Extended Data Fig. 11: Extended analysis of 8CLC chromatin landscape.
From: Rolling back human pluripotent stem cells to an eight-cell embryo-like stage
a. UMAP visualization of gene score for all genes in the scATAC-seq of primed ESC untreated (red) or converted by 4CL (day 12; blue) or stepwise e4CL (day 5; green). b and c. UMAP visualization based on panel a highlighting the gene score for primed (ZIC2), shared naïve pluripotency/8CLC (DPPA3) (b) and totipotency (c) genes projected onto each individual cell in the scATAC-seq of primed ESC untreated or converted by 4CL (day 12) and stepwise e4CL (day 5). d. UMAP visualization based on panel a highlighting the gene score for classical pluripotency genes (OCT4 and SOX2) projected onto each individual cell in the scATAC-seq of primed ESC untreated or converted by 4CL (day 12) and stepwise e4CL (day 5). e and f. UMAP visualization showing the gene score for totipotency (e) and classical pluripotency (f) genes projected onto the individual cells of stepwise e4CL-day 5 of Fig. 2a. g. UMAP visualization showing DNA binding motif deviation scores of the lineage specifiers (GSC, PITX1 and PITX2) and classical pluripotency (SOX2) gene projected onto the UMAP of Fig. 2a. h. Violin plot showing the log normalized expression of the lineage specifiers GATA2, GATA4, GATA6 and PITX2 in human embryonic stages and human ESC passage 1026 (left panel), and in primed ESC untreated or converted by 4CL (day 12) and e4CL (day 5 C5 [8CLC] and nnon-8CLC [all other clusters summed]) (right panel). Data were taken from the droplet-based scRNA-seq time course. All reference datasets used in this study are summarized in Supplementary Table 8. hESC, human ESC; P, passage. i. RT-qPCR showing upregulation of lineage specifiers (GATA6 and PITX2) in stepwise e4CL day 5 cells compared to primed ESC and 4CL-day 12 naïve ESC. H9 ESC were used to generate these data. Data are presented as mean values ± SD. n = 3 biological replicates. D, day. j. Violin plot showing the log normalized expression of indicated lineage specifiers in early mouse embryonic stages23 (left panel) and mouse 2CLC compared to naïve ESC39 (right panel). blast., blastocyst. k. Genome browser tracks showing chromatin accessibility, H3K27ac level42 and transcription factor DNA binding motif location at the naïve pluripotency KLF17 locus, multiple totipotency (TPRX1 and ZNF280A) loci and lineage specifier (GATA6) locus in the indicated cell types.
