Departments of 1Environmental and Occupational Health, 2Cell Biology and Physiology, and 3Radiation Oncology, University of Pittsburgh, Pittsburgh, Pennsylvania 15260
Submitted 14 May 2003 ; accepted in final form 30 July 2003
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ABSTRACT |
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stem cell; Clara; lung; Hoechst 33342
Intrapulmonary stem cell pools exhibit the molecular property of Clara cell secretory protein (CCSP) expression, yet have been distinguished from Clara cells by their resistance to the Clara cell-specific toxicant naphthalene. Properties of these variant CCSP-expressing (vCE) cells include infrequent steady-state proliferation, multipotent differentiation potential, and either low or no expression of CyP4502F2 that may account for their resistance to airway pollutants such as naphthalene (10, 13, 20, 21). The pivotal role of this cell in airway repair was demonstrated in transgenic mice that expressed the procytotoxic gene Herpes simplex virus thymidine kinase (HSVtk) specifically in CE cells (CCtk transgenic mice; see Ref. 21). Exposure of these mice to the HSVtk substrate gancyclovir resulted in ablation of all CCSP-expressing cells and an associated lack of epithelial regeneration (13, 21). Taken together, these findings suggest that both NEB and BADJ-associated vCE cells are airway-specific stem cell populations that are activated to effect epithelial renewal after progenitor (Clara) cell depletion.
In addition to characteristics including infrequent proliferation, multipotent differentiation, and pollutant resistance, Goodell et al. (11, 12) and Scharenberg et al. (22) have identified preferential dye/drug efflux as a characteristic of hematopoietic stem cells. Commonly referred to as the side population (SP) phenotype, this property was initially defined by a low Hoechst 33342 blue/red fluorescent dye signal when compared with the majority of cells within a given population (non-SP). It was subsequently shown that this SP phenotype was the result of ATP-binding cassette (ABC)-dependent transporter activity (17, 22, 26). Moreover, the bone marrow SP was depleted of cells expressing either myeloid or lymphoid lineage-specific markers (Gr-1 or B220, respectively). Fractionation of CD45+ (hematopoietic) bone marrow cell populations using this approach resulted in a SP that was highly enriched for stem cells based on the molecular characteristics of Sca-1 and c-kit expression and the functional criterion of long-term bone marrow engraftment (11).
Recently, it has been shown that populations of CD45-positive (hematopoietic-derived) cells bearing a characteristic SP profile (low Hoechst 33342 blue/red fluorescence) exist in a number of tissue types (1, 16, 19, 32). Tissue-derived, CD45-positive SP cells were similar to bone marrow-derived SP in their sensitivity to ABC transport inhibitors. Functionally, these cells maintained an enhanced ability to form hematopoietic colonies when compared with tissue-derived, CD45-positive non-SP in vitro (1). Thus it appears that numerous tissues in addition to bone marrow harbor hematopoietic cells with properties common to hematopoietic stem cells.
The recent demonstration in both mammary gland and muscle of nonhematopoietic (CD45-negative) SP cells that maintain both functional and molecular properties of tissue-specific stem cells suggests that the property of rapid Hoechst efflux may be applied to purification of stem cells from other organ systems (15, 16, 19, 28). The present study was undertaken to test the hypothesis that intrapulmonary conducting airways harbor a rapid Hoechst effluxing population and that these cells would be enriched for markers of lung-specific epithelial stem cells. We demonstrate that isolated lung cells include a verapamil-sensitive SP that can be further fractionated based on the presence or absence of the hematopoietic cell surface marker CD45. CD45-negative SP expressed molecular markers characteristic of mesenchymal and vCE cells.
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MATERIALS AND METHODS |
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Lung cell isolation. Animals were killed by intraperitoneal injection of 2.5% avertin to achieve a surgical plane of anesthesia followed by exsanguination. Lungs were perfused with HBSS (Invitrogen, Grand Island, NY), and the lungs were lavaged eight times with 1 ml HBSS/200 µM EGTA (Sigma, St. Louis, MO). Lung cells were isolated by the method of Chichester et al. (6) with minor modifications. Briefly, the entire heart-lung unit was removed and suspended in 2 liters of 37°C saline. One milliliter of elastase (3.5 U/ml in HBSS; Worthington, Lakewood, NJ) was instilled in each lung via a tracheal cannula and incubated for 5 min, and the process was repeated five times. After digestion, lungs were finely minced in <1-mm pieces using a razor blade, strained through a 100-µm nylon membrane (Becton-Dickinson, Franklin Lakes, NJ), and resuspended in 35 ml HBSS containing 0.2 mg/ml DNase. Eight milliliters of sterile FBS (Invitrogen) was underlayered, and cells were pelleted by centrifugation at 300 g for 5 min at 4°C. Cells were washed two times in HBSS containing 2% FBS/10 mM HEPES, pH 7.4 (HBSS+), as described above, and resuspended at 1 x 106 live cells/ml in DMEM containing 2% FBS/10 mM HEPES, pH 7.4 (11).
Bone marrow cell isolation. Animals were killed as described above, and the femurs were recovered by blunt dissection. The epiphesial ends were removed, and the lumen of the metaphesis was flushed with 5 ml HBSS. Cell aggregates were dispersed by trituration, washed two times in HBSS containing 2% FBS/10 mM HEPES, pH 7.4 (HBSS+), as described above, and resuspended at 1 x 106 live cells/ml in DMEM containing 2% FBS/10 mM HEPES, pH 7.4.
Hoechst 33342 incubation. Cells were stained with 5 µg/ml Hoechst 33342 (Sigma) alone or in combination with 50 µM verapamil (Sigma) for 90 min at 37°C with intermittent mixing. Immediately after staining, cells were placed on ice, pelleted, and washed two times with ice-cold HBSS+ containing 0.2 mg/ml DNase, as described above.
Staining for cell-specific surface markers was performed at 4°C after incubation with Hoechst 33342. Briefly, directly conjugated mouse anti-CD45 (FITC, clone 30-F11; Becton-Dickinson) and/or mouse anti-Sca-1 (PE, clone D7; Becton-Dickinson) was applied at a concentration of 1 µg antibody/106 cells and incubated for 30 min at 4°C. An isotype-matched control antibody was used to confirm the specificity of Sca-1 antibody staining specificity. Propidium iodide (2 µg/ml; Sigma) was added to the cell suspension 5 min before sorting to discriminate between viable (dye impermeant) and nonviable (dye permeable) cells.
Immunostaining. After elastase digestion, 105 cells were cytospun on slides for immunophenotypic analysis. Cells were fixed overnight in 10% neutral-buffered formalin (Fisher, Pittsburgh PA), washed three times in excess PBS, and blocked using 5% BSA/PBS. Cells were then stained using antibodies for the airway-specific markers CCSP (rabbit-anti-CCSP, 1:8,000; see Ref. 24) and acetylated tubulin (mouse IgG2b-anti-ACT, 1:8,000; Sigma), washed, and subsequently stained with appropriate secondary antibodies donkey-anti-rabbit Alexa 594 (1:500; Molecular Probes, Eugene, OR) and goat-antimouse IgG2b Alexa 488 (1:500; Molecular Probes). Nuclei of cells were counterstained using 1 µg/ml 4',6-diamidino-2-phenylindole dihydrochloride (Sigma).
Flow cytometry. Cells were analyzed and sorted on a MoFlo (DakoCytomation, Fort Collins, CO) high-speed cell sorter equipped with three excitation lines (488 nm, ultraviolet, and 635 nm), eight parameters and subsystems of SortMaster Droplet Control, and Cy-CLONE Automated Cloner. SP cells were identified as described previously (11). Bandpass filters, 670/40 nm and 450/65 nm, were used to measure the red and blue Hoechst 33342 emission, respectively. Red fluorescence resulting from propidium iodide staining was detected using a 613/20 nm bandpass filter. Antibodies used for phenotypic analysis of SP cells were directly conjugated with FITC or PE, and emission of these fluorochromes was detected using a 530/40 nm and 575/26 nm bandpass filter.
RNA isolation and RT-PCR. Six-thousand sorted cells belonging to either total, non-SP, or SP categories (see Fig. 4) were directly sorted in cell lysis buffer (Promega, Madison, WI) and frozen before RNA isolation using a glass fiber system (SV Total RNA Isolation Kit; Promega). All RNA samples were eluted in 60 µl of nuclease-free water. First-strand cDNA synthesis was performed on 20 µl RNA using random hexamer primers in the presence (+RT) or absence (-) of Superscript II RT (Invitrogen). RT-PCR (40 cycles) was carried out using AmpliTaq Gold DNA polymerase (Applied Biosystems, Foster City, CA) and 3 µl cDNA template. Primers used in PCR were as indicated in Table 1. PCR reactions for CCSP, CyP4502F2, plateletendothelial cell adhesion molecule (PECAM), and vimentin were carried out at an annealing and extension temperature of 60°C, 1 min/cycle. PCR reactions for surfactant protein-C (SP-C) and -actin were carried out at an annealing temperature of 51°C (30 s/cycle) and an extension temperature of 72°C (1 min/cycle). Products were separated on 3% agarose gels containing ethidium bromide and visualized using an Alpha Innotech video gel documentation system and digital imaging software (Alpha Innotech, San Leandro, CA).
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RESULTS |
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Cells characterized by rapid efflux of Hoechst 33342 were detected in both the CD45+ and CD45- groups (Fig. 2, A and B, respectively). Generation of each of these subpopulations was inhibited by coincubation with the ABC transport inhibitor verapamil, indicating that Hoechst 33342 efflux is dependent on ABC transporter function (Fig. 2, C and D). Identification of verapamil-sensitive CD45+ and CD45- lung SP cells was independent of both gender and genetic background (FVB/n, C57/Bl6, 129/Sv, hybrid strains; data not shown). Within the FVB/n strain, the average representation of CD45+ (hematopoietic) SP cells was 0.09 ± 0.02% of CD45+ cells, whereas the average representation of CD45- (nonhematopoietic) SP cells was 0.87 ± 0.22%. These data demonstrate that cells from elastase-digested lung tissue contain two populations of cells characterized by rapid efflux of Hoechst 33342: the CD45+ and the CD45- side populations. The overall cellular toxicity associated with 5 µg/ml Hoechst 33342 exposure for 90 min was 60%. Differential susceptibility of the CD45+ and CD45- cell populations to Hoechst 33342 incubation resulted in a shift in the representation of viable cells such that
60% of cells were CD45+ and
40% were CD45-. This finding was highly consistent and did not appear to influence the representation of hematopoietic or nonhematopoietic SP cells identified in the present study. This lineage-dependent toxicity observed after Hoechst 33342 incubation was not entirely unexpected, as previous studies confirm that there is significant time, species, and cell type-specific dependence associated with Hoechst 33342 exposure (7a, 9a, 16a, 26a).
To address whether the Hoechst 33342-stained, PI-negative, sorted cell population remains viable after the initial FACS analysis, CD45- cells were subjected to resorting in the presence of PI 30 min after their initial isolation. Approximately 5% of cells were PI permeable after this time interval, indicating that the vast majority of initially sorted cells retain viability. Similar findings were found evaluating trypan blue dye exclusion up to 90 min after initial sorting (data not shown).
Stem cell antigen-1 is enriched within lung SP cells. To further characterize SP cells released by elastase digestion of lung tissue, expression of stem cell antigen-1 (Sca-1), an antigen highly enriched in SP cells of the hematopoietic lineage (11), was assessed. Lung or bone marrow-derived cells were stained with Hoechst 33342, FITC-conjugated mouse anti-CD45, and PE-conjugated mouse anti-Sca-1 antibody. The minimum fluorescence intensity required for classification of cells as Sca-1 positive (Fig. 3, AC) was established by staining cells with an isotype-matched control antibody (MATERIALS AND METHODS). As has been reported previously, Sca-1-expressing cells were significantly enriched within bone marrow-derived SP (78% positive) and were infrequent within the non-SP fraction (3% positive; Fig. 4A). In contrast, Sca-1 immunoreactivity was not significantly observed among CD45+ SP or non-SP cells (Fig. 3C). The majority of lung CD45- SP cells expressed Sca-1 (80%; Fig. 3B); however, this antigen was also expressed by 30% of cells within the non-SP fraction (Fig. 3B). Although these results demonstrate molecular similarity between SP cells of the bone marrow and CD45- SP cells of the lung, expression of Sca-1 by CD45- non-SP cells limits the utility of this antigen as a positive selectable marker for putative lung stem cells.
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Nonhematopoietic lung SP cells exhibit a unique molecular phenotype. To characterize the molecular phenotype of CD45- lung SP, RNA was purified from pools of 6,000 unfractionated CD45- cells (Fig. 4A), 6,000 CD45- non-SP cells (Fig. 4A), and 6,000 CD45-SP cells (Fig. 4A), and gene expression was assayed by RT-PCR. Expression of gene products detailed in Table 1 was analyzed. A no-RT negative control reaction was performed for all samples, and total lung RNA served as the positive control.
The cell-specific genes CCSP and CyP4502F2 (Clara cells), SP-C (type 2 cells), and vimentin (fibroblasts) were expressed in the unfractionated CD45- cell population (Fig. 4B). PECAM was not detected in this population, suggesting minimal endothelial cell isolation within the lung cell preparation. All genes expressed within the unfractionated CD45- population were also detected in RNA isolated from non-SP cells, indicating that this gate provides an adequate representation of the total CD45- population (Fig. 4B). In contrast, only CCSP and vimentin were detected in the CD45- SP cells. Based on these findings, it is likely that the CD45- lung SP fraction includes at least two populations of cells, one with molecular characteristics of mesenchymal cells (vimentin positive) and the other with characteristics of airway epithelial cells (CCSP positive). The finding that CyP4502F2 mRNA is undetectable within the CD45- SP cells suggests that lung epithelial cells represented within this fraction do not exhibit a molecular phenotype typical of Clara cells. This finding is consistent with our previous identification of an NEB-associated CCSP-expressing cell population that lacks immunoreactive CyP4502F2 protein (20).
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DISCUSSION |
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We have previously shown that NEBs serve as a stem cell niche within the bronchiolar epithelium and that a subpopulation of vCE cells within the NEB contains undetectable levels of immunoreactive CyP4502F2 protein (13, 20, 21). The finding that CCSP-positive, CyP4502F2-negative cells are enriched within the SP further supports the contention that these cells exhibit properties of stem cell populations previously characterized in vivo. In addition to their localization within NEB and BADJ microenvironments within airways, bronchial and bronchiolar stem cells were stimulated to enter the cell cycle after progenitor cell depletion (13). The relative paucity of nonhematopoietic SP (<0.9% of viable, CD45-negative cells) is consistent with the low abundance of stem cells throughout the intrapulmonary conducting airways.
Even though stem cells are greatly enriched within the SP of bone marrow, the applicability of this property to enrichment of stem cells from other sources has been an issue of some debate (4, 11, 12, 32). Although numerous studies satisfy this criterion through a comparison of SP phenotype with that of known stem cell populations (e.g., Sca-1, c-kit expression, molecular markers), by far the most common method used to associate SP and stem cells has been a functional assessment of their engraftment potential in vivo (5, 7, 11). This activity has been demonstrated for tissue-derived SP cells from bone marrow, muscle, mammary gland, and liver (2, 11, 1416, 18, 19, 28, 30). Thus rapid dye efflux is a common property of stem cell populations derived from numerous tissues, although differences in fluorescence intensity and in the shape of the SP profile have been reported (1). In the current study, lung SP cells do exhibit molecular characteristics consistent with their classification as lung stem cells, although future studies are needed to determine the differentiation potential of this population.
It is hypothesized that the genetic integrity of stem cells is critical for maintenance of normal differentiation within a given organ system. Due to their long life and robust differentiation potential, mutations within stem cells could have significant effects on the health status of the entire organism. Although infrequent proliferation and sequestration within a microenvironment are mechanisms for maintenance of this genetic integrity, the expression of ABC transporters (resulting in the SP phenotype) may provide a further level of protection. Targeted deletion of the ABC transporter bcrp1 resulted not only in loss of all SP cells but also increased susceptibility of hematopoietic stem cells to the chemotherapeutic agent mitoxantrone (31). Thus factors directly related to identification of hematopoietic SP cells also serve to protect this cell from toxicant injury. Studies are ongoing to determine the role of ABC transporter activity in maintenance and observed pollutant resistance of lung stem cell populations.
In addition to identification of nonhematopoietic SP, we also confirmed the presence of a subset of CD45+ cells with an SP phenotype in lung. One interpretation of this finding in other organs has been that CD45+ SP cells represent a population of tissue-resident stem cells (16). This hypothesis is contradicted by the recent work of Weissman and colleagues (27, 29) in which parabiotic sex- or CD45 isotype-mismatched mice are used to identify circulating partner-derived HSC within multiple tissues. This has led to a new theory which states that circulating/transiently resident HSC are common throughout multiple tissues and that these cells are distinct from indigenous tissue-specific stem cells. Further support for this hypothesis comes from a recent study by McKinney-Freeman et al. (19) detailing the differentiation potential of muscle-derived SP cells. In this work, the authors demonstrate that heterogeneity in CD45 expression can be used to segregate cells harboring either hematopoietic or myogenic potential within the muscle-derived SP. Although each of these types of muscle SP show enhanced differentiation potential relative to its corresponding non-SP, it was clear that only CD45+ SP cells were capable of colonizing the hematopoietic lineage in vitro (19). Moreover, studies by Asakura and Rudnicki (1) demonstrate that a large number of tissues contain SP with hematopoietic colony-forming potential. As was the case in muscle, only the CD45+ fraction of these cells demonstrates the ability to undergo any hematopoietic differentiation. Therefore, it is most likely that the CD45+ lung SP cells identified in this study are only capable of hematopoietic differentiation. However, because we are primarily interested in determining the cellular and molecular mechanisms of airway-specific injury and repair, this population has not been extensively characterized in the present study.
A recent paper by Summer et al. (25) also describes the identification of lung-derived SP cells with properties similar to bone marrow SP. SP cells identified in their study are predominately CD45 positive, express high levels of PECAM (CD31), and are uniformly Sca-1 positive. In addition to these distinct findings, the SP they identify was significantly less abundant than SP identified in our study (0.05 vs. 0.87% of total cells). This discrepancy likely results from differences in the methods used for lung cell isolation, particularly methods used for enzymatic dissociation of lung tissue. Whereas the SP identified by Summer et al. (25) is predominately positive for PECAM/CD31, endothelial cell-specific mRNA species were not detected in any isolated cell populations (total, non-SP, nor SP) within the current study. These observations support the notion that multiple distinct populations of stem cells likely exist within different lung compartments (3, 8, 10, 13). Future studies are needed to determine whether SP subsets exist within other cellular compartments of the lung that were not adequately sampled with the isolation methods employed in this study.
In summary, studies presented herein demonstrate the existence of rapid Hoechst effluxing cells among isolated lung cell preparations enriched for epithelial cells. Included within the SP identified in elastase digests of lung tissue is a CD45- negative fraction that includes cells expressing marker genes indicative of fibroblast (vimentin) and nonciliated airway epithelial (CCSP) lineages. Expression of CCSP by CD45-negative SP cells was not associated with expression of the mature Clara cell marker CyP4502F2, a property that is consistent with the molecular phenotype of a subpopulation of cells residing within the NEB, a niche that has been shown to harbor stem cells in vivo (13, 20, 21). Future studies may focus on the establishment of methods for stem/SP cell culture and the identification of unique molecular markers for airway stem cells.
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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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