a, Photograph showing microfluidic devices in a six-well plate. Inset shows a top view of the device. Devices were filled with medium containing food colour dyes to illustrate medium reservoirs and microfluidic channels. b, Design of microfluidic device incorporating three parallel channels (80 µm in height) partitioned by trapezoid-shaped supporting posts spaced 80 µm apart. The central channel (gel channel) is preloaded with Geltrex. The other two open channels are used for cell loading (cell-loading channel) and chemical induction (induction channel), respectively. c, Protocol for generating epiblast-like cysts. After cell loading (designated as t = −18 h), a basal medium comprising Essential 6 medium and FGF2 (20 ng ml−1) is supplied to both the cell-loading and induction channels from t = 0 h onwards. d, Schematic showing cell loading, cell clustering and lumenogenesis. After injection of Geltrex into the gel channel, Geltrex gelation and contraction leads to formation of evenly spaced concave gel pockets between supporting posts. After loading human ES cells (hESC) into the cell-loading channel, the microfluidic device is tilted 90° to allow cells to settle into gel pockets (t = −18 h). At t = 0 h, nascent lumenal cavities emerge within cell clusters. At t = 36 h, epiblast-like cysts contain a single central lumenal cavity reminiscent of the pro-amniotic cavity formed in the epiblast of the blastocyst upon implantation. e, Representative bright-field images showing an array of epiblast-like cysts at t = 0 h and 36 h. Experiments were repeated five times with similar results. Scale bar, 80 µm. f, Representative confocal micrographs showing epiblast-like cysts at indicated time points stained for ezrin (top) or E-cadherin (E-cad) and laminin (Lam; bottom). Nuclei were stained with DAPI. Some epiblast-like cysts initiate lumenogenesis with multiple small ezrin+ lumenal cavities, which gradually resolve into a single central lumen, probably through cavity fusion. Scale bars, 40 µm. Experiments were repeated three times with similar results. g, Representative confocal micrographs showing X–Y, X–Z and Y–Z sections of epiblast-like cysts obtained at t = 36 h, stained for E-cadherin and ezrin. Nuclei were stained with DAPI. Scale bars, 40 µm. Experiments were repeated twice with similar results. h, Percentage of epiblast-like cysts with single, multiple or no lumenal cavities at indicated time points. n = 59, 57, 60, 89 and 110 cysts for t = 0 h, 6 h, 12 h, 24 h and 36 h, respectively. Data were pooled from n = 2 (0 h, 6 h, 12 h) or 3 (24 h and 36 h) independent experiments. Data represent the mean ± s.e.m. i, Cell number in each epiblast-like cyst as a function of time. n = 26 cysts for each time point. Data were pooled from n = 2 independent experiments. Red lines represent the median. j, Equivalent epiblast-like cyst diameter as a function of time. n = 56, 45, 50, 115 and 88 cysts for t = 0 h, 6 h, 12 h, 24 h and 36 h, respectively. Data were pooled from n = 3 independent experiments. Red lines represent the median. k, Embedded epiblast-like cyst perimeter percentage as a function of time. n = 33, 35, 26, 28 and 21 cysts for t = 0 h, 6 h, 12 h, 24 h and 36 h, respectively. Data were pooled from n = 2 independent experiments. Red lines represent the median. l, Representative confocal micrographs showing epiblast-like cysts at t = 36 h stained for ZO1 and actin (top) or GM130 and actin (bottom). Nuclei were stained with DAPI. Far right, magnified views of outlined regions. Scale bars, 40 µm. Experiments were repeated twice with similar results. m, Representative confocal micrographs showing epiblast-like cysts obtained at t = 36 h with indicated drugs supplemented into basal medium from t = 0–36 h. IWP2, 5 µM; BMP inhibitor LDN 193189 (LDN), 0.5 µM; TGF-β inhibitor SB 431542 (SB), 10 µM; Caspase 3 inhibitor Z-DEVD-FMK (Cas 3), 10 µM. Cysts were stained for NANOG and E-cadherin. Fluorescently labelled WGA was used to stain plasma membrane. Nuclei were stained with DAPI. Note that assays were also conducted with different concentrations of Z-DEVD-FMK (5 µM, 20 µM and 40 µM), with results compatible with those obtained from 10 µM Z-DEVD-FMK. Scale bars, 40 µm. Experiments were repeated twice with similar results. n, Equivalent epiblast-like cyst diameter at t = 36 h under indicated conditions. n = 142, 37, 44, 40 and 36 cysts for control, IWP2, LDN, SB and Cas 3 conditions, respectively. Data were pooled from n = 2 independent experiments. Red lines represent the median. Source data