Figure 5

Analysis of alveolar epithelial cell-like transdifferentiation of CB-CD34-derived cells. Confocal fluorescence microscopy of lungs subjected to combined anti-human-cytokeratin (green) and anti-mouse/human SP-C (red) immunofluorescence staining. A-B: Representative micrograph of lungs of animal treated with freshly isolated CD34+ cells. SP-C-positive human-derived epithelial cells were readily visualized (arrows). C: Similar double immunofluorescence staining showing one exceedingly rare SP-C-immunoreactive human-derived cell (arrow) observed in an animal treated with expanded cells. A-C. Merging of the images shows colocalization of granular SP-C-positive material in the CK-positive, human-derived epithelial cells. The SP-C-positive material is coarsely granular, consistent with surfactant-containing lamellar bodies. Colocalization persists at all three-dimensional volume angles analyzed. Volume slices along the xz (horizontal) and yz (vertical) axes both show colocalization of green and red signals in the same cell, resulting in a yellow-orange composite signal and confirming the unequivocal presence of SP-C-positive material in this human CK-positive, human cord blood-derived epithelial cell. A-C: Anti-human cytokeratin stain (green) combined with anti-human/mouse SP-C stain (red), DAPI counterstain. Scale bar = 10 μm.