Extended data 10: Characterization of Osx ^flox; Ctsk-cre mice and human periosteal cells.

a, μ-CT images of Osx ^flox; Ctsk-cre mice 12 days post fracture. b, Safranin O staining was performed to detect cartilage in the callus 12 days after fracture. a-b, Representative images from 3 independent experiments. c, Significantly higher amounts of cartilage were detected in Osx^fl/fl ; Ctsk-cre⁺ mice when compared to Osx^fl/+ ; Ctsk-cre⁺ mice (*p=0.035) and Osx ^+/+; Ctsk-cre⁺ (*p=0.04) mice (mean ± S.D; n=3 independent experiment, 3 mice / group; 2-tailed Student’s t-test). d, H and E staining of callus tissue 12 days after fracture. Representative images from 3 independent experiments. e, Significantly lower bone volume (BV) was detected in Osx^fl/fl ; Ctsk-cre⁺ mice when compared to Osx^fl/+ ; Ctsk-cre⁺ mice (***p=0.0002) and Osx^+/+; Ctsk-cre⁺ (**p=0.002) mice (mean ± S.D; n=3 independent experiment, 3 animals / group; 2-tailed Student’s t-test). f, Safranin O staining for callus tissue 3 weeks after fracture. Representative images from 3 independent experiments. g, Significantly higher amounts of cartilage were detected in Osx^fl/fl ; Ctsk-cre⁺ mice when compared to Osx^fl/+ ; Ctsk-cre⁺ mice (***p=0.0005) and Osx ^+/+; Ctsk-cre⁺ (***p=0.0003) mice at 3 weeks post-fracture (mean ± S.D; n=3 independent experiment, 6 animals for control; 5 animals for het and knockout; 2-tailed Student’s t-test. h-i, FACS for CD49f, CD51 (h) Lepr (i) and CD146 (j) in human periosteal cells obtained from the femur. Representative FACS plots from 10 independent experiments. k, In vitro differentiation of h-PSCs (k, i-ii) and h-PP1 cells (k, iii-iv) after 3 weeks of culture. Color coded boxes (green) indicate parent/daughter gates for each cell type. Representative FACS plots from 3 independent experiments. l, Safranin O staining showing an absence of cartilage formation after h-PSC (left), h-PP1 (middle) and h-PP2 (right) transplantation into the kidney capsule of immunocompromised mice. The area containing the transplanted tissue is shown by the dotted yellow line. Representative images from 3 independent experiments.