Fig. 1.1
Schematic representation of epithelial cell types found in airways of the adult mouse lung. The adult airway exhibits compartmentalization of constituent cells along the proximal—distal axis. The proximal bronchial epithelium is pseudostratified due to the presence of basal as well as luminal cells. The latter includes ciliated cells and non-ciliated secretory cells including goblet, serous, and club cells. In contrast to bronchial epithelium, distal bronchiolar airway in adult laboratory mouse strains lack basal cells and are predominantly populated by secretory club and ciliated cells, resulting in structural demarcation between the proximal and distal airways. This is unlike adult human distal airways where basal cells are maintained within distal bronchiolar epithelium. As described in the text, in vivo and in vitro assays have identified multiple stem and progenitor cell pools that include bronchial basal cells, Scgb1a1-expressing club cells and alveolar type II cells in gas exchange zone
The current model of adult tissue regeneration and maintenance supports the idea that regional pools of epithelial stem cells ensure maintenance and timely repair of damaged tissue (Leeman et al. 2014; Rawlins and Hogan 2006). Precedence for this model comes from studies of developing and adult murine lung epithelium. First, the lung epithelium is derived from the anterior foregut endoderm while the mesenchyme is derived from the splanchnic mesoderm. There is no evidence so far for the existence of a common developmental progenitor that gives rise to epithelial as well as mesenchymal cell types of the adult respiratory system, which precludes the idea of a common stem cell that gives rise to epithelial, as well as mesenchymal, cells in mice (Perl and Whitsett 1999; Morrisey and Hogan 2010; Cardoso and Lu 2006). Secondly, during the process of lung development, the epithelial lining of airways is spatially segregated into distinct proximal and distal zones by non-overlapping lineages of cells that are specified very early in gestation (Perl et al. 2002). This results in the spatially diverse distribution of the adult lung epithelium. Lastly, as outlined below, multiple pools of regionally restricted adult stem cells have been identified in the airway epithelium that contribute to repair. One theme that emerges from these studies is that progenitor and stem cell behavior is significantly altered between steady state and following injuries that remodel the tissue microenvironment, highlighting the interdependence of cell phenotypes and behaviors within different regional microenvironments of the lung. Additionally, different progenitor/stem cell pools respond differently to the injury type and pathological condition. For instance, following bleomycin treatment of laboratory strains of mice, resident alveolar progenitors in distal airway namely surfactant protein C expressing type 2 and integrin α6 β4 expressing stem cells are activated to promote tissue regeneration, whereas following naphthalene exposure that ablates most airway epithelial cells, Scgb1a1-expressing naphthalene-resistant variant club progenitors are essential for airway regeneration (Chapman et al. 2011; Barkauskas et al. 2013).
These and related data underscore the existence of distinct stem cell pools to combat tissue damage from a myriad of environmental and pathological insults during the lifetime of terrestrial beings. A related but less explored question is whether there is cross talk between the different lung stem cell pools similar to what has been observed in other organ systems such as rapid and slow cycling stem cells of small intestinal crypts or between luminal and myoepithelial cells of the mammary epithelium (Van Keymeulen et al. 2011; van Es et al. 2012). In that context, recent findings that luminal Scgb1a1-expressing secretory cells can dedifferentiate into basal-like stem cells following ectopic ablation of tracheal basal cells suggest that different stem cell pools in lung can indeed cross talk and influence each other’s stem cell behavior (Tata et al. 2013).
Identification of human lung stem cells has primarily relied upon in vitro culture assays of cells isolated based on expression of putative stem cell markers (Barkauskas et al. 2013; Rock et al. 2009). However, as mentioned later, strategies to study human lung stem cell behavior in vivo have been reported and advances in such approaches will be critical to better understand their roles. A putative c-kit positive pool of human stem cells has been proposed to generate both epithelial and endothelial cells in culture and promote airway and pulmonary vessel repair following cryo-injury in mice (Kajstura et al. 2011). Such studies suggest existence of a single multipotent stem cell pool, but its biological significance and roles in vivo remain unclear (Lung stem cells: looking beyond the hype 2011). Functions of lung mesenchymal cells in postnatal tissue maintenance, repair, and epithelial stem cell function are just beginning to be understood and precise knowledge of their role in epithelial repair and maintenance will be paramount (Sinclair et al. 2013).
1.2 Epithelial Cells of the Conducting Airway and Alveolar Zones
The epithelium lining proximal airways is pseudostratified in composition and composed of basal cells that contact the basement membrane and luminal cells that extend from basolateral contacts with the basement membrane to the airway lumen. Basal cells are attached to the basement membrane by hemi desmosomes and to luminal columnar cells by desmosomes (Evans et al. 1990; Evans and Plopper 1988); these cytoskeletal associations promote airway structural integrity. As discussed later, basal cells behave as multipotent stem cells to promote adult tissue regeneration.
In mice, basal cell distribution tapers along the tracheal-bronchial airway axis and are absent from bronchiolar airways. This is in contrast to adult human airway epithelium that remains pseudostratified from trachea down to small bronchioles of distal lung (Nakajima et al. 1998). The proximal airway luminal cells include ciliated cells and non-ciliated secretory cells that are specialized for mucus clearance and host defense response, respectively, as described later. Additionally, there are neuroendocrine cells that have been particularly implicated in human small-cell lung cancer (SCLC). Their function in steady state tissue maintenance remains unclear (Song et al. 2012).
The bronchiolar airway epithelium in mice is largely maintained by differentiated ciliated and non-ciliated secretory cells. The abundant cilia protruding from the apical membrane of the ciliated cells are each held to the cytoplasmic basal body through an axoneme (Jeffery and Reid 1975; Rhodin 1966). Axonemes are microtubule-based structures that are associated with dynein and kinesin motors to allow synchronous beating of cilia and mobilization of mucus and adsorbed materials. The non-ciliated secretory cells of the bronchiolar airway include morphologically and functionally distinct cells such as the goblet, serous, and club cells. Goblet and serous cells are primarily involved in secretion of mucous glycoproteins, enzymes, and antimicrobial peptides that collectively promote inactivation and clearance of inhaled agents. Defects in mucus production, clearance, and antimicrobial defense can contribute to serious chronic respiratory conditions such as the lung complications associated with cystic fibrosis (Rogers 1994). Club cells are involved in diverse functions including modulation of local inflammatory responses, host defense, xenobiotic metabolism, and tissue maintenance and regeneration (discussed later).
The distal lung gas exchange component is composed of alveolar type 1 (AT1) and type 2 (AT2) cells. Surfactant protein secreting type 2 cells maintain surface tension and prevent cell collapse following exhalation. The thin and lacy morphology of alveolar type 1 cells and their apposition to blood capillaries makes them well suited for gas exchange (Bertalanffy and Leblond 1955).
The diversity in mature epithelial cell types and their functions along the airway axis underscores the need for region-specific mechanisms to maintain and regenerate the epithelium in homeostasis and repair.
1.3 Region-Specific Stem and Progenitor Cells Maintain the Adult Airway
Identification of regional stem and progenitor cell pools has been accomplished using a combination of techniques including in vivo lineage tracing of defined epithelial cell types, in vitro 3-dimensional culture assays to investigate the behavior of flow sorted epithelial cells, and ex vivo transplant assays following isolation of putative stem cells (Barkauskas et al. 2013; Chen et al. 2012; Rock et al. 2009; Flodby et al. 2010; Kretzschmar and Watt 2012; Randell et al. 1991). Technical caveats and limitations notwithstanding these approaches have proved valuable to advance understanding of lung stem cell functions in maintenance of epithelial diversity along the airway tree.
1.3.1 Basal Cells Function as Stem Cells for Maintenance and Repair of the Pseudostratified Epithelium of Tracheal and Bronchial Airways
Identification of stem cell properties in basal cells is obtained from several lines of evidence including lineage analyses of Keratin 14 and 5 expressing cells under steady state and during tissue regeneration following injury. Lineage tracing experiments demonstrated that mouse tracheal basal cells expressing the intermediate filament protein cytokeratin 5 (Krt5) have long-term self-renewal capacity and can generate differentiating progeny that replenish both ciliated and non-ciliated luminal epithelial cells types. These properties held true under conditions of normal epithelial turnover and during renewal following exposure to toxic chemicals like sulfur dioxide and naphthalene. However, there is evidence that not all basal cells of mouse pseudostratified airways share the same molecular characteristics or have the same capacity for multipotent differentiation (Hong et al. 2004; Rock et al. 2009). It is not clear whether these differences between airway basal cells are stochastic or evidence of functionally distinct subsets. In addition to intrinsic differences, cell extrinsic factors such as changes in the microenvironment from injury significantly alter their proliferation dynamics and differentiation fate (Ghosh et al. 2011; Rock et al. 2009). In vivo data are substantiated by in vitro experiments in which isolated basal stem cells can undergo proliferation and differentiation to generate pseudostratified trachea-like structures reminiscent of in vivo tissue architecture. Additional evidence for their stem cell roles comes from their ability to promote regeneration of denuded tissue in xenograft assays using rodent and human tracheal cells (Rock et al. 2010).
Although the current model suggests basal cells as the stem cells of trachea and proximal bronchi, ectopic ablation of these cells in the trachea could trigger dedifferentiation of Scgb1a1-expressing secretory cells into basal-like stem cells to promote regeneration of pseudostratified airway. Such studies substantiate the role of non-cell autonomous factors in the regulation of airway progenitor cell behavior (Tata et al. 2013).
Evidence for their role in human airways is derived from in vivo clonal patch analyses and in vitro growth assays of isolated putative basal cells. In humans, basal cell marker expression was initially reported in stem cell-derived clonal patches from human upper airway epithelium, and their stem cell function supported by in vitro 3D culture assays (Rock et al. 2010). In vivo clonal patch analyses suggest that proliferating basal cells are maintained by a neutral drift model where loss of progenitor cells due to differentiation is compensated by duplication of neighbor cells (Teixeira et al. 2013).
1.3.2 Non-ciliated Club Cells Behave as Bronchiolar Stem Cells
Non-ciliated cells of mouse bronchiolar airways have been defined based upon their expression of the abundant secretory club cell-specific protein (CCSP or Scgb1a1). The Scgb1a1-expressing cells represent a highly heterogeneous cell type whose diversity develops early in embryonic development (Guha et al. 2012, 2014). In vivo lineage tracing experiments under steady state conditions have identified club cells as the progenitor cells of mouse bronchiolar airways, but not of tracheal airways. Thus, the current model supports the idea that basal cells are stem cells of the tracheobronchial airway in mice while Scgb1a1-expressing cells constitute the bronchiolar progenitor pool. However, as mentioned earlier, in the event of a loss of tracheal basal cells, Scgb1a1 cells undergo self-renewal and differentiation to regenerate pseudostratified tracheal airway epithelium (Rawlins et al. 2009; Tata et al. 2013). Their role in bronchiolar regeneration has been studied in the context of various injury models including those triggered by exposure to naphthalene, bleomycin, and viral infection. Whether distinct subsets of Scgb1a1-expressing cells respond to the different kinds of injury remains unexplored. One subset that is resistant to naphthalene exposure due to their lack of Cy2f2 (termed as variant club cells) promotes regeneration following naphthalene-induced epithelial damage and are localized around the neuroendocrine bodies (NEB) of mouse airway and terminal bronchoalveolar duct junctions (BADJ) (Giangreco et al. 2002; Reynolds et al. 2000a, b). Intriguingly, NEB is also associated with precursors of secretory cells such as Scgb3a2 and Upk3a (Guha et al. 2012), and thus resembles a niche for secretory cell development and progenitor function in adults. The mechanism by which these cells promote parenchymal tissue regeneration remains less clear. But in vivo Scgb1a1-expressing club cells have been shown to generate alveolar type 2 and 1 cells following tissue injury such as those from bleomycin treatment and PR 8 influenza virus infection (Rock et al. 2011; Zheng et al. 2012, 2013).
Scgb1a1-expressing cells also constitute the pool of bronchioalveolar stem cells (BASCs) that were identified by their coexpression of Scgb1a1 and alveolar type 2 protein SPC and were located at regions where terminal airway abuts with alveolar epithelial cells (Kim et al. 2005). In vivo lineage tracing analyses support their role in alveolar type 2 cell regeneration following alveolar cell-specific injury (Tropea et al. 2012). In vitro culture assays of BASCs have not only highlighted their ability to undergo differentiation into multiple epithelial lineages including bronchial, alveolar, as well as bronchioalveolar, cells but also underscored the role of surrounding endothelial cell microenvironment in determination of their differentiation fate (Lee et al. 2014).
Additional pools of multipotent adult airway progenitor cells expressing Epcam, CD49f, CD104, and CD24 (low) have been identified by in vitro matrigel-based clonal assays and have been shown to generate airway as well as alveolar cell lineages (Bertoncello and McQualter 2011; McQualter et al. 2010). In humans, Scgb1a1 or CC10 (club cell)-expressing cells represent significant proportion of proliferating cells in distal conducting airways under steady state and are reduced following cigarette smoke injuries. Their contributions to tissue repair and regeneration, however, remain unknown (Boers et al. 1999).
1.3.3 The Bronchioalveolar and Alveolar Stem Cells
Classical BrdU labeling experiments suggest that adult alveolar epithelium undergo slow renewal (Messier and Leblond 1960). In vivo lineage tracing analyses have shown that adult alveolar type 2 cells are progenitor cells that undergo self-renewal, clonal expansion, and differentiation into alveolar type 1 cells (Barkauskas et al. 2013; Desai et al. 2014). Changes in the endogenous microenvironment caused by targeted loss of type 2 cells or widespread alveolar cell loss by bleomycin treatment significantly alter stem cell behavior namely in their proliferation and differentiation rate, implicating role of cell extrinsic factors in regulation of stem cell behavior. In vitro three dimensional alveolosphere formation assays using isolated surfactant type 2 cells substantiate their self-renewal and differentiation properties (Barkauskas et al. 2013). In vitro culture assays of isolated alveolar type 1 cells implicate their role as putative stem cells although in vivo evidence warrants further research (Dobbs et al. 2010; Gonzalez et al. 2009).
Additional pools of distal lung stem cells include laminin receptor α6 β4 integrin expressing facultative stem cells that promote alveolar cell regeneration after parenchymal injury such as those induced following bleomycin treatment (Chapman et al. 2011).
1.4 Summary
Identification and function of stem cells in a slowly regenerating organ like the lung is highly context dependent. The current body of evidence does not support the existence of a single lung stem cell, rather the existence of multiple region-specific stem and associated progenitor cells that can generate all specialized cell types to fulfill adult airway function. It is, however, safe to infer that several pools of stem cells maintain the different compartments of adult respiratory tract, and that their stem cell behavior is intricately regulated by changes in their microenvironment. Existence of multiple adult stem cell pools is therefore serving multiple functions including: (1) maintenance of spatially diverse epithelial cell types of the adult airway to promote tissue homeostasis as well as (2) timely and efficient regeneration of the airway epithelium to combat the diverse injury types to which the adult airway is exposed during its lifetime.
