Fig 2: Gene expression profiling of TSTerm cells and their derivative STBs and EVTs.a PCA plot of the genes expressed in the different cell types. Each of the CTBs, TS cells, and TS cell-derived STBs and EVTs clusters together in first three dimensions. Data were collected from independent preparations of CTBFirst (n = 3), EVTFirst (n = 3), STFirst (n = 3), TSCT & TSblast (n = 4), EVT-TSCT & -TSblast (n = 4), ST-TSCT & -TSblast (n = 4), CTBTerm (n = 3), TSTerm (n = 3), EVT-TSTerm (n = 3), and ST-TSTerm (n = 3) cells and subjected to a PCA. Note that the data from TSCT and TSblast cells are combined (n = 4) and their derivative EVTs and STBs as well in the Okae’s study. b, Volcano plots displaying differentially expressed genes between TSTerm cells and their derivative STBs (left) or EVTs (right). Raw p-values are presented in the plots. c, Pearson correlation coefficients between the gene expression profiles of ITGA6-positive term CTBs, TSTerm, TSCT, and TSblast cells and their derivative STBs (ST-TSCT, ST-TSblast, and ST-TSTerm) and EVTs (EVT-TSCT, EVT-TSblast, and EVT-TSTerm). d, Heatmap representation of relative expression (Z-score) of lineage-specific genes across TSTerm, TSCT, and TSblast cells and their derivative STBs and EVTs. Of note, additional TSTerm-, ST-TSTerm-, and EVT-TSTerm-specific genes that match the CTB, STB, and EVT lineage-specific genes from 3D-cultured human pre-gastrulation embryos are listed in the lower part of the map. e Expression levels of TS, STB, and EVT marker genes in TSTerm, TSCT, and TSblast cells and their derivative STBs and EVTs. Data are presented as mean ± SD of independent preparations of the indicated cell types mentioned in (a). f Functional annotation of DEGs in TSTerm, ST-TSTerm, and EVT-TSTerm cells by ConsensusPathDB. The q-values corresponding to the p values after FDR corrections are smaller than 0.05. Source data are provided in the Source Data file.

Fig 3: Regulation of trophoblast stemness by EGF/CASVY and hypoxia through the ?Np63a-GCM1 antagonism.a BeWo cells were mock-treated or treated with EGF/CASVY (i.e., cultured in incomplete or complete TS medium) for 24 h for analysis of trophoblast stemness or differentiation gene expression. AXIN2, a direct target of WNT signaling, was used as a control. Arrow and arrowhead denote the ?Np63a band and a non-specific band, respectively. b ITGA6-positive term CTBs were cultured in complete TS medium for indicated periods of time and then subjected to immunofluorescence microscopy of GCM1 and ?Np63a. c Expression of ?Np63a, GCM1, and hCGß in ITGA6-positive term CTBs in response to EGF/CASVY treatment and withdrawal. Note that E-cadherin staining reveals formation of hCGß-expressing syncytium after EGF/CASVY withdrawal. d, Suppression of GCM1 expression by hypoxia. BeWo cells and P0 ITGA6-positive term CTBs were cultured under normoxic or hypoxic conditions for three and seven days, respectively. Cells were then harvested for analysis of GCM1, ?Np63a, HTRA4, hCGß, and MKI67 transcripts. Data are presented as mean ± SD of independent experiments (n = 3 in a, d). Differences were assessed by unpaired two-tailed Student’s t-test (a, d). Source data are provided in the Source Data file.

Fig 4: Derivation of TS cells from term placentas.a Images of TSTerm#2 cells and their derivative EVTs and STBs. TSTerm#2 cells derived from ITGA6-positive term CTBs were treated with FSK (ST medium) for five days or A83-01 and NRG1 (EVT medium) for 10 days for differentiation into STBs (ST-TSTerm#2) or EVTs (EVT-TSTerm#2), respectively. Magnification of the boxed syncytium in ST-TSTerm#2 cells is presented at right. b Expression of trophoblast stemness markers in TSTerm#2 cells. c Expression of STB markers and formation of syncytium in ST-TSTerm#2 cells. d Expression of EVT markers in EVT-TSTerm#2 cells. The HLA-G-positive EVT-TSTerm#2 cells were subjected to transwell invasion assays. Scale bar, 100 µm. e Regulation of GCM1 and ?Np63a expression in TSTerm#2 cells and ST-TSTerm#2 by hypoxia. Arrow and arrowhead denote the ?Np63a band and a non-specific band, respectively. f Suppression of GCM1 autoregulation by hypoxia. TSTerm#1 cells were transfected with pGL4-SV40 or E1bLUCGCM1-2K and incubated under normoxia or hypoxia for 48 h. Cells were then harvested for luciferase assays. g Interaction of GCM1 and ?Np63a in TSTerm cells. TSTerm#2 cells were cultured under normoxia for 96 h and then subjected to immunofluorescence microscopy (IFM) or PLA using GCM1 Ab, ?Np63 mAb, and normal rabbit IgG (R-IgG). Arrows point to cells coexpressing ?Np63a and GCM1 in IFM or exhibiting PLA puncta. Average PLA signal per nucleus was calculated as total PLA puncta divided by the number of all nuclei in the field. Scale bar, 20 µm. h Suppression of STB differentiation by ?Np63a. GFP-positive mock or ?Np63a-expressing TSTerm#2 cells were treated with or without FSK for 72 h, followed by cell fusion assays and analysis of GCM1, hCGß and SYN1 transcripts or proteins. Data are presented as mean ± SD of independent experiments (n = 3 in e–h). Differences were assessed by two-way ANOVA with Tukey’s post hoc test (e, h (left)) or unpaired two-tailed Student’s t-test (f, g, h (right)). Source data are provided in the Source Data file.

Fig 5: Inhibition of TPST2 decreases the expression and increases the ubiquitination of ITGB4. (A and B) The protein expression of ITGB4 in sgGFP or sgTPST2 MIA PaCa-2 cells was measured by (A) immunofluorescent staining (scale bars: 10 µm) and (B) Western blot, respectively. Data are presented as means ± SD; N = 3 from biological replicates. *P < .05, **P < .01. Western blot analysis of (C) ITGB4 in MIA PaCa-2 cells transfected with siNegative or siTPST2 and (D) MIA PaCa-2 and PANC-1 cells treated with vehicle or NaClO3 (100 mmol/L) for 72 hours. Data are presented as means ± SD; N = 3 from biological replicates. *P < .05, **P < .01. (E) Immunofluorescence staining of ITGA6 in sgGFP or sgTPST2 MIA PaCa-2 cells. Scale bars: 10 µm. Western blot analysis of (F) ITGB3 in sgGFP or sgTPST2 MIA PaCa-2 cells, or (G) PANC-1 cells treated with PBS or NaClO3 (100 mmol/L) for 72 hours. Data are presented as means ± SD; N = 3 from biological replicates. *P < .05, **P < .01. (H) Western blot analysis of ITGB4 in sgNC or sgTpst2 KPC cells (left) or in KPC cells treated with PBS or NaClO3 (100 mmol/L) for 72 hours (right). Data are presented as means ± SD; N = 3 from biological replicates. *P < .05, **P < .01. (I) Western blot analysis of ITGB4 in sgGFP and sgTPST2 cells transfected with empty vector (EV) or ITGB4 expression plasmids. Data are presented as means ± SD; N = 3 from biological replicates. *P < .05, **P < .01. (J) Migration assay of sgGFP and sgTPST2 cells transfected with empty vector (EV) or ITGB4 expression plasmids; quantifications are shown on the right. Scale bars: 200 µm. N = 3 from biological replicates. Data are presented as means ± SD. (K) sgGFP or sgTPST2 MIA PaCa-2 cells were transfected with His-ITGB4 and HA-ubiquitin. ITGB4 ubiquitination was determined by immunoprecipitation with anti-HA antibody followed by Western blot with the anti-ITGB4 antibody. Arrow indicates the ITGB4 protein bands. Data are presented as means ± SD; N = 3 from biological replicates. *P < .05, **P < .01. (L) sgGFP or sgTPST2 MIA PaCa-2 cells were transfected with His-ITGB4 and treated with vehicle or Na3VO4 for 15 minutes. Phosphotyrosine level of ITGB4 was determined by immunoprecipitation with an anti-His antibody followed by Western blot with the antiphosphotyrosine antibody. Data are presented as means ± SD; N = 3 from biological replicates. *P < .05, **P < .01. DAPI, 4',6-diamidino-2-phenylindole; IB, immunoblotting; siNeg, negative control siRNA; Ub, ubiquitin; Veh, vehicle.