Ethanol alters proliferation and differentiation of normal and chromosomally abnormal human embryonic stem cell-derived neurospheres.

Ethanol is a powerful substance and, when consumed during pregnancy, has significant psychoactive and developmental effects on the developing fetus. These abnormalities include growth retardation, neurological deficits, and behavioral and cognitive deficiencies, commonly referred to as fetal alcohol spectrum disorder. The effect of ethanol has been reported to affect cellular development on the embryonic level, however, not much is known about mutations contributing to the influence of ethanol. The purpose of our study was to determine if mutation contribute to changes in differentiation patterning, cell-cycle regulatory gene expression, and DNA methylation in human embryonic stem cells after ethanol exposure. We exposed human embryonic stem cells (with and without know DNA mutations) to a low concentration (20 mM) of ethanol and measured neurosphere proliferation and differentiation, glial protein levels, expression of various cell-cycle genes, and DNA methylation. Ethanol altered cell-cycle gene expression between the two cell lines; however, gene methylation was not affected in ether lines.

GABRB3 gene expression increases upon ethanol exposure in human embryonic stem cells.

Ionotropic receptors are the target for most mood-defining compounds. Chronic exposure to ethanol (EtOH) alters receptor-mediated responses and the numbers of these channels and specific subunits; as well as induces anxiolytic, sedative, and anesthetic activity in the human brain. However, very little is known regarding the effects of EtOH on ionotropic receptor transcription during early human development (preimplantation). Using two separate human embryonic stem cell lines the study shows that low amounts of EtOH (20 mM) alters transcription of the ionotropic subunit GABRB3. Changes in ionotrophic receptor expression influence the central nervous system development and have been shown to produce brain abnormalities in animal models. These results suggest that low concentrations of EtOH can alter ionotropic receptor transcription during early human development (preimplantation), which may be a contributing factor to the neurological phenotypes seen in fetal alcohol spectrum disorder (FASD).

Heparin binding epidermal growth factor-like growth factor reduces ethanol-induced apoptosis and differentiation in human embryonic stem cells.

Alcohol affects approximately 1% (40,000) of new born infants each year and is the main preventable cause of mental retardation in the US. Ethanol alters cell signaling and promotes apoptosis and differentiation. Heparin-binding epidermal growth factor-like growth factor (HB-EGF), a member of the EGF family of growth factors, has been reported to prevent apoptosis and differentiation. We treated human embryonic stem cells (hESCs) with ethanol (20 mM) to reflect casual drinking, with and without HB-EGF to measure its ability to prevent ethanol-induced apoptosis and differentiation. Apoptosis was measured by DNA fragmentation (terminal dUTP nick-end labeling assays) and activated caspase-3, while differentiation was accessed by SSEA-1 and OCT-3/4; western blotting assessed MAPK signaling. HB-EGF reduced SSEA-1 and elevated OCT-3/4, while reducing the amount of activated caspase-3 and DNA fragmentation. Western blot analysis showed HB-EGF prevents ethanol from altering MAPK phosphorylation. This data suggests that ethanol-induced apoptosis was reduced by HB-EGF, while hESC pluripotency was maintained.

Low ethanol concentration alters CHRNA5 RNA levels during early human development.

Alcohol use is common and consumption during pregnancy has been shown to lead to a myriad of physical and neurologic abnormalities commonly referred to as fetal alcohol spectrum disorder. Substance addiction, which includes alcohol, has been shown to involve the major nicotinic acetylcholine receptor subunit CHRNA5. Using human embryonic stem cells as a model of early human development, we show that low concentrations of ethanol (20mM) can alter the expression of CHRNA5. Changes in CHRNA5 expression is linked to altered GABA and NMDA receptor expression, as well as abnormal development of the frontal cortex. These results suggest that alcohol exposure can alter early neurologic development, which may favor addiction and other developmental abnormalities in unborn children.

Heparin binding epidermal growth factor-like growth factor and PD169316 prevent apoptosis in mouse embryonic stem cells.

Apoptosis or programmed cell death is an important outcome of cell fate and is influenced by several factors. Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a member of the EGF family of growth factors and is synthesized as a membrane-associated precursor molecule (proHB-EGF). Under stressful conditions proHB-EGF is proteolytically cleaved and released as a soluble ligand (sHB-EGF) that activates the EGF receptor. We show that antibody against CD9, a membrane tetraspanin, induces apoptosis in mouse embryonic stem cells through the activation of specific EGF receptor residues (Y-1148 and Y-1173), caspase-3 and MAPK signalling. HB-EGF and the p38 inhibitor PD169316 act in a pro-survival manner by perturbing phosphorylation of EGFR Y-1173, suggesting its importance in inducing apoptosis. Caspase-3 activation was attenuated in the presence of HB-EGF and PD169316. Furthermore, HB-EGF and PD169316 prevent p38 phosphorylation while promoting the phosphorylation of the pro-survival SAPK/JNK and ERK. These results suggest a role for CD9 as an endogenous suppressor of apoptosis, an effect that is mimicked by HB-EGF and PD169316.