The Experimental Study Of Cell Fusion Induced Epithelial Carcinogenesis

Objective:The role of cell fusion in tumor development and progression is worthy of attention and it may induce cancer stem cells. In this study we fuse cultured immortalized human gastric mucosal epithelial cell line (GES-1) and cord blood or cord matrix-derived mesenchymal stem cells (CM-MSCs,CB-MSCs) in vitro. Investigate the the possibility of epithelial malignant transformation induced by cell fusion, explore theorigin of cancer stem cells.Methods:GES-1 cells were cultured and the expression of cytokeratinl8 (CK-18) of GES-1 was determined by immunofluorescence. The growth characteristics of CM-MSCs and CB-MSCs were monitored. Mesenchymal stem cell-specific phenotype of Thy-1 (CD90), SH2(CD105), SH4(CD73), CD45, HLA-ABC, HLA-DR, CD34 were identified by flow cytometry. GES-1 cells were labeled by PKH 26 while CM-MSCs and CB-MSCs were labeled by CFSE. Fusion of CM-MSCs and CB-MSCs with GES-1 was induced with 50%PEG2000. Double positive fusion cells were sorted by flow cytometry. Fusion cells were cultured and their growth characteristics were monitored. The expression of CK-18 and cell surface antigen CD90 were determined by immunofluorescence and flow cytometry. The migration and invasive ability were compared with the unfused parental cells by scratch and transwell migration assay. The ability to form tumour after subcutaneous injection into immunologically incompetent nude mice were also determined.Results:1. CB-MSCs could be isolated from cord blood. The first generation of CB-MSCs is round and spindle. After passaging, cells became polygonal with strong adherent and self-renewal capacity. We observed that CB-MSCs could be cultured for 25 passages in vitro.2. CM-MSCs and CB-MSCs both displayed no expression of hemotopoietic marker CD34, CD45, HLA-DR but expressed the typical MSC marker proteins SH2(CD105), SH4(CD73), HLA-ABC. However, the intensity of expression of CD90 was significantly below that of other markers. 3. Fused cells were sorted by flow cytometry. The fusion rate for CM-MSCs and GES-1 was 3% and for CB-MSCs and GES-1 was 8.7%. The fusion cells were highly positive for labelled red and green fluorescent within three days after fusion. Irregular morphological characters and large fused cells can be seen.4. CK-18 was highly expressed within the cytoplasm of fusion cells. Morphological changes of fusion cells included:greater heterogeneity in the size and shape of cells and nuclei, increased nuclear:cytoplasmic ratio as characteristics of malignant cells. GES-1 did not express CD90 while the fusion cells acquired the markers of CD90. Identification of cell surface antigen CD90 by immunofluorescence further confirmed that the sorted cells were fusion cells.5. DNA ploidy analysis showed that fusion cells consisted of polyploid and aneuploid cells and with the increase of passages the proportion of aneuploid cells increased.6. The growth, migration and invasion ability of the fused cells were increased. Fusion cells were able to form nodules after injected into BaLb/C nude mice. Patholigical analysis confirmed the tumor formation.Conclusion:MSCs and GES-1 can be fused with the stimulation by 50% PEG2000. The resulting fusion cells were morphological different from typical MSCs and GES-1 cells, and showed characheristics of both parental cells. For example:CK-18 was highly expressed and the cell surface marker of CD90 was present. The growth,migration and invasion ability of the fusion cells were increased and they formed tumor nodules in BaLb/C nude mice, indicating that fusion between MSCs and epithelial cells can result malignant transformation.