a–c, Flow cytometry characterization of bone marrow Nes-GFP⁺ cells; representative diagrams were obtained from bone marrow nucleated cells (a, c) or GFP⁺ cells (b); the percentage of the population in each quadrant is indicated. d, Projection image from ~10 µm Z-stack showing fluorescent signals from Nes-GFP (green), CD31⁺ vascular endothelial cells (red) and nuclei counterstained with DAPI (blue). e, f, Nes-GFP⁺ cells are directly innervated by tyrosine hydroxylase⁺ (red) catecholaminergic fibres in the endosteum (e) and the bone marrow parenchyma (f). g, h, Q-PCR from the same number of sorted CD45⁻ Nes-GFP⁺ and CD45⁻ Nes-GFP⁻ cells, and from primary osteoblasts (OB) and osteoclasts (OC; n = 5–10); *P < 0.05, **P < 0.01, ***P < 0.001; unpaired two-tailed t-test. Error bars indicate s.e.m. i, j, Immunostaining for CD150 (red), CD48 and haematopoietic lineages (blue) in femoral sections of Nes-Gfp mice. Stacked images of CD150⁺CD48⁻Lin⁻ cells (arrow) adjacent to Nes-GFP⁺ cells in the endosteum (i) and the sinusoids (j). i, CD150⁺CD48⁺/Lin⁺ megakaryocytes (asterisks). k, Localization and number of CD150⁺CD48⁻Lin⁻ cells relative to Nes-GFP⁺ cells in different bone marrow regions. l, Nes-GFP⁺ cells (green) and cobblestone-forming area (dashed line) in 4-week primary myeloid culture. e, f, l, Overlapped fluorescence and phase contrast images. All scale bars, 50 µm.

a, Number of colony-forming units-fibroblast⁴¹ (CFU-F) in sorted CD45⁻ Nes-GFP⁺ and CD45⁻ Nes-GFP⁻ cells (n = 12). b–h, Differentiation of sorted CD45⁻ Nes-GFP⁺ cells (n = 4). Q-PCR of Gfp and (b) genes associated with osteoblastic (c; alkaline phosphatise (Alpl), Runx2, bone morphogenetic protein-4 (Bmp4), osteoglycin (Ogn), osterix (Sp7), osteocalcin (Bglap), osteoactivin (Gpnmb)), adipogenic (e; adipsin (Cfd), peroxisome proliferator-activated receptor gamma 2 (Pparg)) and chondrogenic (g; aggrecan (Acan)) differentiation is shown. d, f, h, Fully differentiated phenotypes of sorted CD45⁻ Nes-GFP⁺ cells shown by alkaline phosphatase (pink) and Von Kossa (black) staining (d), Oil redO (red) staining (f) and Alcian blue (blue) staining (h). CD45⁻Nes-GFP⁻ cells did not generate any progeny (d, f; inset). i–q, Nes-GFP⁺ cells, but not the remaining CD45⁻ bone marrow population, formed self-renewing and multipotent clonal spheres after 7–10 days in low-density culture. l, m, Adherent bulk-cultured CD45⁻ Nes-GFP⁺ cells (l) or clonal spheres (m) lost GFP expression and differentiated into GFP⁻ adipocytes (refringent lipoid droplets). n, o, Representative mesenspheres showing spontaneous multilineage differentiation into Col2.3-LacZ⁺ osteoblasts (blue) and Oil red O⁺ adipocytes (red). p, q, Chondrogenic differentiation of mesenspheres shown by Alcian blue staining (p; blue) and Q-PCR for sex determining region Y-box 9 (Sox9), aggrecan (Acan), collagen type IIα1 (Col2α1) and collagen type XIα2 (Col11α2) (n = 4–11) (q). i, j, m, p, Bright field; k, Fluorescence. l, Overlapped fluorescence and bright field. Scale bars: 100 µm (f, l, k, n, p); 500 µm (h); 50 µm (l, m, o). Asterisks, where indicated, are: *P < 0.05; **P < 0.01; ***P < 0.001; unpaired two-tailed t-test. All error bars indicate s.e.m.

a–i, In vivo self-renewal of adult bone marrow CD45⁻ Nes-GFP⁺ cells in secondary transplants. a, Scheme showing the experimental paradigm. b–e, Primary ossicles showing numerous β-galactosidase⁺ osteoblasts derived from CD45⁻ Nes-GFP⁺ cells (b, blue; d, e, dark deposits, arrowheads) but none from CD45⁻ Nes-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 β-galactosidase⁺ osteoblasts derived from CD45⁻ Nes-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 µm per square. h, i, Secondary ossicles yielded 8,557 ± 537 GFP⁺ spheres (h) that generated Col2.3⁺ osteoblasts (i; blue precipitates). j–m, Adult nestin⁺ MSCs contribute to endochondral lineages. j–l, Femoral sections from 11-month-old Nes-cre^ERT2/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 α1 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, j–m, o–q, 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 µm (c–f, h, i); 20 µm (j–q).

a, Expression and regulation of core HSC maintenance genes by CD45⁻ Nes-GFP⁺ cells. Q-PCR for Cxcl12, stem cell factor/kit ligand (Kitl), angiopoietin-1 (Angpt1), interleukin-7 (Il7), vascular cell adhesion molecule-1 (Vcam1) and osteopontin (Spp1) in CD45⁻ Nes-GFP⁺ and CD45⁻ Nes-GFP⁻ cells sorted from the bone marrow of mice injected with G-CSF or vehicle (n = 6). b–k, Bone marrow (BM) and spleen nucleated (b, f), Lin⁻ CD48⁻ (c, g), CD48⁻ LSK (d, h) and CD150⁺CD48⁻ LSK (e, i) cells 3–16 days after tamoxifen and diphtheria toxin administration in Nes-cre^ERT2/iDTR double- and control iDTR single-transgenic mice (n = 6–12). j, Long-term culture-initiating cell assay using limiting dilutions of bone marrow cells from Nes-cre^ERT2/iDTR (red) or control iDTR mice (black) 1 month after tamoxifen and diphtheria toxin treatment; the percentage of culture dishes in each experimental group that failed to generate colony-forming units is plotted against the number of test bone marrow cells; bone marrow HSC frequencies are indicated; Pearson chi-squared test (n = 4–6). k–m, Nestin⁺ cells are required for the homing of haematopoietic stem and progenitor cells. k, Bone marrow homing of haematopoietic progenitors (CFU-C) in tamoxifen- and diphtheria-toxin-treated Nes-cre^ERT2/iDTR and control iDTR mice (n = 4–8). l, m, HSCs rapidly home near GFP⁺ cells in the bone marrow of Nes-Gfp transgenic mice. l, Average shorter distances between bone marrow HSCs, Nes-GFP⁺ cells and the bone surface 2 h (n = 16), 48 h (n = 30) and 96 h (n = 14) after HSC transplantation into lethally irradiated mice. m, Representative DyD-stained (red) HSC, Nes-GFP⁺ (green) cell and bone matrix (blue). *P < 0.05, **P < 0.01, ***P < 0.001; unpaired two-tailed t-test. All error bars indicate s.e.m.