Extended Data Fig. 5 |. Overcoming human–mouse primed PSC competition by blocking human cell apoptosis.

a, Western blot analysis confirmed the overexpression of BCL-2 in BCL2^OE hiPS cells. GAPDH was used as a loading control. b, Representative brightfield and immunofluorescence images showing long-term cultured BCL2^OE hiPS cells expressed core (SOX2, green; OCT4, red) and primed (CD24, green) pluripotency markers. Blue, DAPI. Scale bars, 200 μm. c, Representative fluorescence images of day-5 co-cultured and separately cultured BCL2^OE hiPS cells (green) and mEpiSCs (red). Scale bar, 400 μm. d, Dot plot showing the RT–qPCR results confirming knockdown of TP53 transcript in TP53^KD hiPS cells. n = 3, biological replicates. e, Representative brightfield and immunofluorescence images showing longterm cultured TP53^KD hiPS cells expressed core (SOX2, green; OCT4, red) and primed (CD24, green) pluripotency markers. Blue, DAPI. Scale bars, 200 μm. f, Growth curves of co-cultured (blue) and separately cultured (red) TP53^KD hiPS cells and mEpiSCs. n = 3, biological replicates. g, Representative fluorescence images of day-5 co-cultured and separately cultured TP53^KD hiPS cells (green) and mEpiSCs (red). Scale bar, 400 μm. h, Sanger sequencing result showing out-of-frame homozygous 65-bp deletion in TP53^KO hiPS cells. Bold, PAM sequence. i, Representative brightfield and immunofluorescence images showing long-term cultured TP53^KO hiPS cells expressed core (SOX2, green; OCT4, red) and primed (CD24, green) pluripotency markers. Blue, DAPI. Scale bars, 200 μm. j, Representative fluorescence images of day-5 co-cultured and separately cultured TP53^KO hiPS cells (green) and mEpiSCs (red). Scale bar, 400 μm. k, Western blot analysis confirmed the lack of TSC1 protein expression and activation of mTOR pathway (S6K phosphorylation, pS6K) in TSC1^KO hiPS cells. GAPDH was used as a loading control. l, Growth curves of co-cultured (blue) and separately cultured (red) TSC1^KO hiPS cells and mEpiSCs. n = 3, biological replicates. m, Representative fluorescence images of day-5 cocultured and separately cultured TSC1^KO hiPS cells (green) and mEpiSCs (red). Scale bar, 400 μm. Experiments in a and k were repeated independently three times with similar results. For gel source data, see Supplementary Fig. 1. Images in b, c, e, g, i, j and m are representative of three independent experiments. P values determined by unpaired two-tailed t-test (d, l).