Extended Data Figure 8: F/R1-based culture supports self-renewal of human ESCs as well as iPSC generation.

a, Expression of pluripotency markers SOX2, NANOG, TRA-1-60, TRA-1-80 and SSEA-4 in human H1 rsESCs. b, Representative bright-field images showing colonies visualized by alkaline phosphatase (AP) staining after being plated at clonal density (1,000 cells per well) and cultured for 6 days. Y27632 was added at 10 µM. c, Real-time quantitative PCR analysis of expression of pluripotency marker genes (OCT4, SOX2, NANOG and LIN28) and lineage marker genes (T, NKX1-2 and WNT3) in H1 ESCs, human-foreskin-fibroblast-derived iPSCs, H1 rsESCs and human-foreskin-fibroblast-derived rs-iPSCs. Error bars indicate s.d. (n = 3, biological replicates). d, Haematoxylin and eosin staining images of teratomas generated from human H1 rsESCs show lineage differentiation towards three germ layers. e, Karyotype analysis of human H9 rsESCs indicates a normal diploid chromosome content. f, Representative bright-field images showing morphologies of putative iPSC colonies in conventional F/A-based human ESC culture and F/R1-based culture conditions (top). Alkaline phosphatase staining at day 25 post-nucleofection indicates a larger colony size in F/R1-based culture (bottom). g, Efficiency of iPSC generation in conventional F/A-based human ESC culture conditions and F/R1-based culture conditions. Error bars indicate s.d. (n = 3, independent experiments). h, Quality of human iPSC-like colonies generated in F/A-based and F/R1-based culture conditions. Partial and full alkaline-phosphatase-positive iPSC-like colonies were counted separately using the criteria shown on the right. Error bars indicate s.d. (n = 3, independent experiments). i, Phase-contrast image showing morphology of human-foreskin-fibroblast-derived rs-iPSCs. j, Graphic representation of H3K4me3 and H3K27me3 ChIP-Seq signals near the transcription start site (TSS) for Polycomb target genes in H1 ESCs and H1 rsESCs. k, Average H3K27me3 signal at Polycomb target genes in H1 rsESCs (purple) and H1 ESCs (green). i, Cell-cycle profiles of H1 ESCs and H1 rsESCs were analysed by flow cytometry. m, Flow cytometry analysis of OCT4, SOX2, NANOG and TRA-1-60 protein expression in H1 ESCs and H1 rsESCs.