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A multistage model of thyroid gland function for screening endocrine-disrupting chemicals in a biologically sex-specific manner

Periodic Reporting for period 2 - SCREENED (A multistage model of thyroid gland function for screening endocrine-disrupting chemicals in a biologically sex-specific manner)

Periodo di rendicontazione: 2020-07-01 al 2021-12-31

Endocrine disrupting Chemicals (EDCs) are commonly found in our everyday life, but there is growing evidence that EDCs interfere with the functioning of the thyroid and cause changes in thyroid hormone concentrations, the peripheral metabolism of these hormones and the signalling of their receptors. The mechanism by which they act on the thyroid axis is, however, still far from being elucidated, partially due to the limitations of existing tests.
SCREENED aims to develop 3D in vitro tests to characterise the effects of EDCs on thyroid gland function.
SCREENED will deliver highly innovative “Organ-on-a-chip” models, where thyrocyte cells organised in a 3D structure, will be hosted in a microfluidic cell culture device (hereafter called microfluidic bioreactor). This device will mimic the microenvironment of the thyroid gland, by simulating tissue- and organ-level physiology. The first 3D models will consist of thyroid organoids able to recapitulate the thyroid hormone production functionality of the native thyroid (mouse and human models). In addition, we will work on ECM-scaffold-based and on a bioprinted based 3D models, which will represent an even more complex version of the 3D thyroid models, where the “Organ-on-a-chip” will also be supported by a vascularized network.
In period 2, we have improved the protocol to derive thyroid follicles from mouse embryonic stem cells (mESCs). Besides, we have been working in generating a human-derived thyroid tissue using embryonic stem cells (hESCs). Excitingly, our results demonstrate that we can generate, by genetic and chemical manipulation, structures that are capable to organize three-dimensionally in follicles, produce thyroid hormone in vitro, and rescue the levels of TH when transplanted in thyroid-ablated mice. The establishment of a human thyroid follicles from hESCs constitutes a major breakthrough in the field of thyroid research and will be extremely useful to study thyroid gland illnesses beyond EDCs screening.

In parallel, we have successfully developed a microfluidic bioreactor system that is capable of hosting up to eight 3D thyroid tissue constructs in parallel, under flow conditions and that meets the requirements for high throughput screening. We have proved that, in flow conditions, mESC-derived thyroid follicles were able to recapitulate in vivo-like 3D follicular structures, featuring TG expression and a luminal space outlined by ZO-1 expression. Interestingly, follicles cultured in the bioreactor chips revealed an increase in T4 production and storage in the luminal space, as compared to follicles cultured in static conditions. In the same bioreactor platform, we successfully integrated sensors for continuous in situ oxygen measurement. Finally, our bioreactor and battery prototypes for EDCs screening were fabricated and initial experiments revealed that they are capable of providing leak-tight conditions.

We developed few variations of a rat thyroid 3D in vitro model based on 3D collagen scaffolds, 3D Matrigel environments, or decellularized thyroid ECM scaffolds. The rat thyroid progenitor/stem cells that were seeded or encapsulated in these scaffolds showed to be able to repopulate completely the constructs and to self-organize into thyroid follicle morphology able to secrete thyroid hormone. We further developed a 3D bioprinted thyroid construct using a microfluidic bioprinting system. We evaluated the possibility to bioprint not only single cells, but also more complex and physiologically relevant structures, such as thyrocyte spheroids and mESCs-derived thyroid follicles. We verified that the bioprinting procedure does not impair the viability and structure of the bioprinted spheroids and follicles. In addition, we demonstrated that follicles maintain their functionality and express thyroid markers at levels comparable to non-bioprinted conditions. Finally, we developed a procedure of seeding of endothelial cells on the bioprinted constructs that seems functional for the creation of a preliminary vascularization of the constructs. To facilitate the position of either thyroid progenitor/stem cells in the decellularized-ECM scaffolds or endothelial cells in bioprinted constructs, these 2 cell populations were magnetized with biocompatible magnetic nanoparticles, which showed to maintain cell viability and steer cell positioning in space without altering their functionality.

We also worked towards an increased understanding of the mechanism by which EDCs interfere with the thyroid gland. As our work began, it soon became apparent that there is little if no existing data on the impact of EDCs on human thyroid cells on gene and protein expression. We have now completed planned investigation of the effect of several EDCs at different concentrations on a human thyroid cell line. The novel data is being used to support the identification of transcript and proteomic signatures of the impact of EDCs and to model cellular responses to EDCs. In more recent experiments the impact of EDC’s on mouse thyroid follicles is being examined. We are now in the process of using the data to establish targeted measurement of key EDC responsive proteins. In silico models have been parametrized to correlate in vitro data with in vivo data, whenever available in the literature. Preparatory work has been carried out to identify how a molecular initiating event (MIEs) can eventually lead to an adverse effect in the human organisms, in the context of the Adverse Outcome Pathway (AOPs) conceptual framework. Putative MIEs to which SCREENED will eventually work have been identified.
The establishment of a human thyroid in vitro model constitutes a major breakthrough in the field of thyroid research and will be extremely useful to study thyroid gland illnesses beyond EDCs screening. These models bring a new fast, cheaper and animal-free alternative to screen a list of EDCs toxic effect, using a system that resembles what happens in humans.
SCREENED is advancing the field of “Organ-on-a-chip” devices and foster its adoption by industries. Indeed, the development of reversibly sealed bioreactor chips are compatible with high-throughput screening platforms, which are two major requirements for industrial applicability. Moreover, our system is expected to enable integration of sensing technology for continuous monitoring of physical and biochemical parameters during perfusion culture. The progress made in WP3 has led to the successful generation and in vitro characterisation of human thyroid follicles. This gives us the opportunity to progress beyond the current state of the art and undertake the analysis of the effects of EDCs on these in vitro models of the human thyroid at a gene (transcript) and protein level. The data will also support comparative modelling of the effects of EDs on (i) a human 2D model vs. 3D model, and ii) human vs. mouse 3D models. The transcript data is very comprehensive in its coverage (tens of thousands of genes/transcripts) compared to the proteomic data (a few thousand proteins). We intend to develop targeted protein assays to transcripts that have been shown to change and attempt to circumvent the limitation of protein-based discovery experiments.
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