Stem cells are dispensable for lung homeostasis but restore airways after injury Adam Giangrecoa,1, Esther N. Arwerta, Ian R. Rosewellb, Joshua Snyderc, Fiona M. Watta,d, and Barry R. Strippc,1 aCancer Research UK, Cambridge Research Institute, Cambridge CB2 0RE, United Kingdom; bCancer Research UK, London Research Institute, South Mimms EN6 3LD, United Kingdom; cDepartment of Medicine, Duke University Medical Center, Durham, NC 27710; and dWellcome Trust Centre for Stem Cell Research, Cambridge University, Cambridge CB2 1QR, United Kingdom
Edited by Brigid L. M. Hogan, Duke University Medical Center, Durham, NC, and approved April 8, 2009 (received for review January 22, 2009)
Local tissue stem cells have been described in airways of the lung but their contribution to normal epithelial maintenance is currently unknown. We therefore developed aggregation chimera mice and a whole-lung imaging method to determine the relative contributions of progenitor (Clara) and bronchiolar stem cells to epithelial maintenance and repair. In normal and moderately injured airways chimeric patches were small in size and not associated with previously described stem cell niches. This finding suggested that single, randomly distributed progenitor cells maintain normal epithelial homeostasis. In contrast we found that repair following severe lung injury resulted in the generation of rare, large clonal cell patches that were associated with stem cell niches. This study provides evidence that epithelial stem cells are dispensable for normal airway homeostasis. We also demonstrate that stem cell activation and robust clonal cellular expansion occur only during repair from severe lung injury. progenitor 兩 Clara cell 兩 BASC 兩 bronchiole 兩 repair
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t is currently unknown whether airway stem cells contribute significantly to normal epithelial maintenance. Conducting airway cells exhibit significant regional and functional heterogeneity, have a long half life, and overall epithelial proliferation is ⬍1% per day (1–4). This suggests that airways are similar to self-duplicating tissues such as adult pancreatic islets that have been proposed to utilize a single progenitor cell maintenance strategy (5, 6). However, previous studies using murine injury models have also identified populations of local tissue stem cells that can contribute to lung repair and lung cancer (7–9). It is therefore important to determine whether progenitor or stem cell populations govern normal airway homeostasis as these findings may provide insights into human disease etiology. Intrapulmonary airway stem cells reside in unique microenvironments associated with calcitonin gene related peptide (CGRP) expressing neuroepithelial bodies (NEBs) and bronchioalveolar duct junctions (BADJs) (7–9). Both NEB- and BADJ-associated stem cells exhibit a Clara cell secretory protein (CCSP)-expressing phenotype, the capacity to regenerate both secretory and ciliated epithelial cell types that normally reside at these airway locations, and mitotic DNA label retention following injury (8, 10). The critical role played by CCSP-expressing cells in repair was verified by abrogation of epithelial repair following their ablation, using a transgenic suicide gene ablation approach (10, 11). Furthermore, BADJ (but not NEB) -associated stem cells have been implicated as originating cells for bronchioalveolar lung tumors (7). For the purposes of this study, we define airway NEB- and BADJ-associated stem cells as variant CCSP-expressing (vCE) cells that exhibit pollutant resistance, the capacity to regenerate specialized bronchiolar epithelial cell types, and localization to either NEB or BADJ microenvironments. It has been suggested that the existence of self-duplicating progenitor cells in tissues with low intrinsic proliferation and turnover obviates the requirement for tissue stem cells (5, 6). This theory is supported by classical studies in which tissue 9286 –9291 兩 PNAS 兩 June 9, 2009 兩 vol. 106 兩 no. 23
homeostasis was analyzed using aggregation chimera mice (12– 14). More recently, transgenic lineage tracing experiments demonstrated that pancreatic islets are normally maintained by simple progenitor cell self-duplication (5). A similar selfduplication mechanism has also been proposed for maintenance of normal epidermis (15–17). In contrast, intestinal aggregation chimera and lineage tracing studies indicate that stem cells normally maintain this tissue (13, 18, 19). The purpose of this study was to determine whether airway NEB- and BADJ-associated stem cells maintain normal lung homeostasis. An aggregation chimera mouse model and newly developed lung whole-mount imaging methods were used to determine the contribution made by abundant progenitor cells and rare tissue stem cells to epithelial maintenance and repair. We show that airway stem cells do not contribute significantly to lung homeostasis and that their activation is contingent upon epithelial progenitor cell depletion following severe lung injury. Results Defined Airway Stem Cells Do Not Contribute Significantly to Tissue Homeostasis. It remains unknown whether airway stem cells
maintain normal lung homeostasis or if these cells instead represent a normally quiescent cell population. We did not address this question using transgenic lineage tracing models due to the lack of a specific marker gene that identifies the bronchiolar stem cell compartment and hence an inability to uniquely label this cell type (1–3, 20). Instead, we developed green fluorescent protein (GFP) chimeric mice by 8- to 16-cell stage wild-type:GFP (chicken-actin GFP, CAG-GFP) embryo aggregation to track intrapulmonary chimeric epithelial patches [see schematic, supporting information (SI) Fig. S1 A]. A total of 23 adult mice were produced that exhibited good coat color and skin GFP chimerism. We considered 2 possibilities: either that the localization and size of individual chimeric epithelial patches within these adult mice would positively correlate with niches that maintain airway stem cells (Fig. S1B) or that there would be no positive correlation (Fig. S1C). We anticipated that a positive correlation would be observed only if stem cells contributed significantly to normal airway homeostasis. Because of the anticipated difficulty in reliably measuring chimerism by analysis of standard tissue sections, we developed a novel lung whole-mount imaging method. Conducting airways were revealed by microdissection and immunostained, and chimerism was measured as a function of cellular immunophenoAuthor contributions: A.G. and B.R.S. designed research; A.G., J.S., and B.R.S. performed research; A.G., E.N.A., I.R.R., J.S., and F.M.W. contributed new reagents/analytic tools; A.G., J.S., and B.R.S. analyzed data; and A.G. and B.R.S. wrote the paper. Conflict of interest: The authors declare no conflict of interest. This article is a PNAS Direct Submission. Freely available online through the PNAS open access option. 1To
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This article contains supporting information online at www.pnas.org/cgi/content/full/ 0900668106/DCSupplemental.
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Fig. 1. Chimeric epithelial patches do not associate with airway stem cell niches. (A and B) Representative low (A) and high magnification (B) lung whole mount immunostained for GFP (green) to reveal the distribution of chimeric epithelial patches. (C and D) High-magnification images reveal that CGRP-immunoreactive NEBs (red cell cluster, C) and BADJ-associated stem cell niches (arrow, D) do not correlate with GFP-expressing patches. (E) Quantification of chimeric patch correlation with defined airway stem cells. (F and G) Tissue sections stained for CCSP, CGRP, and GFP to visualize GFP chimeric patch distribution with respect to NEB (red, F) and BADJ stem cell niches (right side, G). (H and I) BADJ tissue staining for CCSP (blue), SPC (red), and GFP (green) to compare CCSP/SPC dual-positive cell chimerism (arrows) with adjacent alveolar septae. The proximal– distal axis (P3D) and airway boundaries (white lines) are indicated in A and D. (Scale bars, 50 m.)
type through high-speed resonance scanning confocal imaging (Fig. S1D). Using this approach we compared GFP(⫹) and GFP(–) chimeric patch distribution relative to anatomic localization within conducting airways (low and high magnification, Fig. 1 A and B). Our analysis of 10 adult, normal chimeric lungs consistently revealed no positive correlation between chimeric patches and the spatial distribution of airway stem cell niches (Fig. 1 C–E). The degree of airway chimerism also did not appear to be significantly different between either stem or nonstem cell-containing regions. Similar findings were additionally consistently observed in paraffin-embedded tissue sections (Fig. 1 F and G) and overall closely resembled the hypothetical example shown in Fig. S1C. It has also been suggested that Kras mutation promotes the activation of a bronchiolar stem cell defined by a naphthaleneresistant phenotype and coexpression of CCSP/surfactant protein C (SPC) and that these cells may have the capacity to renew Giangreco et al.
Fig. 2. Epithelial patch size suggests that airways are not maintained by stem cells. (A and B) Expected GFP patch size and abundance if stem (A) or single progenitor cells (B) maintain conducting airways. (C and D) Images of GFPexpressing cell patches within airways before (C) and after image pseudocoloring (D). (Scale bars: C and D, 50 m.) (E) Overall GFP expression within airways of 10 individual mice. (F) Representative pseudocolored chimeric airway used for airway patch size quantification. (G) Analysis comparing GFP-positive and -negative patch size vs. frequency among the lowest (mouse A, black line), highest (mouse J, green line), and average of all chimeric airways (red line). Data represent mean ⫾ SEM (G). P3D indicates proximal– distal axis in F.
alveolar epithelium (21, 22). To determine whether these cells contributed to alveolar homeostasis we compared the chimeric GFP expression patterns of terminal bronchiolar CCSP/SPC dual positive cells with their adjacent alveolar septae. Dualpositive cells were extremely rare in normal airways, and those cells that were identified exhibited no consistent correlation between their GFP phenotype and that of adjacent alveolar epithelium (Fig. 1 H and I). This absence of a clear lineage relationship between CCSP/SPC dual-positive cells and adjacent alveolar epithelium suggests that this bronchiolar stem cell pool does not normally contribute to alveolar tissue maintenance. Abundant Progenitor Cells Maintain Normal Airway Homeostasis. Our data indicated that lungs were not likely to be maintained by airway NEB- and BADJ-associated stem cells. However, we also sought to determine whether chimeric airways could be maintained by an alternative previously uncharacterized stem-like cell population. We postulated that if stem-like cells maintained lung homeostasis, then chimeric airways should predominantly contain large clonal patches with a reproducible interindividual distribution pattern (Fig. 2A). In contrast, if an abundant progenitor cell population (such as airway Clara cells) maintained airway homeostasis through a random, probabilistic (stochastic) process, then chimeric lungs would be more likely to contain numerous patches with a wide distribution of sizes (Fig. 2B). We used Volocity image analysis software to tag and pseudocolor all chimeric patches throughout airways on the basis of GFP intensity and a continuous cellular patch boundary (Fig. 2 C and D). Because all lungs displayed relatively balanced chimerism, our ability to assess true clonality within these epithelial patches was limited and it remains likely that our identification of some large patches was because of fusion among several smaller patches (Fig. 2E). Nonetheless, results revealed that PNAS 兩 June 9, 2009 兩 vol. 106 兩 no. 23 兩 9287
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rate of proliferation in the uninjured adult lung, these results indicate that at least 20–30% of chimeric airway cells will have undergone postnatal proliferation in our 4- to 6-month-old chimeric mice. Thus, although it is clear that our observed epithelial chimerism must involve a developmental component, these results confirm that widely distributed progenitor cells in postnatal airways maintain this high degree of adult chimerism. These data indicate that airway stem cells do not contribute significantly to normal epithelial maintenance and are therefore dispensable for homeostasis. In addition to proliferation we also examined the differentiation potential of small chimeric epithelial patches. Immunostaining revealed that most GFP(⫹) patches contained both CCSP-positive and -negative cells (Fig. 3D). Tissue section staining further revealed that most GFP(⫹)/CCSP(⫺) cells identified by whole-mount imaging were acetylated tubulinpositive ciliated cells (Fig. 3E, arrows). Infrequently, we also observed individual NEBs that were composed of both GFP(⫹) and GFP(⫺) CGRP-expressing pulmonary neuroendocrine cells (Fig. 3F). Injury Severity Determines Stem vs. Progenitor Cell Repair Strategies.
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Fig. 3. Chimeric patches are proliferation competent and multipotent. (A) Immunostaining for GFP (green), CCSP (blue), and Ki67 (red) to identify GFP(⫹) and -(⫺) mitotic cells (arrows). (B and C) Immunofluorescent detection of BrdU (red) plus CGRP (green, B) or CCSP (green, C). White arrows indicate mitotic cells; the yellow line (B) and arrow (C) denote the NEB and BADJ. (D) Wholemount airway immunostaining for GFP (green) plus CCSP (red) to identify multipotent epithelial patches. (E) Tissue CCSP, ACT, and GFP immunostaining reveals GFP-expressing ciliated cells (arrows). (F) Tissue CCSP, CGRP, and GFP immunostaining identifies intrapulmonary neuroepithelial bodies containing GFP(⫹) and -(⫺) neuroendocrine cells (white and yellow arrows). The white line denotes epithelial basement membrane (A–C, E, and F). (Scale bars, 50 m.)
there was no reproducible patch distribution pattern between individual mice regardless of overall chimerism. Rather, chimeric airways were always composed of numerous, randomly distributed GFP-positive and -negative patches of varying size (Fig. 2F). We therefore quantified the abundance and size of each patch (number of cells per patch) to produce size-frequency plots (Fig. 2G). This analysis indicated that the average chimeric patch size was small (ⱕ10 cells) and that there was a wide distribution of overall patch size (Fig. 2G, red line). These results were observed regardless of overall airway GFP chimerism (Fig. 2G, black and green lines). Small Chimeric Patches Are Mitotic and Exhibit Multipotent Differentiation. We next examined chimeric lung epithelial patches for
their proliferative and differentiation capacity. Expression of Ki67 was measured as an index of proliferative activity within GFP(⫹) cell patches. Analysis was limited to airway regions of highly imbalanced chimerism to increase the probability that GFP(⫹) patches were monoclonal. Within these regions we readily identified Ki67-immunoreactive GFP-positive and -negative cells (Fig. 3A, arrows). These data indicate that chimeric patches were capable of intrinsic self-duplication. We also examined airway proliferation following 7-day continuous BrdU exposure in 6-month-old wild-type mice. Results revealed that ⬇1% of airway epithelial cells were mitotic over this period. Mitotic cells were not associated with either NEBs (Fig. 3B) or the BADJ (Fig. 3C). Assuming a relatively constant 9288 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0900668106
Whereas our current findings have shown that abundant progenitor cells regulate epithelial maintenance during normal airway homeostasis, our previous data have established a critical role for NEB- and BADJ-associated stem cells for epithelial repair after injury (7–9). This earlier result suggests that airway injury and epithelial cell loss might regulate stem cell activation and repair. To explore this possibility using our improved airway imaging model we treated 13 chimeric mice with the airway Clara cell-specific cytotoxicant naphthalene. Injured mice were clustered into 2 groups on the basis of their 48-h total body weight loss (Fig. 4A). Three mice from each group were culled 3 days after naphthalene exposure to determine injury severity; the remaining mice were allowed to recover for a total of 30 days. We used CCSP abundance in bronchioalveolar lavage (BAL) fluid to determine both airway injury (3-day recovery) and epithelial repair (30-day recovery). BAL results revealed that mice with ⬎10% body weight loss 3 days postexposure were significantly more injured than those with ⬍10% weight loss (P ⬍ 0.05, Fig. 4B; referred to hereafter as severe and slight injury). Despite this result, both slight and severe injury mice exhibited normal CCSP BAL secretion 30 days after injury (Fig. 4B). This result indicated that naphthalene injury severity does not influence long-term lung regenerative capacity. To determine whether injury severity promotes distinct stem cell- or progenitor cell-mediated repair, we examined GFP chimerism in both slight and severe injury, 3- and 30-day recovered airways (Fig. 4 C–F). Both 3- and 30-day recovered, slight injury airways exhibited GFP(⫹) and -(⫺) patch expression patterns indistinguishable from those in normal, uninjured airways (compare Fig. 4 C and E with Fig. 1D). In contrast, 3-day recovered, severely injured airways contained no detectable GFP-expressing cell patches and only very rare individual GFP(⫹) cells (Fig. 4D). This difference appeared to be due to the destruction of most airway Clara cells. Strikingly, the lungs of all severe injury animals recovered for 30 days exhibited a unique pattern of very large, GFP(⫹) cell patches localized reproducibly at airway branchpoints and terminal bronchioles (Fig. 4F). As before we quantified GFP(⫹) and -(⫺) patch cellularity in naphthalene injured airways and compared their size-frequency plots (Fig. 4G). Three- and 30-day recovered, slight injury plots resembled those of uninjured airways although there was a slight increase in mean patch size (Fig. 4G, dashed and solid green lines). This finding likely reflects the limited proliferation of remaining progenitor (Clara) cells distributed throughout airways. In contrast, 30-day recovered severely injured lungs exGiangreco et al.
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Fig. 4. Injury severity determines epithelial repair modality. (A) Percentage of body weight loss 48 h postnaphthalene administration. Individual chimeric mice were clustered on the basis of low (⬍10%, green) or high weight loss (⬎10%, red). (B) Lavageable CCSP protein abundance in control, 3-day recovered, or 30-day recovered animals. (C–F) Representative images of chimeric epithelial patches within 3-day (C and D) and 30-day (E and F) recovered airways grouped according to slight (C and E) or severe (D and F) injury/48-h weight loss. (Scale bars: C–F, 100 m.) (G) Airway chimeric patch size-frequency plots comparing uninjured (black line), 3- or 30-day slight (dashed and solid green lines), and 30-day severe injury airways (red line). The proximal– distal axes (P3D) and airway boundaries (white lines) are indicated in C–F. Data are represented as mean ⫾ SEM in B and G. We used Volocity image acquisition software to normalize the brightness and contrast on regions acquired during an intermittent laser anomaly.
hibited a significantly increased mean and modal patch size (Fig. 4G, red line). NEB- and BADJ-Associated Stem Cells Repair Severely Damaged Airways. On the basis of the observation that large patches were
reproducibly found in association with airway branchpoints and terminal bronchioles after severe injury, we examined whether large GFP(⫹) patches corresponded to NEB and BADJ microenvironments known to harbor bronchiolar stem cells (Fig. 5 A–C). Our results revealed that, on average, ⬎85% of GFP(⫹) patches observed after severe injury were associated with NEB and BADJ stem cell niches. Although a minority (⬇15%) of chimeric epithelial patches were not associated with NEBs or BADJs, these patches were found only within proximal airways for which repair has previously been shown to involve cytokeratin 14 -positive basal-like progenitor cells (refs. 23 and 24 and data not shown). Examination of 30-day recovered, severely injured airways revealed that branchpoint-associated NEBs typically contained both GFP(⫹) and GFP(⫺) CCSP-expressing regenerative cell patches (Fig. 5A). We also found a number of solely GFP(⫹) NEB-associated regenerating patches (Fig. 5B). Analysis of terminal bronchioles revealed that BADJs always contained a Giangreco et al.
mixture of large GFP(⫹) and GFP(⫺) CCSP-expressing cell patches (Fig. 5C). Together, these results indicate that both NEB and BADJ microenvironments typically contain multiple CCSPexpressing stem cells. Surprisingly, whole-mount airway immunostaining also revealed an abundant yet previously uncharacterized population of non-branchpoint-associated NEBs that lacked regenerating CCSP-expressing epithelial foci (Fig. 5 D and E). This finding suggests that a naphthalene-resistant, NEB-associated stem cell may additionally be defined by its anatomical localization near airway branchpoints. Unlike in NEBs we did not observe any terminal bronchioles that lacked significant airway regeneration. We confirmed these results by paraffin-embedded section analysis, which revealed that large GFP(⫹), CCSP-expressing patches were uniquely associated with branchpoint-associated NEBs and terminal bronchioles 30 days after severe lung injury (Fig. 5 F and G). Discussion This study provides evidence that bronchiolar airways are maintained by self-duplication of abundant lung progenitor cells without measurable contribution from previously identified lung stem cells. This evidence suggests that airway stem cells are dispensable for normal lung homeostasis. We have also demonPNAS 兩 June 9, 2009 兩 vol. 106 兩 no. 23 兩 9289
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H Fig. 5. NEB- and BADJ-associated stem cells define regenerating patches within severe injury airways. (A–C) CGRP (red), GFP (green), and CCSP (blue) immunostaining identifies NEB (red cell clusters, A and B) and BADJ (arrows, C) -associated epithelial regeneration 30 days post injury. (D) Immunostaining also identified numerous non-branchpoint-associated NEBs that lacked associated regeneration. (E) Frequency of regenerative vs. nonregenerative NEBs. (F and G) Tissue section staining confirms epithelial regeneration adjacent to branchpoint-associated NEBs (red cell cluster, F) and terminal bronchioles (arrow, G). (H) Model in which epithelial injury severity is the principal determinant of progenitor cell-dependent homeostasis or stem cell-mediated epithelial repair. Data are represented as mean ⫾ SEM. (Scale bars, 50 m.)
strated that terminal bronchiolar stem cells do not maintain normal lung alveolar tissues. These findings are similar to recent reports describing mechanisms of tissue maintenance in the pancreas, skin, and corneal epithelium and support a hypothesis that many tissues are maintained by stochastic cellular selfduplication rather than stem cell activation (5, 6, 25, 26). Results presented in this study reveal that injury severity determines airway stem vs. progenitor cell activation and epithelial repair strategies. Using whole-mount imaging we have demonstrated that variant CCSP-expressing NEB- and BADJassociated stem cells represent the only stem cell populations within distal conducting airways. Our current observation that regenerating epithelia are composed of both GFP(⫹) and -(⫺) patches suggests that chimeric airway branchpoint NEB and BADJ stem cell niches commonly harbor at least 2 naphthaleneresistant stem cells capable of significant clonal expansion after acute lung injury. Furthermore, our finding that clonogenic cells contributing to repair following severe injury localize exclusively to airway branchpoint-associated NEBs and terminal bronchioles suggests that these microenvironments provide local factors that are necessary for maintenance of airway stem cells. Although participation of either abundant progenitor cells or stem cells can maintain or restore epithelial function, our results demonstrate that repair mediated through activation of local stem cells leads to clonal expansion and loss of chimeric airway progenitor cell diversity. Intriguingly, severe skin injury produces a similar effect whereby hair follicle stem cells undergo significant clonal expansion at the expense of normal tissue heterogeneity (27, 28). On the basis of these results we now propose a model in which tissue injury may be the principal 9290 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0900668106
determinant of stem vs. progenitor cell activation (Fig. 5H). In this model, normal, slight, or moderate injury will result in resident progenitor cell activation to restore tissue homeostasis whereas only severe injury and extensive epithelial cell loss will promote stem cell-mediated repair. We feel this model may be generally applicable to multiple types of lung injury and to injury of other tissue types. Our current model is also consistent with recent reports of a single progenitor maintaining both normal tail skin and endocrine pancreatic tissues, each of which additionally harbor facultative stem cell populations capable of restoring tissues after severe injury (5, 6, 15, 17, 29). Terminal bronchiolar stem cells have been implicated as originating cells for activated Kras-induced lung adenocarcinomas (7, 30). Although various dominant oncogenic Kras mutations can by themselves initiate lung adenocarcinomas (31–33), naphthalene injury potentiates the tumorigenic effects of mutant K-ras (7). We believe that our current findings may help to explain these observations. In the context of normal airways, oncogenic mutations by themselves should not efficiently promote lung tumorigenesis as airway stem cells exhibit a limited contribution to lung homeostasis. In contrast, severe injury, epithelial cell loss, stem cell activation, and clonal cellular expansion subsequent to oncogenic mutation may more efficiently promote lung carcinoma formation. Altogether, we have shown that lung stem cells do not contribute to normal tissue homeostasis and that airways are instead maintained by abundant epithelial progenitors (Clara cells). Although stem cells are present within lungs and become activated following injury to restore lung function, stem cellmediated repair appears to occur at the expense of normal epithelial heterogeneity. These results should have significant practical implications regarding our understanding of airway disease formation and treatment. Experimental Procedures Chimera Generation and Animal Husbandry. Wild-type:GFP chimeric mice (WT:GFP) were generated by 8- to 16-cell embryo aggregation using FVB/nCrl and chicken actin-GFP mouse strains [C57BL/6-Tg(CAG-EGFP)1Osb/J]. Embryos were implanted into pseudopregnant females and adults scored for chimerism on the basis of skin GFP. Mice were housed under specific pathogen-free conditions on a 12-h light cycle and allowed access to food and water ad libitum. All mice were aged between 4 and 6 months at the time of sample collection and showed no signs of ill health. In vivo studies were performed in compliance with Home Office (United Kingdom) and Duke University animal welfare guidelines. Sample Preparation. Mice were killed by i.p. phenobarbitol overdose and lungs were either gently lavaged with PBS to assess secreted CCSP abundance by ELISA followed by fixation (naphthalene exposure experiment) or directly open-cavity inflation fixed at 10 cm H20 using 10% formalin. Whole lungs were fixed overnight in 10% formalin and either switched to 70% EtOH (for paraffin embedding) or washed in PBS/0.02% azide (for whole-mount microdissection). Naphthalene and BrdU Exposure. Eleven chimeric males and 2 females were injected i.p. with 250 mg (males) or 225 mg (females) naphthalene/kg weight dissolved in corn oil. Mice were inspected and weighed before and 48 h postinjection to assess injury severity. Separately, wild-type FVB/n females were given 6 mg/ml BrdU in sterile saline i.p. every 12 h for a total of 7 days. After 7 days lungs were inflation fixed using 10% formalin, degassed in 30% sucrose, and cryopreserved using optimal cutting temperature (OCT) solution. Immunostaining and Whole-Mount Microdissection. Murine CCSP (in house), CGRP (Sigma), acetylated tubulin (ACT) (Sigma), Ki67 (Novocastra), and GFP (Abcam) antibodies were used for immunostaining following citrate buffer antigen retrieval. Secondary antibodies included appropriate species-specific Alexa488, Alexa555, and Alexa633 dyes (Invitrogen). Frozen sections were stained for BrdU plus airway markers as previously described (10). Left lung lobes were inflation fixed and dissected using a brightfield microscope, fine forceps, and microdissecting scissors to expose the airway network to terminal bronchioles and antibody stained. Microdissected lungs
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Image Acquisition and Analysis. Whole lung images were collected using an 8000-Hz resonance detector-equipped Leica TCS confocal microscope. X–Y field size and Z-thickness were determined on the basis of total lung size (⬇2 cm ⫻ 1 cm ⫻ 500 m) to produce a single 3D image projection. High-resolution images were collected at 400 –700 Hz. Digitally flattened, 3D lung images were analyzed using Volocity based on GFP signal intensity and patch boundary continuity to determine airway chimerism and frequency vs. size (square micrometers) of coherent cell patches. Regions of interest (ROI) were drawn on each lung image that encompassed the entire conducting airway and GFP channel intensity was used to distinguish GFP(⫹) and -(⫺) cells. The average
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pixel surface area (square micrometers) of single cells was determined empirically and used to exclude subcellular debris. All coherent, contiguous groups of ⱖ2 cells within our defined ROI were scored automatically using these parameters as single epithelial ‘‘patches.’’ Patch size (in square micrometers) was converted to cell number on the basis of our calculated average epithelial cell size, and patch cell number vs. frequency was plotted using Microsoft Excel frequency/bins parameters. In all figures, error bars represent the standard error of the mean (SEM). Statistical significance was considered when P ⬍ 0.05 based on Student’s t test. ACKNOWLEDGMENTS. We acknowledge Doug Winton and John Stingl for suggestions related to interpretation of epithelial chimerism, Jessica Gruninger for embryo aggregations, Brian Brockway and Jeff Drake for tissue preparation, and Virgilio Failla for assistance with Volocity. This work was supported by grant funding from the National Institutes of Health (to A.G., J.S., and B.R.S.) and Cancer Research UK (to A.G., E.A., I.R., and F.M.W.). A.G., E.A., and F.M.W. acknowledge the support of the University of Cambridge and Hutchison Whampoa.
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were incubated in 10% bovine serum/0.25% fish skin gelatin for 2–3 h to block nonspecific antigen reactivity. Primary antibodies were added in blocking solution overnight (O/N) at room temperature followed by PBS/0.2% Tween20 washes. Secondary antibodies were also added O/N at room temperature and washed extensively. Immunostained lung lobes were imaged up to 4 weeks poststaining.
