Extended Data Figure 4 : Characterization of rsEpiSCs.

From: An alternative pluripotent state confers interspecies chimaeric competency

Extended Data Figure 4

a, Quantitative PCR analysis of expression of pluripotent, naive and primed PSC markers in mouse ESCs, EpiSCs and rsEpiSCs. Error bars indicate s.d. (n = 3, biological replicates); t-test, **P < 0.01, *P < 0.05. b, Expression of OCT4, NANOG, SOX2, DNMT3B and SSEA-1 proteins in mouse rsEpiSCs was analysed by immunofluorescence. Mouse rsEpiSCs also displayed weak alkaline phosphatase activity and showed positive H3K27me3 foci confirming an inactivated X chromosome in female rsEpiSCs. c, Western blot analyses of OCT4, NANOG and SOX2 protein levels in mouse ESCs, EpiSCs and four different lines of rsEpiSCs. β-actin was used as loading control. For NANOG, an additional long-exposure image was shown (without ESC sample loaded). For full scan associated with b, refer to Supplementary Information. d, DNA methylation patterns of Oct4, Dppa5 and Stella promoters in mESCs, EpiSCs and rsEpiSCs. e, Representative bright-field images showing colonies stained by immunohistochemistry for OCT4 expression after being plated at clonal density (500 cells per well), and cultured for 5 days. Y27632 was added at 10 µM. f, Karyotype analysis of mouse rsEpiSCs indicates a normal diploid chromosome content. g, Flow cytometry analysis of OCT4, SOX2 and NANOG expression in mouse ESCs, EpiSCs and rsEpiSCs. h, Cell-cycle profiles of mouse ESCs and rsEpiSCs analysed by flow cytometry. i, Haematoxylin and eosin staining images of teratomas generated by rsEpiSCs show lineage differentiation towards three germ layers. j, Teratomas generated by rsEpiSCs showed tri-lineage differentiation as examined by immunofluorescence analysis using FOXA2 (endoderm), TUJ1 (neuroectoderm) and ASMA (mesoderm) antibodies. k, Teratomas generated by injecting indicated number of cells in testis of NOD/SCID male mice. EpiSCs and three different lines of rsEpiSCs were used for comparison. After one month, mice were euthanized. Teratomas were retrieved and measured in size and weight. (EpiSCs, n = 1, biological replicate, two technical replicates; rsEpiSCs, n = 3, biological replicates, two technical replicates per line; error bars, s.d.) l, Flow cytometry analysis of TUJ1, ASMA and Ep-CAM expression in teratomas generated by EpiSCs and three different lines of rsEpiSCs. m, Bright-field images of isolated non-intact and non-viable E7.25–7.5 mouse embryos before and after in vitro embryo culture.