Human induced pluripotent stem cells to study developmental toxicity in vitro.

Karin Lauschke

Research output: Book/ReportPh.D. thesis

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Abstract

Pregnant women are exposed to an increasing number of chemicals that are potentially hazardous to the developing embryo and fetus. This can have fatal consequences for the unborn life, as developmental damages can be irreversible and might persist during a lifetime. Therefore, safeguarding developing human embryos and fetuses is of utmost importance and suitable toxicity test systems need to be developed. Human induced pluripotent stem cells (hiPSC) are promising for such systems. HiPSC can form embryoid bodies (EBs) and undergo differentiation into most cell types of the human body by processes similar to those during development. In this thesis, I present the development and application of a novel human developmental toxicity assay based on hiPSC, formation of EBs and differentiation into cardiomyocytes. This assay mimics the first weeks of embryonic development in which the heart develops and starts beating, and can be used to test for developmental toxicity. Firstly, we optimized a protocol for differentiation of hiPSC into cardiomyocytes through EBs in a 96-well plate format. This protocol provided the basis for testing chemicals in an assay that we termed the PluriBeat assay, with beating cardiac spheres as a readout. Our data on thalidomide, valproic acid and epoxiconazole suggest that the PluriBeat assay has the potential to detect developmental toxicity of chemicals that affect cardiac development or general developmental pathways. Secondly, we developed an improved readout of the assay in order to substitute the laborious visual inspection of beating cardiac spheres. To this end, we created a luciferase reporter hiPSC line and showed that luminescence intensity followed cardiomyocyte differentiation. The readout of luminescence intensity detected thalidomide with higher sensitivity than the beating cardiomyocytes. Overall, our data suggest that luminescence increases the
throughput, ease of handling and sensitivity of the PluriBeat assay. Thirdly, we investigated how relevant the cardiomyocyte differentiation model was to the in vivo processes in developing embryos. We made use of a mammalian species that develops very similar to the human, namely the pig. By investigating pig embryos of relevant developmental stages before and after the first heartbeat, we found that a number of marker proteins overlap in expression with our in vitro system. These findings indicate that many developmental stages of the heart are recapitulated in our in vitro model of the PluriBeat assay. As a fourth step, we set out to use the developed model of the PluriBeat assay to study the molecular mechanisms of the test chemicals thalidomide, valproic acid and epoxiconazole. To this end, we used a non-targeted approach to study the entire transcriptome, namely RNA sequencing. Upon exposure to the three chemicals, we found numerous genes to be differentially expressed as well as specific cellular pathways to be affected. Studying the effects of epoxiconazole in more detail, we found evidence that it interrupts genes necessary for cholesterol and steroid hormone synthesis, which is in accordance with a presumable endocrine disrupting activity of epoxiconazole. In addition to the development and application of the PluriBeat assay described in the first Chapters, we also attempted to use electrochemical methods to detect the beating of the cardiomyocytes. While we were not able to employ impedance measurements to detect contractions in the cardiac spheres, we found preliminary evidence that amperometry might be suitable for that. This is a potential alternative readout of the PluriBeat assay that might be further developed in future studies. In conclusion, we explored different methodologies to optimize the use of hiPSC in an in vitro model for developmental toxicity and to study the molecular effects of chemicals. The findings presented in this thesis are a valuable contribution to the field of developmental toxicology and promote the use of novel cell sources herein. Moreover, the results might contribute to future risk assessment of chemicals and ultimately to reducing or replacing animal experiments in toxicology.
Original languageEnglish
PublisherTechnical University of Denmark
Number of pages218
Publication statusPublished - 2021

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