Fig. 7: Characterization of hESCs derived from split embryos.

a Representative bright-field developmental progression of human split embryos in vitro. Similar results were obtained in no less than three independently repeated experiments. Scale bar, 100 µm. b Representative IF images of identical split embryos at d.p.f.6 stained for CDX2, OCT4, NANOG, and DAPI (split embryos, n = 5; controls, n = 3). Scale bar, 50 µm (long) and 100 µm (short). c The number of ICM cells compared across split embryos and controls at d.p.f.6, with each dot representing one blastocyst (split embryos, n = 5; controls, n = 3). Data from 3 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. d, e The percentage of OCT4⁺ and NANOG⁺ cells out of the total number of cells (DAPI) (split embryos, n = 5; controls, n = 3). Data from 3 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. f Representative images showing the derivation process of hESCs from split blastocyst. Similar results were obtained in no less than three independently repeated experiments. g Representative IF images of hESCs were stained for pluripotency markers OCT4, SOX2, and NANOG. Similar results were obtained in no less than three independently repeated experiments. Scale bar, 100 µm. h Heatmap showing the expression of pluripotency genes in hESCs and other human PSCs from previous works. The following datasets were used: human conventional ESCs (H9_hESC1, H9_hESC1, H9_hESC3, Chan_hESC1, Chan_hESC2, and Chan_hESC3)²⁸, human naïve ESCs (WIBR2, WIBR3_cl_12, WIBR3_cl_16, WIN1_1 and WIN1_2)²⁹, human other PSCs (AIC-h1-M, AIC-h1-M, 4i_hESC1, and 4i_hESC2)^30,31. Brown-marked cells were generated in our lab. i Heatmap of the correlation coefficient showing a high correlation between hESCs derived from split embryos and other hPSCs mentioned above, based on co-expressed genes in all cells. Brown-marked cells were generated in our lab. Source data are provided as a Source Data file.