Based on its anatomical and functional features, the lung epithelium can be divided into three domains: the proximal cartilaginous airways (trachea and bronchi), the bronchioles (bronchioles, terminal bronchioles, and respiratory bronchioles), and the alveoli. The epithelial cell types in each of these domains are distinguished by their morphology, cellular phenotype (i.e., proteins they express), and function. The proximal airway of the mouse is lined with a pseudostratified columnar epithelium composed mainly of basal, club, goblet, and ciliated cells; the secretory SMGs reside beneath this surface airway epithelium (SAE) and are limited to the proximal trachea in mice (Hansell and Moretti 1969; Pack et al. 1980; Widdicombe et al. 2001; Jeffery 1983; Liu et al. 2006). The major cell types in the human proximal airway differ slightly from those in mice and include basal, intermediate, goblet, non-ciliated columnar, and ciliated cells (Jeffery 1983; Liu et al. 2006; Mercer et al. 1994). Furthermore, in humans the SMGs are present throughout the cartilaginous airways, including the trachea and bronchi. These glands are composed of an interconnecting network of serous acini and mucus tubules, which secrete antibacterial factors, mucous, and fluid into the airway lumen (Wine and Joo 2004). In the distal mouse and human airways (i.e., bronchioles), club, ciliated, neuroendocrine, and goblet cells are the major cell types, and neuroendocrine cells are found both individually and in clusters within NEBs (Mercer et al. 1994; Van Lommel et al. 1999; Plopper et al. 1980; Liu et al. 2006). However, the bronchioles of human lungs have also been shown to contain basal cells, albeit at lower abundance than in the proximal regions (Tamai 1983; Rock et al. 2010). The alveolar epithelium is lined by surfactant-producing cuboidal alveolar type II epithelial cells (AECII) and squamous gas-exchanging alveolar type I epithelial cells (AECI) (Liu et al. 2006). The major epithelial cell types that are present at various locations throughout the airway are listed in Table 3.1.

Stem/progenitor cells are crucial for development, tissue homeostasis, and injury repair in the lung. Studies using epithelial reconstitution assays, murine injury models, and lineage tracing approaches have identified several region-specific stem/progenitor cell populations in the adult lung of mice and humans. Basal cells in the proximal airways, variant club cells in bronchioles, bronchoalveolar stem cells (BASCs) in BADJs, and a subset of AECII in alveolar spaces have all been identified as stem/progenitor cells (Table 3.1).

In the trachea and main-stem bronchi, basal cells are the principal stem cells involved in homeostasis and injury repair and have the capacity to generate all the major cell types found in the proximal airway, including basal, ciliated, goblet, and granular secretory cells (including club cells) (Hong et al. 2004a, b; Hajj et al. 2007; Rock et al. 2009; Schoch et al. 2004; Cole et al. 2010; Engelhardt et al. 1995). The intermediate cells in the human proximal airway are so named because they are generally thought to represent an intermediate state of differentiation from basal cells and to serve as a transient amplifying cell population with the capacity to differentiate into ciliated and goblet cells (Engelhardt et al. 1995; Mercer et al. 1994). Intermediate cells do not exist in the mouse proximal airway, potentially because of the less pseudostratified nature of their smaller diameter airways. Of note, studies of murine lung injury involving BrdU labeling demonstrated that label-retaining cells (LRCs) reside predominantly in the ducts of SMGs, suggesting that these glands serve as a stem cell niche in the proximal airway (Xie et al. 2011; Borthwick et al. 2001; Engelhardt et al. 1995; Engelhardt 2001; Rock et al. 2009). Importantly, the SMG-localized LRCs have the capacity to undergo sequential rounds of cell division despite their slowly cycling phenotype (Xie et al. 2011; Lynch and Engelhardt 2014). Nevertheless, because lineage tracing of glandular LRCs has not yet been possible, the ability of these stem cells to produce specific airway cell types remains unclear. Several cellular markers have been utilized to identify and isolate basal cells. These include cytokeratin 5 (CK5), cytokeratin 14 (CK14), and aquaporin 3 (Rock et al. 2009, 2010; Schoch et al. 2004). Using a CK5-CreERT2 transgenic mouse line, Rock et al. further demonstrated that basal cells are capable of differentiating into club and ciliated cells, both at steady state and during injury repair (Rock et al. 2009). In addition, they identified nerve growth factor receptor (NGFR) and integrin α6 (ITGA6, also called CD49f) as markers on the surfaces of isolated human basal stem cells (Rock et al. 2009). Similarly, Ghosh et al. identified a CD49f^bright/Sca-1⁺/ALDH1⁺ (Aldehyde dehydrogenase 1) subset of tracheal basal cells as region-specific stem cells, and demonstrated that these cells could generate niches in vitro and contribute to tracheal epithelial maintenance and injury repair (Ghosh et al. 2011). These studies suggested that basal cells play key roles in both homeostasis and injury repair of the proximal airway.

In the intralobar bronchiolar airways, a subset of the variant club cells that express club cell secretory protein (CCSP, also called Scgb1a1) but not CyP450-2F2 (CCSP⁺, CyP450-2F2⁻) can self-renew and produce both club cells and ciliated cells (Hong et al. 2001; Rawlins et al. 2009b; Reynolds and Malkinson 2010; Xing et al. 2012; Guha et al. 2012). This CCSP⁺/CyP450-2F2⁻ subset was also found at the BADJ of distal bronchioles, where it contributed to airway epithelial regeneration following naphthalene-mediated depletion of CyP450-2F2⁺ club cells (Giangreco et al. 2002). Kim et al. subsequently identified BASCs as a subpopulation of cells that express CCSP and pro-surfactant protein C (SPC) and serve as region-specific stem cell at the BADJ (Kim et al. 2005). Using naphthalene- and bleomycin-induced murine models of lung injury repair, Kim et al. further demonstrated that these cells possessed the capacity to self-renew and to produce differentiated epithelial cells in vivo, and that BASCs could differentiate into club cells and alveolar epithelial cells in an ex vivo clonogenic assay (Kim et al. 2005). Conversely, an in vivo lineage tracing experiment using a CCSP(Scgb1a1)-CreER™ knock-in mouse line revealed that club cells generated daughter club cells and ciliated cells but not alveolar cells following hypoxia-induced lung injury (Rawlins et al. 2009b). However, subsequent lineage tracing studies following alveolar injury by influenza infection and bleomycin exposure support the finding that CCSP-expressing stem/progenitors can give rise to AECI and AECII cells (Zheng et al. 2013; Rock et al. 2011). These injury-dependent influences on BASC-derived lineages suggest that either specific injury signals may invoke different responses and/or that multiple subsets of BASCs exist with different capacities for differentiation. The later hypothesis is consistent with findings suggesting that BASCs in the distal airways might include a heterogeneous population of progenitor cells (Teisanu et al. 2009, 2011; Chen et al. 2012). A study by Teisanu et al. classified club cells with the surface antigen profile CD45⁻/CD31⁻/CD34⁻/EpCAM⁺/Sca-1^low into two subgroups based on their autofluorescence (AF) profiles and suggested that club cells in the AF^low population are naphthalene resistant, whereas their AF^high counterparts were not (Teisanu et al. 2011). Indeed, mice that were exposed to naphthalene showed significantly greater proliferation in AF^low club cells compared to AF^high club cells, and conversely, mice exposed to ozone showed significantly greater proliferation in the AF^high club cell fraction compared to AF^low club cells (Teisanu et al. 2011). McQualter et al. demonstrated that an EpCAM⁺/Sca-1^low/Integrin α6β4⁺/CD24^low fraction of epithelial stem/progenitor cells was capable of self-renewing and differentiating into a variety of airway epithelial lineages, including alveolar epithelial cells (McQualter and Bertoncello 2012). These studies provide evidence that BASCs play key roles in the repair of injury to both bronchiolar and alveolar cells, as well as in homeostasis.

In the pulmonary alveolus, surfactant-producing AECII cells have long been recognized as stem/progenitor cells for the squamous AECI cells in the adult lung (Adamson and Bowden 1974; Evans et al. 1975). In vitro assays of cell proliferation and clonogenicity, as well as in vivo analyses following epithelial injury and lineage tracing, have produced mounting evidence that a subset of AECIIs have the capacity to proliferate and restore the alveolar epithelium by producing either new AECII cells or their squamous AECI counterparts (Reddy et al. 2004; Barkauskas et al. 2013; Fujino et al. 2011). Equally noteworthy were findings suggesting that integrin α6β4 is a biomarker for a subset of stem/progenitor cells in alveolar epithelia; SPC⁻/integrin α6β4⁺ cells were found resident in the alveolar epithelia, where they were able to regenerate SPC⁺ AECII cells (Chapman et al. 2011). The notion that AECII cells are region-specific stem/progenitors was recently confirmed by work from the Hogan laboratory, which employed a genetic SPC-labeled lineage tracing assay and an in vitro 3D culture model. This study produced convincing evidence that AECII cells were able to maintain the homeostasis of alveolar epithelia during both steady-state turnover and injury repair (Barkauskas et al. 2013). Several strategies that rely on biomarkers to identify and isolate adult lung stem/progenitor cells are listed in Table 3.2.

As discussed above, a vast body of evidence has demonstrated that distinct stem/progenitor cell populations reside in specific anatomical niches (Fig. 3.1), where diverse cell types and signals coordinate the behavior of stem cells during homeostasis and following injury. Stem cell niches are discrete microenvironmental units within a tissue that can provide one or more of the following features important for stem cell control: a unique extracellular matrix; supporting cell types; unique innervation and nearby vasculature; and diffusible factors that allow stem cells to maintain a capacity to self-renewal and control their proliferation and differentiation in the setting of injury (Fuchs et al. 2004). The anatomical sites of airway stem cell niches are typically epithelial structures associated with these unique features described above (e.g., innervation, support cells). Although much remains to be learned about how components of airway niches coordinate stem/progenitor cell behavior and phenotype, data from organ systems that have been studied more extensively suggest that they are likely important in the lung as well.

In the following discussion of stem cell niches in the airway, we focus on the unique anatomic and biologic properties of each niche within a particular region of the lung, and on how these features may contribute to repair following injury. In particular, we concentrate on studies of slowly cycling stem/progenitor cells in the mouse lung, since nucleotide label retention has been one of the most commonly used methods for tracking the anatomic locations of stem cell niches in the lung.