Figure 3.
Figure 3. Empty HSC niches available for engraftment are distant from filled HSC niches. Experimental design (A). Doxycycline-treated nonmyeloablated TetOP-H2B-GFP mice transplanted with 4.0 × 104 CD34−CD48−LSK cells stained with PKH26 (red) (B-C), were analyzed 20 weeks after the administration of doxycycline and 2 days after transplantation. Bone marrow sections from transplanted mice were stained with antibodies against GFP (green, in panel B), c-kit (dark blue, in panels B and C), and S100 (white, in panel C). Arrowheads indicate endogenous H2B-GFP+c-kit+ HSCs (B). Arrows indicate donor PKH26+ HSCs (B-C), which are in contact with S100+ CAR cells (C). The nuclei of cells were labeled with DAPI dye (white, in panel B). Experimental design (D). Doxycycline-treated nonmyeloablated TetOP-H2B-GFP mice transplanted with 1.6 × 108 TetOP-H2B-GFP mouse-derived purified Lin− cells were analyzed 20 weeks after the administration of doxycycline and 27 weeks after transplantation. The numbers of donor (CD45.2+), endogenous (CD45.1+CD45.2+), and total H2B-GFP+c-kit+ HSCs (n = 4) (E). Half bones from transplanted mice were stained with antibodies against CD45.1 (red), GFP (green), and c-kit (dark blue), and representative images are shown (F). Arrows in panel F indicate donor CD45.1−H2B-GFP+c-kit+ HSCs, and arrowheads indicate endogenous CD45.1+H2B-GFP+c-kit+ HSCs. The nuclei of cells were labeled with DAPI dye (light blue). Half bones from transplanted mice were stained with antibodies against GFP (green) and c-kit (red) in panels G and H, S100 (white, in panel G), and CD31 (yellow, in panel H), and representative images are shown. Arrows indicate donor and endogenous H2B-GFP+c-kit+ HSCs (G-H). Arrowheads indicate CD31hi arterioles (H). Distances from donor and endogenous HSCs and random locations to the nearest CAR cells (I) or arteries (J). Using confocal microscopy, tiled Z-stacked optical sections (120-150 μm thick) were acquired from whole mount samples of half femurs (∼450-650 μm thick) (F-J). The statistical significance of differences between HSCs and random locations located within a 5-μm distance from CAR cells was determined by 2-tailed Student t tests (I). The statistical significance of overall differences in cell distribution was determined by Kolmogorov-Smirnov analysis (I-J). All error bars represent standard deviations of the means. **P < .01; ***P < .001.

Empty HSC niches available for engraftment are distant from filled HSC niches. Experimental design (A). Doxycycline-treated nonmyeloablated TetOP-H2B-GFP mice transplanted with 4.0 × 104 CD34−CD48−LSK cells stained with PKH26 (red) (B-C), were analyzed 20 weeks after the administration of doxycycline and 2 days after transplantation. Bone marrow sections from transplanted mice were stained with antibodies against GFP (green, in panel B), c-kit (dark blue, in panels B and C), and S100 (white, in panel C). Arrowheads indicate endogenous H2B-GFP+c-kit+ HSCs (B). Arrows indicate donor PKH26+ HSCs (B-C), which are in contact with S100+ CAR cells (C). The nuclei of cells were labeled with DAPI dye (white, in panel B). Experimental design (D). Doxycycline-treated nonmyeloablated TetOP-H2B-GFP mice transplanted with 1.6 × 108 TetOP-H2B-GFP mouse-derived purified Lin− cells were analyzed 20 weeks after the administration of doxycycline and 27 weeks after transplantation. The numbers of donor (CD45.2+), endogenous (CD45.1+CD45.2+), and total H2B-GFP+c-kit+ HSCs (n = 4) (E). Half bones from transplanted mice were stained with antibodies against CD45.1 (red), GFP (green), and c-kit (dark blue), and representative images are shown (F). Arrows in panel F indicate donor CD45.1−H2B-GFP+c-kit+ HSCs, and arrowheads indicate endogenous CD45.1+H2B-GFP+c-kit+ HSCs. The nuclei of cells were labeled with DAPI dye (light blue). Half bones from transplanted mice were stained with antibodies against GFP (green) and c-kit (red) in panels G and H, S100 (white, in panel G), and CD31 (yellow, in panel H), and representative images are shown. Arrows indicate donor and endogenous H2B-GFP+c-kit+ HSCs (G-H). Arrowheads indicate CD31hi arterioles (H). Distances from donor and endogenous HSCs and random locations to the nearest CAR cells (I) or arteries (J). Using confocal microscopy, tiled Z-stacked optical sections (120-150 μm thick) were acquired from whole mount samples of half femurs (∼450-650 μm thick) (F-J). The statistical significance of differences between HSCs and random locations located within a 5-μm distance from CAR cells was determined by 2-tailed Student t tests (I). The statistical significance of overall differences in cell distribution was determined by Kolmogorov-Smirnov analysis (I-J). All error bars represent standard deviations of the means. **P < .01; ***P < .001.

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