Migration Of Neural Stem Cell Toward Glioma Stem Cell And Effects On The Self-renewal And Tumorigenesis

Objective:Brain tumor stem cell (BTSC) hypothesis posits that a subpopulation of cells within brain tumor have true clonogenic and tumorigenic potential. Especially for glioma, experimental data from different research laboratories have demonstrated the existence of a unique subpopulation of glioma stem cell (GSC). Significantly, a more controversial correlate to GSC is that glioma cells in different culture conditions display distinct stem cell properties. Considering these possibilities, we applied an approach comparing stem cell properties and biologic characteristics of C6 glioma cells under different culture conditions.Methods:C6 glioma cells were cultured under different culture conditions, including conventional culture in serum-containing medium, neurosphere-formation assay and adherent culture in serum-free supplemented medium. Growth characteristics, including morphological differences, growth curve and capacity of self-renewal, were detected contrastively through limiting dilution assay, neurosphere formation and single colony formation assay. Expressions of stem cell and differentiated cell markers were determined by immunofluorescence staining, reverse transcription polymerase chain reaction (RT-PCR), Western Blotting and Flow Cytometry (FCM). Side population cells were analyzed by side population assay via flow cytometry based on the ability to exclude the fluorescent vital dye Hoechst 33342. Cells (105) cultured under different culture conditions were implanted stereotactically and intracranially into the Balb/c nude mice to contrast the capability of tumor initiation and the tumor models were detected by magnetic resonance imaging (MRI) and hematoxylin and eosin (HE) staining. All data analyses were performed with the SPSS software, version 17.0.Results:C6 glioma cells showed distinct growth pattern and different multiplication capacity, which were cultured in different culture conditions, including conventional culture in serum-containing medium (C6-Adh), neurosphere-formation assay (C6-SC-Sph) and adherent culture in serum-free supplemented medium (C6-SC-Adh). Compared to suspending C6-SC-Sph, adherent C6-Adh and C6-SC-Adh displayed higher growth ratio (P<0.05). C6-SC-Sph and C6-SC-Adh under stem cell culture condition were found with enhanced capability of neurosphere formation and self-renewal (P＜0.05). High side population ratio were detected in C6-SC-Sph and C6-SC-Adh, while low in C6-Adh (P<0.05). Determined by immunofluorescence staining, reverse transcription polymerase chain reaction, Western Blotting and FCM, CD 133 was failed to be detected in all three kinds of cells. Conversely, expression of Nestin andβIII tubulin were demonstrated positive in C6-Adh, C6-SC-Sph and C6-SC-Adh, nonetheless with no statistical significance (P>0.05). Interestingly, lower expression of GFAP was demonstrated in C6-SC-Sph and C6-SC-Adh under stem cell culture condition, compared with that in C6-Adh cultured in serum-containing medium (P<0.05). C6-Adh, C6-SC-Sph and C6-SC-Adh were all displayed in situ oncogenicity detected by MRI and HE staining. Nevertheless, statistical differences (P<0.05) of survival time was only demonstrated between C6-SC-Sph and C6-SC-Adh.Conclusion:The C6 glioma cells are endowed with some GSC phenotypes that can be moderately enriched when transferred into neurospheres growth under stem cell culture condition. The resultant tumor spheres, however, may be not a sound source of GSCs because of a lack of increased tumorigenecity in vivo. Adherent growth of C6 cells in stem cell medium proved to be a more optimized culture condition for enriching GSC characteristics and neurosphere-like growth may be not a prerequisite for GSC culturing. Objective:Glioma stem cell (GSC), which is considered to be responsible for tumorigenesis, recurrence and resistance to chemotherapy and radiotherapy, represents a critical therapeutic target. Thus, we induced the differentiation of GSC with ATRA to detect the effect on glioma stem cell phenotype.Method:First, we isolated and enriched glioma stem cell from surgical resection of human glioma specimens by neurosphere assay and then induced the differentiation of GSC with all-trans retinoic acid (ATRA, 1uM) for one week. Markers of GSC and differentiated cells, such as CD133, Nestin and GFAP, were identified through flow cytometry and/or Western Blot. Expression of Notch1 was detected by reverse transcription polymerase chain reaction (RT-PCR). Glioma stem cells and the differentiated cells (1×105, respectively) were implanted stereotactically and intracranially into the Balb/c nude mice to contrast the survival time. All data analyses were performed with the SPSS software, version 17.0.Results:Neurospheres of GSC (NCH421K,NCH441 and NCH644), with high expression of sternness markers, such as CD133 (11.02%—33.55% identified through flow cytometry) and Nestin (detected through Western Blot), were cultured in stem cell medium and could be differentiated in serum-containing medium supplemented with ATRA (1uM). After differentiation, expression of sternness markers in differentiated cells (NCH421 K-diff,NCH441-diff and NCH644-diff) was decreased, while marker of GFAP was increased (P<0.05). Meanwhile, the higher expression of Notchl in GSC was reduced, compared with that in differentiated cells (P<0.05). After differentiation with ATRA, the survival time of nude mice was prolonged obviously (P<0.05).Conclusion:GSC could be differentiated efficiently by ATRA and expression of sternness markers (CD133 and Nestin) was decreased. The Notch signaling pathway was inhibited obviously after differentiation with ATRA and the survival time was prolonged, which suggested that ATRA could be applied for the targeted therapies for glioma stem cell. Objective:Neural stem cell (NSC) has been shown to possess an inherent tropism to glioma, which might highlight a potential and novel therapeutic approach. However, the prognosis of patients with malignant glioma is generally poor after the current traditional treatment due to the presence and chemoradiotherapy resistance of glioma stem cell (GSC). For this purpose, we investigated the targeted migration of NSC towards GSC and effect on stem cell phenotypes and growth.Methods:Glioma stem cells were cultured and expanded in stem cell medium from human glioma cell line U251 and surgical resection of human gliomas specimen. After the identification through immuno fluorescence, flow cytometry, Western Blot and RT-PCR, GSCs were cultured in serum-containing medium to induce differentiation. Migrations of NSC to GSC, differentiated cell and concentration gradient fluid of growth factors were detected by Transwell assay. Concentration of VEGF and bFGF secreted by GSCs and differentiated cells was detected through ELISA assay. The in vitro migration of NSCs (Marked by Dio) to GSCs (Marked by Dil) was observed using fluorescent microscopy. After co-culture with NSC in Transwell chamber for one week, the glioma stem phenotypes were identified through flow cytometry, Western Blot and RT-PCR. The capacity of self-renewal was detected through limiting dilution assay. To investigate the in vivo migration of NSC towards GSC and effect on its growth, marked NSC and GSC (2×105 and 1×105 cells respectively) were simultaneously implanted stereotactically into the unilateral or bilateral cerebral hemisphere of Balb/c nude mice. All data analyses were performed with the SPSS software, version 17.0.Results:Neurospheres of GSCs (U251-SC and primary GSC), with high expression of sternness markers (Nestin and/or CD133), were cultured in stem cell medium and could be differentiated in serum-containing medium. After differentiation, expression of sternness markers was decreased, while marker of GFAP was increased (P<0.05). Meanwhile, the concentration of VEGF and bFGF secreted by GSC was higher than that by differentiated cells. Both GSC and differentiated cell could induce the migration of NSC, while GSC showed significantly stronger chemotactic effect (P<0.05). The migratory ability of NSC might be positively correlated with the concentration of growth factors in chemotactic fluid (P<0.05). NSC could migrate to GSC and surround the neurosphere of GSC, which displays cytostatic effect in vitro. After co-culture with NSC for one week, expression of sternness markers was decreased, while marker of GFAP was increased (P<0.05) and the capacity of self-renewal of GSC was decreased. NSC could migrate to GSC in two weeks in vivo and the survival time of nude mice was prolonged.Conclusion:Gliona stem cell could induce the migration of neural stem cell and show enhanced chemotaxis compared with the differentiated cell. This chemotactic mechanism might be related to high concentrations of growth factors secreted by GSC. In vitro and in vivo migration of neural stem cell to glioma stem cell displays cytostatic effect and improves survival.