IMPACT OF TUMOR-NECROSIS-FACTOR-ALPHA ON MOUSE EMBRYONIC STEM-CELLS

Previous studies have shown the adverse impact of the cytokine tumor n ecrosis factor alpha (TNF alpha) On the development of the inner cell mass in mouse blastocysts. In the present study, two embryonic stem (E S) cell lines were used to further investigate the action of TNF alpha . The expression of TNF alpha receptors in ES cells was tested by reve rse transcription-polymerase chain reaction and Northern blot analysis . Transcripts encoding the two distinct receptor isoforms were detecte d in these cells. Using different approaches, our data showed a TNF al pha dose-dependent decrease in the number of ES cells after 24 h of ex posure. Simultaneous blocking of the two receptors with antagonist ant ibodies was needed to completely abrogate the inhibitory effect of the cytokine. Extensive DNA nicks (visualized by the terminal deoxynucleo tidyl transferase-mediated deoxyuridine triphosphate nick end-labeling [TUNEL] method), but not nuclear fragmentation, was found with a high er incidence in ES cells exposed to TNF alpha. The possibility that TN F alpha may stimulate ES cell differentiation was investigated with a test based on the expression of alkaline phosphatase. The results indi cated that TNF alpha cannot over-ride the negative control exerted by leukemia inhibitory factor on differentiation. The opposite possibilit y, that TNF alpha blocks differentiation, was tested in suspended medi um drops. In this system, TNF alpha was found to decrease the ability of ES cells to differentiate into embryoid bodies. In addition, expres sion of Rex-l, a marker gene for undifferentiated ES cells, was increa sed in ES cells exposed to TNF alpha. Thus our data support the hypoth esis that TNF alpha is a significant (negative) effector of proliferat ion and differentiation in inner cell mass-derived ES cells.