Fig. 2
PCs and BMSCs derive from the Prx1-mesenchymal lineage. a Experimental design for renal capsule transplantations. Femoral cartilages before vascular invasion were isolated from E14.5 Prx1-Cre;YFPfl/+ embryos and transplanted under the renal capsule of wild-type hosts. PCs and BMSCs were isolated from mature skeletal elements 8 weeks post-transplantation as shown in Fig. 1a. b Flow cytometry analyses of PCs and BMSCs isolated from Prx1-Cre;YFPfl/+ mature skeletal elements grown under renal capsule. Both PCs and BMSCs that are negative for endothelial/hematopoietic markers (CD31, CD11b, CD34, and CD45) and positive for Sca1/CD29 are mostly YFP-positive (Prx1-donor-derived) (n = 3 per group). c Localization of Prx1-derived cells in the periosteum and fracture callus of Prx1-Cre;mTmG mouse. The un-injured periosteum is derived from Prx1-lineage (GFP+). In the activated periosteum at day 3 post fracture, some Prx1-derived cells (GFP/pointed by green arrows) colocalize with CD29-positive cells (Merge CD29+ GFP+/pointed by white arrows). In the fracture callus (d14), all chondrocytes and osteoblasts/osteocytes are derived from Prx1-lineage (GFP+). The fractures performed on Prx1-Cre/ERT2,-EGFP mice, where EGFP is expressed under the Prx1 promoter, show no GFP signal in the callus. d Experimental design for cell-lineage analyses of Prx1-derived cells during bone regeneration in renal capsule. Femoral cartilages were isolated from E14.5 Prx1-Cre+/−;LacZfl/+ embryos and transplanted under the renal capsule of wild-type hosts. After 8 weeks, mature femurs underwent osteotomy and were collected at d14 post-fracture for cell-lineage tracing. e TC and Xgal/TRAP double-staining on longitudinal sections of Prx1-Cre+/−;LacZfl/+ fractured femurs in wild-type hosts (top) showing new bone within the callus entirely donor-derived, i.e., LacZ + TRAP− (black arrowheads: osteocytes) and some osteoclasts (TRAP+ LacZ+ with endogenous beta-galactosidase activity). Scale bar: 0.5 mm. TC: Masson’s trichrome, TRAP: Tartrate resistant acid phosphatase, m: muscle, c: cortex, po: periosteum, bm: bone marrow, cal: callus, ca: cartilage, white dashed line: callus, orange lightning bolt: fracture, orange arrow: fracture site, black arrow: points to the periosteum, black arrowhead: osteocytes in new bone
