Extended Data Fig. 4: Extended analysis of scRNA-seq (droplet-based) analysis of human 8CLC generated from PSC.
From: Rolling back human pluripotent stem cells to an eight-cell embryo-like stage
a. UMAP visualization of stepwise e4CL-day 5 cells shows seven clusters. The encircled cluster 5 (8CLC) comprises 11.9% of the whole population. b. Violin plot showing the log normalized expression of representative pluripotency and totipotency genes for each of the seven clusters (n = 111, 109, 99, 97, 92, 70 and 12 cells for clusters 0, 1, 2, 3, 4, 5 and 6, respectively) of panel a. c. Bubble plot representing the frequency of expression and average expression of representative pluripotency and totipotency genes in early human embryonic stages31 and primed ESC untreated or converted by 4CL (days 8 [passage 2] and 12 [passage 3]) and e4CL (day 5 C5 [8CLC] and non-8CLC [all other clusters summed]). D, day. All reference datasets used in this study are summarized in Supplementary Table 8. d. Pseudobulk correlation analysis of the in vitro time course data with the embryo data based on the top 2,000 most variable genes. The average linkage hierarchical clustering of the Pearson correlation is shown. e. Pseudobulk correlation analysis of e4CL-D5 clusters (in Extended Data Fig. 4a) with the embryo data based on top 2,000 most variable genes. The average linkage hierarchical clustering of the Pearson correlation is shown. f. Representative co-immunostaining images of stepwise e4CL-day 5 cells showing mutual exclusivity between SOX2 and TPRX1 (see arrows). Nuclei were counterstained with DAPI. Scale, 20 μm. Representative of three independent experiments. g. UMAP visualization of primed ESC converted by direct e4CL at day 7 shows nine clusters. The encircled cluster 3 (C3) cells resemble 8CLC and comprise 15.6% of the whole population. h. Violin plot showing the log normalized expression of representative pluripotency and totipotency genes for each of the nine clusters (n = 738 cells, 621 cells, 464 cells, 432 cells, 205 cells, 165 cells, 72 cells, 38 cells, and 36 cells for clusters 0, 1, 2, 3, 4, 5, 6, 7, and 8, respectively) of panel g. i. Violin plot showing the log normalized expression pattern of representative early embryo-enriched TE in early human embryonic stages and primed ESC untreated or converted by 4CL (days 8 and 12) or stepwise e4CL (day 5; 8CLC and non-8CLC)
