Extended Data Fig. 1: In vitro formation of blastoids.

a, Non-adherent hydrogel microwell arrays were formed by replica molding using polydimethylsiloxane (PDMS) stamps as previously described^7,8,40. The array used for 12-well plates contains 1,000 cylindrical structures of 200 µm diameter and height. Upon cell seeding, owing to the non-adherent properties of the hydrogel, all cells slide into the microwells. Upon settling, the number of ES cells per microwell follows a distribution. Mean number per microwell: 5.2; half of the microwells contained between 4 and 6 cells per microwell (right, top). Upon aggregation of ES cells (24–36 h depending on the cell line), TS cells were seeded. Mean number per microwell: 12; half of the microwells contained between 10 and 14 cells (right, bottom; time point 0 h). b, After culture in blank medium (see Methods), TS and ES cells mostly arranged into non-organized structures forming trophoblasts cysts. TS cells cultured without ES cells (65 h) formed fewer cystic structures than a co-culture. Rarely, TS cells enclosed ES cells and formed regular cystic structures morphologically similar to the blastocyst (white arrow). n = 4 independent microwell arrays. Structures shown in b were taken out of the microwell array at 65 h. Scale bars, 100 µm. c, d, Optimization of the engulfment of ES cells by TS cells. c, Dosing the number of TS cells. ES cells were seeded at t = −24/36 h. At t = 0 h, different numbers of TS cells were seeded on top of the ES cell aggregates. After 24 h, the engulfment efficiency was defined by measuring the percentage of coverage of TS cells around ES cells. The most efficient yields were observed when more than 12 TS cells were added to ES cell cultures. n = 250 independent microwells. The centre value is the mean, error bars are s.d. d, Left, optimization of the concentration of Y27632. At t = 0 h, different concentrations of Y27632 were added. At t = 24 h, the engulfment efficiency was measured as the percentage of coverage of TS cells around ES cells. The most efficient yields were obtained with 20 µM Y27632. n = 250 independent microwells. The centre value is the mean. Right, optimized engulfment. Images of ES cells (red) engulfed by TS cells (green) using the optimized conditions (mean of 12 TS cells per microwell and 20 µM Y27632, 80% engulfment efficiency). Images are representative of three independent experiments. Scale bar, 100 µm. Error bars are s.d. e, Blastocoel area of blastocysts formed from E2.5 CBA × C57BL/6 morula, selected for initiation of compaction and cultured in M16 medium for 24 h along with antagonists of WNT (XAV939, 15 µM), PKA (H89, 10 µM) or DMSO (1:1,000, control). n = 10 independent blastocysts. P = 0.015 and P = 0.002, two-sided Student’s t-test. The centre values are medians. Errors bars are s.d. f, RNA-sequencing data for Wnt6 and Wnt7b, in E3.25 and E3.5 blastocysts, TS cells (TSC) and ES cells (ESC). TS cells were cultured in serum-free (TX) or serum-rich (TS) medium (see Methods). Differentiation was induced by the removal of growth factors. g, TCF luciferase assay for WNT activity in TS cells (see Methods). WNT secretion was blocked using IWP2 (2.5 µM). n = 4 independent biological samples of TS cell culture. *P = 0.045, **P = 0.0057, two-sided Student’s t-test. The centre values are medians. Errors bars are s.d. h, Induction of cavitation. Blastoids were defined as described in the Methods. ES and TS cells were seeded in serum-free TX medium including Y27632 (20 µM) and a WNT modulator. WNT3A-conditioned medium (50% of the total volume), CHIR99021 (3 µM), or the combination of CHIR99021 (3 µM) and XAV939 (15 µM) was added at the time of TS cells seeding (t = 0 h). n = 3 independent microwell arrays. *P = 0.017, two-sided Student’s t-test. The centre values are medians. Errors bars are s.d. i, Yield of blastoids depending on the initial ratio of TS to ES cells, at t = 0 h, within individual microwells (left). Yield of blastoids formed using three lines of TS cells representative of the scope of efficiency observed (right). Different lines were isolated upon CBA × C57BL/6 matings (F_1-1, F_1-2, derived by N.R.) and ICR × ICR matings (F₄, provided by J. Rossant). n = 3 independent microwell arrays. The centre values are the mean. Error bars represent s.d. The red line represents the median of the three cell lines (5% of the total number of microwell per array). j, Bright-field images of a representative E3.5 blastocyst (top) and a blastoid (bottom). Scale bar, 50 µm.