Establishment a human bone marrow microenvironment in NOD/SCID/IL-2rγ^null mice. Human BM-derived MSCs and ECFCs mixed with Matrigel were subcutaneously injected into the flanks of mice to induce the development of extramedullary bones. (A) Eight weeks after implantation, mice were scanned in a VISEN FMT 2500 imaging system to measure osteoblastic activity using OsteoSene 750, and the fluorescence throughout the extramedullary bones was measured. (B) Representative H&E staining (shown at low magnification) shows an overview of the extramedullary bone with the typical bone structures (i). Representative imagines from mouse femur (ii) and human bone biopsy (iii) were displayed as normal bone control. Scale bar represents 1 mm. (C) Higher magnification of the H&E staining shows different kinds of hematopoietic cells with variety of morphologic appearances in the extramedullary bone cavities (i). Cells were flushed from the extramedullary bone, stained with mouse lineage antibodies, and analyzed by flow cytometry (ii). (D) Staining with the hypoxia probe pimonidazole (i-ii) and HIF1α (iii-iv) was performed to determine the presence of hypoxic conditions. Pimonidazole staining was converted to fluorescent (green) signal using the CRi multispectral system. (E) Phase-contrast (i) and confocal fluorescent (ii) images of extramedullary bone stained positive with human collagen type I (COL-1) antibody. (F) Human vasculatures were observed in the extramedullary bones (i) that displayed similar composition as the vessels in normal human bone biopsy (ii). Human CD31⁺ endothelial cells (red) were surrounded by αSMA⁺ pericytes (green). (G) CD31/αSMA/DAPI (i) and H&E (ii) staining displayed typical blood vasculature and bone structure, the adjacent slides were used for FISH (iii,iv); green spots indicate positive hybridization on human X chromosome (white arrows). The figures show 1 representative result of at least 5 experiments.