Fig. 1: Construction and in vitro development of split embryos.

a Schematic diagram of the generation of aESCs and comparative analysis of genetically matched aESCs, iPSCs, and ntESCs. b, c Representative bright-field analysis of in vitro developmental progression showing that compaction and blastocyst cavity formation in split embryos occurs synchronously with that of control embryos. The 2/4th embryo refers to an embryo reconstructed from any two blastomeres of a 4-cell stage embryo. Similarly, the 5/8th and 3/8th embryos denote embryos reconstructed from any five blastomeres and the remaining three blastomeres of an 8-cell stage embryo, respectively. Similar results were obtained in no less than three independently repeated experiments. Scale bar, 100 µm. d Representative bright-field images of in vitro-split embryos developing from d.p.f.7 to d.p.f.10. Similar results were obtained in no less than three independently repeated experiments. e, f Identical split embryos and control embryos at d.p.f.8 were stained for lineage-specific markers (OCT4, CDX2, and GATA6). Yellow arrowheads represent Hypo cells (GATA6-positive, OCT4-negative, CDX2-negative). Scale bar, 100 µm. The scale bar in the magnified image represents 50 µm. g Percentage of 2/4th embryos that developed into blastocysts with a well-defined cavity at d.p.f.6 (cynomolgus monkey 2/4th embryos, n = 176; identical pairs, n = 56; controls, n = 169). Mean ± SEM presented for n  =  11 independent experiments. Unpaired two-tailed Student’s t-test. h Percentage of 3/8th and 5/8th embryos that developed into blastocysts. (cynomolgus monkey 3/8th and 5/8th embryos, n = 92; identical pairs, n = 40; controls, n = 53). The data for the split embryos and controls represent the mean of 5 and 3 independent experiments, respectively. Results are shown as mean ± SEM. The statistical analysis utilized the unpaired two-tailed Student’s t-test. i Comparison of OCT4⁺ cell numbers between genetically matched 2/4th embryos and control embryos at d.p.f.8. (2/4th embryos, n = 18, 9 pairs; controls, n = 13). Data from three independent experiments. Paired two-tailed Student’s t-test without adjustment. j Comparison of OCT4⁺ cell numbers between genetically matched 3/8th embryos and 5/8th embryos at d.p.f.8. (3/8th embryos and 5/8th embryos, n = 18, 9 pairs; controls, n = 13). Data from three independent experiments. The statistical analysis utilized the paired two-tailed Student’s t-test without adjustment. k Comparison of OCT4⁺ cell numbers among 2/4th embryos, 3/8th embryos, 5/8th embryos, and controls at d.p.f.8. Each dot represents one embryo for (i–k). (cynomolgus monkey 2/4th embryos, n = 18, 9 pairs; 3/8th embryos and 5/8th embryos, n = 18, 9 pairs; controls, n = 13). Data from three independent experiments are shown as mean ± SEM. The upper and lower edges of the box represent the maxima and minima, respectively, while the thick lines in the middle indicate the mean of each sample. The statistical analysis utilized the unpaired two-tailed Student’s t-test without adjustment. Source data are provided as a Source Data file.