Thyroid hormones (TH) play a fundamental role in brain development. Changes on TH availability during pregnancy and early childhood can lead to neurological disorders. Accordingly, concerns are mounting that exposure to environmental compounds capable of interfering with TH action can disrupt neuronal development. Current approaches to categorize chemicals as thyroid hormone system disrupting chemicals (THSDC) are mainly based on determination of changes in circulating TH concentrations in vivo. Since such tests are not adequate to capture alterations at cellular and molecular levels during human brain development, there is an urgent need to establish and validate human in vitro models for THSDC assessment. Human cerebral organoids (hCOs) derived from induced pluripotent stem cell (hiPSC) present a promising model system as hCOs recapitulate tissue complexity and critical developmental processes while providing an infinite source of material. We used single cell hCO transcriptome data to extract lists of TH-responsive genes to be used as molecular markers of TH action in RT-qPCR assays. Since hCOs show dynamic changes in cell composition during their development, these lists included broad response genes as well as cell type-specific markers. We analyzed expression patterns of marker genes following acute 48 h treatment of hCOs at different stages with T3 (3-300 nM). We further characterized expression profiles following co-exposure of hCOs to T3 and the MCT8 inhibitor silychristin (SC) or the pan-deiodinase inhibitor iopanoic acid (IA). In addition, we established a sensitive LC-MS/MS method to determine basal TH concentrations in media and the TH metabolite pattern (rT3, 3,3´-T2; 3,5-T2; 3-T1; T0) following TH treatment and co-exposure experiments. RT-qPCR assays of whole hCOs showed significant gene expression changes in a TH concentration-dependent manner for most of the selected marker genes. SC co-treatment attenuated the T3-induced expression response for a subset of genes (i.e., CADM2, DBP and DIO3) in acute T3 treatment schemes. IA co-treatment enhanced T3-induced expression levels in longer-term T3 treatment schemes. Cell type composition (progenitors, neurons) had a clear influence on T3-induced expression responses as evident from comparison of T3 effects in early and late stage hCOs. hCOs effectively metabolize T3 to 3,3´T2. Notably, metabolic degradation of T3 was reduced upon co-treatment with either SC or IA. Thus, these reference compounds affected both molecular markers and local T3 metabolism. Our study demonstrates the utility of hCOs as a promising platform for THSDC assessment, identifies critical aspects (validation, endpoints, reference compounds) and highlights pitfalls for the assay validation process.
10 Sep 2022 - 13 Sep 2022