Periodic Reporting for period 4 - SCREENED (A multistage model of thyroid gland function for screening endocrine-disrupting chemicals in a biologically sex-specific manner)
Période du rapport: 2023-07-01 au 2024-06-30
Increasing evidence shows that endocrine disrupting chemicals (EDCs) interfere with thyroid function and cause changes in thyroid hormone concentrations, their peripheral metabolism and receptor signalling. However, the mechanisms by which EDCs affect the thyroid axis are not yet elucidated, partly due to the limitations of existing test methods.
SCREENED has developed 3D in vitro tests, supported by modelling, to characterise the effects of EDCs on thyroid function. SCREENED delivers innovative “organ-on-a-chip” models, where thyrocyte cells organised in a 3D structure are hosted in a microfluidic cell culture device (a microfluidic bioreactor). This device mimics 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). The consortium developed also 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 is also supported by a vascularized network.
The SCREENED solutions aim to surpass existing in vitro tests by providing greater sensitivity to low exposure doses and supporting prediction of toxicity that accounts for sex-specific differences in human health.
SCREENED has developed 3D in vitro tests, supported by modelling, to characterise the effects of EDCs on thyroid function. SCREENED delivers innovative “organ-on-a-chip” models, where thyrocyte cells organised in a 3D structure are hosted in a microfluidic cell culture device (a microfluidic bioreactor). This device mimics 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). The consortium developed also 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 is also supported by a vascularized network.
The SCREENED solutions aim to surpass existing in vitro tests by providing greater sensitivity to low exposure doses and supporting prediction of toxicity that accounts for sex-specific differences in human health.
We have developed three 3D in vitro cellular models, able to mimic the microenvironment of a thyroid gland at different levels of anatomical complexity:
• Beyond purified thyroid cells/progenitors from murine origin coaxed into thyroid follicles, we have established the derivation of thyroid follicles from human embryonic (hESC) and induced pluripotent stem cells (iPSC). 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 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 PSC. We also developed a vascularized bioprinted thyroid model that showed the successful formation of a primitive vascular network, which resulted in enhanced thyroid hormone production.
“Magnetic cells” were developed using endothelial cells and a rat thyroid cell line, demonstrating maintained cell viability and morphology. Proof of concept was achieved for cell guiding in vitro using a static magnetic field in both 2D standard condition and a 3D collagen-based scaffold.
In parallel, a modular microbioreactor was developed to host the 3D in vitro assays. It is compatible with organoid culture, sensing technology and plug-and-play microfluidics. The initial microfluidic bioreactor platform has been upscaled to host up to 54 culture chambers for 3D thyroid tissue constructs simultaneously under flow conditions, suited for high throughput screening. It has been successfully used with mESC- and hESC-derived thyroid follicles. Proof-of-concept validation of the microfluidic bioreactor platform, designed to be compatible with industry demands and translatable for commercialization, has thus been achieved.
Using this platform, EDC screening experiments elucidated key thyroid responses to EDC exposure. Comparative studies between the 3D in vitro models have been performed on selected representatives per class of screened chemicals. Such studies showed that bioprinted assays were more sensitive, indicating the need for complex biological biomimicry. Sex-specific experiments revealed significant differences at the single-cell level when simulating a sex-specific environment in the 3D in vitro models.
To help elucidate the interference of EDCs 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);
• 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 documented in the AOP-Wiki. Additionally, mathematical models linking changes in thyroid function to observed changes in circulating thyroid hormones in vivo were developed.
Several biomarkers reflecting the interference of a chemical with thyroid function were identified using LC-MS/MS proteomics. This approach revealed a protein candidate biomarker signature for EDCs affecting the thyroid. By integrating proteomics with transcriptomic data from EDC-exposed samples, differentially expressed genes and proteins were identified as potential biomarkers for the classification of samples based on their exposure to different EDC classes. Machine learning, particularly using a Random Forest model, highlighted the top 20 biomarkers for each class, providing high classification accuracy and detailing the importance and direction of change for each biomarker.
Regular discussions with regulatory and industry representatives through the SCREENED Stakeholder Group and the EURION cluster have clarified the requirements for our assays in regulatory contexts. Efforts have been devoted to ensure that methods are well documented and standardised. Since in vitro assays are typically part of a battery of assays rather than used alone, we are exploring their combination with other assays within EURION.
• Beyond purified thyroid cells/progenitors from murine origin coaxed into thyroid follicles, we have established the derivation of thyroid follicles from human embryonic (hESC) and induced pluripotent stem cells (iPSC). 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 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 PSC. We also developed a vascularized bioprinted thyroid model that showed the successful formation of a primitive vascular network, which resulted in enhanced thyroid hormone production.
“Magnetic cells” were developed using endothelial cells and a rat thyroid cell line, demonstrating maintained cell viability and morphology. Proof of concept was achieved for cell guiding in vitro using a static magnetic field in both 2D standard condition and a 3D collagen-based scaffold.
In parallel, a modular microbioreactor was developed to host the 3D in vitro assays. It is compatible with organoid culture, sensing technology and plug-and-play microfluidics. The initial microfluidic bioreactor platform has been upscaled to host up to 54 culture chambers for 3D thyroid tissue constructs simultaneously under flow conditions, suited for high throughput screening. It has been successfully used with mESC- and hESC-derived thyroid follicles. Proof-of-concept validation of the microfluidic bioreactor platform, designed to be compatible with industry demands and translatable for commercialization, has thus been achieved.
Using this platform, EDC screening experiments elucidated key thyroid responses to EDC exposure. Comparative studies between the 3D in vitro models have been performed on selected representatives per class of screened chemicals. Such studies showed that bioprinted assays were more sensitive, indicating the need for complex biological biomimicry. Sex-specific experiments revealed significant differences at the single-cell level when simulating a sex-specific environment in the 3D in vitro models.
To help elucidate the interference of EDCs 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);
• 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 documented in the AOP-Wiki. Additionally, mathematical models linking changes in thyroid function to observed changes in circulating thyroid hormones in vivo were developed.
Several biomarkers reflecting the interference of a chemical with thyroid function were identified using LC-MS/MS proteomics. This approach revealed a protein candidate biomarker signature for EDCs affecting the thyroid. By integrating proteomics with transcriptomic data from EDC-exposed samples, differentially expressed genes and proteins were identified as potential biomarkers for the classification of samples based on their exposure to different EDC classes. Machine learning, particularly using a Random Forest model, highlighted the top 20 biomarkers for each class, providing high classification accuracy and detailing the importance and direction of change for each biomarker.
Regular discussions with regulatory and industry representatives through the SCREENED Stakeholder Group and the EURION cluster have clarified the requirements for our assays in regulatory contexts. Efforts have been devoted to ensure that methods are well documented and standardised. Since in vitro assays are typically part of a battery of assays rather than used alone, we are exploring their combination with other assays within EURION.
The establishment of human thyroid in vitro models represents a significant breakthrough in thyroid research and will be very useful to study the thyroid gland. These models also offer a fast, cost-effective and animal-free alternative for screening EDCs for toxic effects, closely resembling human physiology.
SCREENED has advanced the field of “organ-on-a-chip” devices, fostering its adoption by industries and regulators. Our reversibly sealed bioreactor chips are compatible with high-throughput screening, a major requirement for industrial applicability. Our culture system enables integration of sensing technology for continuous monitoring of physical and biochemical parameters. 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 allowed us to analyse the effects of EDCs on these in vitro models of the human thyroid at a gene (transcript) and protein level, and cross-omics approaches proved successful in designing biomarkers able to reflect the interference of a chemical product on the thyroid development and function. Elucidating of the underlying MoA and AOPs advanced our understanding of the widespread and interconnecting detrimental actions of environmental chemicals. SCREENED results are being published for societal benefit.
SCREENED has advanced the field of “organ-on-a-chip” devices, fostering its adoption by industries and regulators. Our reversibly sealed bioreactor chips are compatible with high-throughput screening, a major requirement for industrial applicability. Our culture system enables integration of sensing technology for continuous monitoring of physical and biochemical parameters. 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 allowed us to analyse the effects of EDCs on these in vitro models of the human thyroid at a gene (transcript) and protein level, and cross-omics approaches proved successful in designing biomarkers able to reflect the interference of a chemical product on the thyroid development and function. Elucidating of the underlying MoA and AOPs advanced our understanding of the widespread and interconnecting detrimental actions of environmental chemicals. SCREENED results are being published for societal benefit.