Impaired endosteal bone formation and HSC niche function in G-CSF–mobilized mice. (A) Mobilization of CFCs, Lin⁻Sca1⁺KIT⁺ (LSK) HSPCs, and Lin⁻CD41⁻Sca1⁺KIT⁺CD48⁻CD150⁺ phenotypic HSCs in blood following G-CSF administration. Data are means ± SD of 4 mice per group. (B) Histomorphometry for osteoblast surface per unit of bone surface (ObS/BS) and osteoid surface per bone surface (OS/BS) in the trabeculae of the proximal tibial secondary spongiosa from mice mobilized for 6 days with G-CSF. Data are mean ± SEM of both tibias from 3-6 mice per group from one representative experiment of 2 independent experiments. (C) Dynamic histomorphometry in mice mobilized with G-CSF. Mice treated with saline or G-CSF for 6 days were injected every third day alternatively with either calcein or xylenol orange and killed at day 12. Tibial sections were analyzed by confocal laser-scanning micrographs (×20). Green and red lines are areas of bone that mineralized and incorporated calcein and xylenol orange, respectively. BM is slightly red due to nonspecific incorporation of xylenol orange by BM cells, whereas solid bone is dark, except where labeled. Top micrographs are across the cortical bone on the anterofibular side, whereas bottom micrographs are across the secondary spongiosa of the metaphysis. (D) The incorporation of calcein lines was quantified in the secondary spongiosa and at the periosteum; shown are percentage of trabecular bone surface covered by double-calcein lines (dLS/BS), trabecular mineral appositional rate (MAR) between double-calcein lines, trabecular bone forming rate corrected for bone surface (BFR/BS), and periosteal mineral appositional (PsMAR). (E) Expression levels of CXCL-12, KL, and Ang-1 mRNA in endothelial cells (□), mesenchymal stromal cells () and osteoblasts (■) sorted from nonmobilized endosteum, and whole BM cells (▨). All expression levels are relative to β2-microglobulin mRNA and are the average of 2 experiments in which populations were sorted from 10 mice each. (F) mRNA for CXCL-12, KL, Ang-1, and fibronectin were quantified at the indicated time points of G-CSF-mobilization by qRT-PCR. All expression levels are relative to β2-microglobulin mRNA. Each symbol represents the result from an individual mouse. Data are means ± SD. ***P < .001, **P < .01, and *P < .05.