DEHP_1_uM_2
Mus musculus
Protocols: Embryonic stem cells culture and differentiation: Thyroid follicles were differentiated from the A2Lox.Cre_TRE-Nkx2-1/Pax8_Tg-EGFP mouse ESC as previously described [1, 2]. Briefly, cells were initially cultured on gamma-irradiated mouse embryonic fibroblasts (MEFs) feeder using mouse stem cell medium [1, 2] and incubated at 37 °C, 5% pCO2 and >95% humidity. For differentiation into thyroid, embryoid bodies (EBs) were generated by hanging drops culture of ESCs (1000 cells per drop) for 4 days. They were then collected and embedded in growth factor restricted Matrigel (354230, Corning). 50 uL matrigel drops containing around 20 EBs were plated into 12-well plates. Cells were differentiated using a differentiation medium composed of DMEM (31966021, Gibco) supplemented with 15% FBS, vitamin C (50 ug mL-1) (A4544, Sigma), nonessential amino acids (0.1 mM) (11140035, Gibco), sodium pyruvate (1 mM) (11360039, Gibco), penicillin and streptomycin (50 U mL-1) (15140122, Gibco), 2-mercaptoethanol (0.1 mM) (31350010, Gibco). The differentiation medium was supplemented with 1 μg mL-1 of doxycycline (D9891-1G, Sigma) for 3 days for Nkx2-1 and Pax8 induction, followed by two weeks of maturation by using the differentiation medium containing 8-Br-cAMP (0.3 nM) (B 007-500, BioLog). Follicles enrichment protocol At day 21, after complete thyroid maturation, matrigel drops containing the thyroid follicles were washed twice with Hanks's balanced salt solution (HBSS, containing calcium and magnesium) (14025050, Gibco) and incubated in a HBSS solution (1 mL per well) containing 10 U mL-1 of dispase® II (4942078001, Roche) and 125 U mL-1 of collagenase type IV (Sigma) for 30-45 min at 37 °C. The enzymes were then inactivated by adding 10% FBS. Organoids were centrifuged at 1200 rpm for 3 minutes. After rinsing twice with HBSS, the follicles were enriched by filtration using 30 um (to remove single cells) (43-50030, pluriSelect Life Science GmbH) to and 100 um reverse strainer (to remove big follicles aggregates) (43-50100, pluriSelect Life Science GmbH). Resuspended follicles were cultured in subsequent experiments in the differentiation medium described above and supplemented with 8-Br-cAMP (10 µM) and TGF-βRI inhibitor SB431542 (10 µM) (1614, Tocris). [1] Antonica, F., et al., Generation of functional thyroid from embryonic stem cells. Nature, 2012. 491(7422): p. 66-71. [2] Romitti, M., et al., Single-Cell Trajectory Inference Guided Enhancement of Thyroid Maturation In Vitro Using TGF-Beta Inhibition. Frontiers in Endocrinology, 2021. 12. Enriched thyroid follicles were exposed to DEHP (CAS 117-81-7) /DIDP (CAS 26761-40-0)/DINP (CAS 28553-12-0) (1-10-100 nM-1-10 uM), DnOP (CAS 117-84-0) (2-20-200 nM-2-20 uM) (n = 3), 0.5% DMSO (n = 5) or only culture media (n = 2) for 24 hours. The culture media was DMEM (31966021, Gibco) supplemented with 15% FBS, vitamin C (50 μg mL-1) (A4544, Sigma), nonessential amino acids (0.1 mM) (11140035, Gibco), sodium pyruvate (1 mM) (11360039, Gibco), penicillin and streptomycin (50 U mL-1) (15140122, Gibco), 2-mercaptoethanol (0.1 mM) (31350010, Gibco), 8-Br-cAMP (10 µM) and TGF-βRI inhibitor SB431542 (10 µM) (1614, Tocris). At the end of the exposure time, cells were collected in 1.5 mL Eppendorf tubes and washed once with PBS. They were then lysed in QIAzol Lysis Reagent (79306, Qiagen). Total RNA was extracted using the miRNAeasy Micro Kit (217084, Qiagen). 20 ng of total RNA isolated from thyroid organoids samples were used as input for the NEXTFLEX® Combo-Seq™ mRNA/miRNA Kit (NOVA-5139-53, PerkinElmer). All RIN (RNA Integrity Number) values were 8 or higher. To deplete tRNA fragments and Y RNA fragments during the library preparation, the NEXTFLEX® tRNA/YRNA Blocker was used following the manufacturer's instruction. We performed 14 or 16 PCR cycles were performed during library preparation (see samplesheet). RNA-Seq libraries were quantified on a Qubit 2.0 Fluorometer (ThermoFisher) and quality control checked on the 2200 TapeStation System (Agilent) or BioAnalyzer 2100 expert (Agilent).