Fig. 1: Generation and characterization of human somitoids.

a Somitoid protocol. 350 human iPSCs were aggregated in a U-bottom plate and treated with CHIR99021, bFGF, SB431542, and DMH1. The cocktail was washed out on day 2, and Matrigel was added on day 4. b Time-course images of somitoid development. c Classification of morphologies of day 7 somitoids. N = 210 from 12 independent experiments. Only the images with entire somitoid structures were used. d Somite formation on day 6. Arrows indicate the somite boundaries. e Quantification of the number of somite rows. Mean ± SEM. N = 284 (day 5), 113 (day 6), and 77 (day 7). 3–15 independent experiments. f The images of day 7 somitoids were segmented, and the width perpendicular to the detected posterior-anterior axis was plotted (top). The somite-to-somite distance and the somite width (middle and bottom). Only somitoids with single somites were measured. Boxplots show median, 75th and 25th percentiles, and max and min. N = 9 (somite number 1–3), 8 (somite number 4), and 7 (somite number 5). P-values are from two-sided paired t-test. g HCR images of day 6 somitoids. Asterisks indicate the stripes of TBX18 expression. N = 8 samples showed similar expression patterns. h IHC images of day 6 somitoid that displayed mostly single somites. N = 10 samples showed similar expression patterns. i IHC images of day 7 somitoids that displayed typical paired somites. N = 5 samples showed similar expression patterns. Insets: Enlarged images of the regions indicated by the arrows. j Enlarged images of the boxed regions in h. k Cell morphometry of Phalloidin images in j. Boxplots show median, 75th and 25th percentiles, and max and min except for outliers. N of measured cells = 9 (Area 1), 9 (Area 2), and 9 (Area 3). P-values are from two-sided student’s t-test. l Schematic diagram of a human somitoid. NMPs: Neuromesodermal progenitors, PSM: Presomitic mesoderm. Scale bars: 300 µm (b, c, d, g, h, i) and 50 µm (inset of i, j). Microscopes: Opera (b, c, d) FV3000 confocal (h, j), and Light-sheet (g, i). Source data are provided as a Source Data file.