Extended Data Fig. 7 |. Genetic inactivation of P65 and MYD88 in human PSCs overcome human–mouse primed PSC competition.

a, Sanger sequencing results showing out-of-frame homozygous 1-bp insertion in two independent P65^KO hiPS cell clones: 1A3 and 1B1. Bold, PAM sequence. b, Western blot analysis confirmed the lack of P65 protein expression in several independent P65^KO hiPS cell clones. GAPDH was used as a loading control. c, P65^KO hiPS cells (clone 1A3) maintained normal karyotype after long-term passaging (passage 10). d, Representative brightfield and immunofluorescence images showing long-term F/R1-cultured P65^KO hiPS cells maintained stable colony morphology and expressed core (SOX2, green; OCT4, red) and primed (CD24, green) pluripotency markers. Blue, DAPI. Scale bars, 200 μm. e, Representative haematoxylin and eosin staining images of a teratoma generated by P65^KO hiPS cells (clone 1A3) showing lineage differentiation towards three germ layers. Scale bar, 200 μm. f, Representative fluorescence images of day-5 co-cultured and separately cultured P65^KO hiPS cells (green, clone 1A3) and mEpiSCs (red). Scale bar, 400 μm. g, Growth curves of co-cultured (blue) and separately cultured (red) P65^KO hiPS cells (clone 1B1) and mEpiSCs. n = 3, biological replicates. Data are mean ± s.e.m. h, Representative fluorescence images of day-5 co-cultured and separately cultured P65^KO hiPS cells (clone 1B1, green) and mEpiSCs (red). Scale bar, 400 μm. i, Sanger sequencing result showing out-of-frame homozygous 13-bp deletion in MYD88^KO hiPS cells. Bold, PAM sequence. j, Western blot analysis confirmed the lack of MYD88 protein expression in MYD88^KO hiPS cells. k, Representative brightfield and immunofluorescence images showing longterm F/R1-cultured MYD88^KO hiPS cells maintained stable colony morphology and expressed core (SOX2, green; OCT4, red) and primed (CD24, green) pluripotency markers. Blue, DAPI. Scale bars, 200 μm. l, Representative haematoxylin and eosin staining images of a teratoma generated by MYD88^KO hiPS cells showing lineage differentiation towards three germ layers. Scale bar, 200 μm. m, Representative fluorescence images of day-5 co-cultured and separately cultured MYD88^KO hiPS cells (green) and mEpiSCs (red). Scale bar, 400 μm. n, Western blot analyses of IKBA, P65, phospho-P65 (s468), P53 protein expression levels in co-cultured and separately cultured wild-type and mutant (P65^KO, TP53^KO and MYD88^KO) HFF-hiPS cells. Vinculin was used as a loading control. Boxed areas were from separate blots. o, Bar graphs showing the fold changes of protein expression levels (shown in n) in co-cultured versus separately cultured wild-type and mutant HFF-hiPS cells. n = 1, biological replicate. Experiments in b, j and n were repeated independently three times with similar results. For gel source data, see Supplementary Fig. 1. Images in d–f, h and k–m are representative of three independent experiments.