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

Reporting period: 2019-01-01 to 2020-06-30

Endocrine disruptors (EDs) are a class of chemicals used to produce materials commonly found in everyday life, such as plastics, tin cans, cosmetics, pesticides, among others. EDs are not without danger: these molecules interfere with the endocrine system, disrupting the physiological production and the target effects of hormones. Particularly, EDs have proven effects on the reproductive system and an incidence on the occurrence of obesity, type 2 diabetes and cardiovascular diseases during aging. There is also a growing evidence that EDs interfere with the functioning of the thyroid. EDs 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. The tests currently available are limited by the availability of adequate quantities of human thyroid tissue, the ability of currently available thyroid models to recapitulate the complexity of the native gland and by the inability to predict the effects of EDs after low dose exposure. The EU project SCREENED aims to develop three-dimensional (3D) cell-based in vitro tests to better characterize the EDs effects on thyroid gland function. The method will overcome the limitations of existing tests, being more sensitive at low doses of exposure to chemicals and enabling the prediction of their toxicity on human health in a sex-specific manner.
During the project initial 18 months, we designed and fabricated the first working bioreactor prototypes. Current prototypes enabled culture of enriched thyroid follicles in a microphysiological setting that closely resembles the native tissue. Optical sensors were successfully integrated into the bioreactor housing to measure and monitor oxygen levels throughout culture. We improved our stem cell derived thyroid functional in vitro model, in terms of methodology, efficiency and purity, using embryonic stem cells to allow it to be an efficient tool to test the EDs effect on the function of the thyroid gland.
Five decellularization protocols suitable for removal of all cell types from the adult male and female, rat thyroid gland have been developed without hampering the main structural and geometrical 3D organisation of the thyroid lobe matrix: each showing a preferential retention of specific matrix molecules (collagen types, structural proteins, growth factors, glycosaminoglycans). In addition, isolation, expansion, enrichment and characterisation of adult male rat, thyroid stem cells / progenitor (TSC/P) using a simple, reliable and reproducible methodology has been achieved, based on long-term, semi-starvation low-density monolayer subcultures.
We also focused on the optimisation of microfluidic bioprinting for the generation of the bioprinted thyroid model. We evaluated different hydrogel based bioink formulations to assess their printability and the parameters for an optimal bioprinting in terms of fibre dimension and deposition. We verified that bioprinted cells remain viable and metabolically active inside the construct. We also increased bioprinted constructs’ robustness by post crosslinking the construct with a CaCl2 base solution. Finally, we exploited a new core-shell bioprinting technique that allowed to produce hollow fibers containing thyroid and endothelial cells at the same time. As a further method to provide better control of cell spatial positioning for the 3D cellular assays to be developed in SCREENED, we also optimized a method to magnetize cells while maintaining a high viability.
The current lack of available proteomic and transcriptomic information on the thyroid drove us to initiate the first combined proteomic and transcriptomic analysis of ED effects on (non-cancerous) thyroid cells, using state of the art technologies to identify thousands of gene and protein thyroid signatures. Preliminary data on recent research on the in vivo effects of a mixture of polychlorinated biphenyls on the hypothalamic-pituitary-thyroid (H-P-T) axis of adult male rats have provided groundwork for eventual development of theoretical modelling of in vivo EDs action on the rat thyroid gland.
One of the project aims is to focus on the development of reversibly sealed microfluidic bioreactors for long-term culture of thyroid cells. Our bioreactors are expected to enable prediction of thyroid disruption while being compatible with high-throughput platforms, which are two major requirements for industrial applicability. As such, the bioreactors have the potential to advance the organs-on-chip field and foster its adoption by the pharmaceutical industry to reduce or even replace animal models in pre-clinical testing for drug screening and discovery. The establishment of a human thyroid in vitro model also constitutes a major breakthrough in the field of thyroid research and will be extremely useful to study thyroid gland diseases. These models bring a fast, cheaper and animal-free alternative to screen a list of EDs toxic effect, using a system that resembles what happens in humans. Changes in matrix composition due to different decellularization protocols, that we develop, provide different molecular environments ideal to test the role of different matrix proteins in driving differentiation of seeded adult TSC/P from either male or female rats. Differential and sex-specific matrix composition may represent a reference for eventual bioprinting of matrix replica as engineered surrogates of native matrices in a developing microbioreactor. In addition, evidence that adult male TSC/P can give rise to clonogenic colonies with molecular markers of native TSC/P enlighten their usefulness as an innovative experimental tool for developing 3D functional thyroid organoids to be used for the project purposes, promising to be of relevant commercial impact in the field of in vitro assays.
We are also producing a bioprinted 3D model able to mimic the thyroid architecture and functionality. The main models currently available for thyroid studies are based on 2D cell cultures or on the use of animal models. Either of these two solutions do not represent the human thyroid complexity. With bioprinting, we could produce a 3D model able to closely mimic the human thyroid and to offer different advantages over classical 2D models. At the same time, the bioprinted model could reduce the use of animal models for drug screening and discovery purposes, while offering a new platform for studying thyroid physiology and physiopathology. The potential addition of magnetized cells as a further method to control cell spatial deposition in 3D would offer the capacity to precisely locate thyrocytes and endothelial cells in the 3D cellular assays of SCREENED. This method would allow an exquisite control in organoids and decellularized extracellular matrix constructs, as well as a further level of spatial control in bioprinted constructs.
Since there is no existing state of the art for (normal) thyroid cell transcriptomics and proteomics, we will establish a baseline transcriptome and proteome of human thyroid cells and the impact of ED’s on them. By the end of SCREENED we expect to collect comparative and reliable information between the effects of a list of EDs in the in vitro 3D thyroid assays under experimental development and in vivo results on rodent models (male and female rats) used as control systems.