Comparative assessment of toxic responses in 3D embryoid body differentiation model and mouse early embryos treated with 5-hydroxytryptophan.

Abstract

Pluripotent stem cells recapitulate in vitro the early developmental stages and are considered promising cell models for predictive developmental toxicity studies. To investigate the consistency between adverse drug effects on early development and the early stages of embryonic stem cell differentiation in three-dimensional (3D) in vitro culture, the toxic responses to 5-hydroxytryptophan (5-HTP; 0.5-2mM) were evaluated in early mouse embryos and the embryoid body (EB) differentiation model. 3D architectures, developmental and differentiation dynamics and the cell death rates were analyzed in early mouse embryos (E2.5-E5.5) and EBs at 1 and 6days of differentiation using a combination of confocal immunofluorescence microscopy with high content imaging analysis and quantitative gene expression analysis. Comparative analysis of toxic responses in early embryos and EBs revealed a similar dose- and stage-dependent decrease in the 5-HTP toxic effects during development and differentiation. The integral toxic responses in the early embryos and EBs were significantly dependent on their 3D architecture and cellular composition. Treatment with 5-HTP (1mM and above) induced developmental arrest, growth inhibition, and increased cell death in the early embryos without the trophoblasts (E2.5) and those with impaired trophoblasts and in early EBs, whereas later embryos and EBs were more resistant due to the protection of the extraembryonic tissues. This study demonstrates that the EB differentiation model is a relevant 3D-model of early mammalian development and can be useful for the predictive evaluation of toxic and teratogenic effects in embryos at the preimplantation and early post-implantation developmental stages.