The Pulmonary Center, Boston University School of Medicine, Boston, Massachusetts 02118
Submitted 10 January 2003 ; accepted in final form 5 March 2003
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ABSTRACT |
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Hoechst; stem cells; smooth muscle cells; breast cancer resistance protein 1
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.
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METHODS |
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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 <515% 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 (333363 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 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
, 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).
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RESULTS |
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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 (6575%) 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).
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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.
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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.
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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.
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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 (-Sma) as a marker, we determined that 10% of our lung suspension
cells were
-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 -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).
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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 -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
-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).
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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.
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SP cells express lung-related transcription factor HNF-3.
Last, we examined lung SP cells for the expression of the lung-related
transcription factors TTF-1 and HNF-3
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(5,
17,
29). In the adult mouse lung,
both HNF-3
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
, but not TTF-1, is expressed in adult lung SP cells
(Fig. 9). A similar pattern of
expression was identified in marrow SP cells.
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DISCUSSION |
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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 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
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
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.
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ACKNOWLEDGMENTS |
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This work was supported by National Heart, Lung, and Blood Institute Grants RO1 HL-069148-01A1 and R21 HL-072205-01.
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FOOTNOTES |
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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.
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REFERENCES |
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