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Impact of tumor necrosis factor alpha on mouse embryonic stem cells
Y D Wuu1, S Pampfer, I Vanderheyden
1University of Louvain Medical School, Physiology of Human Reproduction Research Unit, Brussels, Belgium.
Abstract:
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.
Insights
Tumor necrosis factor alpha (TNFalpha) inhibits embryonic stem (ES) cell proliferation and differentiation. Blocking both TNFalpha receptors is necessary to prevent these adverse effects on ES cells.
Area of Science:
- Developmental Biology
- Stem Cell Biology
- Molecular Biology
Background:
- Previous studies indicate tumor necrosis factor alpha (TNFalpha) negatively impacts inner cell mass development in mouse blastocysts.
- Embryonic stem (ES) cells, derived from the inner cell mass, are crucial for early development.
Purpose of the Study:
- To investigate the specific actions of TNFalpha on mouse embryonic stem (ES) cells.
- To determine the role of TNFalpha receptors in mediating these effects.
- To elucidate TNFalpha's impact on ES cell proliferation and differentiation.
Main Methods:
- Reverse transcription-polymerase chain reaction and Northern blot analysis to detect TNFalpha receptor expression in ES cells.
- Exposure of ES cells to varying doses of TNFalpha and assessment of cell number.
- Use of antagonist antibodies to block TNFalpha receptors.
- Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) assay to detect DNA damage.
- Alkaline phosphatase expression assays and embryoid body formation assays to assess differentiation.
- Analysis of Rex-1 gene expression as a marker for pluripotency.
Main Results:
- Both TNFalpha receptor isoforms were detected in ES cells.
- TNFalpha exposure resulted in a dose-dependent decrease in ES cell number.
- Complete abrogation of TNFalpha's inhibitory effect required simultaneous blocking of both receptors.
- Increased DNA nicks, but not nuclear fragmentation, were observed in TNFalpha-treated cells.
- TNFalpha inhibited ES cell differentiation into embryoid bodies.
- TNFalpha increased the expression of Rex-1, a marker of undifferentiated ES cells.
Conclusions:
- TNFalpha acts as a significant negative regulator of proliferation in mouse ES cells.
- TNFalpha inhibits the differentiation of ES cells.
- The effects of TNFalpha on ES cells are mediated through its two distinct receptor isoforms.