A 3Rs approach to assess drug toxicity on the mammalian embryo during early pregnancy in vitro

It is important for pregnant women, or women trying to conceive, to be aware of the risks posed by any medication they may have to take during pregnancy. This is especially important during the 1st trimester, when the embryo is at its most sensitive to developmental defects. The general advice given to women is not to take medication at all during their pregnancy & healthcare professionals are advised to prescribe only when the benefit to the mother outweighs the potential effects on the embryo. This demonstrates the need for adequate information on the effect of current & new drugs during pregnancy, especially when treating certain conditions (e.g. epilepsy, HIV) where withholding treatment might not be prudent. As pregnant women cannot be used to test whether drugs are harmful in pregnancy, we must rely on results from animals during pre-clinical testing, where millions of animals (a large proportion of which are rodents) are used & consequently the tests are highly expensive. Unfortunately, the accuracy of these Developmental & Reproductive Toxicity studies (DARTs) in predicting human developmental toxicity is still unknown & the absence of any effect in animal tests does not necessarily mean a drug is safe to take during pregnancy. Although there are validated in vitro alternatives, none of these provide an integrated approach to look at the early developing embryo.

We have developed a novel 3D tissue-culture protocol that uses small numbers of mouse embryonic stem cells (mESCs), grown in suspension. Over time, these “gastruloids” mimic many of the early developmental events seen during mouse development: symmetry-breaking, polarised gene expression, axis elongation & formation of 3 embryonic axes. Gastruloids are highly tractable, allowing precise experimental manipulation, difficult to do with embryos. It is for these reasons that we believe that gastruloids will substitute mice during DARTs.

In this project, we propose to directly target the area of drug development where animals are the predominant model for DARTs. Firstly, we will tailor the standard gastruloid protocol to test whether the parent compound & metabolites of teratogenic drugs (i.e. Thalidomide; THD & Valproic Acid; VPA) effect gastruloid development. Using existing reporter mESC lines (BraGFP for example) & engineering new lines expressing all 3 germ-layer markers, we will measure in real time how these drugs alter gastruloid patterning, gene expression, & morphology. Immunostaining, in situ hybridisation (ISH) & ISH Chain Reaction will provide a complementary & enhanced assessment of the changes in cell fate & patterning at static time-points. By coupling gastruloids to an in vitro flow model of a zonated liver, we will expose gastruloids to a range of concentrations of THD & VPA metabolites generated in a more physiological manner, at precise temporal intervals to establish how much & at what time patterning is disrupted. Focussing on antiepileptics (i.e. VPA, levetiracetam, zonisamide), we will assess the effect of polytherapy on gastruloid development, a treatment regime that may increase the risk of congenital anomalies.

Finally, we will use human embryonic (hESC) & human induced pluripotent (hiPSC) stem cell lines to establish a protocol for generating gastruloids which will allow us to assess the effect of drugs that is more applicable to human development without using embryos. Using a combination of fluorescent confocal & widefield microscopy, will quantify the effect of drugs of interest (identified from the first objective) have on gastruloid patterning both in real time (using existing triple germ-layer reporter hESCs), & at fixed, static time-points. This project will allow us to gain insight into drug-induced developmental toxicity in a model that can be tailored to human development, providing us with an experimental platform that has the potential to greatly reduce the use of animals in DARTs.