Lung stem cells - from an evolving understanding to a paradigm shift?

The ideal cell type to regenerate an acutely injured or chronically diseased lung would be a stem cell population from the patient's own lung. Consequently, extensive research efforts have focused on identifying and characterizing endogenous lung stem cells. Advances in techniques to facilitate cell isolation, labelling and tracking in vivo to determine their fate have led to the identification of several putative stem cell niches. Recently, convincing evidence has emerged for a novel stem/progenitor cell population in the submucous glands of the cartilaginous airways. These findings support the concept that there is no classical stem cell 'hierarchy' but that different progenitor populations within spatially distinct lung regions regenerate the lung epithelium adjacent to its niche. Intriguingly, recent findings challenge this concept; it was reported that the human lung may contain a primitive stem cell capable of differentiating into multiple cells of both endodermal and mesodermal lineage and of regenerating the injured lung. This suggests that a classical stem cell hierarchy may, in fact, exist in the lung. Although caution is needed in interpreting these emerging findings, the implications for our current concepts regarding lung stem cells, the insights into lung repair and regeneration, and the potential therapeutic implications are considerable.


Background
Respiratory diseases such as chronic obstructive pulmonary disease, asthma pulmonary fi brosis, and acute lung injury/acute respiratory distress syndrome (ALI/ARDS) confer an enormous disease burden. Despite exten sive research eff orts, curative therapies for these diseases have proven elusive and current management remains limited to symptom control or supportive strategies or both. A promising therapeutic approach may be the use of stem cells to aid lung regeneration and repair. Although exogenous stem cells, particularly mesenchymal stem cells, have received much attention [1], lung stem cell populations might be the ideal cell to regenerate the lung [2]. Progress in this area has been diffi cult [3], but recent advances have invigorated this fi eld.

Insights from lung development
Th e lung, being derived from cells of endodermal and meso dermal origin, has a complex embryological origin. Elegant studies, in which individual cells in the developing embyro are labeled to allow their descendants (that is, lineage) to be traced, have generated considerable insights. Lung development begins during the fi fth gestational week with the formation of epithelial buds from the primordial endoderm which then grow into the embryonic mesenchyme. Almost all of the epithelial cell types found in the developed lung arise from these earliest lung-committed buds [4]. In a complex but poorly understood process involving much crosstalk between the dermal layers, these buds organize to form the epithelial architecture, whereas the mesoderm forms the supporting stroma and blood vessels [5]. Of importance, there is no evidence from lineage-tracing studies that the embryonic lung -or, indeed, any developing organ -contains a common progenitor for endodermal and meso dermal lineages [6,7]. Th is suggests that separate populations of stem/progenitor cells are necessary for the maintenance of the endodermal-and mesodermal-derived lung components.

Insights from other organs
Mammalian tissue regeneration appears to involve several mechanisms, including compensatory hyperplasia, dediff erentiation, and adult stem cells. Diff erent organs use diff erent strategies to renew themselves, and more diversity and fl exibility underpin these renewal processes than previously imagined. Some organs, such as hair follicles, blood, and gut, which constantly renew themselves through out life, contain adult stem cells that are

Abstract
The ideal cell type to regenerate an acutely injured or chronically diseased lung would be a stem cell population from the patient's own lung. Consequently, extensive research eff orts have focused on identifying and characterizing endogenous lung stem cells. Advances in techniques to facilitate cell isolation, labelling and tracking in vivo to determine their fate have led to the identifi cation of several putative stem cell niches. Recently, convincing evidence has emerged for a novel stem/progenitor cell population in the submucous glands of the cartilaginous airways. These fi ndings support the concept that there is no classical stem cell 'hierarchy' but that diff erent progenitor populations within spatially distinct lung regions regenerate the lung epithelium adjacent to its niche. Intriguingly, recent fi ndings challenge this concept; it was reported that the human lung may contain a primitive stem cell capable of diff erentiating into multiple cells of both endodermal and mesodermal lineage and of regenerating the injured lung. This suggests that a classical stem cell hierarchy may, in fact, exist in the lung. Although caution is needed in interpreting these emerging fi ndings, the implications for our current concepts regarding lung stem cells, the insights into lung repair and regeneration, and the potential therapeutic implications are considerable.

V I E W P O I N T
*Correspondence: john.laff ey@nuigalway.ie 1 Lung Biology Group, Regenerative Medicine Institute, National Centre for Biomedical Engineering Science, National University of Ireland, University Road, Galway, Ireland Full list of author information is available at the end of the article morpho logically unspecialized, have a relatively low rate of division, and are topologically restricted to localized regions known as 'niches' that tightly regulate their behavior [8]. In the intestine, for example, only a few stem cells are present near the base of the crypts, and these stem cells appear to be responsible for replenishing the entire epithelium. In contrast, the adult lung normally regenerates slowly, but following lung injury, rapid repair is essential for survival.

Current concepts of lung tissue regeneration
Current paradigms propose the existence of a 'nonclassical' stem cell hierarchy in the lung, in which relatively quiescent progenitor cells diff erentiate into a restricted number of cells to regenerate specifi c lung epithelial cells and structures [9]. Advances in laboratory techniques that facilitate in vivo cell isolation, labeling, and tracking have identifi ed several candidate stem cell populations ( Figure 1). Th ese putative microenvironmental niches appear to contain cells that can self-renew and generate diff erentiated progeny that regenerate the lung epithelium adjacent to its respective niche [5,10]. In the trachea and main bronchi, it appears that undiff erentiated basal cells can function as classical stem cells, both self-renewing and giving rise to ciliated and secretory cells. Duct cells from submucosal glands (SMGs) located in the cartilaginous airways are also a potential niche [10]. In the more distal lung, Clara cells [2] and subpopulations of Clara cells (termed 'variant' Clara cells) found in the bronchioles and bronchiolar-alveolar junctions, respectively, appear to self-renew and give rise to diff erent cell types after injury [10]. In the alveoli, damaged type I cells can be generated from dividing type II cells, although whether all type II cells have this capacity is not yet known [5].
Th e concept that there are diff erent progenitor populations within spatially distinct regions in the adult lung, rather than a 'master' lung stem/progenitor cell capable of repairing any lost epithelial cell, is further supported by recent fate-mapping studies [11,12]. Th is regeneration model may refl ect the special requirements for lung regeneration and repair, in which the ability of multiple cell types to proliferate and rapidly repair the lung may be advantageous.

The submucosal gland duct stem/progenitor cell
Previous studies have suggested that SMGs might constitute a stem cell niche, and progenitor cells have been identifi ed in the SMGs of other organs, including the breast and submandibular gland [13,14]. Now, Hegab and colleagues [15] provide convincing evidence that SMG duct cells do constitute a stem/progenitor cell population. Th ese investigators developed a novel approach to label and isolate these cells. Th ese SMG duct cells survived in vivo during hypoxic-ischemic injury and were capable of self-renewal and of diff erentiation into SMG duct and tubule cells and surface epithelium cells [15]. Th ough sharing similarities with basal cells, these SMG duct cells appear to be a distinct stem/progenitor cell population.
As we consider these fi ndings, it is important to remem ber that the anatomy and physiology of the murine proximal airway diff er signifi cantly from those of humans [10]. Specifi cally, human SMG cells are more copious and are found throughout the cartilaginous airways rather than in just the upper third of the trachea and larynx. Also, interspecies variation in SMG location between diff erent strains of inbred mice has been reported [16]. Consequently, these fi ndings need to be replicated in the human lung. It is also unclear whether these are true stem or progenitor cells, a common issue in studies such as this. Features that suggest that these are progenitor rather than true stem cells include their limited diff erentiation potential and the fact that, being capable of mucin secretion, they are already specialized.
Notwithstanding these issues, the identifi cation of this novel stem/progenitor cell population is important for a number of reasons. First, given the position of these cells throughout the cartilaginous airways in humans, these cells may play a major role in repair following diff use injury to the lung, such as that seen in ALI/ARDS. Second, dysfunction of these cells may contribute to the pathogenesis of a number of hypersecretory lung diseases, including chronic obstructive airway disease, asthma, and cystic fi brosis. Th ird, these cells express the K14 surface marker, which is seen in pre-malignant lesions and which correlates with poor prognosis in nonsmall cell lung cancer, suggesting that these cells might be the stem cell of origin of these cancers.

The 'true' lung stem cell -a paradigm shift?
Intriguingly, Kajstura and colleagues [17] now provide evidence that the lung may contain 'true' stem cells. In a series of elegant experiments, the authors demonstrate that these c-kit + cells are undiff erentiated, self-renewing, clono genic, and multipotent in vitro. Th ese undiff er entiated cells do not express lung-specifi c proteins or hematopoietic markers. Th e multipotency and apparent plasticity of these cells may arise from concurrent expression of the transcription factors Nanog, Oct3/4, Sox2, and Klf-4. Th e fi nding that, when injected into a mouse model of focal lung injury, these human c-kit + cells appeared to regenerate many diff erent lung components -including bronchioles, alveoli, smooth muscle, and even pulmonary vessels -is as surprising as it is exciting [17].
If confi rmed, these fi ndings constitute a landmark development. Th e implications for our current concepts regarding lung stem cells, the insights into lung repair and regeneration, and the potential therapeutic implications are considerable. Th ese c-kit + lung stem cells could be used to stimulate lung tissue regeneration in a patient with lung diseases that result in loss of alveoli (such as chronic obstructive pulmonary disease) or that are charac terized by widespread loss of lung epithelium (such as ALI/ARDS). Th ese fi ndings could bolster attempts to bioengineer lung tissue in vitro [18]. However, enthu siasm for the fi ndings of the study must not preclude a careful appraisal of its strengths and limitations [7].

Lung stem cells -where are we now?
Th e fi ndings of Kajstura and colleagues [17] pose a serious challenge to our existing concepts by providing evidence for a classical stem cell hierarchy in the adult Figure 1. Schematic diagram of the microenvironmental niches that may contain lung stem/progenitor cells. In the trachea and main bronchi, undiff erentiated basal cells (stained for transcription factor p63) can function as classical stem cells. Duct cells from submucosal glands located in the cartilaginous airways are also a potential niche. In the more distal lung, Clara cells and 'variant' Clara cells (stained red) are found in the bronchioles and bronchiolar-alveolar junctions, respectively, while the alveoli contain type II cells (stained green) that can regenerate type 1 pneumocytes. bv, blood vessel; D, dorsal; H and E, hematoxylin and eosin; V, ventral. Reprinted with permission from The Company of Biologists Ltd. [5].
lung. At the top of this hierarchy are c-Kit + cells, which constitute the ultimate reservoir for self-renewal of epithelial (and mesenchymal) progenitor cells, such as basal cells, Clara cells, and SMG duct cells. Th e novelty of these fi ndings is underlined by the fact that multipotent stem cells that can give rise to both endodermal and mesodermal lineages have not previously been described in the lung or, indeed, in any organ [9]. Furthermore, there is no known interconversion of endodermal and mesodermal cells during embryonic lung development [18].
Not surprisingly perhaps, this study has already generated signifi cant comment in the literature [7,18]. Issues highlighted in a discussion in Nature Medicine [7] include the fact that these studies, though extensive, lacked appropriate controls for certain key experiments; another issue is the need to more clearly defi ne the niche of these cells within the lung and to determine the factors that regulate their quiescence and activation. Th ese studies focused solely on human c-kit + stem cells. Subsequent studies, which will be required to validate these fi ndings, could use more tractable systems, such as genetically modifi ed mouse models. Detailed in vivo lineage-tracking studies of the fates of c-kit + lung stem cells, during normal lung maintenance and in response to lung injury, would provide signifi cant additional insights.

Conclusions
Th is is an exciting time for lung stem cell research. Newer and more robust laboratory techniques that facilitate cell isolation, labeling, and tracking to determine stem cell fate in vivo have considerably advanced our knowledge of these populations of the lung. Although the need for caution in considering the clinical implications of these fi ndings is clear, these studies provide exciting new insights. Th e studies challenge our current concepts and should energize the fi eld of lung stem cell research.