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TRANSLATIONAL PHYSIOLOGY

EDITORIAL FOCUS

Origin and phenotype of lung side population cells

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

Submitted 20 January 2004 ; accepted in final form 21 March 2004

	ABSTRACT

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Side population (SP) cells, a rare cell type identified by their ability to efflux the vital dye Hoechst 33342, are highly enriched for stem cell activity. Bone marrow (BM) SP cells uniformly express the pan-hematopoietic marker CD45, whereas tissue SP cells are heterogeneous in CD45 expression. In previous studies, we found that CD45 is expressed on 75% of lung SP cells. By performing whole BM transplantations, we determined that CD45-positive and CD45-negative lung SP cells are marrow derived. Transplantation of 200 highly purified BM SP cells indicated that both lung SP cell subtypes are derived from this marrow cell type. Morphologically, CD45-positive lung and BM SP cells possess similar features. They are small, round, and contain scant cytoplasm. CD45-negative lung SP cells are larger and contain abundant granular cytoplasm. Gene expression patterns for hematopoietic transcription factors GATA-1, GATA-2, and PU.1 further differentiated SP marrow and lung subtypes. By immunostaining for -smooth muscle actin and cytokeratin, we found significant differences in the relative expression patterns of these markers in lung and marrow SP cell subtypes. In summary, these findings demonstrate that lung SP cells are derived from the BM and that CD45-positive and -negative subtypes can be distinguished by morphological differences and gene expression patterns.

Hoechst; stem cells; CD45; breast cancer resistance protein

Targeted gene ablation in mice indicated that the ATP binding cassette-transporter protein breast cancer resistance protein (Bcrp1) mediates Hoechst efflux; SP cells are not detected in Bcrp1-null mice (24, 25). Despite this, Bcrp1-deficient mice have a normal number of functional HSCs (24). This indicates that Bcrp1 serves as a marker of SP cells but is not required for stem cell function.

Recently, SP cells have been identified in nonhematopoietic tissues, including skeletal muscle, testes, mammary gland, and lung (2, 12, 15, 19, 22). Tissue SP cells share phenotypic features with BM SP cells, such as the expression of Sca-1, and the absence of expression of mature hematopoietic markers. Tissue SP cells can be distinguished from BM SP cells on the basis of their heterogeneity in their expression of the pan-hematopoietic marker CD45 (1). The percentage of CD45-positive and -negative SP cells varies between tissues.

In the lung, SP cells comprise <0.1% of digests, a percentage that remains fixed from postnatal day 5 to 1 yr of age (19). To date, the anatomical location of lung SP cells is unknown. The inability to definitively localize lung SP cells in tissue sections relates to the rarity of the population and the expression of Bcrp1 and Sca-1 in multiple lung cell types (11, 19). Similar to SP cells identified in other tissues, lung SP cells variably express CD45. Previous studies report that 75% of lung SP cells express the pan-hematopoietic marker CD45 (19). This observation is consistent with accumulating data suggesting a marrow origin for some tissue stem cells (13, 14).

In this paper, we set out to examine the origin of lung SP cells and to further characterize the relative differences in CD45-positive and -negative subtypes. Using several BM transplantation models, we found that both lung SP cell subtypes are marrow derived. Despite having a common origin, lung (CD45-positive and -negative) and BM SP cells represent three phenotypically distinct cell populations. These results suggest distinct roles for CD45-positive and CD45-negative SP cells in lung tissue homeostasis.

	METHODS

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Animals. Lung suspensions were prepared from adult mice (4–8 wk of age) of backgrounds C57BL/6J, C57BL/6-TgN (ACT-EGFB), and 10sb/J (Jackson Laboratories, Bar Harbor, MA), and the mixed background 129/O1a/C57BL/6 (Bcrp1-deficient mice). Bcrp1-deficient mice were kindly provided by Dr. Brian Sorrentino (St. Jude Children's Research Hospital, Memphis, TN) (24). Animals were killed by isoflurane anesthesia followed by cervical dislocation. Animal studies were conducted according to protocols approved by the National Institutes of Health and the Boston University Animal Care and Use Committee.

Transplant studies. BM transplantation was performed in lethally irradiated mice (14 gray of -irradiation in split dose) utilizing 20 million whole bone marrow (WBM) cells isolated from 6- to 8-wk-old C57BL/6, C57BL/6-TgN (ACT-EGFB), or Bcrp1-deficient mice and delivered by tail vein injection. Transplant of BM SP cells was performed by injecting 200 BM SP cells isolated from C57BL/6-TgN (ACT-EGFB) mice mixed with 200 x 10³ nucleated WBM cells prepared from 6- to 12-wk-old C57BL/6J mice. Cells were delivered by retroorbital injection. Recipient mice were maintained on acidified or antibiotic water and autoclaved food.

Cell preparations and staining. Cell suspensions were obtained from enzyme-digested lungs as previously described (19). Briefly, before lung extraction, animals were bled by transecting the abdominal aorta. Next, in an attempt to remove circulating blood cells, perfusion of the pulmonary vasculature was performed using ice-cold saline until the lungs bleached white. Lung extracts were then digested by finely mincing tissue with a razor blade in the presence of 0.1% collagenase (Roche Diagnostics, Indianapolis, IN), 2.4 U/ml dispase (Roche Diagnostics), and 2.5 mM CaCl₂ at 37°C for 1 h. Removal of nonspecific debris was accomplished by sequential filtration through 70- and 40-µm filters. Cells were resuspended at a concentration of 10 x 10⁶ cells/ml. Hoechst 33342 (5 µg/ml; Sigma-Aldrich, St. Louis, MO) staining of lung and BM cells was performed at 37°C for 90 min in DMEM supplemented with 2% fetal calf serum (FCS), 10 mM HEPES, and 1% penicillin/streptomycin (5, 19). At the completion of staining, cells were immediately placed on ice. Immunostaining was performed in the dark at 4°C for 30 min using directly fluorochrome-conjugated monoclonal rat anti-mouse antibodies reactive to CD45 (BD Pharmingen, Lexington, KY). After staining, cells were washed twice and resuspended in Hanks' balanced salt solution supplemented with 2% FCS. Dead cells were excluded from flow cytometry analysis based on propidium iodide staining (2 µg/ml). In all studies, dead cells comprised <10–15% of total cells. An isotype control antibody for CD45 was employed as a negative control 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 long-pass filter (Hoechst red). A second 488-nm argon laser was used to excite phycoerythrein, green fluorescent protein (GFP), and propidium iodide. Data analysis was performed using Summit software.

RT-PCR. cDNA was generated from RNA extracts derived from 11,000 CD45-positive and -negative lung SP cells, or BM SP cells, with a reverse transcription kit (Promega, Madison, WI). PCR was performed using the following primers: GATA-1 primers 5'-CCAATGCACTAACTGTCAAACG-3' and 5'-CATGCCTGAATCTCAGTACTCG-3', GATA-2 primers 5'-AACTGCATAAGCTTAACCCGC-3' and 5'-GGTTGACTCAGCACAATCGTC-3', PU.1 primers 5'-ACAGATGCACGTCCTCGATAC-3' and 5'-GGAACTGGTACAGGCGAATCT-3', -Sma (-smooth muscle actin) primers 5'-AGCTTTGGGCAGGAATGATTTGG-3' and 5'-AAGATCATTGCCCCTCCAGAACG-3', keratin 18 primers 5'-GGCCACTACTTCAAGATCATC-3' and 5'-GTACTTGTCCAGTTCCTCGCG-3', keratin 19 primers 5'-CTACAGATTGACAATGCTCGC-3' and 5'-GGATCTTGGCTAGGTCGACAC-3', keratin 23 primers 5'-GACACTGAAGGGACGATGGAT-3' and 5'-GTCGAGACTCACCCATTAGCG-3', and -actin primers 5'-GCTCGTTGCCAATAGTGATG-3' and 5'-AAGAGAGGTATCCTGACCCT-3'. Cycling conditions for -Sma, keratin 18, and -actin were 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min, 35 cycles. Cycling conditions for keratin 19, keratin 23, GATA-1, GATA-2, and PU.1 were 94°C for 1 min, 54°C for 1 min, and 72°C 1 min, 35 cycles.

Immunohistochemistry. Collected lung SP cells were cytospun onto charged slides, fixed with acetic acid/methanol (1:3) for 5 min, and washed in PBS. Hematoxylin (filtered) staining was performed at a 1:9 dilution for 1 min. Immunostaining of SP cells was done using an immunofluorescent detection method. Before staining, cells were quenched with sodium borohydride. Fluorescent staining was carried out using a fluorochrome-conjugated monoclonal antibody against the acidic forms of cytokeratin (Clone C-11; Sigma-Aldrich) and -Sma (Sigma-Aldrich). In parallel slides, isotype control antibodies were used to immunostain cytospun SP cells. After staining, slides were washed in PBS, and nuclear counterstaining was performed using 4',6'-diamidino-2-phenylindole dihydrochloride (Sigma-Aldrich). The percentage of positive cells for each marker was determined from averaging the values obtained from three independent investigators counting 100 cells.

	RESULTS

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Lung SP cells originate from the BM. We employed several different transplantation models to determine whether lung SP cells originate from the BM. Utilizing recipient mice that lacked detectable SP cells (Bcrp1-deficient mice), we performed transplantation studies with wild-type or Bcrp1-deficient BM cells. Six months after transplantation, lungs were isolated, digested, stained with Hoechst dye, and then analyzed by flow cytometry. As anticipated, lung SP cells were not detected in Bcrp1-deficient mice transplanted with Bcrp1-deficient BM cells (n = 2). However, in mice receiving wild-type BM cells (n = 2), a visible SP band was detected (Fig. 1). Importantly, the fraction of SP cells (0.09%) was similar to that detected in previous reports, and both CD45-positive and -negative cells were present in the SP gate.

View larger version (23K): [in this window] [in a new window]   Fig. 1. Reconstitution of lung side population (SP) cells in breast cancer resistance protein (Bcrp1)-deficient mice. Bcrp1-deficient lungs were digested and stained with Hoechst dye 6 mo after transplantation of wild-type (B) or Bcrp1-deficient (A) bone marrow (BM) cells. SP cells (boxed area) were detected in the lungs of mice transplanted with wild-type, but not Bcrp1-deficient, BM cells. C: CD45-positive and -negative cells were present in the SP gate from animals transplanted with wild-type BM. SSC, side scatter; PE, phycoerythrein.

View larger version (30K): [in this window] [in a new window]   Fig. 2. Lung SP cells are BM derived. A: wild-type lungs were digested and stained with Hoechst dye 8 mo after transplantation of green fluorescent protein (GFP)-expressing BM cells. Density dot plot analysis demonstrates the presence of lung SP cells (boxed area). B: analysis of lung SP fraction demonstrates the presence of wild-type and donor cells. CD45-positive (45%) and negative (7%) GFP-expressing cells were detected. C: bar graph demonstrates the average contribution of donor-derived (GFP-expressing) cells to CD45-positive and -negative SP fractions (error bar reflects SD) in all 4 mice.

View larger version (31K): [in this window] [in a new window]   Fig. 3. Reconstitution of lung SP cell fraction from BM SP cells. A: wild-type lungs were digested and stained with Hoechst dye 8 mo after transplantation of 200 GFP-expressing BM SP cells. Density dot plot demonstrates the presence of SP cells (boxed area). B: analysis of lung SP fraction shows the presence of GFP-expressing cells in SP gate. CD45-positive (34%) and -negative (4%) donor-derived (GFP-expressing) cells were detected. C: bar graph shows the average contribution of donor-derived cells to CD45-positive and -negative SP fractions (error bars reflect SD) in all 4 mice.

View larger version (58K): [in this window] [in a new window]   Fig. 4. Analysis of hematoxylin-stained lung and BM SP cells by light microscopy. A: BM SP cells are well circumscribed and uniform in both size and shape. B: CD45-positive lung (L) SP cells have a similar appearance to BM SP cells. Both cell populations had scant cytoplasm and possessed large nuclear to cytoplasmic ratios. C: CD45-negative lung SP cells were larger and possessed abundant cytoplasm with extensive granularity. D: density dot plot demonstrating the differences in cell shape and granularity between CD45-positive and -negative lung SP cells.

View larger version (56K): [in this window] [in a new window]   Fig. 5. RT-PCR of lung and BM SP cells for the hematopoietic transcription factors GATA-1, GATA-2, and PU.1. BM SP cells express GATA-1 alone. CD45-positive lung SP cells express GATA-1 and PU.1. CD45-negative lung SP cells are negative for GATA-1, GATA-2, and PU.1. Actin primers from different exons were utilized to ensure the fidelity of cDNA and the absence of genomic DNA. WBM, whole bone marrow.

View larger version (20K): [in this window] [in a new window]   Fig. 6. A: immunostaining of BM and lung SP cells for -smooth muscle actin (-Sma; left, red), cytokeratin (Cyto; middle, green), and merged images (right, yellow). B: bar graph demonstrates the percentage of BM SP cells (top) and CD45-positive (middle) and CD45-negative (bottom) lung SP cells expressing -Sma only (left), both cytokeratin and -Sma (middle), or neither marker (right). Dbl, double. Error bars reflect SD.

View larger version (59K): [in this window] [in a new window]   Fig. 7. RT-PCR for -Sma and keratins (K) 18, 19, and 23 in SP cells. -Sma was expressed in BM SP and CD45-positive and -negative lung SP cells. Whole lung (WL) digest was used as a positive control. All 3 forms of keratin were detected in WL and CD45-negative lung SP cells. BM and CD45-positive lung SP cells were distinguished on the basis of their differential expression of keratin 18 and keratin 19, respectively. Actin primers from different exons were utilized to ensure the fidelity of cDNA and the absence of genomic DNA.

	DISCUSSION

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To do this, we performed BM transplantation studies and examined for the presence of donor-derived cells within the lung SP gate. These studies found that CD45-positive as well as CD45-negative lung SP cells were BM derived. We also performed transplantation studies utilizing highly purified CD45+, Sca-1+, c-kit+, and lineage negative BM SP cells. These studies showed that CD45-positive and -negative lung SP cells could be derived from marrow SP cells. Although marrow SP cells are uniformly CD45 positive, we cannot definitively exclude the possibility of a contaminating CD45-negative stem cell contributing to our findings. Despite the small percent of donor-derived CD45-negative lung SP cells in our transplantation experiments, these results were reproducible in four separate animals. The differential turnover or sensitivity of CD45-positive and -negative lung SP cells to radiation may contribute to the kinetics of BM-dependent reconstitution. Importantly, the variable expression of GFP in donor cells (75–85%) limits our ability to absolutely quantify the engraftment potential of BM SP cells.

The ability of BM SP cells to reconstitute tissue SP cells has been documented in sites other than the lung. In skeletal muscle, SP cells were found to be 90% BM derived 12 mo posttransplantation, although the relative contribution to CD45-positive and -negative subsets was not characterized in these studies (14, 15). Whether BM SP cells serve as a source for the reconstitution of SP cells residing within other tissues is unclear. A recent study demonstrated that the contribution of BM cells to the testes SP fraction was negligible (<5%) (12). In this study, total body irradiation was not a component of the experimental protocol; this suggests that tissue injury may be required for tissue SP cell reconstitution.

To further examine the relationship of lung and BM SP cells, we compared the morphological characteristics of each population. Except for small differences in cell size, which may be a consequence of enzymatic digestion, CD45-positive lung SP cells and BM SP cells were indistinguishable. These shared characteristics may reflect functional similarities between the two populations or their developmental proximity. Interestingly, CD45-negative lung SP cells were easily distinguished from BM and CD45-positive lung SP cells based on histological analysis. Of note, CD45-negative lung SP cells do not resemble mature lung (i.e., type I, type II cells) or hematopoietic cell types. Attempts to localize SP cells in lung tissue sections have thus far been unsuccessful due in part to the rarity of the population and the absence of SP cell-specific markers. Insight into the physiological role of CD45-negative SP cells may require genetic profiling and the identification of its anatomical niche within the lung.

Having established that lung SP cells were derived from BM, we evaluated whether lung SP cells express genes characteristic of hematopoietic stem/progenitor cells. To do this, we performed RT-PCR to survey expression of the hematopoietic transcription factors GATA-1, GATA-2, and PU.1. As expected, BM SP cells expressed only GATA-2, a factor critical for the maintenance of HSCs (20, 21). In contrast, CD45-positive lung SP cells expressed both GATA-2 and PU.1, an expression pattern characteristic of myeloid progenitor cells, whereas CD45-negative lung SP cells did not express any of the three transcription factors examined (26). On the basis of this, we speculate that CD45-positive lung SP cells are local progenitors for myeloid cells in the lung, such as macrophages, mast cells, and eosinophils.

To begin to examine the expression of epithelial and mesenchymal genes in lung SP cells, we performed immunostaining using monoclonal antibodies to pan-cytokeratin and -Sma. These studies demonstrate that within each population of SP cells, there exists significant heterogeneity. Interestingly, we found that subpopulations of BM SP cells also express cytokeratin and -Sma proteins. These findings are consistent with gene profiling studies on highly purified BM SP cells (17). The relationship of marrow SP cells to local organ progenitor cells is a focus of future study. Cells of a similar phenotype, termed myoepithelial cells, have been identified in various tissues and have been found to play an important role in tissue homeostasis and the pathogenesis of disease (3, 4, 7, 16).

In conclusion, we confirmed that adult lung SP cells arise from the BM and that both the CD45-positive and -negative cell types can be derived from a uniform purified population of HSCs (BM SP cells). These data thus suggest that local environmental cues induce these subtypes into phenotypically distinct cell populations. We speculate that CD45-positive and -negative lung SP cells have unique biological roles within the lung and that dysregulation of either population may contribute to the pathogenesis of various lung diseases.

	GRANTS

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National Institutes of Health Grants RO1-HL-69148, R21-HL-72205, and PO1-AI-50516 and the American Lung Association Research Fellowship Training Award supported this work.

	ACKNOWLEDGMENTS

 
We thank Alan Ho for assistance with cell sorting experiments.

	FOOTNOTES

 

Address for reprint requests and other correspondence: R. Summer, The Pulmonary Center, R-304, 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.

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This Article Abstract Full Text (PDF) All Versions of this Article: 287/3/L477    most recent 00020.2004v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (14) Citing Articles via Google Scholar Google Scholar Articles by Summer, R. Articles by Fine, A. Search for Related Content PubMed PubMed Citation Articles by Summer, R. Articles by Fine, A.