Side population cells and Bcrp1 expression in lung

Ross Summer, Darrell N. Kotton, Xi Sun, Bei Ma, Kathleen Fitzsimmons, and Alan Fine

The Pulmonary Center, Boston University School of Medicine, Boston, Massachusetts 02118

Submitted 10 January 2003 ; accepted in final form 5 March 2003


    ABSTRACT
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Side population (SP) cells are a rare subset of cells found in various tissues that are highly enriched for stem cell activity. SP cells can be isolated by dual-wavelength flow cytometry because of their capacity to efflux Hoechst dye, a process mediated by the ATP-binding cassette transporter breast cancer resistance protein (Bcrp) 1. By performing flow cytometry of enzymedigested mouse lung stained with Hoechst dye, we found that SP cells comprise 0.03–0.07% of total lung cells and are evenly distributed in proximal and distal lung regions. By RT-PCR, we found that lung SP cells express hepatocyte nuclear factor-3{beta}, but not thyroid transcription factor-1. Surface marker analysis revealed lung SP cells to be stem cell antigen 1 positive, Bcrp1 positive, lineage marker negative, and heterogeneous at the CD45 locus. As expected, we did not detect lung SP cells in Bcrp1-deficient animals. We, therefore, employed nonisotopic in situ hybridization and immunostaining for Bcrp1 as a strategy to localize these cells in vivo. Expression was observed in distinct lung cell types: bronchial and vascular smooth muscle cells and round cells within the distal air space. We confirmed the expression of Bcrp1 in primary bronchial smooth muscle cell cultures (BSMC) and in lavaged distal airway cells, but neither possessed the capacity to efflux Hoechst dye. In BSMC, Bcrp1 was localized to an intracellular compartment, suggesting that the molecular site of Bcrp1 expression regulates SP phenotype.

Hoechst; stem cells; smooth muscle cells; breast cancer resistance protein 1


SIDE POPULATION CELLS (SP) have recently been identified in multiple species and have been isolated from various types of tissues, including bone marrow, liver, and muscle (2, 3, 13, 14). These rare cells are highly enriched for stem cell activity, displaying a potent capacity for reconstituting locally damaged tissue. For example, 250 bone marrow-derived SP cells can fully reconstitute lethally irradiated marrow in the mouse (12). In addition, injection of muscle-derived SP cells can replace cardiotoxin-injured skeletal muscle (3). SP cells can also reconstitute tissue types distinct from their site of origin. Injected marrow SP cells can replace injured skeletal muscle and cardiac myocytes, whereas muscle SP cells can fully reconstitute irradiated bone marrow (14, 19). In these studies, injected SP cells were found to assume the morphologic and molecular phenotype of the injured cell type.

SP cells are isolated by high-speed dual-wavelength flow cytometry analysis on the basis of their unique ability to efflux Hoechst dye, a process that requires the action of the ATP-binding cassette (ABC) half-transporter breast cancer resistance protein (Bcrp) 1 (28, 31, 32). Calcium channel blockers inhibit the Bcrp1-dependent efflux of Hoechst dye. Bcrp1 is unique among half-transporter proteins in that it is expressed on the cell surface, rather than only on an internal membrane (27). Although the function of Bcrp1 in SP cells is unknown, its expression on various cancer cell lines mediates resistance to chemotherapeutic drugs (9, 21, 22, 26).

SP cells are not detectable in Bcrp1-deficient mice. Despite this, Bcrp1-deficient mice possess qualitatively normal numbers of functional hematopoietic stem cells (HSC) (30). These findings suggest that Bcrp1 is a marker of HSCs but is not required for stem cell function.

Despite diverse tissue origins, SP cells share a similar surface marker profile; they are stem cell antigen-1 (Sca-1) positive and lineage marker negative (13, 14, 20, 30). Bone marrow-derived SP cells also express the endothelial marker CD31; however, the presence of this marker on other SP populations has yet to be examined (19). Currently, there is little information regarding the existence of lung-derived SP cells. Our primary goals for this study were to identify lung SP cells and to characterize their molecular phenotype. Because Bcrp1 is a marker of SP cells, we also employed immunostaining and in situ hybridization as a strategy to localize Bcrp1-expressing cells in lung tissue.


    METHODS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals. Lung suspensions and tissue blocks were prepared from 4- to 8-wk-old C57Bl/6J mice (Jackson Laboratory, Bar Harbor, ME). Bcrp1-deficient mice were kindly provided by Dr. Brian Sorrentino (St. Jude Children's Research Hospital, Memphis, TN) (31). Animals were euthanized by isoflurane anesthesia followed by cervical dislocation. Before lung extraction, we bled animals by transecting the aorta, and the pulmonary vasculature was perfused with cold saline until lungs turned white. Animal studies were conducted according to protocols approved by the National Institutes of Health and the Boston University Animal Care and Use Committee.

Cell preparations and staining. Cell suspensions were obtained from enzyme-digested lungs and by bronchoalveolar lavage (BAL). We performed lung digestion by finely mincing tissue with a razor blade followed by incubation with 0.1% collagenase (Roche Diagnostics, Indianapolis, IN), 2.4 U/ml dispase (Roche Diagnostics), and 2.5 mM CaCl2 at 37°C for 1.5 h. BAL cells were collected according to published protocols (16). In brief, after cannulation of the trachea, lungs were insufflated with 1 ml of PBS. Wash fluid was removed and placed into a collection tube. This process was repeated until each lung was lavaged with a total of 5 ml of PBS. To remove nonspecific debris, we sequentially filtered cell suspensions through 70- and 40-µm filters and then resuspended them at a concentration of 1 x 106 cells/ml. Hoechst 33342 (5 µg/ml; Sigma-Aldrich, St. Louis, MO) staining of lung and bone marrow cells was performed in the presence and absence of verapamil (50 mM). Staining was performed at 37°C for 90 min in DMEM containing 2% FCS, 10 mM HEPES, and 1% penicillin-streptomycin (13). At the completion of staining, cells were placed immediately on ice. Immunostaining was performed in the dark at 4°C for 30 min with directly fluorochrome-conjugated monoclonal rat anti-mouse antibodies reactive to CD45, CD34, CD31, Sca-1, and c-kit (BD Pharmingen, Lexington, KY). For unconjugated monoclonal rat anti-mouse antibodies to lineage antigens (B220, CD3, CD4, CD8a, CD11b, Gr-1, and Ter119), a labeled secondary antibody was employed. After staining, cells were washed twice and resuspended in HBSS supplemented with 2% FCS. Dead cells were excluded from flow cytometry analysis on the basis of propidium iodide (PI) staining (2 µg/ml). In all studies, dead cells comprised <5–15% of total cells. Isotype control antibodies were employed as negative controls and to establish gating parameters for positive cells.

Fluorescence-activated cell sorting. Flow cytometry analysis of Hoechst-stained cells was performed on a triple laser instrument (MoFlo; Cytomation, Fort Collins, CO). An argon multiline UV (333–363 nm) laser was used to excite Hoechst dye. Fluorescence emission was collected with a 405/30 band pass filter (Hoechst blue) and a 660 ALP (Hoechst red). A second 488-nm argon laser was used to excite phycoerythrin, FITC, and PI. We performed data analysis with Summit software. Flow cytometry experiments not requiring a UV laser were carried out using the Becton Dickinson FACScan and accompanying CELLQuest software (San Jose, CA).

RT-PCR. cDNA was generated from RNA extracts with a reverse transcription kit (Promega, Madison, WI). We performed PCR using the following primers: Bcrp1: 5'-CCAT-AGCCACAGGCCAAAGT-3' and 5'-GGGCCACATGATTCT-TCCAC-3', thyroid transcription factor (TTF)-1 primers 5'-GGTCCTAGTCAAAGACGGCAAAC-3' and 5'-AAGGTAGA-ACAAGACATTGGCGC-3', hepatocyte nuclear factor (HNF)-3{beta} primers 5'-GATGGCTTTCAGGCCCTGCTAG-3' and 5'-GGTCCGGTACACCAGACTCTTAC-3', actin primers 5'-GCTCGTTGCCAATAGTGATG-3' and 5'-AAGAGAGGTATCCTGACCCT-3'. Conditions for Bcrp1 were 94°C 1 min, 56°C 1 min, 72°C 1 min, 35 cycles. Conditions for TTF-1, HNF-3{beta}, and actin were 94°C for 1 min, 55°C for 1 min, 72°C for 1 min, for 35 cycles. For the TTF-1 reaction DMSO was added to the reaction mixture.

Synthesis of probe for in situ hybridization. A 327-bp Bcrp1 cDNA sequence was subcloned into pGEM-T (Promega). For in vitro transcription, linearized plasmid was incubated with T7 or SP6 RNA polymerase, transcription buffer, digoxigenein-labeled UTP, and nucleotides at 37°C for 2 h. Unincorporated nucleotides were removed by ethanol precipitation with glycogen carrier.

Nonisotopic in situ hybridization. The methods are as previously described (10, 11). In brief, rehydrated paraffin-embedded tissue sections were fixed in 4% paraformaldehyde, washed twice in PBS, and treated with a 0.2 N HCl solution for 10 min at room temperature. Slides were again washed in PBS and incubated with proteinase K (5 µg/ml) for 5 min at room temperature before treatment with 1 M triethanolamine-0.25% acetic anhydride. Hybridization with heat-denatured probes at various concentrations was performed overnight in a humidified chamber at 50°C. After hybridization, sections were washed with 50% formamide-2x saline-sodium citrate (SSC) and then with an NaCl (0.5 M)-Tris (10 mM, pH 7.5)-EDTA (1 mM) buffer. Sequential washes were performed with 2x SSC and 0.2x SSC before digestion with RNase A (20 µg/ml) and RNase T1 (1 U/ml). Sections were rewashed and blocked with 2% rabbit serum for 30 min before localization of digoxigenin-labeled cells. This was achieved by application of a sheep polyclonal antidigoxigenin-alkaline phosphatase conjugate (1:250, Roche) applied for 3 h at room temperature. After washing, sections were incubated overnight with a color developing solution (337.5 µg/ml 4-nitro blue tetrazolium chloride, 175 µg/ml 5-bromo-4-chloro-3 indolyl phosphate, and 2 mM levamisole) in a light-free container at 4°C. Color development was terminated by immersion in stop solution (Tris-EDTA). Slides were subsequently counterstained with methyl green. After final washing, slides were dehydrated in graded alcohols and mounted with immersion oil.

Western analysis. To detect Bcrp1 protein during SDS-PAGE and in tissue sections, we generated an affinity-purified rabbit polyclonal antibody to a unique peptide sequence contained within an intracellular region of the mouse Bcrp1 protein. For Western analysis, lung tissue was first homogenized with mortar and pestle. Disrupted lung tissue was incubated in buffer (containing 1% Nonidet P-40, 0.5% sodium deoxycholate, and 150 mM NaCl) in the presence of a cocktail of protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml aprotinin, and 1 µg/ml pepstatin A) for 30 min at 4°C. Lysates were sheared by passing through a 21-gauge needle before centrifugation. We measured the protein concentration in the supernatant using the Bradford assay (4). For analysis, 20 µg of protein were separated through a 10% SDS-polyacrylamide gel before electrophoretic transfer to a nitrocellulose membrane. Membranes were blocked with 5% nonfat dry milk in Tris-buffered saline (TBS) for 2 h. Primary antibody staining (Bcrp1, dilution 1:250) was performed in TBS with 0.1% Tween 20 (TBST) and 5% nonfat milk. Mixture was incubated overnight at 4°C. Secondary antibody staining [anti-rabbit-horse-radish peroxidase (HRP), dilution 1:5,000] was performed for 1 h at room temperature. Membranes were then washed three times with TBST. Chemiluminescence was performed with the ECL Western blotting detection reagent (Amersham Biosciences, Piscataway, NJ).

Immunohistochemistry. Paraffin-embedded tissue sections (5 µm) were incubated with the rabbit anti-mouse Bcrp1 polyclonal antibody at 1:500 dilution (or with preimmune serum) before washing and addition of a secondary goat anti-rabbit antibody conjugated to HRP. Chromogen substrates were subsequently applied, followed by PBS washes and nuclear fast red counterstain.

Primary bronchial smooth muscle cultures. Human primary bronchial smooth muscle cells (Cambrex, East Rutherford, NJ) were cultured according to the manufacturer's recommendations. Cells were passed upon reaching 80% confluence. To suspend adherent cells for flow cytometry analysis cell, we used an enzyme-free cell dissociation buffer (GIBCO, Carlsbad, CA).


    RESULTS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cells with an SP phenotype exist in the adult mouse lung. The ability to detect SP cells depends on their capacity to efflux Hoechst dye, a process that is inhibited by calcium channel blockers. To evaluate the presence of SP cells in the adult murine lung, we Hoechststained enzyme-digested lung suspensions before flow cytometry. Density dot-plot analysis (Hoechst red vs. Hoechst blue) confirmed the presence of SP cells in the lung (Fig. 1A). Notably, the appearance of the SP phenotype was blocked by verapamil (50 mM) (Fig. 1B). The percentage of SP cells ranged from 0.03 to 0.07%, which remained constant over varying mouse age ranges (postnatal day 5 to 1 yr of age).



View larger version (35K):
[in this window]
[in a new window]
 
Fig. 1. Hoechst staining of enzyme-digested lung. Propidium iodide-positive dead cells (far right) are gated from analysis. Red blood cells (RBCs) stain negative due to the absence of DNA. A: side population (SP) cells (boxed area, top arrow) are distinguished from the main population. B: SP cells are undetectable (top arrow) following treatment with calcium channel blockers.

 

Surface phenotype of lung SP cells. To examine the expression of surface markers on lung SP cells, we incubated Hoechst-stained lung suspensions with various antibodies. We found lung SP cells to be heterogeneous, consisting of CD45-positive (65–75%) and CD45-negative cells (Table 1). CD45-positive SP cells were further distinguished from CD45-negative cells by the coexpression of CD31 and the absence of CD34. Both CD45-positive and CD45-negative populations expressed Sca-1 and were negative for lineage markers (B220, CD3, CD4, CD8a, CD11b, Gr-1, and Ter119). We were unable to assess expression of c-kit antigen due to effects of enzymatic digestion (data not shown).


View this table:
[in this window]
[in a new window]
 
Table 1. Surface phenotype of lung SP cells

 

Bcrp1 is required for lung SP detection. Recently, Zhou et al. (31, 32) determined that bone marrow-derived SP cells require surface expression of the ABC transporter protein Bcrp1 for the efflux of Hoechst dye and the resultant detection of SP cells. To assess expression of Bcrp1 on lung SP cells, we produced an affinity-purified anti-mouse Bcrp1 antibody generated to an internal peptide sequence. Using this antibody as a probe during Western analysis of total lung proteins, we detected a single protein band with a molecular mass of ~72 kDa, the expected size of Bcrp1 (Fig. 2A). These findings confirm expression of Bcrp1 protein in lung tissue and the specificity of this polyclonal antibody.



View larger version (44K):
[in this window]
[in a new window]
 
Fig. 2. A: Western analysis of lung lysate using our polyclonal anti-breast cancer resistance protein (Bcrp) 1 antibody demonstrates a single ~72-kDa band (arrow). B: immunoperoxidase staining of lung SP cells following incubation with polyclonal anti-Bcrp1 antibody (top) or preimmune serum (bottom).

 

Next, we collected sorted SP cells onto glass slides and utilized this antibody for immunostaining. In this study, lung SP cells stained positive for Bcrp1 protein in a pattern consistent with surface expression. Staining was absent in cells incubated with preimmune serum (Fig. 2B). Lung SP cells were uniform in size and shape.

To confirm that Bcrp1 expression is required for detection of lung SP cells, we performed Hoechst staining of lung digests from Bcrp1-deficient mice (31). We found that Bcrp1-deficient animals do not display a lung SP population (Fig. 3). Together, these findings confirm the expression of Bcrp1 in lung SP cells and its requirement for establishing the SP phenotype in the lung.



View larger version (31K):
[in this window]
[in a new window]
 
Fig. 3. Hoechst staining of wild-type (A) and Bcrp1-/- (B) lung digests. SP cells (boxed areas) are undetectable in Bcrp1-/- lungs.

 

Localization of Bcrp1 in the lung. To localize Bcrp1-expressing cells, we performed nonisotopic in situ hybridization (NISH) and immunostaining on paraffin-embedded lung sections. As shown in Fig. 4, we determined that Bcrp1 mRNA expression (purple staining) was restricted to two cell populations: smooth muscle cells and a subpopulation of round cells in the alveolar space. Using the affinity-purified polyclonal antibody, immunostaining demonstrated Bcrp1 protein expression (brown staining) restricted to these same cell types (Fig. 5). In these two locations, the pattern of protein staining was suggestive of cytoplasmic localization. Expression of Bcrp1 was not detectable in endothelial cells or epithelial cells.



View larger version (57K):
[in this window]
[in a new window]
 
Fig. 4. Nonisotopic in situ hybridization for Bcrp1. Bcrp1 expression (purple cytoplasmic staining) was detected in 2 populations: smooth muscle cells and a subpopulation of cells in the distal air space. A: staining of vascular (V) smooth muscle. B: airway (A) smooth muscle. C: distal airway cell (AL). Bottom: absence of staining with sense strand in vessel (D) and airway (E).

 


View larger version (64K):
[in this window]
[in a new window]
 
Fig. 5. Immunoperoxidase staining (brown staining) of paraffin-embedded lung sections for Bcrp1. A: blood vessel (V) staining positive for Bcrp1. B: high-power view of blood vessel with endothelium (EN) detached from underlying Bcrp1-expressing smooth muscle cells (SM). C: airway epithelium (E) stains negative, underlying smooth muscle stains positive for Bcrp1. D: Bcrp1-expressing cell residing in alveolar space (AL). Bottom: isotype control sections from vessel (E) and airway (F).

 

Smooth muscle cells express Bcrp1 but do not contribute to the SP phenotype. Our findings indicate a marked discrepancy between the number of Bcrp1-expressing cells within the lung, as noted by histological study and the small percentage of SP cells detected by Hoechst staining. Because tissue digestion may selectively isolate specific cell types, we examined our lung digests for the presence of smooth muscle cells. Using smooth muscle alpha actin ({alpha}-Sma) as a marker, we determined that 10% of our lung suspension cells were {alpha}-Sma positive (data not shown). These findings are consistent with previous morphometric analyses of the lung (7, 15).

To verify expression of Bcrp1 in lung-derived smooth muscle cells, we employed primary human bronchial smooth muscle cells. Utilizing a FITC-conjugated antibody directed to the extracellular domain of human Bcrp1, we stained formalin-fixed bronchial smooth muscle cells. Analysis by deconvolution microscopy indicate that Bcrp1 expression was restricted to an intracellular compartment (data not shown). To confirm intracellular expression, we immunostained permeabilized (saponin-treated) and unpermeabilized smooth muscle cells before flow cytometry analysis. Bcrp1 expression was detected only in permeabilized cells (Fig. 6A). As a control for our permeabilization reaction, we stained cells with an antibody to {alpha}-Sma. Next, we examined whether intracellular expression of Bcrp1 enables smooth muscle cells to efflux Hoechst dye. To do this we Hoechst-stained bronchial smooth muscle cells according to our established protocol. Density dot-plot analysis of stained cells failed to detect a visible SP (Fig. 6B).



View larger version (26K):
[in this window]
[in a new window]
 
Fig. 6. A: Bcrp1 is located within an internal compartment of human bronchial smooth muscle cells (BSMC). Top: flow cytometry analysis of BSMC stained with FITC-conjugated smooth muscle alpha actin ({alpha}-Sma) antibody (diagonal hatched) or isotype control (vertical hatched) in unpermeabilized (left) and saponin-treated cells (right). Bottom: BSMC stained with FITC-conjugated anti-Bcrp1 antibody (diagonal hatched) or isotype control (vertical hatched) in unpermeabilized (left) or saponin (right)-treated cells. B: BSMC do not efflux Hoechst dye. Hoechst staining of BSMC demonstrates the absence of an SP (arrow).

 

Absence of Hoechst efflux in primary cells in culture, however, does not exclude the possibility that SP cells in the lung in vivo represent a rare subpopulation of smooth muscle cells. To investigate this further we examined sorted mouse lung SP cells for the expression of {alpha}-Sma. By flow cytometry analysis of permeabilized SP cells and by immunostaining lung SP cells sorted onto glass slides, we did not detect expression of {alpha}-Sma.

Bcrp-expressing cells in the airway are not SP cells. On the basis of our histology findings, we next examined whether Bcrp1-expressing cells within the distal airway possess the ability to efflux Hoechst dye. To sample distal airway cells, we utilized BAL. First, we confirmed our ability to isolate Bcrp1-expressing cells by this technique. To do this, we pooled BAL samples from 10 mice; analysis of lavaged cells by flow cytometry demonstrates that Bcrp1-expressing cells were recoverable (data not shown). We subsequently examined whether lavaged cells efflux Hoechst dye. Similar to smooth muscle cells, we found that Bcrp1-expressing cells within the distal airway do not efflux Hoechst dye. These findings demonstrate that these cells do not contribute to the SP phenotype (Fig. 7).



View larger version (73K):
[in this window]
[in a new window]
 
Fig. 7. A: SP cells are absent from Hoechst-stained bronchoalveolar lavage cells. B: verapamil had no effect. C: SP cells are detected in bone marrow cells stained at the same time. D: efflux of Hoechst dye by bone marrow cells is blocked by verapamil.

 

SP cells are evenly distributed in the proximal and distal lung. We examined whether SP cells selectively reside in the proximal or distal lung. After gross dissection, we performed separate enzyme digestions and Hoechst staining of proximal and distal regions of the lung (Fig. 8). These results demonstrate equal numbers of SP cells distributed in both compartments.



View larger version (45K):
[in this window]
[in a new window]
 
Fig. 8. Separation of distal (A) and proximal (B) regions of the lung followed by Hoechst staining demonstrated similar percentages of SP cells (boxed areas) in both anatomical regions.

 

SP cells express lung-related transcription factor HNF-3{beta}. Last, we examined lung SP cells for the expression of the lung-related transcription factors TTF-1 and HNF-3{beta}. (5, 17, 29). In the adult mouse lung, both HNF-3{beta} and TTF-1 are expressed in the lung epithelium. These factors are thought to regulate expression of lung-specific genes (5, 6, 17, 29). By RT-PCR, we determined that HNF-3{beta}, but not TTF-1, is expressed in adult lung SP cells (Fig. 9). A similar pattern of expression was identified in marrow SP cells.



View larger version (12K):
[in this window]
[in a new window]
 
Fig. 9. RT-PCR of lung (L-SP) and marrow (BM-SP) SP cells, whole lung (L), and muscle (M) for thyroid transcription factor (TTF)-1, hepatocyte nuclear factor (HNF)-3{beta}, and actin. HNF-3{beta} but not TTF-1 was detected from SP cell cDNA. Both TTF-1 and HNF-3{beta} were present in whole lung and absent in muscle cDNA. Actin primers from different exons were utilized to ensure the fidelity of cDNA and the absence of genomic DNA.

 


    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In this study, we identified SP cells in the adult murine lung. Similar to SP cells detected in other tissues, lung SP cells represent a small fraction of the total cell population. The percentage of SP cells remained constant between various experiments and over a range of animal ages. Notably, in preliminary work, we identified the presence of SP cells in the embryonic lung (data not shown).

The surface characteristic, Sca1 positive and lineage negative, parallels the phenotype of SP cells isolated from other tissues (13, 14, 30). Although c-kit expression is detected on the surface of bone marrow SP cells, the lack of expression in lung, and possibly muscle SP cells, may be due to molecular cleavage during tissue digestion (13, 14). Importantly, we found that dispase and collagenase cleaved c-kit from the surface of marrow cells (data not shown).

Lung SP cells are heterogeneous at the CD45 locus, a feature that may have physiological relevance. Along these lines, McKinney-Freeman et al. (24) demonstrated that Sca-1+, CD45+ muscle cells have hematopoietic potential, whereas Sca1+, CD45-muscle cells have myogenic capacity. Interestingly, muscle SP cells are derived from the bone marrow (23). Lung and bone marrow SP cells also express the endothelial marker CD31. Several recent studies have suggested that endothelial cells and HSC are derived from a common origin (1, 8, 18, 25). The coexpression of the hematopoietic marker CD45 and CD31 on lung SP cells further supports this link. Expression of endothelial markers suggests the possibility that lung SP cells play a role in the homeostasis of the pulmonary vasculature. Notably, injection of marrow-derived SP cells into the injured heart can reconstitute cardiac endothelium (19).

To begin to evaluate lung-related genes in SP cells, we examined HNF-3{beta} and TTF-1 expression by RT-PCR; these factors are thought to play key roles in the transcriptional activation of lung genes (5). In this study, we found that lung and marrow SP cells express HNF-3{beta} but not TTF-1. This finding, along with our data showing a similar surface phenotype, suggests a common origin for these two SP cell populations. Importantly, HNF-3{beta} regulates TTF-1 expression (5, 6, 17, 29). The relevance of these observations is under study.

In an attempt to localize the lung SP cell, we examined Bcrp1 expression by NISH and immunostaining. Both methods showed that Bcrp1 is present in two apparent populations of cells: smooth muscle cells and a subpopulation of cells within the distal air space. Further examination of smooth muscle and airway cells demonstrated that Bcrp1 expression does not confer the SP phenotype in either cell type. Our localization data suggest that Bcrp1 expression may be intracellular in these two cell types. Whether Bcrp1 is mobilized to the surface in these cells remains uncertain. Indeed, the relative function of intracellular Bcrp1 needs to be further elucidated. In contrast, our data and those of others indicate that Bcrp1 is expressed on the surface of SP cells (27, 28).

We found that the percentage of SP cells was the same in digests of proximal and distal lung. These data indicate that SP cells are not selectively localized in the gas-exchange regions of the lung. Overall, we were unable to precisely localize the lung SP cell in vivo. We believe this is due to 1) the rarity of the cell and 2) the unanticipated expression of the Bcrp1 marker in other cell types. Precise localization may require identification of other unique markers in this cell population. A key issue will be deciphering whether this cell resides in a specific parenchymal or vascular niche.

In conclusion, we have confirmed that SP cells reside in the adult mouse lung and possess a surface phenotype similar to SP cells derived from other organs. We speculate that the dysregulation of lung SP cell function may contribute to the pathogenesis of specific lung diseases. Future work will be directed at examining the ability of these cells to differentiate into lung, blood, and other cell types in vitro and in vivo. It should be noted, however, that the inadvertent enzymatic digestion of key surface molecules (i.e., c-kit) may be a confounding factor that adversely affects the outcome of experiments that employ SP cells derived from solid organs.


    ACKNOWLEDGMENTS
 
We thank Alan Ho for assistance with cell sorting experiments.

This work was supported by National Heart, Lung, and Blood Institute Grants RO1 HL-069148-01A1 and R21 HL-072205-01.


    FOOTNOTES
 

Address for reprint requests and other correspondence: R. Summer, The Pulmonary Center, Boston Univ. School of Medicine, 80 E. Concord St., Boston, MA 02118 (E-mail: rsummer{at}lung.bumc.bu.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


    REFERENCES
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Asahara T, Murohara T, Sullivan A, Silver M, van der Zee R, Li T, Witzenbichler B, Schatteman G, and Isner JM. Isolation of putative progenitor endothelial cells for angiogenesis. Science 275: 964-967, 1997.[Abstract/Free Full Text]
  2. Asakura A and Rudnicki MA. Side population cells from diverse adult tissues are capable of in vitro hematopoietic differentiation. Exp Hematol 30: 1339-1345, 2002.[ISI][Medline]
  3. Asakura A, Seale P, Girgis-Gabardo A, and Rudnicki MA. Myogenic specification of side population cells in skeletal muscle. J Cell Biol 159: 123-134, 2002.[Abstract/Free Full Text]
  4. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254, 1976.[ISI][Medline]
  5. Cardoso WV. Transcription factors and pattern formation in the developing lung. Am J Physiol Lung Cell Mol Physiol 269: L429-L442, 1995.[Abstract/Free Full Text]
  6. Cardoso WV. Molecular regulation of lung development. Annu Rev Physiol 63: 471-494, 2001.[ISI][Medline]
  7. Crapo JD, Barry BE, Gehr P, Bachofen M, and Weibel ER. Cell number and cell characteristics of the normal human lung. Am Rev Respir Dis 126: 332-337, 1982.[ISI][Medline]
  8. Demeule M, Labelle M, Regina A, Berthelet F, and Beliveau R. Isolation of endothelial cells from brain, lung, and kidney: expression of the multidrug resistance P-glycoprotein isoforms. Biochem Biophys Res Commun 281: 827-834, 2001.[ISI][Medline]
  9. Doyle LA, Yang W, Abruzzo LV, Krogmann T, Gao Y, Rishi AK, and Ross DD. A multidrug resistance transporter from human MCF-7 breast cancer cells. Proc Natl Acad Sci USA 95: 15665-15670, 1998.[Abstract/Free Full Text]
  10. Fine A, Anderson NL, Rothstein TL, Williams MC, and Gochuico BR. Fas expression in pulmonary alveolar type II cells. Am J Physiol Lung Cell Mol Physiol 273: L64-L71, 1997.[Abstract/Free Full Text]
  11. Gochuico BR, Zhang J, Ma BY, Marshak-Rothstein A, and Fine A. TRAIL expression in vascular smooth muscle. Am J Physiol Lung Cell Mol Physiol 278: L1045-L1050, 2000.[Abstract/Free Full Text]
  12. Goodell MA, Brose K, Paradis G, Conner AS, and Mulligan RC. Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo. J Exp Med 183: 1797-1806, 1996.[Abstract]
  13. Goodell MA, Rosenzweig M, Kim H, Marks DF, DeMaria M, Paradis G, Grupp SA, Sieff CA, Mulligan RC, and Johnson RP. Dye efflux studies suggest that hematopoietic stem cells expressing low or undetectable levels of CD34 antigen exist in multiple species. Nat Med 3: 1337-1345, 1997.[ISI][Medline]
  14. Gussoni E, Soneoka Y, Strickland CD, Buzney EA, Khan MK, Flint AF, Kunkel LM, and Mulligan RC. Dystrophin expression in the mdx mouse restored by stem cell transplantation. Nature 401: 390-394, 1999.[ISI][Medline]
  15. Haies DM, Gil J, and Weibel ER. Morphometric study of rat lung cells. I. Numerical and dimensional characteristics of parenchymal cell population. Am Rev Respir Dis 123: 533-541, 1981.[ISI][Medline]
  16. Hessel EM, Cruikshank WW, Van Ark I, De Bie JJ, Van Esch B, Hofman G, Nijkamp FP, Center DM, and Van Oosterhout AJ. Involvement of IL-16 in the induction of airway hyper-responsiveness and up-regulation of IgE in a murine model of allergic asthma. J Immunol 160: 2998-3005, 1998.[Abstract/Free Full Text]
  17. Ikeda K, Shaw-White JR, Wert SE, and Whitsett JA. Hepatocyte nuclear factor 3 activates transcription of thyroid transcription factor 1 in respiratory epithelial cells. Mol Cell Biol 16: 3626-3636, 1996.[Abstract]
  18. Ivanova NB, Dimos JT, Schaniel C, Hackney JA, Moore KA, and Lemischka IR. A stem cell molecular signature. Science 298: 601-604, 2002.[Abstract/Free Full Text]
  19. Jackson KA, Majka SM, Wang H, Pocius J, Hartley CJ, Majesky MW, Entman ML, Michael LH, Hirschi KK, and Goodell MA. Regeneration of ischemic cardiac muscle and vascular endothelium by adult stem cells. J Clin Invest 107: 1395-1402, 2001.[Abstract/Free Full Text]
  20. Jackson KA, Mi T, and Goodell MA. Hematopoietic potential of stem cells isolated from murine skeletal muscle. Proc Natl Acad Sci USA 96: 14482-14486, 1999.[Abstract/Free Full Text]
  21. Kawabata S, Oka M, Shiozawa K, Tsukamoto K, Nakatomi K, Soda H, Fukuda M, Ikegami Y, Sugahara K, Yamada Y, Kamihira S, Doyle LA, Ross DD, and Kohno S. Breast cancer resistance protein directly confers SN-38 resistance of lung cancer cells. Biochem Biophys Res Commun 280: 1216-1223, 2001.[ISI][Medline]
  22. Litman T, Brangi M, Hudson E, Fetsch P, Abati A, Ross DD, Miyake K, Resau JH, and Bates SE. The multidrug-resistant phenotype associated with overexpression of the new ABC half-transporter, MXR (ABCG2). J Cell Sci 113: 2011-2021, 2000.[Abstract/Free Full Text]
  23. Majka SM, Jackson KA, Kienstra KA, Majesky MW, Goodell MA, and Hirschi KK. Distinct progenitor populations in skeletal muscle are bone marrow derived and exhibit different cell fates during vascular regeneration. J Clin Invest 111: 71-79, 2003.[Abstract/Free Full Text]
  24. McKinney-Freeman SL, Jackson KA, Camargo FD, Ferrari G, Mavilio F, and Goodell MA. Muscle-derived hematopoietic stem cells are hematopoietic in origin. Proc Natl Acad Sci USA 99: 1341-1346, 2002.[Abstract/Free Full Text]
  25. Reyes M, Dudek A, Jahagirdar B, Koodie L, Marker PH, and Verfaillie CM. Origin of endothelial progenitors in human postnatal bone marrow. J Clin Invest 109: 337-346, 2002.[Abstract/Free Full Text]
  26. Robey RW, Medina-Perez WY, Nishiyama K, Lahusen T, Miyake K, Litman T, Senderowicz AM, Ross DD, and Bates SE. Overexpression of the ATP-binding cassette half-transporter, ABCG2 (Mxr/BCrp/ABCP1), in flavopiridol-resistant human breast cancer cells. Clin Cancer Res 7: 145-152, 2001.[Abstract/Free Full Text]
  27. Rocchi E, Khodjakov A, Volk EL, Yang CH, Litman T, Bates SE, and Schneider E. The product of the ABC half-transporter gene ABCG2 (BCRP/MXR/ABCP) is expressed in the plasma membrane. Biochem Biophys Res Commun 271: 42-46, 2000.[ISI][Medline]
  28. Scharenberg CW, Harkey MA, and Torok-Storb B. The ABCG2 transporter is an efficient Hoechst 33342 efflux pump and is preferentially expressed by immature human hematopoietic progenitors. Blood 99: 507-512, 2002.[Abstract/Free Full Text]
  29. Stahlman MT, Gray ME, and Whitsett JA. Temporal-spatial distribution of hepatocyte nuclear factor-3beta in developing human lung and other foregut derivatives. J Histochem Cytochem 46: 955-962, 1998.[Abstract/Free Full Text]
  30. Welm BE, Tepera SB, Venezia T, Graubert TA, Rosen JM, and Goodell MA. Sca-1(pos) cells in the mouse mammary gland represent an enriched progenitor cell population. Dev Biol 245: 42-56, 2002.[ISI][Medline]
  31. Zhou S, Morris JJ, Barnes Y, Lan L, Schuetz JD, and Sorrentino BP. Bcrp1 gene expression is required for normal numbers of side population stem cells in mice, and confers relative protection to mitoxantrone in hematopoietic cells in vivo. Proc Natl Acad Sci USA 99: 12339-12344, 2002.[Abstract/Free Full Text]
  32. Zhou S, Schuetz JD, Bunting KD, Colapietro AM, Sampath J, Morris JJ, Lagutina I, Grosveld GC, Osawa M, Nakauchi H, and Sorrentino BP. The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype. Nat Med 7: 1028-1034, 2001.[ISI][Medline]