In vivo serial transplantation of Nestin-GFP⁺PDGFR-α⁺ CD45^–Ter119^–CD31^– (A) and LepR-YFP⁺CD45^–Ter119^–CD31^– PDCs (B). Single cell–derived colonies from donors’ PDCs were expanded to generate 5 × 10⁶ cells and injected into the femora of five 1-month-old NOD SCID mice at a density of 1 × 10⁶ per injection. GFP⁺ or YFP⁺ cells were sorted from the primary recipients’ bone marrow at 8 weeks after injection and harvested for forming CFU-Fs. The colonies were then transplanted into the secondary recipient mice. FAC analysis of CFU-Fs showed the percentages of GFP⁺ or YFP⁺ cells expressing MSC markers (CD90 and CD105) (bottom panels in A and B). (C–J) Lineage-tracing of periosteal Nestin⁺ cells in Nes-creERT2 R26R-EYFP mice. Samples were collected 2, 7, and 14 days or 2, 14, and 30 days after tamoxifen (100 mg/kg i.p.) administration at P14 or P60, respectively (C, G). Representative images of coronal tibia diaphyseal periosteum sections stained for YFP and Osx (D and H), YFP and CD31 (E and I). Quantification of Nestin⁺ lineage cells’ contribution to periosteal Osx⁺ osteoblasts (F and J, left panel), CD31⁺ endothelial cells (F and J, right panel) (n = 5 mice/group). Representative images from 1- or 3-month-old LepR-cre R26R-EYFP mice stained for YFP and Osx (L), YFP and CD31 (M). Scale bars: 20 μm. (N) Quantification of LepR⁺ lineage cells’ contribution to periosteal Osx⁺ osteoblasts (left panel) and CD31⁺ endothelial cells (right panel). Data are presented as mean ± SEM. *P < 0.05; **P < 0.01. C, periosteal cortical bone; NS, not significant as determined by ANOVA with Bonferroni’s post hoc analysis; P, periosteum.