Fig 2: Examining the response of naïve and primed hPSCs to conditions for hTSC derivation.(A) The experimental scheme for deriving hTSCs from primed and naïve hPSCs. (B) Phase contrast images of H9 and AN_1.1 hTSC-like cells derived from naïve hPSCs, as well as H9 and AN_1.1 primed hPSCs following culture in hTSC medium. All H9 cells were at passage 8, and all AN_1.1 cells were at passage 10. The scale bars indicate 75 µm. (C) Flow cytometry analysis for TSC markers ITGA6 and EGFR in H9 naïve hPSCs, H9 hTSC-like cells derived from naïve hPSCs, H9 primed hPSCs, and H9 primed hPSCs cultured in hTSC medium. (D) Quantitative gene expression analysis for trophoblast marker genes ELF5, KRT7, GATA3, TFAP2C, TEAD4, and CDX2 in H9 primed and naïve hPSCs, H9 hTSC-like cells derived from naïve hPSCs, and H9 primed hPSCs cultured in hTSC medium. Error bars indicate ±1 SD of technical replicates. ‘***' indicates a p-value<0.001. (E) Immunofluorescence staining for TSC markers KRT7, TEAD4, and TP63 in H9 hTSC-like cells derived from naïve hPSCs. The scale bars indicate 75 µm. (F) Flow cytometry analysis for naïve hPSC markers SUSD2 and CD75 in H9 and AN_1.1 naïve hPSCs and hTSC-like cells derived from naïve hPSCs. (G) Quantitative gene expression analysis for naïve hPSC marker KLF17 in H9 primed and naïve hPSCs, hTSC-like cells derived from naïve hPSCs, and primed hPSCs cultured in hTSC medium. Error bars indicate ±1 SD of technical replicates. ‘***' indicates a p-value<0.001. (H) Quantitative gene expression analysis for the embryonic germ layer markers PAX6, VIM, and SOX17 in H9 primed and naïve hPSCs, hTSC-like cells derived from naïve hPSCs, and primed hPSCs cultured in hTSC medium. Error bars indicate ± 1 SD of technical replicates. ‘*' indicates a p-value<0.05, ‘***' indicates a p-value<0.001, and ‘n.s.’ indicates a p-value>0.05.

Fig 3: The IP-based purification of ITGA6-negative iPSC-TM. The ratio of the ITGA6-negative cells after the IP-based sorting by oscillation at different conditions: (A) at 37 °C every 15 min; (B) at 37 °C every 5 min; (C) at 25 °C every 15 min; and (D) at 25 °C every 5 min. (E). Quantification analysis of the ratios of the ITGA6-positive cells (pink) and ITGA6-negative cells (blue).

Fig 4: Differentiation of iPSCs into TM-resembling cells by the conditioned medium. (A) Schematic illustration of iPSC generation and differentiation by the conditioned medium approach. (B) Representative morphology of iPSCs, differentiated iPSCs for 3, 7, and 25 days, and hTM cells of three donors. Scale bars, 100 µm. (C) IHC staining of ITGA6 (green) and nuclei (blue) shows only partial iPSC-TM expressing ITGA6 (pointed by the arrows). Scale bars, 10 µm.

Fig 5: The magnetic platform to purify ITGA6-positive iPSC-TM. (A) Schematic illustration of the ITGA6-positive iPSC-TM purification process. The unlabeled iPSC-TM cells in the red tube were the negative control. iPSC-TM cells after labeling with FITC-conjugated anti-ITGA6 antibodies and anti-FITC microbeads were collected in a blue tube. ITGA6-negative iPSC-TM after the LS-based purification was collected in a purple tube, while ITGA6-positive iPSC-TM after the LS-based purification was collected in an orange tube. (B) A fluorescence density plot (FL1-H vs. SSC-H) depicting the distribution of the negative control cells (red), pre-first sorting cells (blue), and post-first sorting cells (orange). Each dot indicates an individual cell. (C,D) Histogram analyses of the negative control cells (red), pre-first sorting (blue) cells, and post-first sorting (orange) cells. The ratios of ITGA6-positive cells and ITGA6-negative cells are labeled in each plot. (E) A plot (FSC-H vs. SSC-H) indicating the distributions of the ITGA6-positive cells (blue) and ITGA6-negative cells (pink) in the post-first sorting samples. Each dot represents an individual cell. (F) A representative image showing the morphology of the ITGA6-positive iPSC-TM after one round of LS-based sorting. (G–J) Similar to panels (B–E) showing the flow cytometry results after two rounds of LS-based sorting. (K) An image showing the morphology of ITGA6-positive iPSC-TM after the second round of cell sorting. (L–O) Similar to panels (B–E) showing the flow cytometry results after three rounds of LS-based sorting. (P) An image showing the morphology of ITGA6-positive iPSC-TM after the third round of cell sorting. Scale bars, 50 µm. (Q) Quantification analysis of the ratios of ITGA6-positive cells (pink) and ITGA6-negative cells (blue) based on 3–6 experimental repeats. LS-based purification significantly increased the ratios of the ITGA6-positive cells (pre-first sorting vs. post-first sorting: 0.42 ± 0.042 vs. 0.68 ± 0.02, ** p < 0.01; pre-multiple sorting vs. post-multiple sorting: 0.45 ± 0.11 vs. 0.89 ± 0.05, ** p < 0.01). As the round of sorting increases, the ratio of ITGA6-positive cells also increases (post-first sorting vs. post-multiple sorting: 0.68 ± 0.02 vs. 0.89 ± 0.05, * p < 0.05). p values were analyzed by one-way ANOVA.