Effects of ethanol on mouse embryonic stem cells.
BACKGROUND
Fetal alcohol syndrome (FAS) reflects a constellation of congenital abnormalities caused by excess maternal consumption of alcohol. It is likely that interference with embryonic development plays a role in the pathogenesis of the disorder. Ethanol-induced apoptosis has been suggested as a causal factor in the genesis of FAS. Mouse embryonic stem (mES) cells are pluripotent cells that differentiate in vitro to cell aggregates termed embryoid bodies (EBs), wherein differentiation capacity and gene expression profile are similar to those of the early embryo.


METHODS
To investigate the effects of ethanol during differentiation, mES cells were cultured on a gelatin surface in the presence of leukemia inhibitory factor which maintains adherent undifferentiated cells or in suspension to promote formation of EBs. All cells were treated (1-6 days) with 80 mM ethanol. The pluripotency and differentiation of mES cells were evaluated by western blotting of stage-specific embryonic antigen (SSEA-1), transcription factors Oct-3/4, Sox-2, and Nanog, using alkaline phosphatase staining. Apoptosis (early to late stages) was assessed by fluorescence-activated cell sorting using TdT-mediated biotin-dUTP nick-end labelling assay and fluorescein isothiocyanate-Annexin V/propidium iodide staining.


RESULTS
Ethanol increased apoptosis during in vitro differentiation of mES cells to EBs, whereas undifferentiated cells were not affected. Ethanol exposure also interfered with pluripotency marker patterns causing an upregulation of SSEA-1 under self-renewal conditions. In EBs, ethanol delayed the downregulation of SSEA-1 and affected the regulation of transcription factors during differentiation.


CONCLUSIONS
Our findings suggest that ethanol may contribute to the pathogenesis of FAS by triggering apoptotic pathways during differentiation of embryonic stem cells and deregulating early stages of embryogenesis.
Arzumnayan
Alla
A
Department of Pathology, Anatomy and Cell Biology, Jefferson Medical College, Thomas Jefferson University, 1020 Locust Street, Philadelphia, PA 19107, USA.
Arzumanyan
Alla
A
Anni
Helen
H
Rubin
Raphael
R
Rubin
Emanuel
E
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D002492:Central Nervous System Depressants / Q000494:pharmacology*; D003470:Culture Media; D047108:Embryonic Development / Q000187:drug effects; D053595:Embryonic Stem Cells / Q000187:drug effects* / Q000378:metabolism; D000431:Ethanol / Q000494:pharmacology*; D015870:Gene Expression / Q000187:drug effects; D020287:In Situ Nick-End Labeling; D053605:Leukemia Inhibitory Factor / Q000494:pharmacology; D051379:Mice; D008856:Microscopy, Fluorescence
D002492:Central Nervous System Depressants; D003470:Culture Media; D053605:Leukemia Inhibitory Factor; D000431:Ethanol; D000469:Alkaline Phosphatase