Human embryonic stem cell model of ethanol-mediated early developmental toxicity.
BACKGROUND
Fetal alcohol syndrome is an important clinical problem. Human embryonic stem cells (hESC) have not been widely used to study developmental alcohol toxicity. Here we document the phenotype of hESC exposed to clinically-relevant, low dose ethanol (20mM).


METHODS
All cultures were maintained in 3% O2 to reflect normal physiologic conditions. Undifferentiated hESC were expanded with basic fibroblast growth factor (bFGF), with or without ethanol, then differentiated without ethanol. Proliferation and apoptosis in response to ethanol were assayed, and PCR used to examine expression of GABA receptor subunits. Whole cell patch clamping was used to examine GABA(A) receptor function in undifferentiated hESC. Immunocytochemistry and western blotting were used to follow differentiation of early neurons, astrocytes, and oligodendrocytes,


RESULTS
Exposure to 20mM ethanol resulted in larger colonies of undifferentiated hESC despite an increase in apoptosis, because proliferation of the undifferentiated cells (and neuroblasts) was significantly increased. Differentiation of hESC (following a week of ethanol exposure) resulted in decreased expression of GFAP (by western) compared to unexposed cells, suggesting that astrocyte differentiation was reduced, while markers of oligodendrocyte and neuron differentiation were unchanged. At the message level, undifferentiated hESC express all GABA(A) receptor subunits, but functional receptors were not found by whole cell patch clamping.


CONCLUSIONS
Our results in hESC suggest a complex mix of ethanol-induced phenotypic changes when ethanol exposure occurs very early in development. Not only increased apoptosis, but inappropriate proliferation and loss of trophic astrocytes could result from low-dose ethanol exposure very early in development. More generally, these studies support a role for hESC in developing hypotheses and focusing questions to complement animal studies of developmental toxicities.
Department of Anesthesiology, Emory University School of Medicine, Atlanta, GA 30322, USA.
Rodney
R
Nash
Malini
M
Krishnamoorthy
Andrew
A
Jenkins
Marie
M
Csete
D017209:Apoptosis / Q000187:drug effects*; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D002492:Central Nervous System Depressants / Q000633:toxicity*; D051357:Cyclin-Dependent Kinase 2 / Q000235:genetics / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D053595:Embryonic Stem Cells / Q000187:drug effects*; D000431:Ethanol / Q000633:toxicity*; D005333:Fetus; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects*; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D006801:Humans; D020287:In Situ Nick-End Labeling / Q000379:methods; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D011963:Receptors, GABA-A / Q000235:genetics / Q000378:metabolism; D014404:Tubulin / Q000378:metabolism; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
D002492:Central Nervous System Depressants; D005904:Glial Fibrillary Acidic Protein; D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; D012333:RNA, Messenger; D011963:Receptors, GABA-A; D014404:Tubulin; D016222:Fibroblast Growth Factor 2; D000431:Ethanol; D005680:gamma-Aminobutyric Acid; D003577:Cytochrome P-450 Enzyme System; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2