression (P<0.01), down-regulated the Tob expression (P<0.05). The cell surface markers of BTSCs were detected by flow cytometry after sodium valproate treatment for 21 days. In the FCS group, the GFAP or beta-tubulin III positive cells increased significantly (P<0.01), but in the growth factor group, no statistical differences were observed in the GFAP or beta-tubulin III expression (P>0.05). The results of confocal microscopy indicated that the GFAP+ or beta-tubulin III+ cells coexpressed with Nestin., Conclusions: HDAC1 and Tob genes were the potential target molecules in reversion of the differential inhibition of human glioma cells with sodium valproate. The BTSCs undergoing the processes of differentiation were the target cells for sodium valproate.

Understanding of the differentiation profile of brain tumor stem cells (BTSCs), the key ones among tumor cell population, through comparison with neural stem cells (NSCs) would lend insight into the origin of glioma and ultimately yield new approaches to fight this intractable disease. Here, we cultured and purified BTSCs from surgical glioma specimens and NSCs from human fetal brain tissue, and further analyzed their cellular biological behaviors, especially their differentiation property. As expected, NSCs differentiated into mature neural phenotypes. In the same differentiation condition, however, BTSCs exhibited distinguished differences. Morphologically, cells grew flattened and attached for the first week, but gradually aggregated and reformed floating tumor sphere thereafter. During the corresponding period, the expression rate of undifferentiated cell marker CD133 and nestin in BTSCs kept decreasing, but 1 week later, they regained ascending tendency. Interestingly, the differentiated cell markers GFAP and beta-tubulinIII showed an expression change inverse to that of undifferentiated cell markers. Taken together, BTSCs were revealed to possess a capacity to resist differentiation, which actually represents the malignant behaviors of glioma.

Objective: To pursue the changes of cell morphology, expression of differentiation related markers, and proliferation cycles of brain tumor stem cells (BTSCs) after differentiation in vitro., Methods: Tumor stem cells of the line CD133(+) were obtained from two specimens from one clinical case with anaplasia ependymocytoma during operation, one specimen being obtained during the first operation and then second specimen being obtained during the second operation 6 months later on the recurrent tumor. CD133(+) cells were acquired by using magnetic sorting and then cultured to differentiate in medium containing 10% fetal bovine serum. The morphology of the cells was observed under phase contrast microscope. Cells were collected respectively before differentiation and 3, 7, 10, and 21 days after the differentiation. The cell surface markers such as CD133, nestin, glial fibrillary acidic protein (GFAP), and beta-tubulin III were detected with flow cytometry. Proliferation cycles were examined before differentiation and in the 7th day after differentiation. Normal neural stem cells (NSCs) obtained from fetal brain tissues were used as controls., Results: (1) The BTSCs were round shape at the beginning, then changed to short fusiform, polygon and long fusiform. Seven days later the cells reversed to short fusiform and round shape. The cells accumulated into cell spheres and floated in the culture medium again. While the NSCs differentiated along their routine rules. (2) Both the undifferentiated BTSCs and NSCs showed high level expression of CD133 and nestin. After differentiation the BTSCs expressed CD133 and nestin, the expression levels decreased first and then increased. The expression rates of CD133 and nestin were (3.65 +/- 0.17)% and (28.99 +/- 1.26)% in the 7th day, (14.63 +/- 1.16)% and (45.46 +/- 1.27)% in the 21st day. While the positive expression rate of GFAP was higher than that of beta-tubulin III. In the 10th day the NSCs under differentiation lost the expression of CD133 and nestin. The percentage of GFAP positive cells and beta-tubulin III positive cells were (88.94 +/- 1.23)% and (11.94 +/- 0.36)% respectively. (3) All undifferentiated BTSCs were hypodiploid. After differentiation majority of the BTSCs were hypodiploid or hyperdiploid, The percentages of S phase and G(2)-M phase cells in the BTSCs were higher than that in the NSCs. The cell composition of recrudescent BTSCs was more complex than that of the primary BTSCs. All NSCs were diploid whether differentiated or not. Most of the NSCs were G(0)-G(1) phase cells., Conclusion: The differentiation direction of BTSCs is quietly different from that of the NSCs. There is an obvious dysdifferentiation in BTSCs.

Objective: To isolate and culture tumor stem cells from glioma tissues obtained at surgical operation and to study their biological characteristics., Methods: Glioma tissues obtained from surgically resected specimens of 8 patients were fully chopped, trypsinized, and filtered to prepare single cell suspensions. The cells were cultured in serum-free medium with EGF, LIF and bFGF. CD133(+) cells were purified by magnetic cell sorting, and cultured continuously in vitro to obtain tumor cell spheres. Tumor stem cells of the 5th passage were induced to differentiate with 10% FBS, and expression of cell differentiation markers such as Nestin, MAP2, GFAP was evaluated with immunocytochemistry techniques., Results: CD133(+) cells were successfully separated and cultured from one anasplastic mixed astrocyte-ependymocyte type glioma specimen. These cells maintained a sphere-like growth status in vitro (3 months, 14 passages), and can self-renew, proliferate and conditionally differentiate into MAP2(+) and GFAP(+) cells. However, CD133(-) cells did not possess these properties., Conclusion: Glioma tissue contains tumor stem cells. Those cells can be cultured and passaged in vitro for a long term, and therefore to offer new approaches for studying cellular and molecular biology of glioma.