Lung Stem Cells: Do They Exist?1

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Lung stem cells: do they exist?1

Ivan Bertoncello MSc, PhD and Jonathan McQualter PhD

Lung Health Research Centre, Department of Pharmacology, University of Melbourne, Victoria 3010 Australia

Corresponding Author: Ivan Bertoncello PhD Lung Health Research Centre Department of Pharmacology Level 8, Medical Building The University of Melbourne Victoria 3010 Australia Phone: +613 8344 6992 E-mail: [email protected]

RunningArticle Head: Epithelial stem cells in the adult lung

The Authors: Associate Professor Ivan Bertoncello MSc, PhD is the head of the Lung Regeneration Laboratory leading research investigating the organisation and regulation of adult lung stem and progenitor cells and their role in lung regeneration and repair. Dr Jonathan McQualter leads the Lung Stem Cells and Microenvironment Group and has a strong interest in understanding how dynamic interaction of lung epithelial stem cells with their microenvironment regulates their regenerative potential.

Note to typesetters: Please print glossary on first page of the review in shaded text box

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this Accepted version and the Version of Record. Please cite this article as doi: 10.1111/resp.12073 1 © 2013 The Authors Respirology © 2013 Asian Pacific Society of Respirology ABSTRACT

Recognition of the potential of stem cell based therapies for alleviating intractable lung diseases has provided the impetus for research aimed at identifying regenerative cells in the adult lung, understanding how they are organised and regulated, and how they could be harnessed in lung regenerative medicine. In this review we describe the attributes of adult stem and progenitor cells in adult organs, and how they are regulated by the permissive or restrictive microenvironment in which they reside. We describe the power and limitations of experimental models, cell separative strategies and functional assays used to model the organisation and regulation of adult airway and alveolar stem cells in the adult lung. The review summarises recent progress and obstacles in defining endogenous lung epithelial Article stem and progenitor cells in mouse models and in translational studies.

Key Words: airway epithelium; animal models; cell biology; lung regeneration;

adult stem cells Accepted

“Omnis cellula e cellula” Rudolf Virchow (1858)

The identity of stem cells, their location and organisation within tissues, and the regulation of their regenerative potential has been the subject of ongoing debate since articulation of modern cell theory in the mid 19th century which recognised that cells are the fundamental building blocks of all living organisms, and that every cell is derived from a pre-existing cell

(Omnis cellula e cellula). The term “stem cell” was originally coined in the late 19th century1 by embryologists to describe embryonic cells thought to give rise to the specialised tissues

ofArticle multicellular organisms during ontogeny; and by haematologists to describe the hypothesised common precursor of the diverse cell types identified in the bone marrow, peripheral blood and lymphatic system.

Historically, adult organs have been classified as continuously renewing (bone marrow, gut, skin), conditionally renewing (lung, kidney, liver), or essentially non-renewing (nervous tissue, muscle) on the basis of their proliferative capacity and cell turnover in the steady state or following injury2, 3. A continuously renewing tissue like the bone marrow, has the prodigious capacity to generate approximately 200 x 109 erythrocytes, 50 x 109 white blood cells, and 125 x 109 platelets daily in the steady state, and can rapidly augment cell production up to 10-fold in times of stress4. The gut also generates approximately 100 x 109 intestinal epithelial cells daily, replacing the entire intestinal epithelium every 5 days5, 6.

The lung on the other hand, is a conditionally renewing organ. Airway epithelial cell turnover is low (~1% per day) with replacement of the tracheobronchial epithelium Accepted

3 © 2013 The Authors Respirology © 2013 Asian Pacific Society of Respirology estimated at approximately 4 months7. However like kidney and liver, the lung is able to undergo compensatory growth, rapidly increasing its rate of regeneration to replace ablated tissue following severe injury8.

The intrinsic properties of adult stem cells:

The adult stem cell paradigm is largely underpinned by the analysis of cell regeneration in continuously renewing tissues. Accordingly, adult stem cells are defined as rare, morphologically unrecognisable cells endowed with a high proliferative potential and the life-long ability to (a) maintain and replenish their own kind, (b) generate large numbers of functionally differentiated progeny, and (c) replace senescent and damaged cells in the

9 steadyArticle state and following perturbation or injury .

The classical adult stem cell hierarchy comprises an age-structured continuum of stem and transit amplifying progenitor cells of progressively restricted proliferative and differentiative potential which in turn give rise to mature non-dividing functionally differentiated cells

(Figure 1). As a general rule multipotent stem cells at the head of this hierarchy are highly

10 quiescent. Typically, the vast majority are in a G0 state and descendent cell lineages are derived from a small number of active clones11, thus ensuring the preservation of a stem cell reserve while mitigating the risk of error-prone stem cell replication and transformation12.

Day-to-day demands for replacement of senescent and damaged cells are met by more abundant rapidly cycling lineage-restricted transit amplifying progenitors of limited proliferative potential10. According to this paradigm, stem cells divide asymmetrically to homeostatically regulate stem cell pool size while simultaneously replenishing the more committed transit amplifying progenitor cell compartments, but also retaining the ability to divide symmetrically to expand stem cell numbers in order to meet the increased demand Accepted

4 © 2013 The Authors Respirology © 2013 Asian Pacific Society of Respirology for differentiated cells following tissue injury13, 14. Analysis of stem cell compartments in inbred mouse strains15, and the BXD set of recombinant mouse strains derived by crossing

C57BL/6J and DBA/2J mice16, 17, has also shown that stem cell variables including pool size, cell kinetic status, sensitivity to cytokines, irradiation and cytotoxic perturbation are genetically determined.

While the organisation of regenerative cells in continuously renewing tissues conforms to this classical model, it is not precisely recapitulated in all organs and tissues. Regional differences in the developmental potential and organisation of regenerative cells have been documented in different compartments of organs including the prostate, skin and the

18-20 7, 21 gastrointestinalArticle tract and along the proximal-distal axis of the lung . A recent study argues against the obligate requirement for a long-lived oesophageal epithelial stem cell reserve20, 22. There is evidence for and against the existence of regional diversity in neural stem cells in the brain23. Sub-populations of differentiated multifunctional cells able to revert to a (facultative) stem cell state have been identified in lung24, liver and pancreas25, while terminally differentiated cardiomyocytes appear capable of regenerating damaged heart tissue by a process of dedifferentiation and redifferentiation26, 27.

However, it is interesting to note that at a time when the delineation and functional analysis of adult stem cells was in its infancy, Till28 speculated that they would likely be characterised by the expression of multiple markers including differentiation markers characteristic of later stages of development. Likewise, Lajtha10 questioned the imprecision of the concept that stem cells were necessarily the sole precursors of other cell types, and the assumption that they were necessarily undifferentiated10. He speculated: (a) that although stem cells may be less differentiated than their descendents they were nonetheless one of many Accepted

5 © 2013 The Authors Respirology © 2013 Asian Pacific Society of Respirology differentiated cell types; and (b) that renewal is relative rather than absolute with stem and transit progenitor cells part of a recognisable age-structure continuum in which both stem and transit populations could be precursors of other transit populations. In hindsight, these reflections on the nature and properties of stem cells have proven remarkably insightful, suggesting that classical and non-classical models of stem cell organisation may have more in common than might appear at first glance25, 29, 30, and emphasizing the fact that stem and progenitor cells are defined operationally: by what they do rather than how they are decorated.

Stem cells are defined in context – the role of the niche in stem cell specification:

Importantly,Article adult stem cells are not solely defined by their intrinsic properties, but also by the permissive or restrictive microenvironments in which they reside. The importance of the microenvironment in stem cell specification had long been appreciated in both embryonic development31 and adult haematopoiesis32, but the concept of the stem cell niche was only articulated by Schofield in 197833 in modelling the regulation of haematopoietic stem cells.

He proposed that adult stem cells occupy specific anatomical sites which preserve their developmental potential, regulate their replication, inhibit their differentiation and provide a milieu conducive to the preservation of engrafted cells in a stem cell state. The crosstalk between the stem cell and its niche profoundly affects stem cell behaviour and functionality3, 34-37. In the lung, the interdependence of regenerative cells and their niche microenvironment is evident in both the process of lung epithelial regeneration and repair38 and in the aberrant proliferation and differentiation of airway and alveolar epithelial cells as a consequence of the pathophysiological remodelling and scarring of the subepithelial microenvironment in lung fibrosis and asthma39-42. Accepted

Problems and pitfalls in assaying stem cells and modelling stem cell hierarchies:

Because stem cells are fundamentally defined by what they do rather than by their morphology or biomarker profile, the assignment of “stemness” relies on the precise analysis of their developmental potential and regenerative capacity9. For this reason, the modelling of stem and progenitor cell hierarchies has progressed in lockstep with the development and refinement of:

1. specific antibodies, reagents and multiparameter cell separative protocols for the

prospective isolation of highly purified and defined target cells;

2. robust in vitro clonogenic assays for enumerating stem and progenitor cells and Article measuring their proliferative and differentiative potential;

3. transplant models and assays to measure their regenerative potential in vivo;

4. lineage tracing techniques to mark their spatial location in situ and measure their

contribution to tissue maintenance, regeneration and repair; and,

5. genetically-engineered and mutant mouse models to identify specific stem cell regulatory

networks.

The delineation of haematopoietic stem and progenitor cells43 is an outstanding example of the power of these multiparameter strategies to: (a) analyse stem and progenitor cell organisation and regulation, (b) validate assays predictive of their regenerative capacity, and

(c) identify stem cell targets around which therapies can be constructed (Figure 2). The characterisation of multipotent mouse mammary stem cells capable of regenerating a functional mammary gland following transplantation of a single cell44, 45 is another pertinent example. However, because the analysis of the functional attributes of stem cells is context Accepted

7 © 2013 The Authors Respirology © 2013 Asian Pacific Society of Respirology dependent, there are many experimental variables which can confound the assignment of stemness and compromise the ability of assays to discriminate closely related stem and progenitor cell subsets and measure their ability to give rise to descendent lineages.

Tissue disaggregation disrupts the relationship between stem cells and their regulatory microenvironment potentially affecting their viability, developmental potential, and signature profile46, 47. Stem cell biomarkers are commonly shared by multiple cell lineages as well as closely related cells of differing developmental potential and regenerative capacity48,

49. The proliferation and differentiation of stem and progenitor cells in vitro is determined by colligative properties of the culture system including cell seeding density, choice of

50 51 mediumArticle , oxygen tension , pH, and the conditioning of the medium by factors elaborated by the progeny of the cultured stem cells52. Discordance between proteome and transcriptome as a result of post-translational regulation of the proteome must also be considered in assessing the fidelity of biomarker signature profiles used to characterise and order stem and progenitor cells and their descendent progeny53, 54.

The interpretation of in vivo assays can be similarly confounded. Transplant outcomes are contingent on the number of transplanted cells, their route of delivery, and the modality employed to precondition transplant recipients. Evidence from haematopoietic transplant models also shows that stem cell homing and engraftment is influenced by the cell cycling status of transplanted stem cells55. The regenerative capacity of resident stem cells in acute lung injury models can be confounded by disruption and remodelling of the stem cell niche microenvironment7, 56. And, cell lineage tracing in conditional transgenic reporter mouse models can be confounded by the lack of specificity of promoters and the pharmacokinetics of inducers used to elicit gene expression57, 58. Accepted

The lung is a complex organ comprising at least 40 cell lineages of endodermal, mesoderm and ectodermal origin which arises from an out-pouching of the foregut59. While progress has been made in delineating stem and progenitor cells in all lung compartments21, 60 this review focuses on controversies surrounding the nature, properties and organisation of epithelial stem cells in the adult lung.

During development the primordial lung undergoes a precisely orchestrated program of branching morphogenesis culminating in arborisation of the conducting airways and their supporting parenchyma and vasculature, terminating in the gas-exchanging alveolar bed59,

61, 62

Article . The trachea and proximal airways are lined by a pseudostratified epithelium comprising basal, secretory, ciliated and neuroendocrine cells. Distal conducting airways are lined by a simple columnar epithelium comprising club (Clara), ciliated, and pulmonary neuroendocrine cells, while alveoli are populated by cuboidal surfactant secreting alveolar epithelial Type II cells and squamous gas-exchanging alveolar epithelial Type I cells. The cell lineages comprising each of these compartments are maintained by regenerative cells characterised by high proliferative activity during foetal and perinatal life, and slow turnover and the capacity to replace senescent and damaged cells in the adult63-65.

The organisation of endogenous epithelial stem and progenitor cells in the adult lung has been extensively reviewed in recent years7, 21, 60, 62, 66, 67. Briefly, these studies suggest that regional stem and progenitor cells are responsible for the maintenance of specific epithelial cell lineages in the proximal and distal conducting airways and the alveolar bed (Figure 3).

These include p63posKrt5pos and/or Krt14pos basal cells, Scgb1a1pos club (Clara) cells and

Krt14pos submucosal gland (SMG) duct cells of the trachea and upper airways68-72; Accepted

9 © 2013 The Authors Respirology © 2013 Asian Pacific Society of Respirology naphthalene resistant Scgb1a1posCyP450neg and/or scgb3a2pos variant club (Clara) cells and neuroendocrine cells in the bronchiolar airways73-75; CCSPposSP-Cpos bronchioalveolar stem cells (BASC) at the bronchioalveolar duct junction of terminal bronchioles76-78; and the Type

II progenitor of Type I cells in the alveolar bed79, 80. However, the analysis of lung epithelial cell regeneration in genetically engineered mice, and the development of multiparameter cell separative strategies and three dimensional organotypic clonogenic assays for the identification and characterisation of adult lung stem cells in vitro has added another layer of complexity and ambiguity to the understanding of their organisation and spatial localisation.

LungArticle injury models confirm that the regenerative potential of candidate stem cells is context dependent, and that different lung epithelial stem and progenitor cell cohorts are recruited to regenerate specific airway epithelial cell lineages in the steady state and following severe perturbation81, 82. On the other hand, in vitro clonogenic assay of putative epithelial stem cells, and transcription profiling of their progeny, provides evidence of the existence of multipotent epithelial stem cells in the adult lung21, 83-85. While, cell lineage tracing in genetically engineered conditional mutant mice also reveals plasticity in the differentiative potential of specific lung epithelial stem cell cohorts.

Discordant studies notwithstanding86, this context-dependent diversity in the proliferative and differentiative potential of epithelial stem and progenitor cells has been observed in all anatomical compartments of the airway tree. In the trachea, Krt14pos basal cells adopt a different fate in the normal lung and following naphthalene injury71, 87. Likewise, Hegab et al72 have identified multipotent nerve growth factor receptor positive (NGFRpos) α6 integrin positive (α6pos) tracheal SMG duct cells able to regenerate both SMG tubules and secretory Accepted

10 © 2013 The Authors Respirology © 2013 Asian Pacific Society of Respirology and ciliated airway epithelial cells following severe ischaemic injury. In the distal lung, pulmonary neuroendocrine and alveolar cells (but not club (Clara) and ciliated cells) have been shown to share a common CGRPpos neuroendocrine cell lineage origin during development, whereas they function as unipotent neuroendocrine progenitor cells in the normal adult lung but are able to renew and generate both club (Clara) and ciliated cells following naphthalene injury88. Other lineage tracing studies also provide evidence of the derivation of alveolar epithelial cells from Scgb1a1pos airway cells following bleomycin injury89, and from normally absent p63posKrt5posKrt14pos airway cells following H1N1 virus infection90. In the alveolar bed, it appears that alveolar epithelium is not simply repaired by expansion and differentiation of SP-Cpos type II cells following bleomycin injury, but also by Article SP-Cnegα6posβ4pos cells sporadically distributed in the alveolar wall91.

Modelling lung epithelial stem cell hierarchies: Are we there yet?

The development of powerful animal models57 and the refinement of cell separative strategies and in vitro clonogenic assays based on approaches proven effective in characterising haematopoietic stem cells (Figure 2) have underpinned the significant progress made in identifying putative stem cells in the adult lung and the critical factors and pathways regulating their regenerative potential. However, the lack of definitive lung epithelial stem cell biomarkers; the co-expression of cellular and molecular biomarkers by cells of differing regenerative potential, cell lineage origin or maturational age; the limitations of flow cytometric sorting47 and in vitro organotypic assays92; and the lack of gold-standard in vivo assays akin to those that have enabled the validation and modelling of the haematopoietic stem cell hierarchy93, 94, have conspired to blur the identity, spatial location and organisation of lung epithelial stem cells in health and disease. In particular, it Accepted

11 © 2013 The Authors Respirology © 2013 Asian Pacific Society of Respirology is still unclear whether the heterogeneous behaviour of seemingly homogeneous target cells is indicative of co-fractionation of heterogeneous cells expressing common biomarkers, or the stochastic commitment of homogeneous cells to different fates in response to microenvironmental cues. This is especially the case in injury models where modulation of biomarker signature profiles is not necessarily indicative of the ability of regenerative cells to give rise to descendent lineages.

Translational studies and the identification of therapeutic targets in the human lung

As yet relatively few studies have attempted to isolate and characterise adult epithelial stem cells in the human lung. These have mostly analysed tracheal and proximal airway epithelia

utilisingArticle various in vitro assays including air-liquid interface culture and serial propagation of spheroid-forming cells, and the generation of mucocilliary epithelium in devitalised tracheal implants xenografted in immune-compromised mice (reviewed in66). More recent studies also suggest that human homologues of murine lung epithelial stem cells are also amenable to analysis and characterisation employing similar experimental strategies to those utilised in mice.

Both immortalised human bronchial epithelial cells95 and freshly isolated human proximal airway epithelial cells70, 96-98 generate complex 3-dimensional organoids of differentiated airway epithelial cells when cloned in matrigel. Multiparameter flow cytometric analysis shows that these putative human airway epithelial stem cells share a biomarker profile similar to that of their murine counterparts. These include NGFRposα6posp63pos basal cells which generate cystic organoids of Krt14posp63posbasal cells, Krt8pos luminal cells and ciliated cells70; and, NGFRposα6pos basal and SMG duct cells which can be further resolved on the basis of their aldehyde dehydrogenase (ALDHhi), CD166, CD44, and α6 integrin signature Accepted

Krt14pos and mucous and serous secretory epithelial cells97.

Others have propagated dispersed, heterogeneous human lung cell suspensions in liquid culture to pre-enrich lung epithelial stem cells prior to their prospective isolation and characterisation by flow cytometric cell sorting and functional analysis 99, 100. Using this approach, Oeztuerk-Winder et al99 have cloned putative E-CadposLgr6posα6pos lung epithelial stem cells that can be serially passaged in liquid culture, and they have shown that both freshly isolated and propagated E-CadposLgr6pos cells are able to generate differentiated human bronchioalveolar epithelial tissue following transplantation of a single cell under the

kidneyArticle capsule of recipient immune-deprived (CD-1 nude) mice. While lung cells expressing the stem cell associated antigen c-Kit did not exhibit stem cell potential in this study, another study using this approach100 has described a putative multipotent c-kitpos human lung stem cell with broader developmental potential, seemingly capable of generating lung epithelial, endothelial and mesenchymal cell lineages in different contexts100 but its status is as yet uncertain101.

This notwithstanding, the delineation of human lung epithelial stem and progenitor cell hierarchies is beset with practical difficulties likely to slow progress. Translational studies aimed at identifying stem cell homologues of those in mice will need to consider differences in the distribution of epithelial cell lineages along the proximal-distal axis of the murine and human airway tree62. Murine cells also have extremely long telomeres102, endowing them with a far more prodigious replicative capacity than human cells. Consequently, human homologues of putative mouse lung epithelial stem cells may have a more restricted proliferative potential and may not be appropriate therapeutic targets in clinical contexts. Accepted

13 © 2013 The Authors Respirology © 2013 Asian Pacific Society of Respirology Practical issues related to the acquisition of human lung tissue biopsies suitable for stem cell experimentation will impact the development and refinement of optimal strategies for stem cell isolation and characterisation. The modelling of lung epithelial stem cell organisation and regulation will require access to normal lung tissue from different anatomical regions along the proximal-distal axis of the airway tree, and its processing in a timely manner.

Regular access to normal lung is rare, and resected “normal” lung tissue and bronchoscopy samples are invariably acquired from patients undergoing procedures for diverse clinical indications. These clinical indications are likely to affect the biomarker profile and attributes of putative stem and progenitor cells and also amplify the intrinsic biological variability of measured human parameters necessitating larger sample sizes for statistical validation of Article stem cell attributes.

That said, recent clinical studies demonstrating the therapeutic efficacy of tracheobronchial reconstruction using bioengineered recellularised human tracheal matrix scaffolds103 is cause for optimism for the future of stem cell based therapies in lung regenerative medicine.

ACNOWLEDGEMENTS

The authors are supported by grants from the Australian National Health and Medical

Research Council (Grant 1009374) and the Victorian Cancer Agency. Accepted

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Accepted

FIGURE LEGENDS

Figure 1: In the steady state, the classical stem cell hierarchy comprises an age-structured

continuum of stem cells, transit amplifying progenitor cells and their progeny of

progressively restricted proliferative and differentiative capacity. The majority of

stem cells in the hierarchy are highly quiescent, have a low probability of

entering cell cycle, and are randomly recruited to replenish transit amplifying

progenitor cell pools. Day-to-day requirements for functional differentiated cells

are met by the more abundant pool of rapidly cycling committed transit

Article amplifying cells, while the highly quiescent stem cell pool is activated and

recruited into cycle to regenerate descendent lineages following severe

perturbation or injury.

Figure 2: A haematopoietic “roadmap” illustrating the iterative experimental process for

determining and validating the immunophenotypic signature profile of putative

stem and progenitor cell sub-populations, and the robustness of surrogate in

vitro assays to predict the incidence and regenerative potential of candidate

stem cells in the steady state and in perturbation and disease models.

Figure 3: A schematic representation of the spatial location, regional distribution and

differentiation potential of lung epithelial stem and progenitor cells along the

proximal-distal axis of the airway tree. Regenerative pulmonary neuroendocrine

and neuroepithelial cells are scattered or clustered throughout the proximal and

distal airways, and there is evidence for the existence of multipotent

Accepted stem/progenitor cells in all compartments. 26 © 2013 The Authors Respirology © 2013 Asian Pacific Society of Respirology Note to typesetters: please print glossary on first page of review in shaded textbox

Glossary

Adult stem cell: Rare, undifferentiated cells within a tissue which are able to renew and to maintain the life-long production of progenitor cells and the diverse mature functionally differentiated non-dividing cells which comprise that tissue, in both the steady-state and following perturbation or injury.

Progenitor cell: Relatively undifferentiated transit amplifying cells which arise from stem cells and have a finite lifespan with a more limited proliferative capacity and a more restricted ability to give rise to descendent cell lineages within a tissue. Article

Facultative stem cell: This term describes a class of normally functional differentiated cells in some adult organs which are able to revert to an undifferentiated stem cell state and regenerate diverse damaged cell lineages in response to severe perturbation or injury.

Regenerative cells: In this review, this is a collective term used to describe any cells in a tissue or organ which are able to proliferate and replenish descendent cell lineages in the steady state or following injury.

Pluripotent/multipotent/unipotent stem cells: These terms describe the relative ability of a stem cell to give rise to diverse differentiated cell lineages. The term pluripotent stem cell is reserved for embryonic stem cells which give to differentiated cells of endodermal, mesodermal and ectodermal origin which comprise the organism. Multipotent stem cells give rise to a multiple, but limited number of cell lineages, usually of a single germ layer origin. Unipotent stem or progenitor cells are only able to differentiate into a single cell type. Accepted

Non-dividing Functionally Differentiated Cells

G0 Article Potential for renewal

Probability of cycling Proliferative potential

Lineage commitment/differentiation Fig 1 Accepted Cytotoxic Perturbation . and Phenotype C-kit Mutant Mouse Models Sca-1 Correlation Co-expression Co-fractionation Does assay readout predict in vitro “Gold Standard” stem cell Clonogenic Transplant dysregulation ? Assay Assay 100 80 60 40 20 Percent donor donor Percent 0

Article 25 50 100 200 Cells transplanted Fig 2 Accepted Article

Fig 3 Accepted

Minerva Access is the Institutional Repository of The University of Melbourne

Author/s: Bertoncello, I; McQualter, JL

Title: Lung stem cells: Do they exist?

Date: 2013-05-01

Citation: Bertoncello, I. & McQualter, J. L. (2013). Lung stem cells: Do they exist?. RESPIROLOGY, 18 (4), pp.587-595. https://doi.org/10.1111/resp.12073.

Publication Status: Accepted manuscript

Persistent Link: http://hdl.handle.net/11343/43880