Formation of hPSC-TS cells. A, confocal images of H9 hPSC-TS in TSCM, staining for CDX2, TFAP2C, GATA3, YAP, TEAD4, and P63. Nuclei were stained with DAPI. The scale bars represent 200 μm. B, flow cytometry histogram of KRT7 expression of H9 hPSC-TS cells in TSCM compared with an isotype control. C, confocal images of EVTs from H9 hPSC-TS cells, staining for HLA-G and VE-Cadherin. Nuclei were stained with DAPI. The scale bars represent 200 μm. D, flow cytometry histogram of HLA-G expression of EVTs from H9 hPSC-TS cells compared with an isotype control. E, gene expression of CGβ, SDC1, CSH1/2, HLA-G, MMP2, TEAD4, and TP63 of EVTs from H9 hPSC-TS- and placenta-derived TS #1 (CT30) and TS #2 (CT29) cells. Four biological replicates were used. (Error bars, SE, ∗p < 0.05 from TS cells). F, confocal images of STB from H9 hPSC-TS, staining for hCG and KRT7. Nuclei were stained with DAPI. The scale bars represent 100 μm. G, gene expression of CGβ, SDC1, CSH1/2, HLA-G, MMP2, TEAD4, and TP63 of STB from H9 hPSC-TS and placenta-derived TS #1 (CT30) and TS #2 (CT29) cells. Four biological replicates were used. (Error bars, SE, ∗p < 0.05 from TS cells). DAPI, 4′,6-diamidino-2-phenylindole; EVT, extravillous trophoblast; hPSC, human pluripotent stem cell; STB, syncytiotrophoblast, TS, trophoblast stem; TSCM, trophoblast stem cell medium.