Isolation, Culture And Characterization Of Cancer Stem-like Cells From Human Choriocarcinoma

Objective:In this study, we prepare to isolate and culture chorionic cancer stem-like cells from human choriocarcinoma JEG-3cell lines and to identify their biological properties with serum-free suspension culture.Methods:(1) The human choriocarcinoma JEG-3cell lines were dissociated into single-cell suspension, incubated in serum-free medium to get choriocarcinoma suspended cells spheres;(2) The expression of tumor stem cells surface markers CD44、 CD133and ABCG2was detected with flow-cytometry;(3) Transwell invasive method was utilized to analyze the migration ability between JEG-3parent cells and suspended cells in vitro;(4) Trypan blue staining proliferation experiment was utilized to compare cell proliferation of JEG-3parent cells with suspended cells, and draw the growth curve;(5)1000copies of single-cell suspension contained the suspended cells and parent cells, was incubated in the SFM to observe and calculate tumor sphere formation rate;(6) We use limited dilution method to observe the suspended cells formation of single-cell;(7) the cells of the JEG-3suspended cells were placed in serum-containing medium to induce differentiation and observe its morphological changes.Results: (1) Human choriocarcinoma JEG-3cell lines can survive, proliferate and suspended cells with self-renewal capacity in the serum-free medium,and have the possibility of continuous passage;(2) CD44, CD133and ABCG2in the JEG-3suspended cells were significantly higher than those in the parent cells with flow cytometry (P <0.05);(3) In the Transwell little chamber testing, JEG-3suspended cells had a stronger migratory ability than parent cells in vitro (F=8.140, P=0.008, P<0.05);(4) In the trypan blue proliferation experiment, JEG-3suspended cells had a stronger cell proliferation compared to the parent cells (F=2775.601, PO.001);(5) The tumor sphere formation rate of JEG-3parent cells, the third generation of JEG-3spheres and the sixth generation were respectively (7.57±0.80)%,(7.23±0.33)%,(7.52±0.62)%(P>0.05).So the stem cell sphere formation rates among the three periods cells have no significant differences;(6) We used limited dilution method in96-well plate to get a single suspended cell, and observed that the single-cell proliferated into suspended cells sphere under the microscope.(7) Seeded in serum supplemented medium, the JEG-3suspended cells could be adherent again and differentiate, and there was no significant difference in the morphology between the differentiated cells and JEG-3parent cells.Conclusion:(1) Choriocarcinoma stem-like cells can be obtained by serum- -free suspension culture;(2) The JEG-3cancer stem-like cells can high expression of cancer stem cell surface markers of ABCG2, CD133CD44;(3) The JEG-3cancer stem-like cells have high ability of migration, self-renewal, proliferation and multi-differentiation.