Impact of Tumor Necrosis Factor a on Mouse Embryonic Stem Cells

Previous studies have shown the adverse impact of the cytokine tumor necrosis factor (TNFa) on the development of the inner cell mass in mouse blastocysts. In the present study, two embryonic stem (ES) cell lines were used to further investigate the action of TNFot. The expression of TNFea receptors in ES cells was tested by reverse transcription-polymerase chain reaction and Northern blot analysis. Transcripts encoding the two distinct receptor isoforms were detected in these cells. Using different approaches, our data showed a TNFot dose-dependent decrease in the number of ES cells after 24 h of exposure. Simultaneous blocking of the two receptors with antagonist antibodies was needed to completely abrogate the inhibitory effect of the cytokine. Extensive DNA nicks (visualized by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling [TUNEL] method), but not nuclear fragmentation, was found with a higher incidence in ES cells exposed to TNFta. The possibility that TNFao may stimulate ES cell differentiation was investigated with a test based on the expression of alkaline phosphatase. The results indicated that TNFot cannot over-ride the negative control exerted by leukemia inhibitory factor on differentiation. The opposite possibility, that TNFa blocks differentiation, was tested in suspended medium drops. In this system, TNFot was found to decrease the ability of ES cells to differentiate into embryoid bodies. In addition, expression of Rex-1, a marker gene for undifferentiated ES cells, was increased in ES cells exposed to TNFot. Thus our data support the hypothesis that TNFot is a significant (negative) effector of proliferation and differentiation in inner cell mass-derived ES cells.