Figure 1.
Characterization and heterogeneity of BMSCs
(A and B) Tile-scan confocal images of 3-week-old Pdgfra-GFP (green) femur in a longitudinal section (A) and transverse section through diaphysis (B), immunostained for PDGFRβ (red) and the EC marker EMCN (blue). Epiphysis (epi), metaphysis (mp), growth plate (gp), cortical bone (cb), bone marrow (bm), central vein (cv; white arrowheads), and arteries (a; yellow arrowheads) are indicated. Dashed lines illustrate borders of growth plate and metaphysis. White arrowheads indicate Pdgfra-GFP+ PDGFRβ+ double-positive cells in metaphysis, bone marrow, growth plate, and epiphysis, whereas white arrows point at Pdgfra-GFP+ PDGFRβ− cells, including osteocytes in cortical and trabecular bone as well as chondrocytes in the growth plate and articular cartilage (top).
(C) Representative maximum intensity projections and optical z stack single plane (center) of metaphyseal region showing Pdgfra-GFP (green, arrow) expression in PDGFRβ (red) perivascular cells (arrowheads). (Right) Higher magnification of vessel with lumen (lu) lined by EMCN+ (blue) ECs.
(D) Confocal image showing Osterix+ (OSX, gray) osteoprogenitor cells together with Pdgfra-GFP (green) and PDGFRβ (red) staining in metaphysis. OSX+ and Pdgfra-GFP double-positive cells are marked by arrowheads, whereas cells with only Pdgfra-GFP+ signal are shown by arrows. ECs, EMCN+ (blue). High-magnification image on the right highlights Pdgfra-GFP+ PDGFRβ+ OSX+ triple-positive cells (yellow arrowhead).
(E) Quantification of Pdgfra-GFP+ PDGFRβ+ double-positive cells and Pdgfra-GFP+ PDGFRβ− cells in metaphysis. Pdgfra-GFP expression (mean intensity) is higher in single-positive cells compared with Pdgfra+β+ cells. (n = 5–8; data are presented as means ± SEM, p values, two-tailed unpaired t test).
(F and G) High-magnification confocal images of Pdgfra-GFP (green) femoral diaphysis and marrow immunostained for PDGFRβ (red) and EMCN (blue). In endosteum (eo) next to cortical bone (cb) and marrow, PDGFRβ+ cells co-express Pdgfra-GFP, whereas Pdgfra-GFP+ cells in periosteum lack PDGFRβ (F) but express OSX+ (red) (G).
(H) Representative image of perivascular (arrowheads) Pdgfra-GFP+ PDGFRβ+ BMSCs in metaphysis and BM co-expressing Runt-related transcription factor 2 (RUNX2) (red). ECs, EMCN (blue).
(I and J) Electron micrographs of 3-week-old wild-type metaphysis (I) and marrow (J). Arrows highlight ECs, perivascular MSCs (PVC), hematopoietic cells (HC), and osteoblasts (OB). Collagen bundle in metaphysis and thin fiber structures in BM (yellow arrowheads) are indicated in high-magnification images.