Loss of HSPC attachment to endosteal zone and ectopoic BM hematopoietic loci upon myelosuppression in Cxcl12-deficient mice. (A) Hematoxylin-eosin staining of BM section of Cxcl12-WT (i-iv) and Cxcl12-cKO (v-viii) mice at various time point after 5-FU treatment. Hematopoietic cell clusters from endosteal surface (arrow) or from perisinusoidal space (arrowhead) are indicated. The magnified pictures (iv,viii) show the ectopic site of BM regeneration in Cxcl12-cKO mice. Scale bar = 100 μm. (B) Representative figure of the regenerated perisinusoidal cell cluster in Cxcl12-cKO BM stained with CD150/CD41/CD48 and endothelial marker MECA-32. CD150⁺CD41⁻CD48⁻ cells (red, arrow) were identified, and they were associated with CD150⁺CD41⁺CD8⁺ (yellow) and CD150⁻CD41⁺CD48⁺ (green) progenitors. Scale bar = 50 μm. (C) Four days after 5-FU treatment, CD45.2⁺Tie2⁺ HSCs (arrow) can be seen along the endosteal surface in Cxcl12-WT BM, but they were scattered in perisinusoidal region in Cxcl12-cKO BM. Dotted line indicates the endosteal surface. Scale bar = 50 μm. For statistics, CD45.2⁺Tie2⁺ cells per BM section were measured for their distance (in microns) to the nearest endosteal surface. The percentage in respect of such distance was analyzed and is presented in the right penal (mean ± SEM; **P < .005, ***P < .001; n = 4). (D) Representative figures of CD150/CD41/CD48/osteocalcin immunofluorescence on Cxcl12-WT (i-ii) and Cxcl12-cKO (iii-iv) BM at homeostatic state. The CD150⁺CD41⁻CD48⁻ cells are indicated by arrow. The inset is the higher magnification for CD150⁺CD41⁻CD48⁻ cells in Cxcl12-cKO samples. In Cxcl12-WT BM, some of them were associated with CD150⁻CD41⁺CD48⁺ and CD150⁺CD41⁺CD48⁺ progenitor cluster along the bone lining surface (white bracket), but in Cxcl12-cKO BM, these hematopoietic clusters were not detected. Scale bar = 50 μm; inset scale bar = 20 μm. (E) Immunostaining for cKit/Sca-1 combined with osteocalcin on Cxcl12-WT (i-ii) and Cxcl12-cKO (iii-iv) BM at homeostatic state. cKit⁺Sca-1⁺ cells (arrow) can be seen along the bone-lining surface, usually within a cKit⁺ cell cluster, but it was absent at bone-lining surface of Cxcl12-cKO samples. Scale bar = 50 μm.