Enhancing Immunogenicity of Embryonic Stem Cells by Expression of GM-CSF
As the previous vaccine contained whole STO fibroblasts and ES-D3 stem cells, it cannot be tested in humans due to the risk of teratoma development and other side effects. The objective of this research was to eliminate these whole cells and work towards a final version that could eventually be injected into humans. To this purpose, we expressed GM-CSF in ES-D3 cells by two different approaches, eliminating the need for GM-CSF-expressing fibroblasts.
To improve the vaccine, we stably overexpressed GM-CSF in ES-D3 cells through two methods: transfection (selected by GFP) and lentiviral infection (selected by puromycin) to eliminate the need for STO fibroblasts. GM-CSF expression was confirmed by ELISA, which confirmed that appreciable concentrations of GM-CSF were obtained through these methods. Transfected cells showed GM-CSF concentrations only slightly lower than concentrations from the original STO fibroblasts, while lentivirally infected cells showed concentrations slightly higher than the STO fibroblasts. Additionally, we confirmed that ES-D3 cells maintained their pluripotency, and therefore their shared tumor antigens, through immunofluorescence and flow cytometry evaluating levels of SSEA-1, a murine stem cell marker for pluripotency, Oct-3/4, a murine transcription factor, and SSEA-4, a murine stem cell marker for differentiation. GM-CSF expression did not appear to modulate pluripotency of ESD3 cells, as shown by high-level expression of SSEA1 and Oct3/4 as well as low-level expression of SSEA4 in GM-CSF-expressing ESD3 cells.
Vaccine adjuvant GM-CSF was stably over-expressed in ES-D3 cells by two different approaches: transfection (selected by GFP) and lentiviral infection (selected by puromycin). Over-expression was very successful on two fronts, with GM-CSF-expressing ES-D3 cells showing appreciable concentrations of GM-CSF as well as maintained pluripotency. These results indicate that GM-CSF-expressing ES-D3 cells could be used to produce a new generation of anti-cancer vaccines.
This work was supported by the James Graham Brown Cancer Center Summer Research Internship Program.