Figure 5
Detection of BM-derived cells in nonhematopoietic tissues after transplantation. Various tissues and organs were imaged intact at day 30 after transplantation of Lin− cells cotransduced with 5FP vectors (A-D,G-I) or Mix 5FP (E-F). Images are presented as extended depth of focus sections in x-y and x-z and y-z directions after computationally removing the outer layers of collagen fibers. In the heart viewed from the epicardial side, (A) numerous individual large fluorescent cells with macrophage-like morphology originating from multiple clones based on the diverse colors seen are visible along the superficial collagen fibers (SHG at 920 nm, white) and also (B) interspersed deeper between cardiomyocytes (white) visualized by their intrinsic 2-photon autofluorescence (at 780 nm). No FP fluorescent cardiomyocytes were observed. In skeletal muscle, (C) fluorescent cells were found within the external fibrous network (SHG, white) but also intercalating between individual striated muscle fibers (SHG, white). In the lung, (D) numerous fluorescent cells with a diverse palette of colors were scattered throughout at all depth levels, with variable morphologies suggesting dendritic cell, macrophage, and type 2 pneumocyte identities. An abdominal skin flap was imaged from the epidermal side (E) and the dermal side (F); fluorescent cells of diverse colors and morphologies were seen, most with large size and morphology suggesting Langerhans or dendritic-like identity, lying under elastin fibers (autofluorescence at 780 nm, white; E) and along collagen (SHG at 920 nm, white) muscle fibers and hair follicles (F). Groups of nearby cells of the same color suggest in situ proliferation of at least some of these cells. In the liver, (G) fluorescent cells with morphology suggestive of stellate cells or macrophages (Kupffer cells) of various colors were aligned along the collagen fiber network (SHG, white) delineating hepatic lobular structures. Nearby cells of the same color suggest in situ proliferation and short-distance migration. Adipose tissue (H) displayed individual cells or clonal clusters of fluorescent macrophage-like cells or small round lymphocyte-like cells interspersed along fibers (SHG, white) and outlining the large adipose cells. In the kidney, (I) examined from the capsule (collagen-SHG at 920 nm, white), rare small fluorescent cells were visible along collagen fibers in the interstitial space between tubules (golden) imaged via 2-photon autofluorescence (780 nm). Scale bars represent 100 μm (A-F), 50 μm (G-I). Related images shown in supplemental Videos 9-15.

Detection of BM-derived cells in nonhematopoietic tissues after transplantation. Various tissues and organs were imaged intact at day 30 after transplantation of Lin− cells cotransduced with 5FP vectors (A-D,G-I) or Mix 5FP (E-F). Images are presented as extended depth of focus sections in x-y and x-z and y-z directions after computationally removing the outer layers of collagen fibers. In the heart viewed from the epicardial side, (A) numerous individual large fluorescent cells with macrophage-like morphology originating from multiple clones based on the diverse colors seen are visible along the superficial collagen fibers (SHG at 920 nm, white) and also (B) interspersed deeper between cardiomyocytes (white) visualized by their intrinsic 2-photon autofluorescence (at 780 nm). No FP fluorescent cardiomyocytes were observed. In skeletal muscle, (C) fluorescent cells were found within the external fibrous network (SHG, white) but also intercalating between individual striated muscle fibers (SHG, white). In the lung, (D) numerous fluorescent cells with a diverse palette of colors were scattered throughout at all depth levels, with variable morphologies suggesting dendritic cell, macrophage, and type 2 pneumocyte identities. An abdominal skin flap was imaged from the epidermal side (E) and the dermal side (F); fluorescent cells of diverse colors and morphologies were seen, most with large size and morphology suggesting Langerhans or dendritic-like identity, lying under elastin fibers (autofluorescence at 780 nm, white; E) and along collagen (SHG at 920 nm, white) muscle fibers and hair follicles (F). Groups of nearby cells of the same color suggest in situ proliferation of at least some of these cells. In the liver, (G) fluorescent cells with morphology suggestive of stellate cells or macrophages (Kupffer cells) of various colors were aligned along the collagen fiber network (SHG, white) delineating hepatic lobular structures. Nearby cells of the same color suggest in situ proliferation and short-distance migration. Adipose tissue (H) displayed individual cells or clonal clusters of fluorescent macrophage-like cells or small round lymphocyte-like cells interspersed along fibers (SHG, white) and outlining the large adipose cells. In the kidney, (I) examined from the capsule (collagen-SHG at 920 nm, white), rare small fluorescent cells were visible along collagen fibers in the interstitial space between tubules (golden) imaged via 2-photon autofluorescence (780 nm). Scale bars represent 100 μm (A-F), 50 μm (G-I). Related images shown in supplemental Videos 9-15.

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