a, Representative images of monkey blastoids derived from different passage 4CL naive ESCs on D3 and D7 in aggrewell. Scale bars, 100 μm. b, Monkey blastoid formation efficiency at indicated passages of monkey 4CL naïve ESCs (P4-P7, n = 13; P8-P12, n = 5 from 3 independent experiments). Each dot represents a sample. Two-sided unpaired t-test, *** = P ≤ 0.001 (P = 5.12E-10). c, Relative expression levels of naive marker genes in different passage 4CL naive ESCs (n = 3 biologically independent samples). Each dot represents one repeat. d, Representative brightfield image of monkey 4CL naive ESCs at different passages. Scale bars, 100 μm. e, Cell proliferation rate analysis of 4CL naive ESCs at different passages. P4-P7: n = 12 and P8-P12: n = 8 from 3 independent experiments. Each dot represents a sample. Two-sided unpaired t-test, ** = P ≤ 0.01 (P = 0.001467). f, Representative images of monkey embryoids from D0 to D9 in aggrewell. Scale bars, 100 μm. g, Representative images of IF staining of monkey blastoid on D7 with OCT4 (Green) for epiblast, GATA4 (Red) for hypoblast and GATA3 (Cyan) for trophoblast. Scale bars, 100 μm. h, Representative images of IF staining of monkey pre-gastruloid on D9 with OCT4 (Red) for epiblast, SOX17 (Cyan) for hypoblast and GATA3 (Green) for trophoblast. Scale bars, 100 μm. i, Representative images of monkey embryoids in each group during in vitro culture from D9 to D17. The blue arrow represents group of succeeded developed monkey embryoids with embryonic disc and properly formed amnion/yolk sac cavity; the green arrow represents group of failed monkey embryoids with embryonic disc but no amnion/yolk sac cavities; the red arrow represents another group of failed monkey embryoids without embryonic disc. Scale bars, 200 μm. j, Statistical analysis of formation efficiency of each group in i (n = 8 biologically independent experiments for D9; n = 10 biologically independent experiments for D11, n = 8 biologically independent experiments for D13; n = 11 biologically independent experiments for D17). k, Developmental efficiency of monkey embryoids from D9 to D17 (n = 8 for D9; n = 6 for D11; n = 11 for D13; n = 11 for D17). Optimal formed monkey embryoids with embryonic disc under standard range (Diameter <0.1 mm for D9; 0.1–0.2 mm for D11; 0.15–0.5 mm for D13; 0.15–0.5 mm for D17) were calculated for statistical analysis. Each dot represents one independent experiment. l, Measurement of long diameter of whole embryo and embryonic disc of monkey embryoids from D9 to D17 respectively (n = 35 for each group from 5 independent experiments). m, Measurement of embryonic disc diameter of natural human embryos from CS6 (n = 7) and CS7 (n = 12) (Data from Carnegie stage, O’Rahilly et al. 1987) and monkey embryoids from D13 and D17, respectively (n = 35 for each group from 5 independent experiments). Each dot represents a sample. Two-sided unpaired t-test was used for D13 versus CS6-H and D17 versus CS7-H groups respectively, ns = P > 0.05 (P = 0.8599 for D13 versus CS6-H group; P = 0.0900 for D17 versus CS7-H group). n, Ratio of length of PS to length of embryonic disc between groups of monkey embryoids at different stages, natural monkey embryos at E16-18 and natural human embryos at CS7 stage. N = 5 for D13; n = 8 for D15 and D17 groups from 3 independent experiments; n = 4 for E16-18 group; n = 12 for CS7 group. Each dot represents a sample. One-way ANOVA with Dunnett’s T3 multiple comparisons test, ns = P > 0.05; * = P ≤ 0.05. D13 versus D15: P = 0.0203; D15 versus D17: P = 0.0256; D17 versus E16-18-M: P > 0.9999; D17 versus CS7-H: P = 0.8756; E16-18-M versus CS7-H: P = 0.8321. b-d, j-n, Data are presented as mean ± SEM.
Source data