We show that Sca-1, a previously described cell surface marker for the bronchioalveolar stem cell [10
], is a common cell surface marker for a broad population of bronchiolar epithelial progenitor cells that include the abundant pool of facultative TA (Clara) cells and naphthalene-resistant bronchiolar stem cells. We found that bronchiolar progenitor cells could be distinguished from many other epithelial and mesenchymal cell types on the basis of their Sca-1low
phenotype. Bronchiolar cells could be further subdivided on the basis of their AF characteristics: facultative TA (Clara) cells displayed an AFhigh
phenotype, whereas bronchiolar stem cells exhibited an AFlow
phenotype. This fractionation approach was validated using strategies involving targeted cell ablation, lineage tagging, and stem cell expansion and provides a robust set of criteria for further investigation of isolated bronchiolar stem cells at molecular and functional levels.
Our demonstration that bronchiolar stem cells can be enriched for within dissociated mouse lung preparations on the basis of their CD45neg
phenotype differs from the previously described characteristics of isolated bronchioalveolar stem cells detailed by Kim et al. [10
]. Differences between our findings are that in our study Sca-1 did not distinguish between subsets of CCSP-expressing cells (facultative TA vs. stem cells), that the epithelial Sca-1low
fraction was a relatively abundant component of the total cell preparation, and that epithelial cells defined by a CCSP-Cre activated lineage tag were negative for CD34. The basis for these differences may be related to methods used for cell isolation and/or antibodies used to define cell surface phenotype. Lung cell preparations used for analysis by Kim et al. were generated using dispase/collagenase digestion coupled with methods optimized for the isolation of alveolar type II pneumocytes [10
]. In contrast, lung cell preparations used herein were generated through use of Elastase and methods optimized for inclusion of a broad population of epithelial cells from the conducting airway and alveolus [15
]. Importantly, bronchiolar stem cells from both bronchioalveolar duct junction and neuroepithelial body microenvironments in addition to the abundant pool of facultative TA (Clara) cells should be represented among dissociated cell preparations used in this study. This would not be the case using preparations that largely exclude epithelial cells from conducting airways. Furthermore, differences in cell surface CD34 reactivity observed herein and that of Kim et al. [10
] remain to be determined. The possibility that absence of CD34 reactivity among lung epithelial cell types in this study results from conditions used for proteolytic dissociation of cells would seem unlikely, as this antigen is preserved on the surface of other nonepithelial cell types.
Stem cells are commonly thought to exhibit a less-differentiated character than their transit-amplifying progeny. Whereas this distinction is difficult to make for rapidly renewing tissues such as the epithelium of the small intestine, for which both populations appear to proliferate frequently and lack characteristics of specialized epithelial cells [17
], it is more apparent within the progenitor cell hierarchy of the bronchiolar epithelium. We demonstrate that Clara cells, an abundant facultative TA cell type of bronchiolar airways, exhibit the distinguishing characteristic of high autofluorescence. This characteristic of Clara cells is supported through (a) loss of the AFhigh
fraction of lung cells following ablation of CCSP-expressing cells (including Clara and stem cells) in GCV treated CCSP-HSVtk transgenic mice, and (b) loss of AFhigh
cells following airway potentiation of β
-catenin signaling. Autofluorescence is a well-known characteristic of the airway epithelium [18
]. In human patients autofluorescence bronchoscopy is used as a screening tool for lung cancer [18
]. In lung cancer, a patient's malignant cells, which are poorly differentiated in character, are detected as low-level autofluorescence fields within a high autofluorescent background of healthy epithelial cells [18
]. Collectively, these data argue that stem cells can be distinguished from their more differentiated derivatives on the basis of autofluorescence characteristics and that this property is common between airway stem cells and tumor cells.
Bronchiolar epithelial cells observed in airways of mice following constitutive potentiation of β
-catenin signaling lack differentiated features (cytoplasmic organelles and differentiation markers) typical of facultative TA (Clara) cells, are resistant to naphthalene injury, and show a CCSP/Pro-SPC dual-expressing phenotype, all characteristics of the bronchiolar stem cell [10
]. However, even though coexpression of CCSP and Pro-SPC has the potential to distinguish bronchiolar stem cells from more abundant facultative TA (Clara) cells in vivo, our data suggest that this may not be the case following enzymatic dissociation and sorting of lung cells. We find that all CCSP-immunoreactive cells present within the Sca-1low
fraction (both AFhigh
populations) show a Pro-SPC-immunoreactive phenotype. These data suggest that Pro-SPC immunoreactivity does not distinguish between facultative TA (Clara) cells and bronchiolar stem cells following enzymatic dissociation of lung tissue and subsequent fractionation of isolated cells. This is consistent with our observation of a similar frequency of CCSP/Pro-SPC dual-positive cells between isolated wild-type and ΔE3 cell preparations. We conclude that use of CCSP/Pro-SPC dual positivity alone as a basis to investigate the impact of signaling pathways on isolated bronchiolar epithelial cells does not provide a basis for discrimination between rare bronchiolar stem cells and abundant facultative progenitor (Clara) cells [21
]. We show that despite upregulation of Pro-SPC within mature Clara cells, their distinguishing morphological features coupled with unique high autofluorescence characteristics provide a basis for their separation from low-autofluorescent bronchiolar stem cells.
Standard methods in stem cell biology include in vivo and in vitro assays to demonstrate self-renewal and differentiation potential of the proposed stem cell population. Even though in vitro models have been developed allowing propagation of tracheobronchial epithelial cells [24
], bronchiolar epithelial cells are extremely difficult to maintain and propagate in vitro. Moreover, no in vivo transplantation studies have been reported that allow faithful establishment of bronchiolar epithelium from fractionated preparations of bronchiolar cells. Fractionation methods allowing enrichment of bronchiolar stem cells described herein will allow further analysis of gene expression to define a unique molecular phenotype and will provide a basis upon which to build in vitro and transplantation models to assess mechanisms of self-renewal. Together with transgenic animal models for identification and manipulation of the stem cell compartment, these assays will provide critical tools to unravel the bronchiolar stem cell phenotype and mechanisms governing the behavior of these cells in normal and diseased lung.