In situ visualization of human MSCs and hematopoietic cells in the murine BM compartment at 4 weeks after IBMT or intravenous transplantation. Transduction efficiencies of eYFP into CBCD34 cells and eGFP into MSCs, determined by flow cytometric analysis at the day of transplantation, were approximately 65% (A) and 99% (B), respectively. The shaded histograms indicate nontransduced cells. (C-E) The presence of human hematopoietic cells was determined by eYFP fluorescence and immunofluorescence staining with an anti-human CD45 antibody followed by Alexa-fluor 594 goat anti-mouse immunoglobulin secondary antibody. Cell nuclei were visualized by staining with TOTO3. Similar amounts of eYFP-cells expressing human CD45 (red) are present in BM after intravenous transplantation (C) or IBMT (D). (E) No eYFP- or CD45-reactive cells are present in the BM of noninjected mouse. (F) eGFP-MSCs are rarely seen in BM after intravenous transplantation (white arrowhead). After IBMT, eGFP-MSCs are easily identified in the BM cavity by eGFP fluorescence (G) and immunohistochemistry (H). (G-H) The same section was stained with an anti-GFP antibody after examining for eGFP fluorescence. All bars represent 10 μm. Images in panels C-G were obtained using an LSM510 META confocal microscope; image in panel H was obtained using an Olympus AX80 microscope and a 20 ×/0.7 NA UPlanApo objective lens.