Extended Data Fig. 4: Human–mouse primed PSC competition depends on cell–cell contact.

a, Growth curves of H9-hES cells (left) and mEpiSCs (right) plated at different ratios (mouse:human = 1:1, 1:2, 1:4 and 1:8) in separate and co-cultures. The seeding cell number of H9-hES cells was fixed at 1 × 10⁴ cm⁻², whereas seeding cell numbers of mEpiSCs were adjusted according to different seeding ratios. n = 3, biological replicates. b, Growth curves of H9-hES cells (left) and mEpiSCs (right) plated at high and low densities (high, 1.25 × 10⁴ cm⁻²; and low, 0.625 × 10⁴ cm⁻²; 4:1 ratio) in separate and co-cultures. n = 3, biological replicates. c, Quantification of AC3⁺ cells in day-3 co-cultured and separately cultured H9-hES cells (blue) and mEpiSCs (red), plating ratio of 1:1 (human:mouse), n = 10, randomly selected 318.2 × 318.2 μm² fields examined over three independent experiments. d, Representative fluorescence images of day-5 co-cultured and separately cultured H9-hES cells (green) and mEpiSCs (red) in transwell. Scale bar, 400 μm. Images are representative of three independent experiments. e, Live H9-hES cells (cell numbers per cm²) at day 5 after treatments with different dosages (50%, 33% and 10%) of conditioned medium (CM) collected from co-cultures of H9-hES cells and mEpiSCs (cCM), or separate cultures of mEpiSCs (mCM). n = 3, biological replicates. P values (co-cultures compared with separate cultures), unpaired two-tailed t-test (a, b), or one-way ANOVA with Tukey’s multiple comparison (c). All data are mean ± s.e.m.

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