Figure 5
Figure 5. Homing of CD166−/− cells to the hematopoietic niche. (A) Homing of CD166−/− hematopoietic cells to the BM of lethally irradiated recipients. Recovery of BM-homed cells 16-hours PT (corrected for total body BM). Cells were stained with CTV, injected into lethally irradiated recipients and then recovered from flushed central (C BM) or from digested endosteal (E BM) regions (n = 3-4 mice per group). *P < .05 compared with WT. (B) Intravital images of CFSE-stained Lin− cells in the calvariae of nonirradiated recipients 16-hours PT. Panels on the left depict WT cells in WT recipients and those on the right show KO cells in KO recipients. Top 2 panels show CFSE-stained cells (green) in the BM microenvironment. Images of vessels (collected at 605 nm) that were stained with tetramethyl rhodamine isothiocyanate (TRITC)–conjugated dextran appear in red. Images in the middle row depict 2 different areas of the calvarium with the bone rendered silver to facilitate visualization of marrow-homed cells. The bottom row shows the same areas depicted in the middle row after removal of the images of the vasculature. Distances from each cell to the surface of the endosteum was measured using Amira 3D Visualization and Image Analysis software, version 4.1, using a 3-dimensional caliper tool. Additional information can be found in supplemental Figure 7. (C) Average distances between WT or KO Lin− and LSK cells and the endosteum. Distances between cells and the bone surface (reconstructed with a constant threshold value, visible in gold) were measured using a 3-dimensional caliper tool. *P < .05 compared with WT. Data shown are from 1 representative experiment from a total of 3. At least 1 recipient mouse per condition was used in each experiment. In the analysis shown between 8 and 25 cells per condition were measured for the Lin− group and 5 to 7 for the LSK group. Similar data were obtained in the other experiments. (D) Cell cycle analysis of BM-homed cells for 16-hours PT. BM homed CTV+ cells were isolated by cell sorting, stained with propidium iodide, and flow cytometrically analyzed for cell cycle status to determine cells in G0, G1, and G2+M (n = 3-4 per group). (E) Frequencies of WT and KO marrow-derived LSK and LSK48-150+ cells expressing CXCR4 (BD, clone 2B11, cat# 561734).

Homing of CD166−/− cells to the hematopoietic niche. (A) Homing of CD166−/− hematopoietic cells to the BM of lethally irradiated recipients. Recovery of BM-homed cells 16-hours PT (corrected for total body BM). Cells were stained with CTV, injected into lethally irradiated recipients and then recovered from flushed central (C BM) or from digested endosteal (E BM) regions (n = 3-4 mice per group). *P < .05 compared with WT. (B) Intravital images of CFSE-stained Lin− cells in the calvariae of nonirradiated recipients 16-hours PT. Panels on the left depict WT cells in WT recipients and those on the right show KO cells in KO recipients. Top 2 panels show CFSE-stained cells (green) in the BM microenvironment. Images of vessels (collected at 605 nm) that were stained with tetramethyl rhodamine isothiocyanate (TRITC)–conjugated dextran appear in red. Images in the middle row depict 2 different areas of the calvarium with the bone rendered silver to facilitate visualization of marrow-homed cells. The bottom row shows the same areas depicted in the middle row after removal of the images of the vasculature. Distances from each cell to the surface of the endosteum was measured using Amira 3D Visualization and Image Analysis software, version 4.1, using a 3-dimensional caliper tool. Additional information can be found in supplemental Figure 7. (C) Average distances between WT or KO Lin− and LSK cells and the endosteum. Distances between cells and the bone surface (reconstructed with a constant threshold value, visible in gold) were measured using a 3-dimensional caliper tool. *P < .05 compared with WT. Data shown are from 1 representative experiment from a total of 3. At least 1 recipient mouse per condition was used in each experiment. In the analysis shown between 8 and 25 cells per condition were measured for the Lin− group and 5 to 7 for the LSK group. Similar data were obtained in the other experiments. (D) Cell cycle analysis of BM-homed cells for 16-hours PT. BM homed CTV+ cells were isolated by cell sorting, stained with propidium iodide, and flow cytometrically analyzed for cell cycle status to determine cells in G0, G1, and G2+M (n = 3-4 per group). (E) Frequencies of WT and KO marrow-derived LSK and LSK48-150+ cells expressing CXCR4 (BD, clone 2B11, cat# 561734).

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