Periodic Reporting for period 3 - SCREENED (A multistage model of thyroid gland function for screening endocrine-disrupting chemicals in a biologically sex-specific manner)
Reporting period: 2022-01-01 to 2023-06-30
There is growing evidence that endocrine disrupting chemicals (EDCs) interfere with thyroid function and cause changes in thyroid hormone concentrations, their peripheral metabolism and the signalling of their receptors. Yet, the mechanism by which EDCs act on the thyroid axis is still far from being elucidated, in part due to the limitations of existing tests.
SCREENED aims to develop 3D in vitro tests, supported by modelling, to characterise the effects of EDCs on thyroid function. SCREENED will deliver innovative “organ-on-a-chip” models, where thyrocyte cells organised in a 3D structure will be hosted in a microfluidic cell culture device (a microfluidic bioreactor). This device will mimic the microenvironment of the thyroid gland, by simulating tissue- and organ-level physiology. The first 3D models consist of thyroid organoids able to recapitulate the thyroid hormone production functionality of the native thyroid (mouse and human models). We work also on ECM-scaffold-based and bioprinted 3D models, which 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.
Our solutions have the potential to overcome the limitations of existing in vitro tests by being more sensitive at low doses of exposure and supporting the prediction of toxicity on human health in a sex-specific manner.
SCREENED aims to develop 3D in vitro tests, supported by modelling, to characterise the effects of EDCs on thyroid function. SCREENED will deliver innovative “organ-on-a-chip” models, where thyrocyte cells organised in a 3D structure will be hosted in a microfluidic cell culture device (a microfluidic bioreactor). This device will mimic the microenvironment of the thyroid gland, by simulating tissue- and organ-level physiology. The first 3D models consist of thyroid organoids able to recapitulate the thyroid hormone production functionality of the native thyroid (mouse and human models). We work also on ECM-scaffold-based and bioprinted 3D models, which 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.
Our solutions have the potential to overcome the limitations of existing in vitro tests by being more sensitive at low doses of exposure and supporting the prediction of toxicity on human health in a sex-specific manner.
Work on the three 3D in vitro cellular models, able to mimic the microenvironment of a thyroid gland at different levels of anatomical complexity, continued:
- Beyond the purified thyroid cells/progenitors from murine origin coaxed into thyroid follicles, we have established the derivation of thyroid follicles from both human embryonic (hESC) and induced pluripotent stem cells (iPSC). Excitingly, our results demonstrated that we can generate structures capable to organise three-dimensionally in follicles, produce thyroid hormone in vitro, and rescue the levels of TH when transplanted in thyroid-ablated mice.
- Recellularization of decellularized scaffolds with these 3D organoids has been accomplished and allows to reproduce the biological composition of a native thyroid. We have also developed two simpler models based on the repopulation of a collagen scaffold and of a cell-laden hydrogel “cookie” configuration. Both models allowed to recreate functional rat thyroid function.
- Bioprinted constructs comprising the 3D organoids have shown successful maintenance of murine and human thyroid follicles derived from pluripotent stem cells. In addition, vascularization strategies to obtain a vascularized bioprinted thyroid model have been finalised showing the successful formation of a primitive vascular network, able to mimic the spatial and geometrical architecture of a native thyroid.
In parallel, we continued to develop the modular microbioreactor, compatible with organoid culture, sensing technology and plug-and-play microfluidics, able to host the 3D in vitro assays. In period 2, the microfluidic bioreactor platform developed could host up to eight 3D thyroid tissue constructs in parallel, under flow conditions and meeting the requirements for high throughput screening. In the current period, the platform has been successfully used with mESC- and hESC-derived thyroid follicles, and has been upscaled to host up to 54 culture chambers in parallel. This supports the validation of the microfluidic platform, designed to be compatible with industry demands and translatable for commercialization.
Using this platform, we completed screening of 16 different EDCs for 24h and the top three compounds for 10 days. We were able to elucidate key thyroid responses to EDC exposure. Representatives per class of screened chemicals were selected, and comparative studies between the 3D in vitro models are now being performed on the representatives of the class. Sex-specific experiments have also been performed by simulating a sex-specific environment in the 3D in vitro models, resulting in significant differences at the single cell level. These results are now being further analysed.
To help elucidate the capacity of EDCs to interfere with thyroid development and function, two mechanisms of action (MoA) have been identified:
- activation of the aromatic hydrocarbon receptor by PAHs (a class of chemicals that occur naturally in, and from the burning of, coal, crude oil and petroleum products) and planar PCBs (industrially-generated fat-soluble substances that persist in the environment and living organisms), and
- inhibition of succinate dehydrogenase by phthalates and phthalate esters (a group of chemicals used to make plastics more durable or to help dissolve other materials).
Three adverse outcome pathways (AOPs) initiated by these MoAs have been created, and are under development in the AOP-Wiki. Additionally, mathematical models have been developed linking changes in thyroid function to observed changes in circulating thyroid hormones in vivo.
We are now working on the development of a biomarker able to reflect the interference of a chemical product on the thyroid function. Unbiased liquid chromatography with tandem mass spectrometry (LC-MS/MS) proteomics is used to identify a protein candidate biomarker signature of EDC action on the thyroid proteome. We used the literature and the Human Protein Atlas to identify thyroid-specific proteins and targeted protein multiple reaction monitoring (MRM) assays developed to measure the proteins in both mouse and human thyroid cells. Analytical validation of the MRM assays is in progress.
Regular discussions with regulatory and industry representatives through the SCREENED Stakeholder Group and the EURION cluster have given us a better understanding of the requirements for the use of our assays for regulatory purposes. To support this aim, efforts have been devoted to ensure that methods are well documented and standardised. As an in vitro assay is rarely used as stand-alone test method but rather in a battery of assays, this possible combination with other assays is further investigated in the frame of EURION.
- Beyond the purified thyroid cells/progenitors from murine origin coaxed into thyroid follicles, we have established the derivation of thyroid follicles from both human embryonic (hESC) and induced pluripotent stem cells (iPSC). Excitingly, our results demonstrated that we can generate structures capable to organise three-dimensionally in follicles, produce thyroid hormone in vitro, and rescue the levels of TH when transplanted in thyroid-ablated mice.
- Recellularization of decellularized scaffolds with these 3D organoids has been accomplished and allows to reproduce the biological composition of a native thyroid. We have also developed two simpler models based on the repopulation of a collagen scaffold and of a cell-laden hydrogel “cookie” configuration. Both models allowed to recreate functional rat thyroid function.
- Bioprinted constructs comprising the 3D organoids have shown successful maintenance of murine and human thyroid follicles derived from pluripotent stem cells. In addition, vascularization strategies to obtain a vascularized bioprinted thyroid model have been finalised showing the successful formation of a primitive vascular network, able to mimic the spatial and geometrical architecture of a native thyroid.
In parallel, we continued to develop the modular microbioreactor, compatible with organoid culture, sensing technology and plug-and-play microfluidics, able to host the 3D in vitro assays. In period 2, the microfluidic bioreactor platform developed could host up to eight 3D thyroid tissue constructs in parallel, under flow conditions and meeting the requirements for high throughput screening. In the current period, the platform has been successfully used with mESC- and hESC-derived thyroid follicles, and has been upscaled to host up to 54 culture chambers in parallel. This supports the validation of the microfluidic platform, designed to be compatible with industry demands and translatable for commercialization.
Using this platform, we completed screening of 16 different EDCs for 24h and the top three compounds for 10 days. We were able to elucidate key thyroid responses to EDC exposure. Representatives per class of screened chemicals were selected, and comparative studies between the 3D in vitro models are now being performed on the representatives of the class. Sex-specific experiments have also been performed by simulating a sex-specific environment in the 3D in vitro models, resulting in significant differences at the single cell level. These results are now being further analysed.
To help elucidate the capacity of EDCs to interfere with thyroid development and function, two mechanisms of action (MoA) have been identified:
- activation of the aromatic hydrocarbon receptor by PAHs (a class of chemicals that occur naturally in, and from the burning of, coal, crude oil and petroleum products) and planar PCBs (industrially-generated fat-soluble substances that persist in the environment and living organisms), and
- inhibition of succinate dehydrogenase by phthalates and phthalate esters (a group of chemicals used to make plastics more durable or to help dissolve other materials).
Three adverse outcome pathways (AOPs) initiated by these MoAs have been created, and are under development in the AOP-Wiki. Additionally, mathematical models have been developed linking changes in thyroid function to observed changes in circulating thyroid hormones in vivo.
We are now working on the development of a biomarker able to reflect the interference of a chemical product on the thyroid function. Unbiased liquid chromatography with tandem mass spectrometry (LC-MS/MS) proteomics is used to identify a protein candidate biomarker signature of EDC action on the thyroid proteome. We used the literature and the Human Protein Atlas to identify thyroid-specific proteins and targeted protein multiple reaction monitoring (MRM) assays developed to measure the proteins in both mouse and human thyroid cells. Analytical validation of the MRM assays is in progress.
Regular discussions with regulatory and industry representatives through the SCREENED Stakeholder Group and the EURION cluster have given us a better understanding of the requirements for the use of our assays for regulatory purposes. To support this aim, efforts have been devoted to ensure that methods are well documented and standardised. As an in vitro assay is rarely used as stand-alone test method but rather in a battery of assays, this possible combination with other assays is further investigated in the frame of EURION.
The establishment of human thyroid in vitro models constitutes a major breakthrough in thyroid research and will be extremely useful to study the thyroid gland. These models also bring a new fast, cheaper and animal-free alternative to screen EDCs for toxic effects, using a system that resembles human physiology.
SCREENED is advancing the field of “organ-on-a-chip” devices, fostering its adoption by industries and regulatory bodies. The development of reversibly sealed bioreactor chips is compatible with high-throughput screening platforms, a major requirement for industrial applicability. Our system enables integration of sensing technology for continuous monitoring of physical and biochemical parameters during culture. The progress made on the 3D in vitro cellular assays has led to the successful generation and characterisation of functional human thyroid follicles, derived from mESC, hESC and iPSC. This gives us the opportunity to analyse the effects of EDCs on these in vitro models of the human thyroid at a gene (transcript) and protein level, and by elucidating the MoA and AOPs caused by the interference of an EDC with thyroid development and function. Besides, we started to develop targeted protein assays to transcripts that have been shown to change and attempt to circumvent the limitations of protein-based discovery experiments.
SCREENED is advancing the field of “organ-on-a-chip” devices, fostering its adoption by industries and regulatory bodies. The development of reversibly sealed bioreactor chips is compatible with high-throughput screening platforms, a major requirement for industrial applicability. Our system enables integration of sensing technology for continuous monitoring of physical and biochemical parameters during culture. The progress made on the 3D in vitro cellular assays has led to the successful generation and characterisation of functional human thyroid follicles, derived from mESC, hESC and iPSC. This gives us the opportunity to analyse the effects of EDCs on these in vitro models of the human thyroid at a gene (transcript) and protein level, and by elucidating the MoA and AOPs caused by the interference of an EDC with thyroid development and function. Besides, we started to develop targeted protein assays to transcripts that have been shown to change and attempt to circumvent the limitations of protein-based discovery experiments.