STEM-19. THE ISOLATION AND IDENTIFICATION OF GLIOMA STEM CELLS AND MESENCHYMAL STEM CELLS FROM HUMAN GLIOMA SPECIMENS

Abstract

Abstract INTRODUCTION Glioma is the primary malignancy with the highest incidence in central nervous system. Studies have shown that glioma stem cell (GSC) is the important origin for recurrence and closely associated with tumor microenvironment. Glioma-associated human mesenchymal stem cells (GA-hMSCs), as an important part of the tumor microenvironment, might promote proliferation and invasion of GSC. We try to isolate GSCs and GA-hMSCs through glioma primary culture, and investigate the relationship between GSC and GA-hMSCs, as well as their roles in the recurrences of glioma. MATERIALS AND METHODS Five surgical specimens from patients with human high-grade glioma were collected for primary culture. The surgical specimens were cut up and digested with Dispase II (collagenase), terminated by adding plenty of DNase II. Cell suspension was collected and resuspended in conditional medium for MSC or GSC. After being cultured for several passages, GSCs were identified by immunofluorescence, and GA-hMSCs were identified by flow cytometry and in vitro differentiation assays. RESULTS Cell cultures were observed with an inverted phase-contrast microscope after 24 hours. Adherent GA-hMSCs were collected and the supernatant was discarded with non-adherent cells. GSC was cultured as neurospheres. The single-cell suspension was prepared from spheres of GSC to identify their surface expression profile by immunofluorescence assays after several passages. The results confirmed that GSCs positively expressed CD133, Nestin and SOX2, but did not express GFAP and Tublin III. GA-hMSCs positively expressed CD105, CD73, CD90 and negatively expressed CD45 and CD34. GA-hMSCs could differentiate into adipocytes, chondrocytes and osteocytes. CONCLUSION We isolated and identified GSCs and GA-hMSCs from glioma primary culture. Our future work will focus on the relationship between the two types of cells and their roles in the process of tumor recurrence based on the successful isolation of GSC and GA-hMSCs. Keywords: Glioma; Mesenchymal stem cells; Glioma stem cells; Recurrence