a, Histogram of surface expression of MHC class I using FACS analysis on the indicated naive, primed and differentiated cell lines. These results show that naive human ES cells and human iPS cells express rudimentary or low levels of MHC class I on their cell surface, whereas primed cells upregulate MHC class I expression to intermediate levels (in comparison to differentiated 293 cells that express high levels of MHC class I). These results are identical to patterns observed in murine naive and primed cells7. b, Representative confocal immunostaining images for the expression of E-CADHERIN and OCT4 on genetically matched naive and primed human ES cells. The samples were processed simultaneously and analysed under identical conditions. Insets represent enlargements of boxed areas. Whereas E-CADHERIN is expressed in primed human ES cells, its expression becomes homogenously distributed and more enhanced in naive human ES cells expanded in NHSM conditions. c, Box and whisker plots of nuclear/cytoplasmic TFE3 ratios in naive and primed mouse and human ES cells are shown. Quantitative unbiased imaging analysis for preferential nuclear localization was conducted on randomly selected 200 cells from independent image frames per sample. Naive human ES cells showed distributions similar to those in naive mouse ES cells, and the nuclear enrichment was lost in primed human and mouse ES cells. *t-test P values < 1 × 10−100. d, Representative immunostaining for OCT4 and DNMT3B in human primed and naive pluripotent cells, showing DNMT3B down regulation in the human naive ground state of pluripotency. This effect was highly dependent on efficient p38 inhibition (data not shown). e, Representative western blot analysis for total and phosphorylated levels of the indicated proteins in genetically matched WIBR3 naive and primed human pluripotent cells (passage 25). The results indicate blocked and reduced activity for ERK1/2, p38 and JNK in naive pluripotency. Consistent with the presence of LIF in NHSM, phosphorylated STAT3 levels accumulate in naive human ES cells. Samples were loaded in triplicates for demonstrating consistency. Similar results were obtained when analysing naive and primed C1 human iPS cells and WIS2 human ES cells (data not shown). SB202190 was used in NHSM conditions when p38 MAPK phosphorylation was tested. f, Naive WIS1 human ES cells expanded in NHSM conditions were expanded after omitting the indicated factors from NHSM conditions for 72 h. qRT–PCR of lineage commitment genes is shown. The results indicate that TGF-β1 blocks the SOX1 inductive effect induced by the presence of ERKi in the medium. Error bars indicate s.d. (n = 3). *t-test P value < 0.05.