Fig. 1. Transient overexpression of NKX2-1 and PAX8 promotes differentiation of human ESCs into thyroid follicular cells.

a Schematic representation of the protocol leading to thyroid follicle differentiation from human ESCs. b NKX2-1 and PAX8 co-staining at day 9. c Quantification by flow cytometry of NKX2-1^GFP and PAX8 expressing cells after Dox stimulation, at days 9 and 16 (n = 4). d qRT-PCR analysis of exogenous NKX2-1 and PAX8 and endogenous PAX8, FOXE1, TG, and TSHR genes after Dox stimulation (day 9) (n = 5 for NKX2-1 exog, PAX8 exog and PAX8 endo; n = 3 for FOXE1, TG and TSHR). e Proportion of NKX2-1^GFP cells expressing BrdU during the differentiation protocol (n = 3 per time point). f Quantification by flow cytometry of NKX2-1^GFP cells during the differentiation protocol (n = 3 for −Dox, Day 23, 45 and 58; n = 4 for Day 9, 11, 14, 16, 30, and 37). g Heatmap of normalized bulk RNA-Seq of thyroid genes expression among NKX2-1^GFP cells at different stages of the thyroid differentiation protocol. Rows represent markers and columns represent specific time points. Color values in the heatmap represent mean expression levels. h qRT-PCR analysis of PAX8, NIS, TSHR, TG, and TPO at thyroid organoids from day 45 of differentiation protocol compared to un-induced control (−Dox) (n = 3). i Immunostaining for NKX2-1 and TG at D30 and TG at day 37, 45, and D58 showing thyroid differentiation and cell organization overtime. Data from at least three independent experiments are shown. Statistical analyses were performed using two-sided unpaired Mann–Whitney (p values are presented in the graphs; data presented as median (IQR)). The experiment was performed at least three times with similar results (b, i). Scale bars 20 μm. Source data are provided as a Source Data file.