Effects of the environmental contaminant, TCDD, on differentiation and epithelial to mesenchymal transition in human embryonic stem cells.

摘要

Pluripotent human embryonic stem (ES) cells have great potential to provide a consistent human in vitro model for the investigation of contaminant-induced changes in cell fate and developmental processes. Therefore, there is a growing need for the mechanistic evaluation of common pathways to fully characterize these cells for use in toxicological applications. The aryl hydrocarbon receptor (AhR) pathway is central to xenobiotic toxicity observed in various tissues and critical to proper development. When exposed in utero to 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD), an environmental contaminant and potent AhR pathway agonist, rodents exhibit developmental abnormalities, such as cleft palate, possibly due to alteration in extracellular matrix remodeling and epithelial to mesenchymal transition (EMT).;Using a fibroblast feeder-based culture system, we showed AhR pathway functionality by exogenous and endogenous induction of CYP1A1. While TCDD did not induce morphological changes in undifferentiated human ES cells, TCDD exposure in spontaneously differentiating cells resulted in an inhibitory effect on differentiation, as well as an augmentative effect on the expression of the pluripotency marker, Oct4. These results suggest that TCDD may forestall differentiation in human ES cells cultured with fibroblast feeders, while allowing cells to maintain properties of pluripotency and/or self-renewal.;To eliminate undefined interactions with feeder cells, we employed a feeder-free culture system which was found to be non-permissive for differentiation. Therefore, differentiation was induced by addition of the morphogen, retinoic acid (RA). While RA-treated human ES cells exhibited morphological differentiation in a dose-dependent manner, as well as a reduction of Oct4 protein expression, cells co-treated with TCDD and RA showed fewer signs of morphological differentiation and maintained intermediate levels of Oct4 protein.;In addition, TCDD treatment attenuated EMT-like differentiation in cells grown with fibroblast feeders, as shown by reduced mRNA expression of EMT markers, as well as lower incidence of the mesenchymal marker, N-cadherin, in differentiating cells. However, when human ES cells were grown in feeder-free culture and treated with the EMT-inducing agent, BMP4, EMT-like differentiation was not observed. Together, these findings suggest that TCDD exposure inhibits differentiation of human ES cells; however the extent of inhibition is dependent upon culture conditions.