Impact of tumor necrosis factor alpha on mouse embryonic stem cells

Previous studies have shown the adverse impact of the cytokine tumor necrosis factor alpha (TNFalpha) 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 TNFalpha. The expression of TNFalpha 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 TNFalpha 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 TNFalpha. The possibility that TNFalpha may stimulate ES cell differentiation was investigated with a test based on the expression of alkaline phosphatase. The results indicated that TNFalpha cannot over-ride the negative control exerted by leukemia inhibitory factor on differentiation. The opposite possibility, that TNFalpha blocks differentiation, was tested in suspended medium drops. In this system, TNFalpha 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 TNFalpha. Thus our data support the hypothesis that TNFalpha is a significant (negative) effector of proliferation and differentiation in inner cell mass-derived ES cells.