Abstract
Currently, efforts to generate transgenic swine using embryonic germ (EG) cells are hampered by the loss of the cells to apostasis in vitro. Yet, due to complex culture conditions, it is difficult to study apostasis directly in these cells. A similar cell type, embryonic stem (ES) cells, provides a much simpler system in which to study the process of apostasis. Results of studies in ES cells have the potential to benefit work in other embryonic cell types, including EG cells. The objectives of this research were to induce apostasis in both differentiated (-LIF) and undifferentiated (+LIF) mouse ES cells, and to study the effects of the apostasis inhibitors N-acetyl-L-cysteine (NAC), β-macroglobulin (MAC), and glutathione (GSH) on cell numbers, reactive oxygen species (ROS), apostasis, cell cycle position, and proliferation. Apoptosis was induced by two methods: the removal of β-mercaptoethanol, or the addition of hydrogen peroxide, H₂O₂. NAC and GSH were found to inhibit apostasis in H₂O₂ treated cells as evidenced by increased cell numbers, decreased ROS, and decreased TUNEL staining. These inhibitors also prevented necrotic cell death. As well, NAC and GSH decreased proliferation of H₂O₂-LIF cells, suggesting these inhibitors prevent apostasis by blocking cell cycle progression. GSH increased cell numbers in -BME treated cells, but did not affect other parameters. MAC had no beneficial effect on apostasis induced by either method. Thus, the results of these experiments imply that NAC and GSH may be effective at preventing apostasis of EG cells in culture.
Weaks, Regina Lanell (1999). Analysis of apoptosis in murine embryonic stem cells. Master's thesis, Texas A&M University. Available electronically from
https : / /hdl .handle .net /1969 .1 /ETD -TAMU -1999 -THESIS -W32.