Analysis of hESC stages of pluripotency. Analysis of hESC stages of pluripotency. (A) MicroRNA analysis associated with pluripotency. (B) XIST labeling shows that Elf1-3iLs (Left) do not display a XIST cloud by FISH, whereas Elf1s primed have two XIST signals and cells differentiated for 10 d (Right) gain a single XIST signal (red dot) within the nucleus. When the nucleus is highlighted using DAPI and the field magnified XIST remains undetectable in naïve Elf1 (Lower Left), whereas the XIST signal can be detected upon differentiation on one or both X chromosomes (red dots, white arrows, Lower Right two panels). (C) Bisulfite sequencing of the XIST promoter using the 11L-11R primers shows that XIST remains methylated throughout the naïve and primed stages. Using the 7L-7R primers, methylation seems to diminish in naïve relative to primed cells, 10-d in vitro-differentiated cells and in the 98-d teratoma. Note that the 1, 9, and 11 primer sets did not vary from data shown for set 11 upon differentiation. Open circles indicate unmethylated and filled circles indicate methylated CpGs. (D) Graphs representing cloning efficiency (percent) and doubling times (hours) of Elf1 naive (green), Elf1 primed (yellow), H1 naïve (dark blue), and H1 primed (light blue). (E) Electron microscopy of mitochondria. The left panels show the mitochondrial shape difference between Elf1-3iL and Elf1-AF. This is quantified in the graph on the right, where increased ratio indicates a rounder mitochondrial population (± SEM). ***P < 0.001, **P < 0.01, and *P < 0.05 as determined by t test. Carol B. Ware et al. PNAS 2014;111:12:4484-4489 ©2014 by National Academy of Sciences