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Author contributions: J.L.M. and I.B.: conception and design, data analysis and interpretation, manuscript writing, financial support, and final approval of manuscript.
Disclosure of potential conflicts of interest is found at the end of this article.
First published online in STEM CELLSEXPRESS February 13, 2012; available online without subscription through the open access option.
Despite burgeoning interest in the potential of cellular therapies in lung regenerative medicine, progress in delivering these therapies has been confounded by a lack of knowledge about the identity of appropriate targets which can be harnessed to repair the lung, and the cellular and molecular factors which regulate their regenerative potential. While systematic analysis of lung development and cell lineage tracing studies in normal and perturbed animal models provides a framework for understanding the complex interplay of the multiple cell types, biomatrix elements and soluble and insoluble cytokines and factors that regulate lung structure and function, a reductionist approach is also required to analyze the organization of regenerative cells in the adult lung and identify the factors and molecular pathways which regulate their capacity to generate descendent lineages. In this review we describe recent progress in identifying and characterizing endogenous epithelial, mesenchymal and endothelial stem/progenitor cells in the adult lung using multiparameter cell separative strategies and functional in vitro clonogenic assays. STEM CELLS2012;30:811–816
By 2020, 12 million of 68 million deaths worldwide will be attributed to lung disease, and actuarial data collated by international bodies including the World Health Organisation ranks lung diseases second in incidence, prevalence, mortality, and cost . While current therapies have significantly improved the quality of life and survival of lung disease patients by attenuating inflammatory responses or selectively targeting molecular pathways, receptors, and ligands which exacerbate disease, they are essentially palliative, providing symptomatic relief without reversing the damage to airways, alveolar epithelia and the microvasculature, which compromises lung function, airflow, and gas exchange capacity. Consequently, chronic lung diseases are prime candidates for stem cell-based therapies, but progress in lung regenerative medicine has been confounded by the complexity of the adult lung and the lack of knowledge about the identity, organization, and regulation of candidate stem and progenitor cell targets around which regenerative therapies can be tailored.
Much of our understanding of adult lung epithelial cell maintenance, regeneration and repair has been deduced from systematic analysis of normal lung development and histomorphometric and epithelial cell lineage tracing studies in normal, perturbed, and gain-of-function and loss-of-function animal models. However, the aggregate effect of diverse microenvironmental factors has limited our ability to identify the defining features of lung epithelial stem cells at the whole organ level. This review describes recent developments using reductionist approaches to establish the precise identity, organization and regenerative potential of endogenous lung epithelial stem cells, and to identify the cellular and molecular factors required for their maintenance and fate specification, and the regulation of their ability to generate descendent lineages.
UNMASKING ADULT LUNG EPITHELIAL STEM/PROGENITOR CELLS IN THE MOUSE LUNG
The adult lung epithelium comprises a variety of epithelial cell lineages with regional distribution along the proximal-distal axis of the respiratory tree (Fig. 1). The tracheobronchial airways of the adult mouse lung are lined by a pseudostratified epithelium containing basal, ciliated, neuroendocrine, goblet, and Clara cells interspersed with ciliated and collecting duct cells that extend into submucosal glands (SMGs) lined with serous and mucous cells.
The bronchiolar airways of the distal lung are lined by a columnar epithelium containing predominately Clara and ciliated cells with discrete foci of neuroendocrine cells (neuroepithelial bodies) and few goblet cells, and the alveoli contain Type I (squamous) and Type II (cuboidal) cells. Not surprisingly, the adult lung is also believed to harbor a variety of epithelial stem/progenitor cells with functional specificity throughout the respiratory tree.
In the trachea and proximal bronchial airways, fate-mapping studies have revealed that basal cells (p63pos, Krt5pos, and/or Krt14pos), Clara cells (Scgb1a1pos), and SMG duct cells (Krt14pos) function as renewable stem/progenitor cells capable of differentiation along the ciliated and secretory lineages [2–6]. In the bronchiolar airways, a subset of Clara cells (Scgb1a1pos, CyP450neg, and/or scgb3a2pos) juxtaposed to neuroepithelial cells is able to give rise to Clara cells and ciliated cells but not alveolar cells [7–11]. More distally, a population of CCSPpos SP-Cpos bronchioalveolar stem cells (BASCs) at the bronchioalveolar duct junction has the capacity for division and expansion in response to injury [12–14], and it has long been proposed that Type II cells function as progenitor cells for Type I cells in the alveoli [15, 16]. In addition, it has recently been reported that p63pos Krt5pos Krt14pos cells, normally absent from the distal mouse lung, are capable of alveolar-lineage differentiation and contribute to the regenerative process following H1N1 influenza infection .
These studies clearly show that different stem/progenitor cell populations are responsible for the maintenance of epithelial cell lineages in different compartments of the adult lung. The remarkably low rate of cell turnover in adult lung epithelium in the steady state  has imposed a dependence on selective injury and perturbation models to reveal the identity, fate and specificity of adult stem/progenitor cells responsible for maintaining epithelial cell homeostasis in vivo. Using this approach, Giangreco et al.  have shown that stem and progenitor cells of differing proliferative and regenerative potential are responsible for the maintenance of the airway epithelium in the steady state and following perturbation or injury. However, it remains unclear whether the spatial discrimination of lineage relationships observed in these models reflects the lineage potential of discrete epithelial stem/progenitor cells or temporospatial changes in the microenvironmental cues instructing their fate. Distinguishing the precise hierarchical organization of stem/progenitor cells and understanding the defining properties of stem/progenitor cells at the individual cell level will have significant implications for therapeutic application.
Prospective Isolation of Endogenous Mouse Lung Epithelial Stem/Progenitor Cells
The first candidate self-renewing distal lung epithelial stem/progenitor cell population identified by ex vivo clonogenic analysis of flow cytometrically sorted nonendothelial (CD31neg), nonhematopoietic (CD45neg) cells was characterized as Sca-1pos . This Sca-1pos lung cell cohort comprises a heterogeneous population of epithelial and mesenchymal stem/progenitor cells and differentiated cells [12, 20–22]. Improved sorting strategies based on the differential expression of EpCAM and Sca-1 have now resolved distinct epithelial (EpCAMpos Sca-1low) and mesenchymal (EpCAMneg Sca-1hi) cell lineages [23, 24]. Using this approach, we have shown that epithelial stem/progenitor cells are enriched in the EpCAMpos Sca-1low α6 integrin (α6)pos β4 integrin (β4)pos CD24low cell fraction, whereas ciliated cells exhibit high levels of CD24 expression, and Type II cells lack β4 expression . When seeded in a three-dimensional culture assay, epithelial stem/progenitor cells within this fraction were able to self-renew and give rise to colonies comprising airway, alveolar, or mixed lung epithelial lineage cells, suggesting that an epithelial stem/progenitor cell hierarchy exists in the adult mouse lung . Parallel studies confirmed α6β4 integrin expression as a defining biomarker for lung epithelial stem/progenitor cells and further showed that SP-Cneg α6pos β4pos cells sporadically distributed in alveolar walls had the capacity to replenish SP-Cpos Type II cells in the alveoli, suggesting that maintenance of the alveolar epithelium involves proliferation and differentiation of interspersed progenitor cells and not simply expansion of Type II cells . In addition, Zacharek et al.  have recently reported that BASCs are also enriched in the EpCAMpos Sca-1low CD24low fraction (Fig. 1).
The overlapping immunophenotypic profile of these independently identified distal lung stem/progenitor cell populations suggests that each preparation may contain a variably enriched population of common stem/progenitor cells likely to exhibit context-dependent differentiation depending on their localization. Likewise, spatially distinct basal cells and SMG duct cells that exhibit the capacity to generate mucous and ciliated lineages have both been isolated from the proximal airways on the basis of NGFRpos α6pos expression [4, 6, 26]. Accordingly, characterization of isolated cell populations should recognize their heterogeneity and we encourage further investigation into the lineage relationships between isolated cell populations. The interrogation of sorted cells at a single cell level in a controllable and reductionist manner in vitro will enable a detailed investigation of lineage relationships, and the analysis of the way in which individual elements of the complex multifaceted lung microenvironment regulates epithelial stem/progenitor cell function.
DELINEATING REGENERATIVE CELLS OF THE EPITHELIAL STEM/PROGENITOR CELL NICHE MICROENVIRONMENT
The adult lung comprises at least 40–60 different cell types of endodermal, mesodermal, and ectodermal origin, which are precisely organized in an elaborate 3D structure with regional diversity along the proximal-distal axis. In addition to the variety of epithelial cells, these include cartilaginous cells of the upper airways, airway smooth muscle cells, interstitial fibroblasts, myofibroblasts, lipofibroblasts, and pericytes as well as vascular, microvascular, and lymphatic endothelial cells, and innervating neural cells (Fig. 1). It is likely that the regenerative ability of lung epithelial stem/progenitor cells in the different regions of the lung is determined not only by their intrinsic developmental potential but also by the complex interplay of permissive or restrictive cues provided by these intimately associated cell lineages as well as the circulating cells, soluble and insoluble factors and cytokines within their niche microenvironment.
The crosstalk between the different cell lineages is reciprocal, multidirectional, and interdependent. Autocrine and paracrine factors elaborated by mesenchymal and endothelial cells are required for lung epithelial cell proliferation and differentiation [27, 28], while endothelial and epithelial cell-derived factors also regulate mesenchymal cell proliferation and differentiation, extracellular matrix deposition and remodeling, and adhesion-mediated signaling [29, 30]. Chemotactic factors elaborated by these cell lineages also orchestrate the recruitment of inflammatory cells, which participate in the remodeling of the niche and the regulation of the proliferation and differentiation of its cellular constituents. So, while lung epithelial stem/progenitor cells have been a prime focus of research in lung regenerative medicine, the delivery of cellular therapies will likely require a precise understanding of the organization, regulation and regenerative potential of lung mesenchymal and endothelial cells and how these compartments are altered in disease initiation and progression.
Lung Mesenchymal Stem/Progenitor Cells
It has long been known that tracheal and distal embryonic lung mesenchyme have inductive properties for the regional specification of the embryonic epithelium . During lung development, mesenchymal stromal cells at the distal tip of the branching epithelium are known to secrete fibroblast growth factor (FGF)-10, which influences the fate and specificity of early lung epithelial progenitor cells [32, 33]. FGF-10 is a critical component of a multifaceted epithelial-mesenchymal cell signaling network involving Bmp, Wnt, and Shh pathways which coordinate the proliferation and differentiation of progenitor cells in the developing lung (reviewed in ). Lineage tracing studies have also revealed that FGF-10pos mesenchymal cells residing at the branching tip of the epithelium function as stem/progenitor cells for smooth muscle cells, which become distributed along the elongating airways [35, 36]. In other studies, mesenchymal stromal cells adjacent to the trachea and extrapulmonary bronchi have also been shown to give rise to bronchiolar smooth muscle cells . Collectively, these studies suggest that at least two distinct populations of mesenchymal stromal cells endowed with epithelial modulating properties emerge during development.
Several studies have since identified resident mesenchymal stromal cells in adult lungs with the capacity for adipogenic, chondrogenic, osteogenic, and myogenic differentiation. These cells have been clonally expanded from heterogeneous populations of mixed lineage cells defined by their ability to efflux Hoechst 33342 [38, 39], by their capacity for outgrowth from lung explant cultures  or by their characteristic expression of Sca-1 [21, 22]. In addition, further enrichment of CD45neg CD31neg Sca-1pos mesenchymal stromal cells has been achieved based on their lack of EpCAM expression, which selectively labels epithelial lineage cells . Importantly, resolution of the mesenchymal and epithelial lineages has revealed that the endogenous lung mesenchymal stromal cell population is necessary and sufficient to support the proliferation and differentiation of bronchiolar epithelial stem/progenitor cells in coculture . This suggests that adult mesenchymal stromal cells share similar epithelial inductive properties to their embryonic counterparts and are an important element of the epithelial stem/progenitor cell niche in the adult lung. This concept is also supported by recent in vivo studies showing that following naphthalene injury of Clara cells, parabronchial mesenchymal cells secrete FGF-10 to support epithelial regeneration from surviving epithelial stem/progenitor cells .
Lung Endothelial Progenitor Cells
Endothelial-epithelial cell interactions and angiogenic and angiocrine factors elaborated in the lung epithelial stem/progenitor cell microenvironment also play an integral role in the regulation of endogenous lung epithelial stem/progenitor cell regeneration and repair [27–29]. For example, a recent study has reported that the coculture of human vascular endothelial cells with a human bronchial epithelial cell line promotes the generation of branching bronchioalveolar epithelial structures in a 3D culture system . While considerable progress has been made in understanding the heterogeneity, functional diversity, and pathophysiological behavior of lung vascular and microvascular endothelial cells, the immunophenotypic profiling, quantitation, and functional analysis of lung endothelial progenitor cells (EPC) lags far behind. As for EPC derived from human umbilical cord blood, bone marrow, and mobilized peripheral blood , the rarity of EPC in the lung, their lack of distinguishing markers, and the inability to discriminate circulating EPC and tissue resident EPC have been major impediments in assessing the contribution of endogenous lung EPC in lung vascular repair, and lung regeneration and remodeling [44, 45].
Lung macrovascular and microvascular endothelial cells can be resolved on the basis of their preferential binding to the lectins Helix pomatia and Griffonia simplicifolica, respectively , but there are no other cell surface markers that can discriminate mature lung endothelial cells and EPC . In addition, the rarity of EPC has also necessitated the ex vivo expansion and passaging of adherent heterogeneous rat  or mouse  lung endothelial cells in liquid culture prior to quantitation and flow cytometric and functional analysis of lung-derived EPC in in vitro assays. These assays suggest that the lung microvasculature is a rich source of EPC. However, it is important to note that the incidence, immunophenotypic and functional properties of EPC in the primary explanted endothelial cells compared with their ex vivo manipulated, selected, and expanded counterparts remains indeterminate. The ability of these endogenous lung EPCs to contribute to vascular repair and remodeling in vivo is also unproven . Although, recent studies suggest it likely that both circulating EPC and resident lung EPC contribute to endothelial cell regeneration and repair [49–51].
CHALLENGES IN DELIVERING CELL-BASED THERAPIES FOR LUNG DISEASE
The reductionist approaches described above have identified endogenous lung epithelial, mesenchymal, and endothelial stem/progenitor cell candidate populations around which regenerative therapies for lung disease could be tailored. However, there are many significant challenges and practical considerations that must be addressed in delivering stem cell-based therapies in lung regenerative medicine.
Different lung diseases have different pathologies targeting different cell lineages. Some will require replacement or correction of proximal or distal epithelial cell lineages, while other conditions will require manipulation or regulation of dysregulated subepithelial-mesenchymal and microvascular endothelial cell lineages. Importantly, the functional organization of the lung as trophic units will also require the precisely coordinated recruitment and proliferation of epithelial, mesenchymal, and endothelial cell compartments for lung regeneration and repair to proceed in an ordered fashion, in order to avoid the formation of inefficient respiratory units with impaired gas exchange and persistent abnormalities .
The plasticity of the stem cell niche during disease progression will also affect the engraftment and regenerative potential of endogenous epithelial stem/progenitor cells as well as the epithelial stem/progenitor cell supportive properties of mesenchymal and endothelial cell elements comprising their niche. Preconditioning protocols designed to optimize the reparative potential of engrafting and endogenous stem cells may require the coordinated delivery or cotransplantation of mesenchymal and/or EPCs to repair the niche in order to normalize the behavior of the regenerating epithelium.
Recent prototype experiments analyzing the engraftment, proliferation, and differentiation of defined lung cell populations seeded in decellularized lung biomatrix scaffolds [53–55] will provide valuable models for defining critical microenvironmental variables which specify endogenous lung stem/progenitor cell fate and regenerative capacity. While this approach has proven effective in bioengineering tracheal and large bronchus implants for clinical transplantation [56, 57], the challenges posed in applying this approach to the bioengineering of the distal lung are formidable.
CONCLUSIONS AND FUTURE DIRECTIONS
The cell separative strategies and clonogenic assays we have described provide powerful tools for monitoring the status of regenerative cells in the normal, diseased or injured lung and for identifying the factors, cytokines, and molecular pathways that can be manipulated to harness their potential in lung regenerative medicine. However, much still remains to be done in order to establish the origins, organization, and role of clonogenic cells identified in the surrogate stem/progenitor cell assays described in this review in maintaining the functional integrity of the adult lung throughout life.
Key issues that remain to be resolved in order to move the field forward include: the cross-referencing of candidate stem/progenitor cell targets prospectively isolated by different laboratories using similar but not identical biomarker repertoires and functional assays; the delineation of the properties of the permissive and/or restrictive stem/progenitor cell niche in which they reside; the development of “gold-standard” in vivo assays to determine the relationship between candidate stem/progenitor cells identified in in vitro clonogenic assay readouts and regenerative cells in vivo; and, the identification of biomarkers for the characterization of human homologs of candidate stem/progenitor cells identified in mouse models. These objectives will entail rigorous interrogation of well-characterized lung injury and loss-of-function and gain-of-function models combining lineage tracing and clonogenic analysis to understand the lineage relationship between candidate stem/progenitor cells identified in different contexts.
Because adult lung stem/progenitor cells are characterized by the expression of multiple markers, which collectively specify the stem cell state but are not unique to any individual candidate stem progenitor cell type it will also be important to establish whether the heterogeneous behavior of immunophenotypically homogeneous target cells in different functional in vitro assays is indicative of cofractionation of heterogeneous cells expressing common biomarkers, or the stochastic commitment of homogeneous stem/progenitor cells to different fates in response to different microenvironmental cues. In this context, the lack of gold-standard transplantation assays for evaluating the regenerative potential of candidate stem/progenitor cells in vivo means that their ability to contribute to lung regeneration and repair is unproven.
Studies thus far have largely focused on the analysis of candidate stem/progenitor cells in mouse models. Relatively few studies describe the prospective isolation and characterization of homologous human endogenous stem/progenitor cell targets, and these have largely analyzed unfractionated cells in liquid or air-liquid interface culture systems or devitalized tracheal xenograft models, which are not amenable to clonogenic analysis (reviewed in ).
Translational studies have also been impeded by the lack of biomarkers for the characterization of homologous targets in different species. For example, the Sca-1 antigen which has been used extensively to identify and prospectively isolate both epithelial and mesenchymal stem/progenitor cell targets in the mouse has no human homolog . The analysis and prospective isolation of adult human lung stem/progenitor cell targets have also been impeded by the inherent biological variability encountered in processing human tissue as well as the difficulty in obtaining adequate amounts of normal human tissue to identify and prospectively isolate rare cell subpopulations. Importantly, human lung tissue representative of the normal steady state is often obtained from patients with diverse respiratory diseases. This will likely affect the relative expression of stem/progenitor cell associated biomarkers compromising the fidelity of cell separative strategies, the validation of surrogate stem/progenitor cell assays, and the identification and quantitation of candidate human lung stem/progenitor cell targets. While a recent study  has described the isolation of candidate human multipotent endogenous lung stem cells on the basis of their expression of the c-kit cell surface antigen, significant reservations have been raised about the rigor of the experimental designs used to characterize these cells and evaluate their regenerative capacity . Their role in lung regeneration and repair awaits confirmation and validation in independent studies.
This work was supported by grants from the Australian Stem Cell Centre, the Australian National Health and Medical Research Council (GNT 1009374), and a University of Melbourne early career research grant to JL McQualter. We apologize to our colleagues whose work we have been unable to cite comprehensively because of space constraints.
DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
The authors indicate no potential conflicts of interest.