The Cytotaxis Of Mice Embyoid Body Cells In A Glioma Model

Purpose: To generate nestin-positive embryoid precursor cells from embryonic stem (ES) cells in the absence of LIF in the culture medium and study cell migration while injecting the precursor cells to C6 glioma. In this study, we compare our results with other reports used either different types of ES cells or ES cells under different differentiation stages. The results provide information for further usage of embryoid precursor cells as shuttle in cancer gene therapy.Methods: The first step was to prepare nestin-positive precursor cells for cell migration experiments. Recovered embryonic stem (ES) cells from mice and trypsinized the cells after two passages in culture. The non-differentiated ES cells were confirmed by staining cells with antibody against SSEA-1 antigen. To initiate differentiation ES cells were incubated in the ES cells medium with 10% FBS in the absence of LIF. The cells grew from single cells to appear as a ball of cells (i.e. embryoid body) suspended in the non-tissue culture treated plates. Then, the ES cells were trypsinized and transferred to regular tissue culture plates allowing the cells to adhere to the plates for 2 days. The SSEA-1 immunostaining was performed and the expression level of ES cell marker, transcription factor Oct-4, and the embryoid body marker, nestin, were determined by RT-PCR. The GFP-lentivirus was prepared in 293T cells, then ES cells and NIH3T3 cells were transduced with GFP-lentivirus, but C6 glioma cells was labeled with fluorescent dye PKH26. All GFP and PKH26 labeled cells expressed fluorescence allowing us to study cell migration in vivo. In the in vivo experiment the GFP-labeled nestin-positive precursor cells were transplanted to the glioma site and its counterpart. The cytotaxis of nestin-positive precursor cells were monitored by the movement of GFP cells. In the control experiment, the migration of GFP labeled NIH3T3 cellswas observed. To test the cytotaxis effect in vitro, the experimental and control cells were compared in the transwell migration plates in three different media, C6 medium, regular medium, and Hank's balanced salt solution (HBSS).Results: Twelve days post differentiation the level of Oct-4 was barely detected in the nestin-positive cells. The GFP-labeled precursor cells migrated into the glioma and distributed inside of the tumor. The precursor cells injected into the counterpart site also migrated toward and invade into glioma. In contrast, the 3T3 cells did not migrate into glioma in neither tumor nor its counterpart sites. In the transwell cell-migration experiments, the C6 medium promotes the migration of precursor cells in the transwell plates. The data is statistically significant. However, C6 medium has no effect on 3T3 migration.Conclusions: 1. the early stage (12-day old) of nestin-positive precursor cells derived from ES cells in the absence of LIF in the culture medium showed the ability of migration toward C6 glioma in SD rats. 2. This precursor cells can trace and migrate to glioma such as NSCU MSC and ES -drived precursors . 3. we propose that the glioma cells might secret some cytotaxins to attract the nestin-positive precursor cells. 4. Therefore, the nestin-positive cells could be used as shuttle cells in glioma therapy.