Fig. 1. Triply inhibited naive PSCs efficiently form human blastocyst-like structures comprising analogues of the three founding lineages.
a, A schematic of the time window of human peri-implantation development modelled by blastoids (days 5–7). M/MC, morula/morula compacted; B, blastocyst. b, One-step protocol of human blastocyst-like structure formation. N2B27, serum-free medium; PALLY, PD0325901 + A83-01 + LPA + hLIF + Y-27632. c, Phase-contrast image of human blastocyst-like structures formed on a non-adherent hydrogel microwell array after 96 h. Each microwell is 200 μm in diameter. Scale bars, 400 μm. d, Phase-contrast images of representative human blastocyst-like structures harvested from microwells. Scale bars, 200 μm (top) and 100 μm (bottom). e, Percentages of microwells including a human blastocyst-like structure for different naive PSC lines cultured in the PALLY condition with optimized LPA concentration compared with a H9 control (Ctrl) deprived of the three inhibitors. The morphometric definition of blastocyst-like structures is provided in Methods. n = 3 microwell arrays; mean ± s.d. f, g, Immunofluorescence of the epiblast (EPI) markers NANOG (yellow) (f) and OCT4 (yellow) (g), the TE markers CDX2 (cyan) (f) and GATA3 (cyan) (g), and the PrE markers SOX17 (magenta) (f) and GATA4 (magenta) (g) in human blastocyst-like structures. Scale bars, 100 μm. h, Absolute number of cells positive for OCT4, GATA3 and GATA4 (left) and ratios of cells belonging to individual lineages represented as percentage of total number of cells (right) in blastocyst-like structures (96 h) based on immunofluorescence. i, Representative immunofluorescence of the tight junction molecule ZO-1 (yellow), the adherence junction molecule CDH1 (magenta) and the apical domain molecule aPKC (cyan) in a representative human blastocyst-like structure. Scale bar, 50 μm.