The application of mouse and human embryonic stem cells with transcriptomics in alternative developmental toxicity tests : A bridge from model species to man

During life humans are exposed to diverse hazardous compounds, which can have toxicological effects. Reproductive and developmental toxicology are research areas dedicated to the study of the potential of a compound to affect male and female fertility, and development of the embryo and fetus during pregnancy, respectively. An important developmental process is the development of the neural system. The developing brain is more susceptible to toxic interference and can be affected at doses much lower than those affecting adult brain function. Alterations in these processes can result in severe congenital abnormalities of the nervous system of humans. Developmental neuro toxicity testing (DNT) is therefore an important toxicological area in the hazard and risk assessment of chemicals. Regulatory toxicity testing requires an enormous number of test animals. These in vivo tests are time consuming, expensive and give rise to many ethical concerns. To reduce the number of test animals used for reproductive toxicity testing, there is a need for high throughput alternative test methods. The use of embryonic stem cells (ESC) offers a great alternative, since they are able to differentiate into all cell types originating from the three germ layers in vitro. Therefore, ESC offer the ability to mimic early in vivo embryonic development. We studied the use of both mouse ESC (mESC) differentiation towards both myocardial and neural cells, and human ESC (hESC) differentiation towards neural cells, with transcriptomics to study gene expression as an indicator of developmental toxicity. Using mESC within the Embryonic Stem cell Test (EST) we demonstrated that the use of transcriptomics as a read-out after 24 hours of exposure enables ranking of compounds of one compound class of phthalates based on their potency, corresponding to existing in vitro and in vivo data. To lower the number of genes normally involved in whole genome array analysis, we described a statistical method to obtain smaller gene sets usable for potency ranking. Subsequently, a mESC based neural differentiation assay (mESTn) was developed as an alternative method for DNT. In the mESTn the effects of methyl mercury on neural outgrowth was demonstrated. However, mouse based test systems have a limited ability to the human toxicity response, due to differences in biological mechanisms, pathways and pharmacokinetics. Therefore, human based test systems are preferred for human risk analysis. We have developed a neural differentiation assay based on hESC cells (hESTn), as an alternative for DNT. The effects on neural differentiation of two anticonvulsant drugs, valproic acid and carbamazepine were studied. Neural differentiation and developmental processes were affected by drug exposure. Ultimately, with a comparative study between the mESTn and the hESTn it was shown that there were common genes regulates in both assays, involved in neural differentiation and affected by compound exposure