Subcutaneous transplantation of human BMMSCs reconstitutes active hematopoiesis in adult immunocompromised mice. (A) Micro-computed tomography analysis revealed that bone (B) and bone marrow elements (BM) regenerated in 8-week BMMSC transplants. Original magnification ×200. (B,C) Long-term (12 months) engraftment of BMMSC-mediated bone/marrow formation in subcutaneous transplants showing organized bone (B) and bone marrow components (BM) by hematoxylin and eosin staining (B) and BrdU label retaining assay showing BrdU-positive cells (arrows) in the bone marrow (BM) compartment for 14 weeks after labeling (C). Original magnification ×200. (D) A scheme of eGFP⁺ mouse bone marrow (BM) cells homed to the BMMSC-generated bone/marrow organs. BMMSCs were transplanted subcutaneously into immunocompromised mice (top panel). Eight weeks after transplantation, eGFP⁺ BM cells were injected through the tail vein of the primary transplant recipient (second top panel). The eGFP⁺ BM cells homed to the bone marrow niche in the primary BMMSC transplants (third top panel). Four weeks later, the primary BMMSC transplants were removed as donor transplants for secondary transplantation (fourth top panel). Four weeks after secondary transplantation, peripheral blood was collected for flow cytometric analysis (bottom panel). (E) The secondary transplantations were capable of supplying hematopoietic cells in the circulation of the recipients (n = 3) at 4 weeks after transplantation. Flow cytometric analysis revealed the existence of eGFP lymphocytes, monocytes, and polymorphonuclear leukocytes in peripheral blood leukocytes (PBLs). PBLs of the nontransplanted mice were used as negative controls. An average of 3 mice per group was used in the analysis of each cell subset. Comparative analysis of each cell subset was determined and statistically analyzed (Control, n = 3; Transplant, n = 3; leukocyte percentage, P < .01; R1, P < .005; R2, P < .05; R3, P < .005). Data are representative of 3 independent experiments.