Fig. 4: Generation and characterization of jawbone-like organoids.

a, Stepwise induction strategy of jawbone-like organoids. b, Image of 1231A3-derived d38 cluster. c, Monitoring ALP activity and calcium content in clusters during osteogenic induction (n = 6 from 3 biologically independent experiments). d, Alizarin Red and von Kossa staining in serial sections of d38 clusters. Dashed boxes magnified on the right (Alizarin Red, n = 12; von Kossa, n = 6 from 3 biologically independent experiments). Scale bars, 100 μm. e, The expression of mature osteocyte marker (SOST) in clusters during osteogenic induction (n = 6 from 3 biologically independent experiments). f, HE and Azan staining in serial sections of d38 clusters (n = 52 from 12 biologically independent experiments). Scale bars, 100 μm. g, Triple-colour immunofluorescence staining for COLI, OCN and OPN in d38 tissues. Nuclei stained with DAPI (n = 9 from 3 biologically independent experiments). Scale bars, 100 μm. h–k, Immunofluorescence staining for SP7 and ALPL (h), PDPN (i), PHEX (j) and SOST (k) in d38 tissues. F-actin stained with phalloidin. Images represent the z-stack projection of 40 confocal optical sections (step size, 0.5 μm) (i–k) (n = 6 (h), n = 12 (i), n = 6 (j) and n = 6 (k) from 4 biologically independent experiments). Scale bars, 25 μm. l, Schematic model of generated jawbone-like organoids. m, Image of a kidney of NSG mice at 4 weeks after jawbone-like organoid transplantation. n, 3D reconstructed micro-CT image of the renal region at 4 weeks after transplantation. The dashed circle indicates an ectopic mineralized tissue. Scale bar, 1.0 mm (n = 6). o, HE and Azan staining in serial sections of transplanted tissues (n = 6). Scale bars, 100 μm. p, Immunofluorescence staining for human Vimentin in the transplanted tissues. F-actin stained with phalloidin. Images represent the z-stack projection of 40 confocal optical sections (step size, 0.5 μm) (n = 6). Scale bars, 25 μm. c,e, Box limits, upper/lower quartile; centre line, median; whiskers, max/min. P < 0.05 by one-way ANOVA with Tukey’s multiple comparisons test. In b, d, f–k and m–p, representative data are shown. All specimens stained after decalcification (except for d).