Vasculature-associated cells expressing nestin in developing bones encompass early cells in the osteoblast and endothelial lineage

Abstract

Nestin-positive (Nes(+)) cells are important hematopoiesis-supporting constituents in adult bone marrow. However, how these cells originate during endochondral bone development is unknown. Studies using mice expressing GFP under the direction of nestin promoter/enhancer (Nes-GFP) revealed distinct endothelial and nonendothelial Nes(+) cells in the embryonic perichondrium; the latter were early cells of the osteoblast lineage immediately descended from their progenitors upon Indian hedgehog action and Runx2 expression. During vascular invasion and formation of ossification centers, these Nes(+) cells were closely associated with each other and increased in number progressively. Interestingly, cells targeted by tamoxifen-inducible cre recombinase driven by nestin enhancer (Nes-creER) in developing bone marrow were predominantly endothelial cells. Furthermore, Nes(+) cells in postnatal bones were heterogeneous populations, including a range of cells in the osteoblast and endothelial lineage. These findings reveal an emerging complexity of stromal populations, accommodating Nes(+) cells as vasculature-associated early cells in the osteoblast and endothelial lineage.

Figure 1. Development of endothelial and non-endothelial nestin⁺ cells during endochondral ossification

a–c. E10.5 proximal limb bud of Nes-GFP mice stained whole-mount for CD31 and nuclei. x40 (a, b), x630 confocal (c), green: EGFP, red: Alexa546, blue: DAPI. Scale bars: 400μm (a, b), 10 μm (c). Note GFP is cytoplasmic, and CD31 is membraneous. d. Flow cytometry analysis of E10.5 Nes-GFP limb bud cells stained for CD45 and CD31 (n=3, data represented as mean ± S.D.). Upper panel: nucleated singlets gated for CD45⁻, blue line: GFP⁻ control limb bud cells, lower panel: CD45⁻ fraction gated for Nes-GFP⁺, blue line: GFP⁺ cells, unstained control. e–i. E11.5–15.5 femur sections of Nes-GFP mice stained for CD31 and nuclei. x200 confocal (e–i), green: EGFP, red: Alexa546, gray: DAPI. Dorsal halves of growth cartilage are shown. Perichondrium is on top. Arrows: CD31⁺Nes⁺ cells (e, f), arrowheads: CD31⁻ Nes⁺ cells in perichondrium (g, h). Asterisks in vascular invasion front: intimate association of CD31⁺Nes⁺ and CD31⁻ Nes⁺ cells (i). Scale bars: 50μm. j. Quantification of CD31⁺Nes⁺ cells (white bars) and CD31⁻ Nes⁺ cells (black bars) in perichondrium and primary ossification center (n=3–6).

Figure 2. Non-endothelial nestin⁺ cells encompass early cells of the osteoblast lineage

a–c. Nes- GFP; Col2-cre; R26R^Tomato femur sections stained for CD31 and nuclei. E12.5, x200 confocal (a) (right panel: CD31 single-color) and E13.5, x200 (b), x630 confocal of dotted area (c). Arrows: CD31⁻ Nes⁺ Tomato⁺ cells, allowheads: CD31⁺Nes⁺Tomato⁻ cells. d. Nes-GFP; Col2-creER; R26R^Tomato E13.5 femur sections stained for CD31 and nuclei. Pregnant mice received 2mg tamoxifen at E12.5. x200 confocal. e, f. Nes-GFP; Osx-creER; R26R^Tomato E13.5 femur sections stained for CD31 and nuclei. Pregnant mice received 2mg tamoxifen at E12.5. x200 (e), x630 confocal of the osteogenic perichondrium (f) (lower panel: CD31 single-color). Arrows: CD31⁻ Nes⁺ Tomato⁺ cells, arrowheads: perivascular Tomato⁺ cells. Green: EGFP, red: tdTomato, blue: Alexa633, gray: DAPI. Scale bars: 50μm (a, b, d, e), 20μm (c, f). g. Flow cytometry analysis of E13.5 Nes-GFP; Osx-creER; R26R^Tomato limb cells (tamoxifen at E12.5) stained for CD45. CD45⁻ fraction gated for Tomato⁺, blue line: Nes-GFP⁻ Tomato⁺ limb cells (n=3, data represented as mean ± S.D.). h. Nes- GFP; Osx-creER; R26R^Tomato E16.5 femur sections (tamoxifen at E13.5) stained for nuclei. Arrowheads: Tomato⁺ cells disappearing from the osteogenic perichondrium. x100. Green: EGFP, red: tdTomato, blue: DAPI. i, j. Nes- GFP; Osx-creER; R26R^Tomato E17.5 femur sections (tamoxifen at E16.5) stained for CD31 and nuclei. x100 (i), x630 confocal of dotted area (j). Arrows: CD31⁻ Nes⁺Tomato⁺ cells on the innermost layer of the osteogenic perichondrium. Scale bars: 200μm (h, i), 50μm (j). k–m. Nes-GFP; Col2-creER; R26R^TomatoP0 femur sections (tamoxifen at E13.5) stained for CD31 and nuclei. x100 (k), x630 confocal of dotted area (l: perichondrium, m: primary spongiosa). Arrows: CD31⁻ Nes⁺ Tomato⁺ cells. Green: EGFP, red: tdTomato, blue: Alexa633, gray: DAPI. Scale bars: 200μm (k), 20μm (l, m). o–q. E13.5 femur sections stained for CD31 and nuclei. Control (o), Ihh^{−/ −} (p) and Runx2^{−/ −} (q), x200 confocal. Green: EGFP, red: Alexa546, gray: DAPI. Scale bars: 50μm. r. Quantification of CD31⁺Nes⁺ cells and CD31⁻ Nes⁺ cells in perichondrium. Control (while bars) and Ihh^{−/ −} (black bars), n=4 per group, *p<0.05. s. Quantification of CD31⁺Nes⁺ cells and CD31⁻ Nes⁺ cells in perichondrium. Control (while bars) and Runx2^{−/ −} (black bars), n=4 per group, *p<0.05. t. Ptch1^LacZ/+ femur stained for β-galactosidase activity. E13.5, x200. Scale bar: 50μm.

Figure 3. Nes-creER preferentially targets nestin⁺ endothelial cells in developing bone marrow

a. Pregnant mice received 2mg tamoxifen at indicated points (E11.5, 13,5 or 16.5), and Nes-creER; R26R^Tomato mice were chased until indicated postnatal days (P0, P7 or P21). Distal femur sections, x40. Red: tdTomato. Scale bars: 500μm. b. Nes-creER; R26R^Tomato mice received 0.1mg tamoxifen at P3 and were chased for 1 month (left panels) and 6 months (right panels). Upper panels: distal femur, metaphysis, x40, scale bars: 500μm, lower panels: diaphysis, x100, scale bars: 200μm. Red: tdTomato. c. Flow cytometry analysis of epiphyseal/metaphyseal cells from Nes-creER; R26R^Tomato mice received tamoxifen at P3 and chased for indicated periods. Cells were stained for CD45 and CD31. Upper panel: CD45⁻ fraction after 2 weeks of chase gated for Tomato⁺, blue line: Tomato⁺ cells, unstained control. Lower panel: Mice were chased for 2 days (n=4), 1 week (n=6), 2 weeks (n=2) and 4 weeks (n=4). The percentage of CD31⁺ cells within CD45⁻ Tomato⁺ fraction is shown. d. Nes-creER; R26R^Tomato (tamoxifen at P3) femur sections. Left panels: diaphysis after 1 month of chase stained for CD31 and nuclei. Upper left: x200 confocal, arrows: Tomato⁺ osteocytes in cortical bone; Lower left:, x630 confocal of boxed area from upper left, rotated perpendicularly, bone marrow sinusoid. Red: tdTomato, blue: Alexa633, gray: DAPI. Upper right: growth plate cartilage after 2 months of chase. Note Tomato⁺ columnar chondrocytes. x400 confocal, Red: tdTomato, gray: DAPI. Lower right panel: diaphysis after 4 months of chase stained for lipid. x200 confocal, Red: tdTomato, blue: LipidTOX Deep Red. Scale bars: 50μm. e. Flow cytometry analysis of epiphyseal/metaphyseal cells from Col1(2.3 kb)-GFP; Nes-creER; R26R^Tomato mice received tamoxifen at P3 and chased for indicated periods. Cells were stained for CD45. Upper panel: CD45⁻ fraction after 2 days of chase gated for Tomato⁺, blue line: Tomato⁺ cells, unstained control. Lower panel: Mice were chased for 2 days (n=4), 1 week (n=5), 2 weeks (n=2), 3 weeks (n=4) and 4 weeks (n=4). The percentage of Col1(2.3 kb)-GFP⁺ cells within CD45⁻ Tomato⁺ fraction is shown. f, g. Cxcl12-GFP; Nes-creER; R26R^Tomato mice received tamoxifen at P3 and chased for 1 week. Flow cytometry analysis of epiphyseal/metaphyseal cells (f); CD45⁻ fraction gated for Tomato⁺ (n=6, blue line: Cxcl12-GFP⁻ Tomato⁺ cells), sections of femur diaphysis bone marrow stained for CD31 and nuclei (g), x630 confocal. Green: EGFP, red: tdTomato, blue: Alexa633, gray: DAPI. Scale bars: 50μm. All data represented as mean ± S.D.

Figure 4. Nestin⁺ cells in developing postnatal bones are heterogeneous stromal cell populations

a, b. Distal femur sections of Nes-GFP mice at 1 week of age (a) and 2 months of age (b) stained for nuclei. x100 confocal, metaphysis (left panels) and diaphysis (right panels). Arrows: Nes-GFP^high cells in the primary spongiosa adjacent to the growth plate, arrowheads in (a): pericytes of bone marrow arterioles, asterisks: osteoblasts on bone surface and osteocytes, arrowheads in (b): osteocytes in cortical bone. Scale bars: 100μm. c, d. Nes- GFP; Nes-creER; R26R^Tomato mice received tamoxifen at P3 and chased for 48 hours. (c) Femur sections of metaphysis (left panel) and diaphysis bone marrow (right panel) stained for CD31 and nuclei. Asterisks: CD31⁺Tomato⁺ cells, arrows: pericytes of bone marrow arterioles, x630 confocal, green: EGFP, red: tdTomato, blue: Alexa633, gray: DAPI. Scale bars: 20μm. (d) Flow cytometry analysis of epiphyseal/metaphyseal cells stained for CD45 and CD31; CD45⁻ fraction gated for Tomato⁺ (left panel and lower right panel), Nes-GFP⁺ (upper right panel) (n=4), blue line: Nes-GFP⁻ Tomato⁺ cells (left panel) and unstained controls (right panels). e. Flow cytometry analysis of epiphyseal/metaphyseal cells from Nes-GFP; R26R^Tomato mice that also carry Osx-cre (left panel), Tie2-cre (center panel) or LepR-cre (right panel). Cells were harvested at P3 (Tie2-cre) or P7 (Osx-cre, LepR-cre). f. Flow cytometry analysis of epiphyseal/metaphyseal cells from Nes-GFP; R26R^Tomato mice that also carry Osx-creER (left panel), Col1-creER (center panel) or Ocn-creER (right panel). Tamoxifen was administered at P3 and cells were harvested a week later. CD45⁻ fraction gated for Tomato⁺ (Osx-cre: n=3, Tie2-cre: n=4, LepR-cre: n=3, Osx-creER: n=6, Col1-creER: n=4, Ocn-creER: n=3), blue lines: GFP⁻ control. All data represented as mean ± S.D.