Fig 2: 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 CGß, 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 CGß, 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.

Fig 3: hPSC-TSCDX2and hPSC-TS generated from hiPSCs. A, confocal image of SC102A-1 hPSC-TSCDX2 in TM4, staining for CDX2, TFAP2C, GATA3, YAP, TEAD4, and P63. Nuclei were stained with DAPI. The scale bars represent 200 µm. B, confocal images of SC102A-1 hPSC-TS in TSCM, staining for CDX2, TFAP2C, GATA3, YAP, TEAD4, and P63. The scale bars represent 200 µm. C, flow cytometry histogram of KRT7 expression of SC102A-1 hPSC-TS cells in TSCM compared with an isotype control. D, proliferation of SC102A-1 hPSC-TS, placenta-derived TS #1 (CT30), and TS #2 (CT29) in TSCM. A total of 1 x 105 cells were seeded and cells were counted after 3 days. Four biological replicates were used (error bars, SD). E, flow cytometry histogram of HLA-G expression of EVTs from SC102A-1 hPSC-TS cells compared with an isotype control. F, gene expression of CGß, SDC1, CSH1/2, HLA-G, MMP2, TEAD4, and TP63 of EVTs compared with TS cells from SC102A-1 hPSC-TS and placenta-derived TS #1 (CT30) and TS #2 (CT29). Four biological replicates were used (error bars, SE, *p < 0.05 for differential expression relative to TS cells). Data for placenta-derived TS cells is the same as used in Figure 4. G, confocal images of EVTs from SC102A-1 hPSC-TS, staining for HLA-G and VE-Cadherin. The scale bars represent 100 µm. H, gene expression of CGß, SDC1, CSH1/2, HLA-G, MMP2, TEAD4, and TP63 of STBs compared with TS cells from SC102A-1 hPSC-TS and placenta-derived TS #1 (CT30) and TS #2 (CT29). Four biological replicates were used (error bars, SE, *p < 0.05). I, confocal images of STB from SC102A-1 hPSC-TS, staining for hCG and KRT7. The scale bars represent 100 µm. EVT, extravillous trophoblast, hiPSC, human induced pluripotent stem cell; hPSC, human pluripotent stem cell; STB, syncytiotrophoblast; TS, trophoblast stem.

Fig 4: Optimizing timing of hESC differentiation enables derivation of hPSC-TSCDX2cells. A, schematic of differentiation protocol for establishment of hPSC-TSCDX2 and hPSC-TS from hESCs. B, confocal images of 3 days treated H9 and H1 hESCs, staining for CDX2, P63, and TEAD4. Nuclei were stained with DAPI. The scale bars represent 100 µm. C, confocal images of 6 days treated H9 and H1 hESCs, staining for CDX2. Nuclei were stained with DAPI. The scale bars represent 100 µm. D, quantitative analysis of cells expressing nuclear CDX2 after 3- and 6-day differentiation treatment of H1 (day 3, 5455 cells in three images; day 6, 2448 cells in two images) and H9 (day 3, 5552 cells in four images; day 6, n = 6448 cells in five images) hESCs. Data points represent fraction of CDX2+cells in individual images from at least two biological replicates. Analysis was performed in MATLAB and at least two biological replicates were used. (Error bars are SD, ***p < 0.05). E, confocal images of H9 hPSC-TSCDX2 in TM4, staining for CDX2, TFAP2C, GATA3, YAP, TEAD4, and P63. Nuclei were stained with DAPI. The scale bars represent 200 µm. F, confocal images of STB from H9 hPSC-TSCDX2 staining for hCG and KRT7. Nuclei were stained with DAPI. The scale bars represent 100 µm. G, confocal images of EVTs from H9 hPSC-TSCDX2, staining for HLA-G (red) and VE-Cadherin (green) as well as KRT7 (red) and hCG (green). Nuclei were stained with DAPI. The scale bars represent 200 µm. DAPI, 4',6-diamidino-2-phenylindole; hESC, human embryonic stem cell; hPSC, human pluripotent stem cell; STB, syncytiotrophoblast; TS, trophoblast stem.

Fig 5: LSD1 Inhibition Promotes Differentiation and Represses Squamous Cell Carcinoma(A) NOTCH3, GRHL3, KLF4, and AP2-g (TFAP2C) are upregulated in GSK-LSD1-treated EPs.(B) GSK-LSD1-treated EPs display significantly reduced growth compared with DMSO.(C) GSK-LSD1-treated 3D human OTCs on collagen rafts prematurely cornify (arrows) as compared with DMSO.(D) GSK-LSD1-treated oncogenic 3D human OTCs on human dermis prematurely cornify as compared with DMSO (H&E).(E and F) GSK-LSD1-treated (2, 10, or 20 mM) oncogenic 3D human OTCs established on human dermis display significantly less epidermal area (E) and dermal protrusions (F) than DMSO. Error bars represent SDs.(G) IF of oncogenic 3D OTCs for Krt14 (red), Collagen VII (green), or DAPI (blue) (original magnification 320; scale bar, 50 mm).(H) Overlap of genes upregulated by GSK-LSD1 in EPs and genes downregulated in cSCC compared with normal skin (top).(I) LSD1 expression negatively correlates with expression of epithelial differentiation TFs in HNSCC data from TCGA.