Transcriptomics-based identification of teratogens in differentiating hiPSCs: A novel in vitro test system

Abstract

The knowledge about the developmental toxicity potential of substances is a matter of high concern in toxicology and medicine, especially if pregnant women need to take potentially teratogenic drugs to treat severe infections or diseases. The regulatory risk assessment of such substances is, however, very difficult, costly and time-consuming and relies currently, due to a lack of appropriate alternatives, solely on animal studies. During the last decades, much effort was put into the development and establishment of trustworthy in vitro-based test systems for the risk assessment of teratogenic substances, but not even one was approved for this purpose yet. In this PhD project, a new attempt was undertaken to develop such a system. The here presented novel system is based on the UKN1 assay, in which pluripotent human embryonic stem cells were differentiated to neuroepithelial precursor cells, and made considerable innovations to it by utilizing human induced pluripotent stem cells (hiPSCs), whole-transcriptome analyses and a thoroughly chosen set of 39 teratogenic and non-teratogenic substances that each were applied at two on human in vivo data-based concentrations to differentiating hiPSCs in this “UKN1 6-day” assay. In contrast to the non teratogenic compounds, the teratogens induced either significant gene expression alterations or a high toxicity in almost all cases, so that this test system was able to predict substances with a high accuracy of 90 %, a robust sensitivity of 83 % and an outstanding specificity of 100 %, what makes it therefore to a very high performing in vitro test system. Even a variation of the system, the UKN1 one-day assay, which shortened the total time requirements of the assay and replaced the whole-transcriptome analyses by a four biomarker-based RT-qPCR, could predict the compounds with an accuracy, sensitivity and specificity of 77 %, 63 % and 100 %, respectively. In addition to the core set of 39 substances, the UKN1 6-day assay was used to determine the health risks of parabens which are widely used as preservatives in cosmetics. No significant gene alterations were found in differentiated, paraben-exposed hiPSCs, and the obtained negative in vitro results could now support the existing in vivo data of paraben-exposed animals in accordance with a read-across-approach for ethylparaben, so that no further animal experiments would be needed for the regulatory risk assessment of ethylparaben. In the near future, steps for a further optimization of the test system will be investigated like the application of targeted instead of whole-transcriptome analyses and multiple concentrations instead of just two. Furthermore, the number of tested compounds shall be increased. Although there is still a long way to go and many hurdles to overcome until this or another test system will be approved for the regulatory risk assessment of teratogens, the UKN1 6-day test system can already provide a tool for an in vitro teratogenicity screening of compounds and aid authorities and companies to assess the health risks of potentially teratogenic substances.