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Metabolic Biomarkers of Prenatal Alcohol Exposure in Human Embryonic Stem Cell–Derived Neural Lineages


Reprint requests: Susan Smith, Ph.D., Department of Nutritional Sciences, University of Wisconsin-Madison, 1415 Linden Drive, Madison, WI 53706; Tel.: 608-263-4316; Fax: 608-262-5860; E-mail:; Gabriella G. Cezar, Ph.D., D.V.M., Department of Animal Sciences, University of Wisconsin-Madison, 1675 Observatory Drive, Madison, WI 53706; Tel.: 608-263-4307; Fax: 608-262-5157; E-mail:



Fetal alcohol spectrum disorders (FASD) are a leading cause of neurodevelopmental disability. The mechanisms underlying FASD are incompletely understood, and biomarkers to identify those at risk are lacking. Here, we perform metabolomic analysis of embryoid bodies and neural lineages derived from human embryonic stem (hES) cells to identify the neural secretome produced in response to ethanol (EtOH) exposure.


WA01 and WA09 hES cells were differentiated into embryoid bodies, neural progenitors, or neurons. Cells along this progression were cultured for 4 days with 0, 0.1, or 0.3% EtOH. Supernatants were subjected to C18 chromatography followed by ESI-QTOF-MS. Features were annotated using public databases, and the identities of 4 putative biomarkers were confirmed with purified standards and comparative MS/MS.


EtOH treatment induced statistically significant changes to metabolite abundance in human embryoid bodies (180 features), neural progenitors (76 features), and neurons (42 features). There were no shared significant features between different cell types. Fifteen features showed a dose–response to EtOH. Four chemical identities were confirmed: l-thyroxine, 5′-methylthioadenosine, and the tryptophan metabolites, l-kynurenine and indoleacetaldehyde. One feature with a putative annotation of succinyladenosine was significantly increased in both EtOH treatments. Additional features were selective to EtOH treatment but were not annotated in public databases.


EtOH exposure induces statistically significant changes to the metabolome profile of human embryoid bodies, neural progenitors, and neurons. Several of these metabolites are normally present in human serum, suggesting their usefulness as potential serum FASD biomarkers. These findings suggest the biochemical pathways that are affected by EtOH in the developing nervous system and delineate mechanisms of alcohol injury during human development.