Extended Data Fig. 2: FACS analysis of microdissected periosteal tissue and characterization of PSCs.

a, Flow cytometry of CTSK–mGFP cells microdissected from the periosteum of P7 mouse long bones, showing the distribution of PSC, PP1 and PP2 cells. b, c, Flow cytometry showing the distribution of PSCs, PP1 and PP2 cells in mouse long bones at day 15 (b) and day 32 (c). Plots in a–c are representative of results from 10 independent experiments. d, Schematic representation of the strategy used for FACS analysis of periosteal PSC, PP1 and PP2 cell populations. e, FACS plot showing the distribution of CD146 (i) and CD140α (ii) expression in bone marrow stromal cells. f, FACS plots displaying the distribution of CD140α (i) and CD146 (ii) in mouse periosteum obtained through periosteal microdissection. Plots in e and f are representative of results from 5 independent experiments. g, Mouse bone marrow immunostained for CD146 (cyan) and CD140α (magenta). Scale bar, 100 µm. Representative images from 3 independent experiments. h, Clonogenic cells detected in the periosteum (top and middle, white arrows) and perichondrium region (bottom, white arrows) of mouse femur 2 weeks after induction of β-actin-Cre with tamoxifen. Enlarged views of outlined regions are shown. Scale bar, 50 µm. Representative images from 3 independent experiments. i, FACS plots showing in vitro differentiation for PP1 (left) and PP2 (right) cells after 15 days of culture. Representative FACS plots from 3 independent experiments. j, Alizarin red staining (red) of bone 5 weeks after transplantation of non-CTSK MSCs (left) and PSCs (right) into the kidney capsule. Representative images from 5 independent experiments. Scale bar, 50 µm. k, Relative gene expression for bone- (Runx2) and cartilage-specific genes (Col2a1, Comp, Chad) 5 weeks after transplantation of PSCs and non-CTSK MSCs. Non-CTSK MSC-derived cells display significantly higher expression of cartilage specific genes than PSCs. *P = 0.003 (Col2a1), *P = 0.002 (Comp), *P = 0.002 (Chad); two-tailed Student’s t-test. Data are mean ± s.d., n = 3. l, CTSK–mGFP⁺ PSCs (green) were immunostained for Runx2 (magenta, top) and osteocalcin (magenta, middle and bottom) 3, 4 or 5 weeks after transplantation into the kidney capsule. Scale bar, 20 µm. Representative images from 3 independent experiments.