Fig. 1. Derivation of hTS^PS cells from primed hPSCs.

(A) Overview of conversion strategy to generate hTSCs (hTS^PS cells) from PSCs (hPSCs). hPSCs were maintained in mTeSR1 medium in monolayer culture. The TS-like colonies appeared at days 7 and 8 and were picked at days 10 to 12 for subculture expansion as purified hTS^PS cells (see Materials and Methods). (B) Morphology of converted cells derived from H1 hESCs under TS medium. Scale bar, 200 μm. (C) Morphology of hTS^PS cells derived from H1 or HN10 hESCs at early (P5) or late passage (P30). Scale bar, 200 μm. (D) Quantitative real-time polymerase chain reaction (qRT-PCR) expression of TS, epithelial, and ES marker genes in hTS^PS cells derived from H1 or HN10 hESCs. H1 and HN10 hESCs serve as negative controls. hTS^blast serves as a positive control. **P < 0.01. Error bars represent means ± SD from three independent replicates. Results represent two samples, H1-hTS^PS and HN10-TS^PS (n = 2). (E) Immunostaining of TS marker genes GATA3/KRT7/TP63/TEAD4 in hTS^PS cells derived from H1 or HN10 hESCs. hTS^blast serves as a positive control. Scale bar, 100 μm. (F) Growth curve of H1 hTS^PS cells at early (P10) or high passage (P30). Error bars represent means ± SD from three independent replicates. Similar results were obtained using another independent cell line HN10-TS^PS (fig. S2H). (G) EdU incorporation assay for hTS^PS cells and their parent hESCs. (H) Apoptosis assay in hTS^PS cells and their parent hESCs. PI and/or annexin V–positive cells were analyzed by fluorescence-activated cell sorting (FACS). The apoptotic cells include late apoptotic cells (PI⁺/annexin⁺) in Q2 and early apoptotic cells (PI⁻/annexin V⁺) in Q4. (I and J) Phase-contrast images showing growth of a single hTS^PS cell. Scale bar, 50 μm. The results shown in (E), (G), and (H) are representative of three independent experiments from two samples, H1-hTS^PS and HN10-TS^PS (n = 2). See figs. S1 to S3 for more details.