To identify and analyze the fusion cells between glioma stem cells with bone marrow mesenchymal stem cells (BMSCs) or macrophages (Mφ) in vitro, and research the biological phenotypes of the fusion cells.

Red fluorescent protein (RFP) gene was stably transfected into human glioma stem cell line SU4. BMSCs and Mφ were harvested from Balb/c nude mice with enhanced green fluorescent protein (EGFP) expression. SU4 cells were co-cultured with BMSCs and Mφ, respectively, in the dual-color tracing platform. RFP/EGFP double positive cells with high proliferation ability, named F-BMSCs or F-Mφ, were monocloned. The gene and protein expressions of RFP and EGFP were detected by Western blotting and fluorescently-labeled in situ hybridization (FISH) in SU4-RFP, F-BMSCs, F-Mφ and BMSCs; immunocytochemistry was employed to detect the molecular markers. Chromosome karyotype was used to analyze the mitotic cell division phase; the biological characteristics of SU4-RFP, F-BMSCs, F-Mφ and BMSCs were further analyzed by colony formation, CCK8 and Transwel invasion assay.

Two kinds of fluorescences expressed in the fusion cells observed by confocal microscopy. FISH and Western blotting showed that F-BMSCs, F-Mφ had gene and protein co-expressions of RFP and EGFP. Immunocytochemistry displayed nestin positive expression only in the SU4-RFP cells, CD105 and CD44 strongly positive expression only in the BMSCs, and CD68 positive expression only in the Mφ cells; co-expressions of nestin, CD105 and CD44 were noted in the F-BMSCs; co-expressions of nestin and CD68 were noted in the F-Mφ. Karyotype analysis showed that mice terminal centromere chromosomes were mainly the F-BMSCs karyotype, while a small number of people metacentric chromosomes could be noted. The clone formation rate of F-BMSCs and F-Mφ was significantly higher than that of SU4-RFP (P<0.05). CCK8 assays showed that the growth rate of F-BMSCs and F-Mφ was significantly higher than that of SU4-RFP (P<0.05); Transwell invasion assay showed that the invasion number of F-BMSCs and F-Mφ (429.4±17.9 and 421.6±14.6) was significantly larger than that of SU4-RFP (216.2±22.6, P<0.05).

Glioma stem cells can fuse with BMSCs or Mφ spontaneously, and the fusion cells are more malignant than their parent cells, indicating the new possibility of malignant progression of gliomas.

Glioma stem cell; Bone marrow mesenchymal stem cell; Macrophage; Cell fusion; Two-color fluorescence tracing