Fig. 5: Generation of 3D trunk organoid model with notochord and ongoing ventral patterning.

a, Notoroid protocol. RA, all-trans retinal. b, Elongated structures with TBXT⁺ cells surrounded by SOX2⁺ epithelial cells. c, Optical sections (at position of dashed line) highlighting TBXT and FOXA2 co-expression. d, Combined 2D embedding of notoroid single-cell data for different days (3, 5 and 7) and initial inhibition delays (18, 24 and 30 h post-induction). The plot highlights cell types present. prog., progenitors. MN, motor neurons. e, Proportion of cell types present in each inhibition delay condition normalized to total proportion across all conditions. f, Left: estimation (GastrUnet) of maximum notoroid length at day 7 per inhibition delay using bright-field images. Right: Ratio of notochord (TBXT) and somitic mesoderm (FOXC2) volume fraction for each trunk organoid generated with 12-, 18-, 24-, 30- and 36-h inhibition delay (n = 15, 20, 46, 19 and 16 samples, respectively, over 2 independent experiments) through quantification of immunofluorescence images. Data are shown as a box plot identical to Fig. 3d overlaid with a plot containing individual observations. g, Immunofluorescence staining showing SHH in TBXT⁺ notochordal cells. Dashed line indicates position of optical section in adjacent image. h, Notoroid cryosection showing ventral neural patterning associated with notochord: floor plate (FOXA2⁺) and motor neuron (OLIG2⁺) progenitors. i, Bright-field image of notoroid with TNE–ZsGreen reporter. j, Heat map showing the maximum TNE-ZsGreen signal along the standardized length of each notoroid (n = 96; see Extended Data Fig. 10c). k, Distributions of maximum length and area proportions of ZsGreen signal in control (Ctrl) and vismodegib (Vismo)-treated notoroids. l, Optical section of TBXT⁺SOX9⁺ notochordal cells showing extensive ECM deposition. Blue arrowhead marks putative lumen, and green arrowheads highlight enlarged cytoplasm. m, Left: 3D view of micro-computed tomography (micro-CT) of notoroid, showing representative transverse (green, m) and longitudinal (blue, n) orientations for EM imaging. Middle: SBF SEM image showing a transverse section of notoroid with neuroepithelium (blue), mesenchymal cells (green) and putative notochord (red). Right: representative SBF SEM image of notochordal cells. n, Left: micro-CT and TNE–ZsGreen overlay of longitudinal notoroid section. Middle: representative SBF SEM image of TNE–ZsGreen⁺ notochord cells. Right: representative SBF SEM image of ECM fibres (f) surrounding the notochord and mitochondria (orange arrowheads) in adjacent neural cells (n). Notochordal cells (in m and n) were closely packed, had abundant cytoplasmic glycogen (magenta asterisks), desmosomes (purple arrowhead in inset in m, and in n middle), lipid droplets (yellow asterisks), endolysosomal structures (blue arrowheads), vacuoles (cyan arrowheads) and covered by basal lamina (yellow arrowheads), rough ER (dark green arrowhead in inset in m) was also observed. o, SEM images of a freeze-fractured notoroid showing lipid droplets (yellow stars) and a sheath of ECM fibres (yellow arrowheads). Images in b,c,h are representative of three independent experiments across two human ES cell lines; images in g,i,j,l are representative of at least two independent experiments. Electron microscopy and micro-CT images in m–o are representative of at least three trunk organoids analysed. Scale bars, 0.5 mm (b(right)), 0.4 mm (i), 100 μm (b(left),m(left),n(left)), 50 μm (c,g(left),h,m(middle),o(left)), 30 μm (g(right)), 10 μm (l,o(middle)), 5 μm (n(middle),n(right)) and 3 μm (m(right),o(right)).