Fig. 8

1 The impact of 2i culture on pluripotent cells in mice and humans. In panel A, mouse pluripotent colonies were subjected to alkaline phosphatase staining. The two plates on the left show that the addition of 2i to mEpiSC colonies leads to differentiation and a loss of alkaline phosphatase positive cells. However, the four plates on the right demonstrate that when mEpiSCs are grown in butyrate plus SAHA before introducing 2i, pluripotent colonies thrive. In panel B, human embryonic stem cells (H1) were either directed back to 2i culture through butyrate exposure or pushed forward towards differentiation without prior exposure to B/S. This indicates that 2i culture must follow B/S exposure to prevent differentiation. The scale bars in the figure represent a length of 100 μM. 2 The genomic analysis conducted on naïve human embryonic stem cells (hESCs). In panel (A), a heat map displays the RNA expression levels of target genes regulated by HIF2α (EPAS1) in H1-2iF cells compared to the parent H1 cells. The comparison was performed using quadruplicate samples and the cells were cultured in TeSR2. Panel (B) presents a principal component analysis (PCA) plot comparing mouse whole genome Agilent array data. The left side of the plot shows embryo data from Hunter et al. (8), while the right side shows mouse embryonic stem cell (mESC) equivalents, including R1p22 (mESC-2iL, naïve), mEpiSC7p24AF (mEpiSC-AF, primed), and mEpiSC7p55(AF7,B/S1)2iL20 (mEpiSC-2iL, toggled to naïve). The expression data of naïve (3iL, green squares) and primed (AF, blue squares) Elf1 cells are also compared with in vivo mouse embryo data in the plot on the left. In panel (C), a comparison is shown between in-house Elf1 expression array data and data generated by Hanna et al. (5). The comparison includes naïve and primed cell lines, represented by dark blue dots and orange dots, respectively. The lines tested on the left side of the graph are grouped identically on the Elf1 primed side. Panel (D) displays DNase I hypersensitivity analysis of the enhancer regions for the POU5F1 gene in Elf1 and H1 cells. The lower black line represents Elf1, and the blue line above represents H1. The first exon of POU5F1 is shown above the H1 data, along with a 2-kb size bar indicating the proximal enhancer (PE) and distal enhancer (DE). In panel (E), a comparison of ChIP-seq H3K27me3 data is presented. The orange line represents primed hESCs (data from Gafni et al., 6), while the blue line represents naïve Elf1-2iL cells. The comparison focuses on the subset of genes from panel (C) that are associated with Gene Ontology “developmental genes” (n = 648). 3 The analysis of different stages of pluripotency in human embryonic stem cells (hESCs). Panel (A) shows the results of microRNA analysis related to pluripotency. Panel (B) demonstrates the labeling of XIST (X-inactive specific transcript) using a technique called fluorescence in situ hybridization (FISH). In the left image, Elf1-3iLs cells do not show a cloud-like XIST signal, whereas Elf1s primed cells exhibit two XIST signals. Furthermore, cells differentiated for 10 days display a single XIST signal (represented by a red dot) within the nucleus. When the nucleus is highlighted using DAPI staining and the field is magnified, XIST remains undetectable in naïve Elf1 cells (lower left). However, upon differentiation, the XIST signal becomes detectable on one or both X chromosomes (red dots, white arrows, lower right). Panel (C) presents the results of bisulfite sequencing of the XIST promoter using different primer sets. The figure shows that XIST remains methylated throughout the naïve and primed stages. However, using specific primers, methylation appears to decrease in naïve cells compared to primed cells. This decrease is also observed in in vitro-differentiated cells and in a teratoma at day 98. The circles represent CpG sites, where open circles indicate unmethylated and filled circles indicate methylated CpGs. Panel (D) consists of graphs representing the cloning efficiency (percentage) and doubling times (hours) of Elf1 naive, Elf1 primed, H1 naive, and H1 primed cells. Panel (E) displays electron microscopy images of mitochondria. The left panels highlight the difference in mitochondrial shape between Elf1-3iL and Elf1-AF cells. This difference is quantified in the graph on the right, where an increased ratio indicates a rounder population of mitochondria. The error bars represent the standard error of the mean (SEM). 4 The developmental capacity of teratomas derived from different types of cells. Panel A displays sections of teratomas labeled with H&E staining. Specifically, it shows sections from Elf1p17-2iL10 teratoma (naïve; 42 days) and Elf1p15T8 teratoma (primed, 67 days). Panel B focuses on endoderm-specific labeling of the Elf1 teratomas shown in Panel A. The upper two panels of both tumors represent sequential sections. The upper panel highlights liver development using red (albumin), green (α-fetoprotein), and blue (E-cadherin) labeling. The second set of panels highlights pancreatic development using red (PDX1), green (SOX9), and blue (E-cadherin) labeling. The next three panels (descending) in both tumors are different sequential sections. The first set represents liver development, the second set represents pancreatic development, and the third set represents liver development using alternative markers (labeled as above and red, CYP3A and green, HNF4A). The lower panel of Elf1p17-2iL10 (naïve) is included to emphasize the level of organization of endodermal development within these tumors, labeled with red (FOXA2), green (SOX9), and blue (E-cadherin). The bottom right panel (Elf1p15T8) serves as a negative control. Panel C displays H&E sections of an H1 naïve teratoma (44 days). The graph below the H&E sections quantifies the areas stained for E-cadherin (epithelial cells) or PDX1 (pancreatic progenitors) in primed (H1p44-AF9), naïve [H1p49(B/S3)2iF10], and naïve reverted to primed [H1p49(B/S3, 2iF4)AF5] H1 generated teratomas. The graph indicates that both the overall epithelial developmental potential and the pancreatic subset are enhanced in the naïve state compared to the primed state. Panel D shows the top three panels with H&E sections of an mESC teratoma (naïve, 13 days). The lower panels display immunofluorescent labeling of sections from this mESC teratoma. The scale bars in panels A and D define the scale for all H&E-stained sections and are set at 100 μM. Reprinted from [477] with permission from the PNAS