Ectopic and orthotopic transplantation of cultured cells into immunodeficient mice. Multiclonal cultures generated from CD271⁺/CD45⁻/CD146^−/low and CD271⁺/CD45⁻/CD146⁺ primary BM-MNCs were transplanted either subcutaneously (with HA/TCP particles) or intrafemorally into immunodeficient mice. (A-B) Representative section of hematoxylin/eosin–stained transplanted cells and HA/TCP carrier particles 8 weeks after subcutaneous transplantation of CD271⁺/CD45⁻/CD146⁺–derived MSCs. (A) Bone (b), adipocytes (a), fibroblastic tissue (ft), and capillaries (*) are indicated. Dark brown areas in the left photograph indicate HA/TCP carrier particles. Black square in the left photograph is shown as a magnification (original magnification 10×). (B) Invading hematopoietic cells could be detected in the transplanted cells (black arrow). Original magnification was 10×. Scale bar indicates 50 μm. (C) GFP⁺ cells generated from either CD271⁺/CD45⁻/CD146^−/low (top row) or CD271⁺/CD45⁻/CD146⁺ (bottom row) cells analyzed 8 weeks after intrafemoral transplantation. GFP⁺ cells (green) could be detected as bone-lining cells, some of which expressed N-cadherin (N-Cad, red) (white arrows, left panels), and in perivascular regions surrounding the endothelium (CD31, red) (white arrowheads, right panels). (D) Reticular GFP⁺ cells (green) could be detected independently from vessels in the marrow space (white arrowheads). The photograph shows reticular GFP⁺ cells generated from CD271⁺/CD45⁻/CD146^−/low cells. (E) Bone marrow section from a control mouse (injected with Dulbecco PBS only) stained with anti–GFP and CD31 antibody. Nuclei were stained with TO-PRO3 (blue). Cortical bone (b) is indicated. Scale bars indicate 25 μm. The photographs were taken with a confocal microscope (DMRE; Leica).