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Bone marrow-derived cells as progenitors of lung alveolar epithelium

The Pulmonary Center, Boston University School of Medicine, 715 Albany Street, Boston, Massachusetts 02118, USA

View larger version (28K): [in a new window]   Fig. 1. Confirmation of donor origin of lung-engrafted X-gal-positive cell cluster. Bone marrow cells from a donor mouse transgenic for a lacZ/neomycin resistance fusion gene (lacZ/neo) were injected into a wild-type recipient with bleomycin-injured lungs. Recipient lungs were harvested at one month and stained with X-gal to identify injected cells. (A) Donor-derived lacZ-positive cells can be seen under a dissecting microscope as a diffuse area of blue staining. (B) The blue cell cluster seen in A was microdissected for DNA extraction and PCR for the lacZ/neo fusion gene. Donor lung DNA served as a positive control whereas wild-type lung DNA served as a negative control. To confirm fidelity of PCR, the mouse surfactant C gene (SP-C) was also amplified in each sample. (–), Negative control; R, recipient lung; +, positive control. Bar, 100 µm.

View larger version (115K): [in a new window]   Fig. 2. Paraffin and plastic sections of X-gal-stained mouse lungs 30 days after cell injection. (A) Low magnification (40x) and (B) high magnification (1000x) views of a representative recipient lung showing engrafted donor-derived cells as thin, flat blue cells consistent with a type I pneumocyte morphology. (C) For comparison, an X-gal-stained negative control lung (lacZ-negative marrow injected into a lacZ-negative recipient) shows no blue cells. (D) A thin plastic section (2 µm) of recipient mouse lung confirms the type I pneumocyte morphology of engrafted donor-derived cells. Sections are counter-stained with Nuclear Fast Red. Bars, 200 µm (A); 30 µm (B-D).

View larger version (26K): [in a new window]   Fig. 3. Summary of lung engraftment in animals sacrificed 30 days after injection of lacZ-positive or wild-type (Wt) cultured plastic-adherent bone marrow cells (BM). Injections were performed 5 days after intra-tracheal (IT) bleomycin (Bleo) or control vehicle (PBS) exposure. There was a statistically significant increase in the proportion of mice showing lung engraftment following bleomycin treatment (*P<0.02). No animals that received wild-type (non-lacZ) cells displayed positive staining (0/9).

View larger version (82K): [in a new window]   Fig. 4. Co localization of Lycopersicon esculentum lectin and X-gal staining of engrafted type I pneumocytes. (A) Type I pneumocytes (arrows) lining an alveolus (Alv) stain blue indicating their origin from lacZ-expressing bone marrow cells. No X-gal staining was found in the adjacent type II cell (T2). (B) This same section after binding to L. esculentum lectin shows that the brown lectin staining co-localizes with the blue cells. The lectin staining stops abruptly at the junction of positively stained type I cells and the negatively stained type II cell. Bar, 7 µm.

View larger version (85K): [in a new window]   Fig. 5. Double labeling for T1 (brown) and lacZ expression (blue) in donor (A-C) and recipient (D,E) lungs. A-C show donor lungs from lacZ-positive transgenic mice that constitutively express lacZ in all cells. These panels demonstrate that: (1) in the lacZ mouse X-gal histochemistry stains all lacZ-expressing lung cells blue; (2) immunohistochemical brown staining of a type I pneumocyte membrane protein produces a characteristic linear staining pattern; (3) X-gal staining does not alter the specificity of the T1 antibody. Consistent with the specificity of this type I cell marker, type II cells (A, arrow), endothelium (B, arrow), and macrophages (C, arrow) are T1 negative. D and E show sections from an experimental wild-type recipient mouse that was injected with donor lacZ-positive marrow-derived cells. A representative alveolus contains blue lacZ-positive flattened engrafted cells (D). This section was then re-stained with the T1 monoclonal antibody (E; brown staining). All donor-derived blue cells are also positive for T1 protein staining (arrows) identifying them as type I pneumocytes. An endogenous type I pneumocyte (lacZ negative, T1 positive) is also indicated (arrowheads). Bars, 20 µm (A-C); 9 µm (D,E).

View larger version (100K): [in a new window]   Fig. 6. Paraffin section of recipient injured lung 5 days after injection of lacZ-positive marrow-derived cells. Linear blue staining indicates engraftment of lacZ-positive donor marrow-derived cells as type I pneumocytes as in the 30 day recipients. No blue type II pneumocytes were observed. Bars, 40 µm (A); 20 µm (B).

View larger version (106K): [in a new window]   Fig. 7. Donor bone-marrow-derived cells (blue) in recipient bleomycin-injured lungs 1 and 2.5 days after injection. (A,B) Ovoid donor cells against a pulmonary blood vessel wall (arrow; A) and in the injured lung parenchyma (arrow; B) 1 day after injection. (C,D) By 2.5 days, donor-derived cells can be found in the lung parenchyma as elongated cells (arrows) with attenuated cytoplasmic extensions (arrowheads). As shown in A-D, engrafted cells do not exhibit the location and shape of type II cells. (E) Phase microscopy of D demonstrating that engrafted cells do not contain lamellar bodies. For comparison, two neighboring endogenous type II cells (lacZ negative; open arrows) with characteristic lamellar bodies are shown. (F) PCNA immunostained lung section with a blue donor-derived cell 2.5 days after injection. This nucleus (arrow) does not stain for proliferating cell nuclear antigen (PCNA, brown). In contrast, a neighboring lacZ-negative cell stains positively for PCNA (open arrow; nuclei counter stained purple with Haematoxylin).

View larger version (122K): [in a new window]   Fig. 8. Histological analyses of adherent bone marrow cultures. (A) Three cell morphologies are discernible: larger polygonal fibroblast-like cells (black arrow), smaller stellate myeloid cells (red arrow), and small round endothelial-like cells (purple arrow). (B-D) Immunostaining of cultures with anti-mouse T1 antibody (brown staining; B, arrows). (C) At higher magnification, the T1-positive cell demonstrates fibroblast-like morphology. (D) Staining within a cell colony is heterogeneous. Bars, 20 µm (A); 200 µm (B); 15 µm (C); 25 µm (D).

View larger version (23K): [in a new window]   Fig. 9. Nested RT-PCR analysis for lung epithelial markers in plastic-adherent cultured marrow cells (7 days) and fresh bone marrow. Positive expression of T1 and aquaporin-5 (AQ5) was noted under both conditions. Surfactant protein-C (SPC) was not expressed.

View larger version (81K): [in a new window]   Fig. 10. Immunostaining for T1 in whole decalcified mouse bone marrow. (A) Low power and (B) high power view of elongated T1-positive cells (arrows) on interior surface of bone, facing the marrow cavity. Cells within the marrow cavity were negative. bn, bone; BM, bone marrow cavity; Fast Nuclear Red counter stain; Bar, 25 µm.