Research Of Differential Inhibition, Autophagy And Transdifferentiation In Glioma Stem/Progenitor Cells

Partâ… Ultrastructural Mechanism of Differential Inhibition in Glioma Stem/Progenitor CellsObjective: In previous study, it has been demonstrated that the most significant difference of glioma stem/ progenitor cells (GSPCs) cultivated in vitro is consecutive differential inhibition comparing with neural stem/ progenitor cells (NSPCs), even occasional retrodifferentiation. In order to explore the underlying mechanism at subcellsular levels, we study the similarity and difference of ultrastructures between GSPCs and NSPCs.Methods: GSPCs were kept by our laboratory. NSPCs were isolated from human fetal brain tissue, and were cultivated, amplificated in vitro under the same conditions as that of GSPCs. After being identified, NSPCs, as well as GSPCs, were collected and prepared for the ultrastructural detections under scanning electron microscope (SEM) and transmission electron microscope (TEM), and their ultrastructures were compared qualitatively or quantitively.Results: Their common characteristics were undeveloped organelles, neurofilaments and microtubules in minority of cells, high nuclear-cytoplasmic ratio, and more euchromatin and less heterochromatin; some certain kinds of cell junctions could be seen between adjacent cells in both kinds of cellular spheres with lucent and dark cells according to electron densities. Differentia: a GSPC, which was bigger than a NSPC, had more microvilli, more developed ribosomes, endoplasmic reticulums, Golgi apparatus and mitochondrias, all of which were closely related to protein synthesis and secretion. Autophagosomes could be seen in some NSPCs but hardly in GSPCs. A GSPC frequently had two or more chromatospherites in a bigger nucleus than that of a NSPC. And the relative quantity of heterochromatin in a GSPC was higher than that in a NSPC. Synapsis-like structures and cellular membrane fusion could be found in neural spheres, but not in the spheres of GSPCs, where existed desmosome-like structures.Conclusions: Between GSPCs and NSPCs there were extensive similarities, as well as a lot of difference, which were the structural bases for the differential inhibition of GSPCs, and were also probably the mechanisms of retrodifferentiation of GSPCs, especially the different quantities of autophagosomes was worth of further study.Partâ…¡Differential Inhibition of Glioma Stem/Progenitor Cells Were Related to Autophagy DepletionObjective: In Partâ… , we found that there were much less autophagosomes in GSPCs than in NSPCs. In this part, we studied whether the autophagic activities between GSPCs and NSPCs were also different, as well, whether the difference was related to differential inhibition of GSPCs.Methods: MDC staining, immunofluorescence staining against microtubule-associated protein light chain-3 (LC3) and the expression of LC3 measured by western-blot were used to detect the autophagic activities of both GSPCs and NSPCs. The autophagic activities of GSPCs induced to differentiate were also detected. As well, autophagy inhibitors including 3- methyladenine (3-MA) and Bafilomycin A1 (BFA) were exerted to inhibit the autophagy of GSPCs when being induced to differentiate to study the effect of inhibition of autophagy on GSPCs'differentiation. Rapamycin (RPM), an autophagy promoter, was used to study its effects on the differentiation of GSPCs.Results: By comparative studies, we found that the autophagic activities of GSPCs were significantly lower than that of NSPCs. However, the autophagic activities increased markedly after GSPCs were induced to differentiate, and autophagy inhibitors, 3-MA or BFA, could inhibit the adherence and differentiation of GSPCs, and western-blot showed that the expressions of GFAP and MAP-2 were much lower than that of control group without autophagy inhibitors. What's more, under special condition, RPM could promote the differentiation of GSPCs.Conclusion: The autophagic activity in NSPCs was much higher than that in GSPCs, which probably leads to the differential inhibition of GSPCs.Partâ…¢Deactivation of PTEN in GSPCs is related to Autophagy DepletionObjective: PTEN, an anti-oncogene, has a close relationship with autophagy, and often deactivated in tumors of nervous system. In partâ…¡, we demonstrated the correlativity of differential inhibition of GSPCs and autophagy depletion. In this part, we want to explore the autophagy depletion in GSPCs was related to the deactivation of PTEN.Methods: The genetic sequences of PTEN, as well as the sequence of PTEN protein, in both GSPCs and NSPCs were compared with that reported by genbank. The wild-type PTEN was introduced into GSPCs by adenovirus, and then the autophagic activities of GSPCs were detected.Results: There were a lot of base mutations of PTEN in GSPCs comparing with the sequence of PTEN reported by genbank, but no base mutations in NSPCss. As well, for the pten protein sequences in GSPC, there were several mutations of amino-acid residues (8th-14th) locating at the N-end, the 238th and the 398th amino-acid residue. After the wild-type PTEN were introduced into GSPCs, the autophagic activities increased significantly.Conclusion: The mutation of PTEN in GSPCs is one of the mechanisms of low atuophagic activities in GSPCs, which is valuable for the further studies on target molecules related to autophagy.Partâ…£Endothelial Cell Transdifferentiation of Glioma Stem/ Progenitor Cells In vitroObjective: Differential inhibition of GSPCs is a malignant characteristic of glioma. And in Partâ… toâ…¢, we have demonstrated, from cellular, subcellular, and molecular levels, that cellular autophagy depletion of GSPCs was involved in differential inhibition. Meanwhile, affluent blood supply and active angiogenesis are also malignant characteristics of glioma. In this part, we aimed to investigate whether glioma stem/ progenitor cells (GSPCs) have the capabilities to participate in angiogenesis by trans-differentiating into vascular endothelial cell-like cells.Methods: we cultivated GSPCs in endothelial differentiation medium for 10 days and to observe the changes of appearance. We also cultivated GSPCs on Matrigel for 10 days, and the process of morphological changes was observed. Ten days later, part of the Matrigel containing cells was detected for the expression of CD31 by immunohistochemistry, and part of them were used for the ultrastructural study. GSPCs were also cultivated under hypoxia or oxygen-glucose deprivation (OGD) for 4 hours, and the transcriptions and expressions of numerous types of molecular markers of vascular endothelial cell (VEC), including CD31, CD34, KDR and vWF were detected respectively with RT-PCR and immuocytochemistry.Results: Ten days after GSPCs were cultivated in endothelial differenetiation medium they present to be the typical "flagstone" appearance of VEC; when cultured on Matrigel, GSPCs gradually formed tubular-like structures in vitro, and cells, which formed the tubular-like structures, were positive to CD31 (a marker of VEC), and had similar ultrastructural characteristics of VEC under a transmission electron microscope. What's more, when cultured in hypoxia or OGD for 4hours, the transcriptions and expressions of these VEC markers increased a lot.Conclusion: GSPC could transdifferentiate into VEC-like cells in vitro, which enlightened the further studies on the independent formation of new vessels by GSPCs in orthotopic implantation tumors, and was significant for the illumination of angiogenesis mechanism in tumors.