Extended Data Fig. 18: Extraembryonic differentiation potency of primed PSC converted by 4CL and e4CL and sorted 8CLC.
From: Rolling back human pluripotent stem cells to an eight-cell embryo-like stage
a. Representative phase contrast images showing TSC derived from sorted 8CLC in e4CL-day 5, stepwise e4CL-day 5 cells or 4CL-day 12 naïve PSC. H9 ESC were used for these experiments. Scale, 40 μm. D, day. Representative of four independent experiments. b. Representative immunostaining images for GATA3 (green) and KRT7 (red) in TSC derived from primed ESC converted by 4CL (day 12) or stepwise e4CL (day 5). Nuclei were counterstained with DAPI. Scale, 50 μm. D, day. Representative of three independent experiments. c. RT-qPCR for the indicated genes in TSC derived from primed ESC converted by 4CL (day 12) or stepwise e4CL (day 5). Data are presented as mean values ± SD of fold-change compared to PSC in 4CL or stepwise e4CL. n = 3 biological replicates. d. DNA methylation plots for the ELF5 promoter in primed ESC untreated or converted by 4CL (day 12) and 4CL-day 12 naïve ESC-derived TSC. Percentages are the proportion of methylated (closed circles) to non-methylated (open circles) CpG sites. A representative experiment is shown. e. Hierarchical clustering of the bulk RNA-seq of primed ESC converted by 4CL (day 12), 4CL-day 12 naïve ESC-derived TSC and a primary human TSC dataset54. All reference datasets used in this study are summarized in Supplementary Table 8. Our dataset was generated using H9 ESC. f. Representative immunostaining images for CGB and SDC1 in SCT differentiated from 4CL-day 12 naïve PSC-derived TSC. Nuclei were counterstained with DAPI. Scale, 50 μm. Representative of three independent experiments. g. ELISA assay detecting the concentration of hCG secreted from 4CL-day 12 naïve ESC-derived TSC and SCT differentiated from 4CL-day 12 naïve ESC-derived TSC. Representative of two independent experiments.
