Fig 2: Trophoblast potential of different hPSC states under spontaneous and BMP4-mediated differentiation conditions.(A) The experimental scheme for assessing spontaneous trophoblast differentiation potential of primed and naïve hPSCs by using EB formation assay. (B) Quantitative gene expression analysis for trophoblast marker genes ELF5, KRT7, TFAP2C, and GATA3 in H9 primed and naïve hPSCs and EBs generated from the respective hPSCs. Error bars indicate ±1 SD of technical replicates. ‘*' indicates a p-value<0.05, ‘**' indicates a p-value<0.01, and ‘***' indicates a p-value<0.001. (C) The experimental scheme for assessing BMP4-mediated trophoblast differentiation potential of naive hESCs (top). Phase contrast image of H9 naïve hESCs and H9 naïve hESCs following the 6 day step I protocol for BMP4-mediated CTB differentiation (bottom). The scale bars indicate 75 µm. (D) The experimental scheme of re-priming naïve hESCs and assessing their BMP4-mediated trophoblast differentiation potential (top). Phase contrast images of H9 re-primed hESCs and H9 re-primed hESCs following the 6 day step I protocol for BMP4-mediated CTB differentiation (bottom). The scale bars indicate 75 µm. (E) Immunofluorescence staining for CDX2, ZO1, and KRT7 in CTBs generated from H9 primed and re-primed hESCs. The scale bars indicate 75 µm. (F) The experimental scheme of capacitating naïve hESCs and assessing their BMP4-mediated trophoblast differentiation potential (top). Phase contrast images of H9 capacitated hESCs and H9 capacitated hESCs following the 6 day step I protocol for BMP4-mediated CTB differentiation (bottom). The scale bars indicate 75 µm. (G) Quantitative gene expression analysis for trophoblast marker genes GATA3, CDX2, KRT7, MMP2, hCGB, and SDC1 in H9 primed and capacitated hESCs and trophoblasts differentiated from the respective hPSCs. Error bars indicate ± 1 SD of technical replicates. ‘***' indicates a p-value<0.001.

Fig 3: eSTBs are highly susceptible to SARS-CoV-2 infection among trophoblasts(A) Schematic diagram of SARS-CoV-2 infection of in vitro trophoblasts.(B) qRT-PCR detection of SARS-CoV-2 genome copy numbers (per mL) in the supernatants of virus-co-cultured cells at different time points. Data are mean ± SD; n = 3 biological replicates. Student’s t test, ***p < 0.001.(C) Immunofluorescence image of 24 hpi EPSC-TSCs stained for SARS-CoV-2 N protein. Scale bar, 100 µm. Right: percentages of N-protein-positive cells. Data are mean ± SEM; n = 36, quantification of 36 random images.(D) Immunofluorescence detection of KRT7, ACE2, and N protein in the infected EPSC-TSCs. Scale bars, 50 µm.(E) Representative immunofluorescence images of N protein and ENPEP at 24 hpi in EPSC-TSCs. Scale bar, 50 µm.(F) Bar plot for expression (cpm) of ACE2 and TMPRSS2 in hEPSCs, EPSC-TSCs, and STBs and EVTs differentiated from EPSC-TSCs at indicated time points.(G) Detection of ACE2 protein in EPSC-TSCs and during their differentiation toward STBs.(H) qRT-PCR detection of SARS-CoV-2 genome copy numbers (per mL) in the supernatants of virus-co-cultured EPSC-TSCs and (day 4) STBs. Data are mean ± SD; n = 3. Student’s t test, ***p < 0.001.(I) qRT-PCR analysis of SARS-CoV-2 genome copy number in cell lysates of 48 hpi EPSC-TSCs and STB-D4. Data are mean ± SD; n = 3. Student’s t test, ***p < 0.001.(J) Immunofluorescence staining of 48 hpi STB-D6 for ACE2, CGB, and N protein and for early STB markers CD46 and SSEA4. Scale bars, 50 or 100 µm as specified.(K) Immunofluorescence staining of 48 hpi STB-D6 for ACE2, CGB, and N protein, and percentages of mono-, bi-, and multinucleated CGB+ cells in the infected STB-D6. Arrowheads indicate that some CGB-low or mono- or binucleated cells were infected by SARS-CoV-2. DAPI stains the nucleus. Scale bars, 100 µm.(L) qRT-PCR detection of SARS-CoV-2 genome copy number (per mL) in the supernatants of virus-co-cultured EPSC-TSCs and eSTBs (STB-D2). Data are mean ± SD; n = 3. Student’s t test, ***p < 0.001.(M) Representative immunofluorescence images of 24 hpi EPSC-TSCs and eSTBs (STB-D2) stained for N protein. Scale bars, 200 µm.(N) qRT-PCR detection of SARS-CoV-2 gene expression in cell lysates of the infected cells at 48 hpi. Data are mean ± SD; n = 3. Student’s t test, ***p < 0.001.(O) Plaque formation assay to detect SARS-CoV-2 virus in supernatants from the infected eSTBs (48 hpi). Data are mean ± SD; n = 3 three independent experiments; ***p < 0.001 (Student’s t test). PFUs, plaque-forming units.(P) Bubble plot for RNA-seq analysis of SARS-CoV-2 genes (E, M, N, S) in the cell lysates of 48 hpi eSTBs.(Q) Representative immunofluorescence images of 24 hpi eSTBs stained for ACE2 and N protein and for STB markers CD46 and CGB. The dotted area shows an uninfected multinucleated CGB+ cell. Arrows indicate an infected binucleated STB. Scale bars, 100 µm.(R) Quantification of the percentages of eSTB marker CD46 in 24 hpi eSTBs. Data are mean ± SEM.(S) Quantification of the percentages of mono-, bi-, and multinucleated cells in 24 hpi eSTBs. SARS-CoV-2 preferentially infects mononucleated cells.

Fig 4: Directed differentiation of EVTs and STBs from naïve hPSC-derived hTSCs.(A) The experimental scheme for EVT differentiation from naïve hPSC-derived hTSCs. (B) Phase contrast image of H9 and AN_1.1 hTSC-like and EVT-like cells. The scale bars indicate 75 µm. (C) Flow cytometry analysis for EVT marker HLA-G in H9 and AN_1.1 hTSC-like cells and EVT-like cells. (D) Levels of MMP2 secreted by H9 and AN_1.1 hTSC-like cells and EVT-like cells as measured by ELISA. Error bars indicate ±1 SD of technical replicates. (E) Quantitative gene expression analysis for EVT marker gene MMP2 in H9 and AN_1.1 hTSC-like cells and EVT-like cells. Error bars indicate ± 1 SD of technical replicates. ‘***' indicates a p-value<0.001. (F) Matrigel invasion assay of H9 and AN_1.1 hTSC-like cells and EVT-like cells. (G) The experimental scheme for STB differentiation from naïve hPSC-derived hTSCs. (H) Phase contrast image of H9 and AN_1.1 hTSC-like and 3D STB-like cells. The scale bars indicate 75 µm. (I) Quantitative gene expression analysis for STB marker genes CGB and SDC1 in H9 and AN_1.1 hTSC-like cells and 3D STB-like cells. Error bars indicate ±1 SD of technical replicates. ‘*' indicates a p-value<0.05, and ‘**' indicates a p-value<0.01. (J) Immunofluorescence staining for STB markers hCG and SDC1 in H9 3D STB-like cells. The scale bars indicate 75 µm. (K) Immunofluorescence staining for STB markers hCG and SDC1 in AN_1.1 3D STB-like cells. The scale bars indicate 75 µm. (L) Quantitative gene expression analysis for TSC marker gene TEAD4 in H9 and AN_1.1 hTSC-like cells and 3D STB-like cells. Error bars indicate ± 1 SD of technical replicates. ‘***' indicates a p-value<0.001.

Fig 5: Generation of human trophoblast stem cells (TSCs) and trophoblast subtypes from hEPSCs(A) Schematic diagram of sequential generation of hTSCs, STBs, and EVTs from hEPSCs.(B) Bright-field images of hEPSCs and EPSC-TSCs. Scale bars: 100 µm.(C) Immunofluorescence-stained EPSC-TSCs and human blastocyst-derived hTSCs (BST-TSC).35 Scale bars, 100 µm.(D) Left: RNA-seq analysis of human trophoblast and putative AME signature gene (Table S1) expression in EPSC- and BST/CT-TSC and five human placenta-derived CT-TSCs.36 Right: Scatterplot for gene set scores for human trophoblast and putative AME signature genes (Table S1) in hTSCs of various origins.(E) PCA for comparison of hTSCs of in vivo (BST/CT) and in vitro origins (naive, primed, and EPSC). Datasets include in vitro differentiation of naive hPSCs to TSCs (D0–D5), primed hPSCs to AME-L (late)-like cells (D0–D3), and partially primed hPSCs to AME-E (early)-like (D0–D5).37 The dashed lines and arrows indicate the differentiation trajectories.(F) Bright-field images of EPSC-TSCs differentiating toward STBs (STB-D6). Scale bars, 100 µm.(G) Gene expression (qRT-PCR) during EPSC-TSC differentiation toward STBs. Data are mean ± SD; n = 3 biological replicates. Student’s t test, *p < 0.05, **p < 0.01.(H) Immunostaining of STBs-D6 for GCM1 and CGB (top) and GATA3 (bottom). Arrows indicate the lack of GATA3 in a multinucleated CGB+ mature STB. Scale bars, 50 or 100 µm as indicated.(I) Bright-field images of EPSC-TSCs differentiating toward EVT-D8. Scale bars, 100 µm.(J) Immunostaining of EVT-D8 for KRT7 and HLA-G. Scale bars, 50 or 100 µm as indicated.(K) Gene expression (qRT-PCR) during EPSC-TSC differentiation toward EVTs. Data are mean ± SD; n = 3 biological replicates. Student’s t test, ***p < 0.001.(L) Flow cytometric analysis of HLA-A, -B, -C and HLA-G on EVT-D8. EPSC-TSCs were used as the control.(M) Invasiveness of EVT-D8 in the transwell assay. The invading/migrating cells on the lower surface were stained with crystal violet. Scale bar, 100 µm.