Effect of Arsenic Trioxide on E14 Murine Embryonic Stem Cells In Vitro.

The ability of pluripotent murine embryonic stem (muES) cells to differentiate into all cell types makes them a very promising tool for cell therapy. The understanding of the molecular mechanisms underlying the growth and differentiation of the ES cells is a pre-requisite for selecting adequately the cell conditions which will be accessible for future use of ES cells in treating hematological malignancies. Pharmacological agents such as arsenic trioxide (As₂O₃) have shown to be an effective anticancer agent for acute promyelocytic leukemia but its effect on murine embryonic stem cells prior to differentiation and subsequent target organ toxicity is yet to be determined. This study was done to gain insight into the biological effects of As₂O₃ on muES cell line, E14, with respect to differentiation, proliferation, cytotoxicity and cell cycle status in vitro. MuEs cells were cultured in complete DMEM in the presence of LIF on gelatin coated plates for 24 hours prior to the addition of varying concentrations of As₂O₃ (0, 0.5,1, 5, and 10 μmol/L, respectively), and the cells were incubated for an additional 48 hours. The differentiation potential of As₂O₃ was determined by measuring Oct-4 levels by flow cytometry. Proliferation was measured by trypan blue exclusion. Cytotoxicity of As₂O₃ was determined by MTT assay. Cell cycle status was measured by Propidium Iodide using flow cytometry. Our results demonstrated that As₂O₃ at different concentrations did not induce differentiation. However, the proliferation of muES cells treated with As₂O₃ was inhibited in a concentration and time dependent manner. The cell-killing rate of As₂O₃ on muES cells was both dose and time dependent with an inhibitory concentration (IC50) of ~ 5±0.2 μM and ~3.75±0.7 μM at 24 and 48 hours respectively. Propidium iodide DNA staining revealed that after 48 hour incubation with arsenic, cells in S phase remained relatively constant (~62–67%) at low doses of As₂O₃ but this decreased by half (33%) at a high dose of 10uM As₂O₃. The percent of cells in the G2 phase of the cell cycle increased to 41±9% in 10μM treated cells compared to 6±3% in control. In conclusion, As₂O₃ does not induce the differentiation of muES cells into different lineages, but does inhibit growth and alter the cell cycle kinetics of these cells in a dose dependent manner.