Fig. 1

Self-assembly of different combinations of distinct stem cells into reconstructed embryoids. a Schematic of the protocol to generate self-assembled embryoids. Single-cell populations of ESCs and TSCs were mixed and transferred into a dish containing reconstructed embryo medium on an orbital and horizontal shaker (1), and embryo-like structures (ETS-embryoids) emerged within 24 h (2) but did not undergo lumenogenesis in further culture (3). Similarly, a combination of ESCs and XENCs produced EXE-embryoids within 24 h (4, 5), and these embryoids formed cavities (6). ETX-embryoids were generated within 36 h by combination of the three types of stem cells (7, 8), and these embryos also formed cavities (9). b, c Immunostaining of EXE-embryoids and EPI-explants after culture of 60 and 24 h: DAPI, blue; Gata4, green; and Oct4, red. n = 45 EXE-embryoids; n = 45 EPI-explants. d, e Immunostaining of ETX-embryoids assembled after culture of 72 h from wild-type XENCs and ESCs, EGFP-TSCs: DAPI, blue; TSC-derived tissues, green; Oct4 and Gata4, red. n = 30 ETX-embryoids. f, g Immunostaining of embryos cultured in vitro for 60 h from the E4.5 blastocyst and E5.5 embryos: Oct4, red; Eomes, green; and DAPI, blue. n = 20 embryos. h Box-plots of expression levels of Pou5f1, Tfap2c and Gata6 in cells from ESC-, TSC-, and XENC- compartments, respectively, of the ETX-embryoids. Each cell type is shown in a separate color. The boxed region represents the middle 50% of expression values, the bar in the boxed region indicates the median values, and the whiskers indicate the maximum and minimum values. Cells with outlying expression values are depicted as dots. A background of Ct = 24 was used to obtain expression levels. Two-tailed Student’s t-test, n = 11, 11, and 11 single cells from ESC-, TSC-, and XENC-compartments, respectively; **P < 0.01, ***P < 0.001. i Mean tissue volumes of embryonic and extra-embryonic structures are similar between ETX-embryoids after 84 h and wild-type E5.75 embryos. Two-tailed Student’s t-test, n = 34 (ETX-embryoids) and 10 (E5.75 embryos); ns, not significant, source data are provided as the Source Data file. Scale bar, 20 µm (b–g). Columns are means ± s.e.m. Experiments were repeated at least three times (b–g)