Figure 2. Nes-GFP⁺ cells are mesenchymal stem cells.

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.