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. 2024 Jan 22;15:668. doi: 10.1038/s41467-024-44969-x

Fig. 1. Self-renewing hnESCs form blastoids spontaneously.

Fig. 1

a Representative phase-contrast images of spontaneous cysts (red arrow) in the hnESCs cultures. Scale bars, 50 μm. (n > 10). b Schematic of spontaneous blastoids formation from hnESCs in suspension plates (top) and in AggreWell (bottom) with 5iLAF medium (components are listed). Created in Adobe Illustrator. c Representative phase-contrast images of blastoids at indicated times points in suspension plates (top) and in AggreWells (bottom). Scale bars, 50 μm. (n > 10) d Representative phase-contrast images of day6-blastoids formed spontaneously in suspension plate (top) and AggreWells (bottom). Scale bars, 200 μm. e Quantification of diameter of day6-blastoids (suspension plate, n = 43(number of blastoids measured), 178.5 ± 46.5 μm; AggreWell, n = 47, 216.2 ± 46.1 μm; mean ± s.d.). f Spontaneous blastoid formation efficiency related to starting cell number per AggreWell. n = 2 (technical replicates). g–j Representative immunofluorescence staining images of blastoids formed in suspension plate (g) or in AggreWell (h–j). Scale bars, 20 μm. k Quantification of SOX2 and GATA3 cell number per blastoid. n = 11 blastoids. l and m, blastoid generation efficiency in AggreWells upon removal of single factors of 5iLAF medium. l indicates representative images and m indicates the number of blastoids generated upon removal of indicated factor. n = 2 technical replicates; mean; unpaired two-tailed t-test; **P < 0.01. Scale bars, 200 μm. n Proliferation of hnESCs cultured in 5iLAF minus each indicated factor. Two rounds independent experiments (top and bottom) with parallel control groups cultured in 5iLAF. n = 3 technical replicates; mean ± s.d.; unpaired two-tailed t-test; *P = 0.0119; ***P = 0.0003 (-MEKi); ***P = 0.00000139 (-ROCKi). Source data are provided as a Source Data file.