Study On The Modeling Of Glioma Stem Cell Generation Based On Cell Reprogramming

Glioma,the most common human primary central system tumor,is characterized by proliferation and invasive capabilities.Glioma seriously endangers human health due to that it is difficult for surgical resection,not sensitive to radiochemotherapy and easy to relapse.Tumor stem cells are closely related to the proliferation,metastasis and drug resistance of tumors.It is of great significance to study the pathogenesis of glioma based on glioma stem cells for the selection of therapeutic targets and prognosis evaluation.However,there is still no reliable cell model to study the mechanism of glioma stem cell development and tumorigenesis.Cell reprogramming technology can be used to effectively establish a variety of disease models for the research of tumor pathogenesis and treatment.Tumorigenesis may drived by the activation of oncogenes and inactivation of tumor suppressor genes accompany with the abnormal regulation of multiple signaling pathways.Ras/Raf/MAPK signaling pathway plays an important role in the development of glioma.To establish a model of glioma stem cell genenration for the study of pathogenesis of glioma,we constructed lentiviral vector plasmids containing EGFR,KRAS?G13D mutant?,BRAF over-expression,which are the key genes of Ras/Raf/MAPK signaling pathway,and PTEN knockdown.By lentiviral infection,we regulated the expression of these genes in embryonic stem cells?H1 cells?alone or in combination,and established stable H1 cells carrying different gene changes,including H1-KRAS^G13D,H1-shPTEN,H1-BRAF-shPTEN,H1-KRAS^G13D-shPTEN and H1-BRAF-EGFR-shPTEN.We further obtained the neural progenitor cells?NPC?carrying different gene changes named NPC-KRAS^G13D,NPC-KRAS^G13D-shPTEN,NPC-BRAF-shPTEN and NPC-BRAF-EGFR-shPTEN by inducing H1 cells neural differentiation.These induced NPCs with different gene changes were identified to express the neural stem cells relatd makers by RT-PCR,immunofluorescence staining and Western blotting,and can differentiate to astrocyte and neurons.Through CCK-8,transwell and cell clone formation assays we found that the induced NPCs carrying tumor-related genes,especially NPC-KRAS^G13D owned elevated proliferation and agressive ability compare to the normal NPCs derived from H1 cells?NPC-H1?.Through transcriptome sequencing?RNA-seq?and bioinformatics analysis,we found that the gene expression profile of NPC-KRAS^G13D13D cells was different from that of NPC-H1,and similar to that of glioma stem cells in the cell proliferation,migration and cell metabolism related genes.We detedcted the cell metabolic ability by Seahorse and DCFH-DA assays,and results showed the enchanced metabolic ability of NPC-KRAS^G13D cells.NPC-KRAS^G13D cells were showed tumorigenic growth in vivo.Based on the characteristic and functional indentification,we speculated that NPC-KRAS^G13D cells were glioma stem cell-like cells and named them as induced glioma stem cells?iGSCs?.To sum up,we have established a new approach for obtaining induced glioma stem cells from pluripotent stem cells,and preliminarily constructed a model of human glioma stem cells generation.It is of great significance for the study on pathogenesis and targeted therapy of glioma.