Subsequently, the CD133? cells were separated from the A2B5+ cells and A2B5? cells, according to the manufacturer’s instructions, using A2B5 Micro Beads. In vitro invasion assay The CD133?/A2B5+and CD133?/A2B5? cells were transferred onto Matrigel-coated invasion chambers (24-well insert, 8-sphere formation assay was used to examine whether the expression of A2B5 was involved in cell renewal upon serial passaging. A2B5+ cells was Rabbit Polyclonal to PPIF higher than that of the A2B5? cells. Taken together, the results of the present study suggested that there are different cell subpopulations in GSCs, and each subpopulation has its own properties. (5) and Wang (11) found the existence of CD133? cells in CSCs. In a previous study, it was reported that A2B5+ cells from glioblastma also exhibit cancer stem-like properties (8). Compared with A2B5? cells from glioblastoma tissue, A2B5+ cells exhibit more marked tumorigenic potential (7). However, in CSC lines, the differences between A2B5? and A2B5+ cells remain to be fully elucidated. In the present study, the differences between A2B5? cells and A2B5+ cells from the SHG139s GSC line were compared. A SHG139s GCS line possessing the molecular phenotype of CD133low/A2B5high was cultured and developed in a previous study (12). In order to rule out the effect of the expression of CD133, the CD133+ cells were first excluded using magnetic-activated cell sorting (MACS). As A2B5? and A2B5+ cells from CD133? SHG139s possess stem cell properties the aim of the present study was to investigate whether expression of A2B5 affects proliferation, invasion, and angiogenesis of CD133? SHG139s. Materials and methods Cell culture The SHG139s GSC line was developed and provided by the Neurosurgery and Brain and Nerve Research Laboratory, The First Affiliated Hospital of Soochow University, (Suzhou, China). The SHG139s cell line was maintained in stem-cell permissive medium [Dulbecco’s modified Eagle’s medium (DMEM)-F12 containing 20 ng/ml epidermal growth factor, basic fibroblast growth factor (bFGF; R&D Systems, Inc., Minneapolis, MN, USA), nitrogen gas (dilution, 1:50) and B27 (dilution, 1:50; Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA)]. MACS The cells were dissociated using 0.25% trypsin (Beyotime Institute of Biotechnology, Haimen, China) and resuspended in phosphate-buffered saline (PBS). All reagents and supplies for MACS Fluopyram separation were purchased from Miltenyi Biotec GmbH (Bergisch-Gladbach, Germany). Selection of CD133? SHG139s cells was performed, according to the manufacturer’s instructions, using CD133/1 Micro Beads. Subsequently, the CD133? cells were separated from the A2B5+ cells and A2B5? cells, according to the manufacturer’s instructions, using A2B5 Micro Beads. In vitro invasion assay The CD133?/A2B5+and CD133?/A2B5? cells were transferred onto Matrigel-coated invasion chambers (24-well insert, 8-sphere formation assay was used to examine whether Fluopyram the expression of A2B5 was involved in cell renewal upon serial passaging. It was found that the high expression level of A2B5 not only affected the size of the spheres, but also led to the reduction in the numbers of spheres in subsequent generations (Fig. 2A and B). To investigate whether the expression of A2B5 affected the proliferation of cells and using IHC. A small number of CD34+ cells were involved in the formation of tumors in the two groups. Tumors formed by A2B5?-derived cells exhibited higher expression levels of VEGF and VEGFR2 (Fig. 5C). Open in a separate window Figure 5 Expression of A2B5 promotes the expression of markers associated with angiogenesis and (10) demonstrated that 100 CD133+ cells from glioblastoma multiforme (GBM) were able to form a tumor in mice, which was similar to the original patient tumor, suggesting that CD133+ cells from GBM exhibit GSC properties (16). However, Beier reported the existence of CD133? GSCs in a later study (5). The results of the present stud also confirmed the existence Fluopyram of CD133? GSCs. A2B5 is a type of multi monosialoganglioside, which is expressed on the cell surface. It is also a marker of progenitors of oligodendrocyte-type-2-astrocyte (O-2A). Tchoghandjian (7) reported that A2B5+ cells isolated from GBM can form spheres. Previous flow cytometric characterization of A2B5+-derived spheres revealed three distinct populations of cells: A2B5+/CD133+, A2B5+/CD133? and A2B5?/CD133? cells (7). CD133+/A2B5+ and Fluopyram CD133?/A2B5+ cells exhibit CSC properties, and it has been shown that A2B5+ cells are crucial for the initiation and maintenance of GBM, whereas the expression of CD133 is more involved in determining tumor behavior (7). Ogden (13) reported that the majority of gliomas can be divided into the three subpopulations described above; and it has been demonstrated that the tumorigenic potential of the CD133+/A2B5+ and CD133?/A2B5+.