ai, In vivo self-renewal of adult bone marrow CD45Nes-GFP+ cells in secondary transplants. a, Scheme showing the experimental paradigm. be, Primary ossicles showing numerous -galactosidase+ osteoblasts derived from CD45Nes-GFP+ cells (b, blue; d, e, dark deposits, arrowheads) but none from CD45Nes-GFP cells (c); haematopoietic areas (b, e; circled by dashed line) were detected only in the former group and frequently associated with GFP+ cells (e, green). f, Secondary ossicle showing numerous -galactosidase+ osteoblasts derived from CD45Nes-GFP+ cells (blue) and also haematopoietic areas (circled by dashed line). g, CD45+ haematopoietic cells (red) localized near Nes-GFP+ (green) cells in the ossicles; cell nuclei have been stained with DAPI (blue); grid, 50 m per square. h, i, Secondary ossicles yielded 8,557 537 GFP+ spheres (h) that generated Col2.3+ osteoblasts (i; blue precipitates). jm, Adult nestin+ MSCs contribute to endochondral lineages. jl, Femoral sections from 11-month-old Nes-creERT2/RCE:loxP double-transgenic mice 8 months after tamoxifen treatment showing the contribution of adult nestin+ cells to bone-lining osteoblasts (j), osteocytes (k; asterisks indicate GFP+ cells, arrowheads indicate GFP osteocytes) and collagen 1 type 2+ (red) chondrocytes (l). m, GFP+ (green) perivascular cells (asterisk) identical in frequency, morphology and distribution to Nes-GFP+ cells and osteoblasts (arrowhead) co-stained with anti-GFP antibodies (red). n, o, Bone marrow section of Nes-Gfp/Col2.3-cre/R26R triple-transgenic mouse showing X-gal+ osteoblasts (dark precipitates), GFP+ (green) and CD31+ vascular endothelial cells (red); Col2.3+ osteoblasts localized near Nes-GFP+ perivascular cells are indicated with arrowheads. p, q, Immunostaining for osterix (red) in trabecular bone section of a 5-week-old Nes-Gfp (green) mouse. g, jm, oq, Nuclei have been stained with DAPI (blue). j, l,m, p, q, Bone (b) margins are indicated with dashed lines. b, c, f, i, n, Bright field; g, j, k, p, q, fluorescence; d, e, h, l, m, o, overlapped fluorescence and bright field. Scale bars: 100 m (cf, h, i); 20 m (jq).

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