Detection of CB-derived endothelial cells at the site of thymic implant. (a) Cryostat section from a human female thymic transplant engrafted into a NOD/SCID mouse reconstituted with male CB, stained by hematoxylin/eosin showing the sites of tissue sampling used for DNA preparation. PCR of these DNA samples for human male Y chromosome–specific sequences (b) shows the presence of Y chromosome in tissue within the graft, as well as in the mouse kidney, at the interface with the graft, but not in the mouse kidney distant from the thymic transplant. The DNA products amplified from triplicate samples of the indicated anatomical regions obtained from 3 consecutive sections are shown. The product from a positive control PCR using male CB cells (♂) is also shown on the left. The far left lane represents a 100-bp DNA ladder. The lane on the far right shows a control PCR using Y chromosome–specific primers and no template DNA. (c through f) Confocal microscopy of female thymic grafts from CB/Thy-NOD/SCID mice reconstituted with male CB stained by 2-color FISH using human Y chromosome–specific probes (green fluorescence) and a human specific anti-CD34 MoAb (c and d, red fluorescence) or an anti–PECAM-1 polyclonal antibody (e, red fluorescence). (f) Background fluorescence obtained using the FITC-conjugated antidigoxigenin antibody and isotype-matched control antibodies for the red fluorescence. CD34⁺ cells bearing the male Y chromosome are detected at the interface of the grafts with the mouse kidney (c, arrowheads), and in the pericapsular connective tissue (d, arrowheads). The star in (c) indicates the thymic graft. Y chromosome–bearing cells comprised within vascular structures, unequivocally identified by the PECAM-1 staining (e, red fluorescence, arrowheads), as well as other nonendothelial stromal elements (e, arrows) are also observed at the interface of the grafts with the mouse kidney. Bar in c, d, and f, 63.5 μm; bar in e, 31 μm.