Development of anIn VitroHuman Thyroid Microtissue Model for Chemical Screening

Abstract

Thyroid hormones (TH) are essential for regulating a number of diverse physiological processes required for normal growth, development, and metabolism. The US EPA Endocrine Disruptor Screening Program (EDSP) has identified several molecular thyroid targets relevant to hormone synthesis dynamics that have been adapted to high-throughput screening (HTS) assays to rapidly evaluate the ToxCast/Tox21 chemical inventories for potential thyroid disrupting chemicals (TDCs). The uncertainty surrounding the specificity of active chemicals identified in these screens and the relevance to phenotypic effects on in vivo human TH synthesis are notable data gaps for hazard identification of TDCs. The objective of this study was to develop a medium-throughput organotypic screening assay comprised of reconstructed human thyroid microtissues to quantitatively evaluate the disruptive effects of chemicals on TH production and secretion. Primary human thyroid cells procured from qualified euthyroid donors were analyzed for retention of NK2 homeobox 1 (NKX2-1), Keratin 7 (KRT7), and Thyroglobulin (TG) protein expression by high-content image analysis to verify enrichment of follicular epithelial cells. A direct comparison of 2-dimensional (2D) and 3-dimensional (3D) 96-well culture formats was employed to characterize the morphology, differential gene expression, TG production, and TH synthesis over the course of 20 days. The results indicate that modeling human thyroid cells in the 3D format was sufficient to restore TH synthesis not observed in the 2D culture format. Inhibition of TH synthesis in an optimized 3D culture format was demonstrated with reference chemicals for key molecular targets within the thyroid gland. Implementation of the assay may prove useful for interpreting phenotypic effects of candidate TDCs identified by HTS efforts currently underway in the EDSP.

A rodent thyroid-liver chip to capture thyroid toxicity on organ functional level

“…The successfully established rat thyroid follicle culture remained viable and maintained a follicular architecture with correct cellular polarity for at least 14 days. Expression of characteristic basolateral and apical specific proteins, including ZO-1, ezrin, pendrin, MCT8, and thyroglobulin, evinced correct follicular polarization, similar to previous reports (Delmarcelle et al, 2014;Deisenroth et al, 2020;Khoruzhenko et al, 2021). Moreover, these rat follicle cultures restored their capability to synthesize and secrete THs, predominantly T4, in the presence of TSH.…”

“…Recent developments illustrate different approaches to rebuilding complex thyroid morphology and function in vitro, highlighting the relevance of an interplay between thyrocytes and extracellular matrix (Samimi et al, 2021). For example, Saito et al and Deisenroth et al successfully reconstructed thyroid follicles from isolated primary thyrocytes of mouse and human origin following Matrigel embedding (Saito et al, 2018;Deisenroth et al, 2020). These follicles developed correct cell polarity and, importantly, were able to produce THs.…”

“…These follicles developed correct cell polarity and, importantly, were able to produce THs. Deisenroth et al also demonstrated that their human 3D thyroid model allowed the recapitulation of chemical-induced TH disruption on a functional level (TH synthesis) (Deisenroth et al, 2020). Alternative approaches described a successful establishment of 3D thyroid in vitro cultures by isolation of intact follicles from tissue explants, i.e.…”

Towards a science-based testing strategy to identify maternal thyroid hormone imbalance and neurodevelopmental effects in the progeny – part II: how can key events of relevant adverse outcome pathways be addressed in toxicological assessments?

“…For example, when testing 19 reference compounds (Dong et al 2020), the Amplex-UltraRed assay yielded similar results for strong or negative compounds using rat microsomes or recombinant human TPO; however, variable responses were seen with weak-acting TPO inhibitors across studies. Generally, these in vitro TPO assays, as well as recently described three-dimensional in vitro models of the thyroid gland (Deisenroth et al 2020) may provide useful testing opportunities while minimising animal use.…”

Stem Cells for Next Level Toxicity Testing in the 21st Century

“…[217,218] In order to increase the applicability for toxicological screenings, Deisenroth et al developed a medium-throughput organotypic screening assay in a 96-well plate format. [219] Functional follicular structures expressing genes of mature thyrocytes (NIS, TPO, TSHR, Tg), capable of iodide uptake and TH production, were derived from human thyrocyte tissue. Of note, screening of reference compounds identified in established HTS assays for thyroid disruption (i.e., NIS and TPO enzyme activity) revealed both similar effects and potencies in the microtissue model, highly indicating its applicability for a tiered screening approach.…”