Figure 5.
Figure 5. Fluorescent images of marrow stromal-cell culture grown in a 6-well plate. Each panel is formed from × 25 to × 2.5 original magnification images that are tiled together, and represents approximately 60% of the surface area of a cell-culture well. The same well is recorded at increasing days of culture (days 10-28) and the fluorescent spots that develop over time are produced by the strong GFP expression within the bone nodule. At day 21 the nodules that have mineralized are visualized by the XO staining that colocalized with the GFP nodules (Aiv,Biv). (A) Profile of developing bone nodules from an irradiated 3.6-blue transgenic mouse to illustrate the bone nodule-forming potential of the osteoprogenitor population in a bone marrow stromal culture. Images from days 14 (Ai), 18 (Aii), and 21 (Aiii) show nodule progression and the extent of mineralization at day 21 is shown by XO staining (Aiv). (B) Stromal-cell culture derived from a 2.3-green recipient mouse that was rescued by WBMT from a 3.6-blue donor. Although the number of host-derived 2.3-green bone nodules is diminished relative to nonirradiated controls (not shown), only host-derived nodules are observed. Images from days 14 (Bi), 18 (Bii), and 21 (Biii) show nodule progression and the extent of mineralization at day 21 is shown by XO staining (Biv). (C) Fluorescent image (original magnification, × 50) of a 2.3-green nodule as seen in the × 25 (original magnification) tiled image in panel B. The 2.3-green nodule (Cii) shows strong mineralization (Ciii). Panel Ci shows the cluster of 3.6-blue donor-derived cells (arrowheads) that happened to form adjacent to this 2.3-green nodule. It has a distinctly different morphology than the bone nodule (BN) that in this image is blue in color because the 2.3-green signal seen in panel Cii spills through the GFPcyan filter. Panel Civ shows the composite image of the 3.6-blue cell cluster and 2.3-green/XO bone nodule. (D) Fluorescent and phase-contrast image (original magnification, × 100) of 3.6-blue cells that develop in a stromal-cell culture derived from a recipient mouse rescued with TBM from a 3.6-blue donor mouse. The expanding cluster of cells does not become a multilayered nodule and does not stain with XO. Images from days 10 (Di), 21 (Dii), and 28 (Diii) show the cellular progression. The overlay (Div) with the phase-contrast image shows the 3.6-blue cells relative to the nonfluorescent cells in the field of view.

Fluorescent images of marrow stromal-cell culture grown in a 6-well plate. Each panel is formed from × 25 to × 2.5 original magnification images that are tiled together, and represents approximately 60% of the surface area of a cell-culture well. The same well is recorded at increasing days of culture (days 10-28) and the fluorescent spots that develop over time are produced by the strong GFP expression within the bone nodule. At day 21 the nodules that have mineralized are visualized by the XO staining that colocalized with the GFP nodules (Aiv,Biv). (A) Profile of developing bone nodules from an irradiated 3.6-blue transgenic mouse to illustrate the bone nodule-forming potential of the osteoprogenitor population in a bone marrow stromal culture. Images from days 14 (Ai), 18 (Aii), and 21 (Aiii) show nodule progression and the extent of mineralization at day 21 is shown by XO staining (Aiv). (B) Stromal-cell culture derived from a 2.3-green recipient mouse that was rescued by WBMT from a 3.6-blue donor. Although the number of host-derived 2.3-green bone nodules is diminished relative to nonirradiated controls (not shown), only host-derived nodules are observed. Images from days 14 (Bi), 18 (Bii), and 21 (Biii) show nodule progression and the extent of mineralization at day 21 is shown by XO staining (Biv). (C) Fluorescent image (original magnification, × 50) of a 2.3-green nodule as seen in the × 25 (original magnification) tiled image in panel B. The 2.3-green nodule (Cii) shows strong mineralization (Ciii). Panel Ci shows the cluster of 3.6-blue donor-derived cells (arrowheads) that happened to form adjacent to this 2.3-green nodule. It has a distinctly different morphology than the bone nodule (BN) that in this image is blue in color because the 2.3-green signal seen in panel Cii spills through the GFPcyan filter. Panel Civ shows the composite image of the 3.6-blue cell cluster and 2.3-green/XO bone nodule. (D) Fluorescent and phase-contrast image (original magnification, × 100) of 3.6-blue cells that develop in a stromal-cell culture derived from a recipient mouse rescued with TBM from a 3.6-blue donor mouse. The expanding cluster of cells does not become a multilayered nodule and does not stain with XO. Images from days 10 (Di), 21 (Dii), and 28 (Diii) show the cellular progression. The overlay (Div) with the phase-contrast image shows the 3.6-blue cells relative to the nonfluorescent cells in the field of view.

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