Single cell RNA Sequencing (scRNA-Seq) on human embryonic stem cell-derived thyroid organoids exposed for 3 days to sex hormones + 1 day to benzo[a]pyrene (BaP) or PCB153
Single cell RNA Sequencing (scRNA-Seq) on human embryonic stem cell-derived thyroid organoids exposed for 3 days to sex hormones + 1 day to benzo[a]pyrene (BaP) or PCB153
At the end of the 24 hours incubation period, we performed Matrigel digestion of the human follicles culture and prepared single-cell suspensions as described for the mouse follicles. Cells were manually counted using Trypan blue and resuspended in PBS + 0.04% BSA at a concentration of 1,000 cells/µL. Human thyroid follicles were differentiated from the modified HES3 human embryonic stem cell line following the protocol described by Romitti et al. (2022) (Romitti et al. 2022). Briefly, HES3 cells were infected with lentivirus containing the sequences of the doxycycline (Dox)-inducible system, TRE_NXK2.1/PAX8_Ubc_rtTA_Neo. Modified cells were then initially cultured as Embryonic Bodies (EBs) and then embedded in Matrigel. Differentiation into endoderm was induced by Activin A (AA) incubation, followed by NKX2.1 and PAX8 overexpression promoted by Dox treatment. Sequential treatment with cAMP, hrTSH, dexamethasone and TGF-beta inhibitors promoted thyroid differentiation and maturation. By day 58, organized and functional thyroid follicles were observed. Differentiated human ESC-derived thyroid follicles were extracted from Matrigel and plated in fresh Matrigel droplets at a density of 1,500 follicles in 20 µL Matrigel and cultured in serum-free human medium (for composition see Supplementary Methods) for 3 days. After 3 days, the medium was changed with fresh serum-free human medium, without hormones or with “female” or “male” sex hormones mixtures (at the same concentrations described above) and cells were cultured for 3 additional days. The media were further changed after 24 hours (7 days post follicles reseeding), this time adding also 10 µM BaP or PCB153 or DMSO only as solvent control (final [DMSO] in well = 0.5%) and cells were incubated for 24 hours.  The single cell suspension was directly used for single cell transcriptome libraries preparation using the Chromium Next GEM Single Cell 3' Reagent Kit v3.1 (Dual Index) from 10X Genomics. Therefore, no preliminary RNA extraction was performed. Cells were used to prepare single cell RNA-Seq libraries using the Chromium™ Next GEM Single Cell 3” Kit v3.1 (PN-1000269, 10X Genomics) and Dual Index Kit TT Set A, 96 rxns (PN-1000215, 10X Genomics) following the manufacturer's instructions.Prepared libraries were quantified on the Qubit 2.0 Fluorometer (ThermoFisher) and quality control performed on a BioAnalyzer 2100 expert (Agilent).
European Bioinformatics Institute
E-MTAB-13502:UNT_p