Fig. 3.

Induced expression of GATA2, GATA3, TFAP2C and KLF5 for 20 days programs hESCs to hTSC-like cells. (A) Schematic of the generation of GATA2, GATA3, TFAP2C, KLF5 and MYC inducible hESCs by lentiviral transduction. (B) Schematic of the strategy for transdifferentiation of 5F-hESCs to hTSCs. 5F-hESCs were plated in mTeSR1 media for 24 h, after which media was replaced with hTSC media. Doxycycline was administered daily for 20 days in hTSC media for transgene induction. (C) Time-course RT-qPCR analysis for the detection of endogenous GATA2, GATA3, TP63, NR2F2, ENPEP, EGFR and ITGA6 in 5F-hESCs across 20 days of doxycycline induction in hTSC media. Relative expression is reflected as fold change over uninduced 5F-hESCs cultured in hTSC media normalized to GAPDH. Data are mean±s.e.m. of n=3 biological replicates analysed with a one-way ANOVA with Dunnett's post-hoc test (*P<0.05, **P<0.01, ****P<0.001). (D) Brightfield images of 5F-hESCs cultured in hESCs cultured in hTSC media in the presence of doxycycline for 20 days (+ dox). (E) Brightfield images of 5F-hESCs cultured in hTSC media in the absence of doxycycline for 20 days (− dox). (F) Brightfield images of stable 5F-iTSC lines derived from transgene overexpression grown for 15 passages. (G) Immunofluorescence analysis for the detection of KRT18, GATA3, TFAP2C and TP63 (green) and DAPI nuclear staining (blue) in stable 5F-iTSCs and previously established control hTSCs. Scale bars: 200 µm (D-F); 50 µm (G).