Fig. 6 |. Identification of human DDR2⁺ CSCs.

a, H&E (n=14) and Alcian Blue (n=7), b, immunostaining (n=7), c, d, immunofluorescence, human SAG craniosynostosis specimens. e, In vivo serial transplantation of hDDR2⁻ CSCs (left)and hDDR2⁺ CSCs (right), FACS plots showing the self-renewal and differentiation. FACS from initial donor (top), primary transplants (middle), and secondary transplants (bottom). Black and red arrows indicate parent/daughter gates and transplanted populations, respectively. μCT, bone organoids derived from the tertiary transplants. f, Flow cytometry histograms of dye dilution proliferation analysis. g, CFU-F, hDDR2⁻ CSCs, hDDR2⁺ CSCs, and other human calvarial non-stem populations (n≥3 independent experiments). h, Bright-field, initially seeded, primary, secondary, and tertiary mesenspheres (left). Quantification of cell numbers per sphere (right, n≥9). i, Gross image of xenograft bone organoid in the renal capsule of NSG-EGFP host (left). μCT timecourse, hDDR2⁻ CSC or hDDR2⁺ CSC-derived bone organoids (right). j, Endochondral ossification in xenograft bone organoids from hDDR2⁺ CSCs by von Kossa and Safranin O. k, Immunostaining, on xenograft bone organoids derived from hDDR2⁺ CSCs, (human nuclear antigen, HNA) implanted into the renal capsule NSG-EGFP hosts. (i) Confocal image showing the graft stained as indicated. (ii) High magnification of i. (iii) COL2A1⁺ chondrogenic cells (left) and MMP13⁺ apoptotic cells (right) in xenograft organoids derived from hDDR2⁺ CSCs. ****P < 0.0001. Mean ± s.d., one-way (g), two-way (h) ANOVA with Tukey’s posttest. FACS plots (e, f) and images (a-d, g-k) are representatives of a minimum of three independent experiments. Scale bars, 200 μm, a (left); 25 μm, a (middle and right), b; 100 μm, c, d, j, k; 20 μm, d (inset); 500 μm, g; 50 μm, h; all μCT images, 1.0 mm.