WO2010085842A1 - Lung epithelial progenitor cells, uses thereof and processes for production - Google Patents

Abstract

The present invention provides an isolated lung epithelial progenitor cell expressing a protein individually or collectively selected from the group consisting of EpCam, CD104, CD49f and CD24 or progeny thereof or a cell differentiated therefrom. The present invention also provides a population of cells enriched for said cells. The present invention also provides methods for isolating lung epithelial progenitor cells and uses of lung epithelial progenitor cells.

Description

LUNG EPITHELIAL PROGENITOR CELLS, USES THEREOF AND PROCESSES FOR PRODUCTION

Incorporation By Reference This application claims priority from Australian Patent Application No.

2009900297 filed on 27 January 2009 and entitled "Lung epithelial progenitor cells, uses thereof and processes for production", the entire contents of which are hereby incorporated by referenced.

Field of the Invention

The present invention relates to the field of cell biology, for example to lung progenitor cells and uses thereof, e.g., in therapy or screening and/or to methods for isolating and/or growing and/or culturing lung progenitor cells,

Background of the Invention

The lung is a complex organ made up of several lineages that interact to maintain structure and function. Even within cells arising from a single germ layer, cells become subdivided into separate cell lineage "zones". The adult lung can be functionally and structurally divided into three epithelial domains, i.e., proximal cartilaginous airway (trachea and bronchi), distal bronchioles (bronchioles, terminal bronchioles and respiratory bronchioles) and gas exchange spaces (alveoli). Predominant epithelial cell types in the trachea and bronchi of humans include basal cells, Goblet cells, nonciliated nonsecretory columnar cells and secretory cells. Predominant epithelial cells in the bronchioles of humans include Clara cells and ciliated columnar cells. Predominant epithelial cells in the alveoli of humans include Type I pneumocytes and Type II pneumocytes. Additional cell types in the lung include airway smooth muscle, fibroblasts, and vasculature. Airway and alveolar architecture and function, result from interaction among epithelium, smooth muscle, fibroblasts, and vascular cells, all within an elaborate structural matrix of connective tissue. Disruption of the lung architecture, e.g., by cell death and/or scarring results in lung disease, such as, for example, inflammatory lung disease, e.g., chronic obstructive pulmonary disease (COPD), cystic fibrosis and pulmonary fibrosis.

COPD is the fourth leading cause of death in the United States, claiming the lives of 122,283 Americans in 2003. In 2004, 11.4 million adults over the age of 18 were estimated to have COPD. However, over 24 million U.S. adults have evidence of impaired lung function, indicating an under-diagnosis of COPD (American Lung Association, 2006, Trends in Chronic Bronchitis and Emphysema Morbidity and Mortality). The cost to the nation for COPD in 2004 was approximately $37.2 billion dollars, including healthcare expenditures of $20.9 billion in direct health care expenditures, $7.4 billion in indirect morbidity costs, and $8.9 billion in indirect mortality costs (American Lung Association, 2006, Trends in Chronic Bronchitis and Emphysema Morbidity and Mortality). COPD is comprised primarily of two related diseases: chronic bronchitis and emphysema. Chronic bronchitis is characterized by inflammation and eventual scarring of the lining of the bronchial tubes. When the bronchi are inflamed and/or infected, less air is able to flow to and from the lungs and a heavy mucus or phlegm is produced. The condition is defined by the presence of a mucus-producing cough most days of the month, three months of a year for two successive years without other underlying disease to explain the cough. Emphysema begins with the destruction of air sacs (alveoli) in the lungs where oxygen from the air is exchanged for carbon dioxide in the blood, due in part, by an abnormal inflammatory response of the lung to noxious particles or gases, e.g., cigarette smoke. The walls of the air sacs are thin and fragile. Damage to the air sacs is irreversible. As air sacs are destroyed, the lungs are able to transfer less oxygen to the bloodstream, causing shortness of breath, and the lungs lose their elasticity, which is important to keep airways open. In both chronic bronchitis and emphysema the obstruction is generally permanent and progressive.

Pulmonary fibrosis is an illness in which the alveoli, or air sacs, of the lungs become inflamed, and are gradually replaced by scar tissue. As the disease progresses, the scar tissue impairs breathing and oxygen transfer. There are a number of known causes, such as cancer, chronic infection or inflammation, industrial dusts, e.g., asbestos, certain drugs, and the like.

Cystic fibrosis (CF) is one of the most common fatal autosomal recessive diseases affecting Caucasian populations. CF has an incidence in neonates of about 0.05%, indicating a carrier frequency of about 5% of the population. Biological parents of subjects with CF are, by definition, obligatory carriers. Carriers are clinically normal and their detection prior to the birth of an affected child has been precluded by the absence of detectable effects of the gene in single dose. CF is caused by mutation of the cystic fibrosis transmembrane conductance regulator which causes the airway mucus of CF patients to becomes viscous and dehydrated, disrupting the mucociliary clearance of inhaled pathogens. This leads to recurring infections in the airways by pathogens such as Pseudomonas aeruginosa, Hemophilus influenzae and Staphylococcus aureus. The resulting inflammation causes fibrosis and a gradual deterioration in lung function resulting in a shortening of mean life span for CF patients to 37 years.

Other lung diseases that involve an undesirable inflammatory component include bronchiolitis (inflammation that involves the bronchioles), alveolitis (inflammation that involves the alveoli), vasculitis, sarcoidosis and acute respiratory distress syndrome (ARDS).

For many lung diseases, main line therapy includes corticosteroid drugs (antiinflammatory drugs). However, for many diseases, they are not effective. For example these drugs improve symptoms in only about one in five people with interstitial lung disease. Chronic obstructive pulmonary disease (COPD) is mainly resistant to steroid therapy. Furthermore, these drugs treat the symptoms of the diseases, rather then the underlying cause. Moreover, long term treatment with steroids can result in serious side effects ranging from immunosuppression to bone loss (Molinoff et ah, Goodman and Gihnan's The Pharmacologic Basis of Therapeutics, MacMillan Publishing Company, New York NY, 1995).

Another treatment option for lung disease is a lung transplant, however this option is obviously hampered by the shortage of suitable donor organs.

One line of treatment currently being pursued for treatment of lung disease is cell-based therapies. Such treatments generally administering a lung cell and/or cells capable of differentiating into lung cells to replace removed and/or damaged tissue. However, such cell based therapies have been hampered by difficulties in identifying lung progenitor cells, i.e., cells capable of giving rise to differentiated lung cells, preferably several different types of lung cells, and more preferably lung epithelial cells. One population of lung epithelial progenitor cells is brochioalveolar stem cells

(BASCs) (US Pub. No. 20070025914), which are characterized as expressing at least stem cell antigen (SCA)-I and CD34. According to the inventors of US Pub. No. 20070025914, BASCs are capable of differentiating into Clara-like cells, AT2-like cells and ATI -like cells. However, a disadvantage of these cells is the requirement to isolate the cells using Sca-1. This marker is expressed on murine cells, however no human equivalent has been identified to date. Accordingly, the cells disclosed in US Pub. No. 20070025914 have little utility in the human context. Moreover, two recent publications have suggested that Sca-1 expression does not distinguish lung epithelial progenitor cells (McQualter et ah, Stem Cells 2008, and Teisanu et ah, Stem Cells 2008) and demonstrate that cells expressing Sca-1 are predominantly fibroblastic in nature, e.g., expressing the cell surface markers Thy-1 and PDGFRα (McQualter et ah, Stem Cells 2008). Thus, the Sca-1 population described in US Pub. No. 20070025914 may not be a lung epithelial progenitor cell. Instead such cells, may be a stromal (e.g., mesenchymal) cell population.

Other cell populations suggested to be lung epithelial progenitor cells include Clara cells in submucosal glands, variant Clara cells residing in neuroepithelial bodies, alveolar type II epithelial cells and "side population" cells (Liu et al, Methods Enzymol.419:285-321, 2006 and references cited therein). Despite identification of these cell populations, a single lung stem cell that can give rise to multiple epithelial lineages in both the proximal and distal airways of the lung has not been identified (Liu et al, supra).

Researchers have also looked to embryonic stem (ES) cells to produce lung tissue, e.g., for therapeutic purposes. For example, Coraux et al., Am. J. Physiol. Lung Cell MoI. Biol, 32: 87-92, 2005 describe a process for producing pseudostratifϊed lung epithelium. However, there are several disadvantages or limitation of ES cell-based systems. For example, to date there is little data obtained showing that human ES cells can be induced to differentiate into lung tissue, let alone lung epithelium. Furthermore, the methods used to direct differentiation of ES cells into lung tissue are inefficient and the amounts of lung cells produced using current methods are inadequate and insufficiently pure for therapeutic or screening purposes. There are also risks associated with therapies produced from ES cells, e.g., teratoma formation by residual ES cells in a therapeutic formulation.

There has also been a suggestion that adult stem cells from other regions of the body can populate and regenerate the lung. For example, Krause et al. Cell 105: 369- 77, 2001; and Herzog et al. Stem Cells, 24: 1986-92, 2006 suggest that cells from the bone marrow can populate the lung. However, the methods described in these reports require that the lung is injured, e.g., by radiation or chemotherapy to cause the bone marrow cells to engraft in the lung. Clearly, this is not a desirable method for treating any subject, let alone a subject having reduced lung function. Furthermore, recent reports have questioned whether or not epithelial engraftment does actually occur, and suggested that initial results may have been misinterpreted (Kotton et al, Am. J. Respir. Cell MoI. Biol, 33: 328-334, 2005 and Chang et al, Am. J. Respir. Cell MoI Biol, 33: 335-342, 2005).

It will be apparent to the skilled artisan based on the foregoing that there is a need to identify lung epithelial progenitor cells, e.g., to provide the basis of therapeutic or prophylactic compositions for treating lung disorders and/or to provide screening tools to identify compounds to treat or prevent lung disease. Preferably, the lung epithelial progenitor cells have the ability to give rise to or produce or differentiate into multiple epithelial lineages of the lung, preferably lineage(s) from the proximal and distal airways of the lung.

Summary of Invention

In work leading up to the present invention, the inventors sought to identify and isolate a population of lung epithelial progenitor cells, preferably with the ability to differentiate into multiple epithelial lineages of the lung. To identify these cells, the inventors initially developed a cell culture system in which prospective lung epithelial progenitor cells are cultured within a matrix, e.g., within a three-dimensional matrix at the air-liquid interface of culture medium comprising suitable growth factors and/or conditioned with factors secreted from stromal cells, such a lung stromal cells or bone marrow stromal cells (e.g., by growing the potential lung epithelial progenitor cells in the same culture medium as lung stromal cells or bone marrow stromal cells). Using the culture method, the inventors grew a population of cells, and subsequently determined that those cells are capable of differentiating into epithelial cells of the proximal lineages (e.g., expressing MUC-I and/or MUC5AC) and/or the distal lineages (e.g., expressing surfactant protein (SP-C). Using gene expression analysis, the inventors demonstrated that they had isolated cells capable of giving rise to populations of lung epithelial cells at various stages of differentiation. For example, the inventors isolated a population of cells capable of giving rise to airway cells (e.g., mucous secretory cells and/or ciliated lung cells and/or Clara cells and/or anion secretory cells) and/or to alveolar cells (e.g., to type I alveolar cells and/or to type II alveolar cells). Such cell populations are useful for regenerating specific cell populations and/or lineages of lung populations (e.g., airway cells and/or alveolar cells) or all lung epithelial cells generally.

The inventors also demonstrated that these cells were capable of proliferation without differentiation, i.e., self-renewal, and that the progeny cells retained the ability to differentiate into epithelial cells of the proximal lineages and/or distal lineages. Accordingly, the cells identified by the inventors have the functional characteristics of a lung epithelial stem/progenitor cell.

The inventors then sought to characterize the structure of the isolated cells. As exemplified herein, the inventors have determined that the lung epithelial progenitor cells isolated by them express one or more of EpCam, CD 104, CD49f or CD24. Preferably, the cell expresses at least EpCam and/or CD 104. Preferred populations of cells isolated by the inventors, e.g., having higher differentiation potential (i.e., the ability to produce more distinct epithelial lineages and/or comprising a higher percentage of cells capable of differentiating into distinct epithelial lineages) include cells expressing high levels of EpCam and/or cells expressing EpCam (or expressing high levels of EpCam) and CD49f and/or cells expressing EpCam (or expressing high levels of EpCam) and CD24 and/or cells expressing EpCam (or expressing high levels of EpCam) and CD 104 and/or cells expressing CD 104 and CD49f and/or cells expressing CD 104 and CD24 and/or cells expressing EpCam (or expressing high levels of EpCam) and CD 104 and CD49f and CD24. The inventors have also found that selecting cells expressing low levels of CD24 cells further enriches for lung epithelial progenitor cells.

The inventors have isolated cells having similar phenotypic profiles from mice and from humans, demonstrating the applicability of their methods for isolating lung epithelial progenitor cells to a variety of organisms, preferably mammals.

The inventors have also demonstrated that they can culture a population lung epithelial progenitor cells such that they maintain at least a plurality of those cells in an undifferentiated state, i.e., capable of differentiating into epithelial cells of the proximal lineages and/or distal lineages using fibroblast growth factor (FGF)-IO and/or hepatocyte growth factor (HGF).

As exemplified herein, the inventors have further demonstrated that the cells isolated by their method are useful for screening for compounds that modulate lung epithelial progenitor cell proliferation and/or differentiation.

The population of cells identified by the inventors provide the basis for therapeutic compositions and methods, e.g., for the treatment of lung disease and/or for screening for compounds to treat lung disease. The findings of the inventors also provide the basis for methods for isolating and/or culturing lung epithelial progenitor cells.

Accordingly, the present invention provides an isolated lung epithelial progenitor cell expressing a protein individually or collectively selected from the group consisting of EpCam, CD 104, CD49f and CD24, or progeny thereof or a cell differentiated therefrom. The skilled artisan will be aware that such a cell will be considered to have a phenotypic profile EpCam⁺ and/or CD104⁺ and/or CD49f⁺ and/or CD24⁺, and the reference herein to a cell defined by such characteristics shall be taken to apply mutatis mutandis to a cell expressing the recited protein.

The present invention also provides an isolated population of cells enriched for lung epithelial progenitor cells, wherein cells in said population express a protein individually or collectively selected from the group consisting of EpCam, CD 104, CD49f and CD24 and/or a population of cells enriched for lung epithelial progenitor cells that have a phenotypic profile EpCam⁺ and/or CD104⁺ and/or CD49f⁺ and/or

CD24⁺ and/or progeny thereof and/or cells differentiated therefrom.

The present invention also encompasses mutants and variants of the claimed cell(s).

Preferably, the lung epithelial progenitor cell(s) express at least EpCam and/or

CD 104.

Preferably, the lung epithelial progenitor cell(s) express a high level of EpCam, e.g., have a phenotypic profile EpCam^hl. Preferably, the lung epithelial progenitor cell(s) express a low level of CD24, e.g., have a phenotypic profile CD24^lQ.

In one embodiment, the lung epithelial progenitor cell(s) as described herein according to any embodiment express low levels, or do not express detectable levels

(e.g., detectable using fluorescence activated cell sorting; FACS) of a marker of a hematopoietic cell (preferably CD45) and/or a marker of an endothelial cell

(preferably, CD31) (e.g., are CD45^" and/or CD31^").

In one embodiment, the lung epithelial progenitor cell(s) have a phenotypic profile selected individually or collectively from the group consisting of: i) EpCam⁺ and CD49f ; ii) EpCam¹¹¹ and CD49f; iii) EpCam⁺ and CD24⁺; iv) EpCam¹¹¹ and CD24⁺; v) EpCam⁺ and CD24^lG; vi) EpCam¹¹¹ and CD24^lG; vii) EpCam⁺ and CD104⁺; viii) EpCam¹¹¹ and CD 104⁺: ix) EpCam⁺, CD 104⁺ and CD49f ; x) EpCarn¹", CD 104⁺ and CD49f; xi) EpCam⁺, CD 104⁺ and CD24⁺; xii) EpCam¹¹¹, CDl 04⁺ and CD24⁺; xiii) EpCam⁺, CD 104⁺ and CD24^lG; xiv) EpCam¹¹¹, CD 104⁺ and CD24^lc; xv) EpCam⁺, CD 104⁺, CD49f and CD24⁺; xvi) EpCam¹¹¹, CD 104⁺, CD49f and CD24⁺; xvii) EpCam⁺, CD104⁺, CD49f and CD24^lo _; xviii) EpCam¹¹¹, CD104⁺, CD49f and CD24^lG; xix) any one of i) to xviii) and CD45^"; and xx) any one of i) to xix) and CD31 ^".

In another embodiment, the lung epithelial progenitor cell(s) have a phenotypic profile selected individually or collectively from the group consisting of: i) CD104⁺ and CD49f; ii) CD104⁺ and CD24⁺; iii) CD104⁺ and CD24^l0; iv) CD 104⁺, CD49f and CD24^lG; v) CD104⁺, CD49f and CD24⁺; vi) any one of i) to v) and CD45^"; and vii) any one of i) to vi) and CD31 ^"

In a particularly preferred embodiment, the lung epithelial progenitor cell(s) have a phenotypic profile EpCam⁺, CD104⁺, CD49f", CD24⁺, CD45^" and CD31 or EpCam^hl, CD104⁺, CD49f , CD24⁺, CD45^" and CD31 or EpCam⁺, CD104⁺, CD49f , CD24^b, CD45^" and CD31 or EpCarn¹", CD104⁺, CD49f , C24^lc, CD45^" and CD31^". In one example of the invention, the propgenitor cell(s) is (are) Sca-l^neg/1° (e.g. for a mouse progenitor cell). In one embodiment, a lung epithelial progenitor cell of the present invention or progeny thereof or cell derived therefrom is non-tumorigenic and/or is not derived from or isolated from a tumor. Methods for determining tumorgenicity of a cell will be apparent to the skilled artisan and/or described herein and include administering a population of the cells to a test subject and determining whether or not a tumor forms. In one embodiment, a lung epithelial progenitor cell as described herein according to any embodiment or progeny thereof is capable of differentiating into a lung epithelial cell. Preferably, the lung epithelial progenitor cell or progeny thereof is capable of differentiating into a plurality of distinct lung epithelial cells, e.g., the lung epithelial progenitor cell is multipotential. Preferably, the lung epithelial progenitor cell or progeny thereof is capable of differentiating into one or more cells of the proximal lung and/or of the distal lung, more preferably, of both the proximal lung and the distal lung. For example, the lung epithelial progenitor cell or progeny thereof is capable of differentiating into a cell that expresses a marker of a proximal lung epithelial cell, e.g., MUC-I and/or MUC5AC. Exemplary proximal lung epithelial cells will be apparent to the skilled artisan and include basal cells and pulmonary endocrine cells. Alternatively, or in addition, the lung epithelial progenitor cell or progeny thereof is capable of differentiating into a cell that expresses a marker of a distal lung cell, e.g., SP-C. Exemplary distal lung epithelial cells include alveolar type I pneumocytes and alveolar type II pneumocytes. In another embodiment, the lung epithelial progenitor cell or progeny thereof is capable of differentiating into a Clara cell, which occurs in both the proximal and distal lung.

In another embodiment, a lung epithelial progenitor cell as described herein according to any embodiment or progeny thereof is capable of dividing and/or proliferating in culture when serially passaged in culture, e.g., for at least 1 passage or at least 2 passages or at least 3 passages or at least 4 passages. Preferably, progeny of the lung epithelial progenitor cell have the ability to differentiate into a lung cell as described herein. This does not mean that all progeny cells have this ability, merely that at least one progeny of the lung epithelial progenitor cell has this ability.

The present invention also provides a method for isolating a lung epithelial progenitor cell or population enriched for said cells, said method comprising identifying a cell expressing a protein individually or collectively selected from the group consisting of EpCam, CD 104, CD49f and CD24 or population of said cells and isolating said cell(s).

Preferably, the method comprises contacting a population of cells comprising lung epithelial progenitor cells with a compound that binds to a protein individually or collectively selected from the group consisting of EpCam, CD 104, CD49f and CD24 for a time and under conditions sufficient for a compound-protein complex to form and isolating the complex and/or a cell on which the complex has formed. Preferably, the compound is an antibody or antigen binding fragment thereof, optionally labelled with a detectable or selectable marker (e.g., a fluorophore or a magnetic or paramagnetic moiety). Suitable methods for isolating cells will be apparent to the skilled artisan and include, for example, FACS or magnetic-activated cell sorting (MACS).

Suitable combinations of proteins for isolating a lung epithelial progenitor cell are described herein and are to be taken to apply mutatis mutandis to the present embodiment of the invention. Preferably, the method comprises identifying and isolating a cell or population thereof expressing EpCam, CD 104, CD49f and CD24 and expressing little or undetectable levels of CD45 and CD31. In this respect, it is not necessary to identify and/or isolate cells based on expression of each of the proteins simultaneously. Rather individual proteins or groups thereof may be detected sequentially. In one embodiment, the cell(s) are isolated from a tissue sample from a subject's lung and/or from a secretion from a lung or sample comprising same (e.g., bronchoalveolar lavage fluid; BALF). Such a method may be performed ex vivo or in vitro, e.g., with a sample previously isolated from a subject.

In one embodiment, the method comprises isolating a lung epithelial progenitor cell or population enriched for same from non-tumorigenic tissue, e.g., from a tissue that does not comprise or is not known to comprise a tumor or from a region of a tissue that does not comprise a tumor and/or from a normal and/or healthy subject.

In one embodiment, the method additionally comprises culturing the isolated cell(s), e.g., to produce progeny of isolated lung epithelial progenitor cells and/or cells differentiated therefrom. Alternatively, or in addition, the method additionally comprises differentiating the isolated cell(s) and/or progeny thereof to produce a lung epithelial cell or population thereof.

The present invention also provides a cell isolated by a method as described herein according to any embodiment.

The present invention also provides a composition comprising a lung epithelial progenitor cell and/or progeny and/or differentiated cell and/or population of cells as described herein according to any embodiment and a pharmaceutically acceptable carrier and/or excipient. Such a composition may comprise additional components, e.g., an anti -inflammatory drug and/or an immunosuppressant drug and/or another cell (e.g., a cell having immunosuppressive properties and/or capable of supporting growth of a lung epithelial progenitor cell, progeny thereof and/or cells differentiated therefrom, e.g., a stromal cell or a mesenchymal stem cell)

Preferably, the composition comprises an effective amount of said progenitor cells and/or progeny and/or differentiated cells and/or population of cells. More preferably, the amount is a therapeutically or prophylactically effective amount. The present invention also provides a method for producing a pharmaceutical composition, said method comprising combining or otherwise mixing a lung epithelial progenitor cell and/or progeny and/or differentiated cell and/or population of cells as described herein according to any embodiment and a pharmaceutically acceptable carrier and/or excipient. The present invention also provides a method of treating or preventing a disease comprising administering to a subject in need thereof a lung epithelial progenitor cell and/or progeny and/or differentiated cell and/or population of cells or a composition as described herein according to any embodiment.

In one embodiment, the subject suffers from, is developing or is at risk of developing a lung disease and/or has undergone surgery to remove at least part of a lung. Preferably, the lung disease is a lung disease associated with inflammation and/or scarring. For example, the lung disease is COPD or cystic fibrosis or pulmonary fibrosis or bronchiolitis or alveolitis or vasculitis or sarcoidosis.

In one embodiment, the lung epithelial progenitor cell and/or progeny and/or differentiated cell and/or population of cells is isolated or derived from the subject to be treated (i.e., are autologous) or from a related subject, e.g., a family member. The present invention also encompasses administration of cells from unrelated subjects and/or from subjects of a different species.

In an example of the invention, the cell and/or progeny and/or differentiated cell and/or population of cells is administered together with a stromal cell, such as, a mesenchymal stem cell or a mesenchymal progenitor cell or a lung fibroblast cell.

The present invention also provides a method of improving lung function or producing lung tissue in a subject, said method comprising administering to the subject a lung epithelial progenitor cell and/or progeny and/or differentiated cell and/or population of cells or a composition as described herein according to any embodiment. The present invention also provides a lung epithelial progenitor cell and/or progeny and/or differentiated cell and/or population of cells as described herein according to any embodiment for use in medicine, e.g., to treat a lung disease and/or to repair or replace damaged or removed lung tissue and/or for improving lung function in a subject and/or for producing lung tissue in a subject. The present invention also provides for use of a lung epithelial progenitor cell and/or progeny and/or differentiated cell and/or population of cells as described herein according to any embodiment in medicine. For example, the present invention provides for use of a lung epithelial progenitor cell and/or progeny and/or differentiated cell and/or population of cells as described herein according to any embodiment in the manufacture of a medicament for the treatment of a lung disease and/or to repair or replace damaged or removed lung tissue and/or for improving lung function in a subject and/or for producing lung tissue in a subject.

The present invention also provides a method for identifying a compound that directs differentiation of a lung epithelial progenitor cell, said method comprising: i) contacting a lung epithelial progenitor cell or progeny thereof or population of cells enriched for same as described herein according to any embodiment with a test compound and determining the amount of lung epithelial cells differentiated therefrom; ii) determining the amount of lung epithelial cells differentiated from lung epithelial progenitor cell or progeny thereof or population of cells enriched for same as described herein according to any embodiment in the absence of the compound, wherein an increased amount of differentiated cells at (i) compared to (ii) indicates that the compound directs differentiation of the lung epithelial progenitor cell.

Preferably, the method comprises determining the amount of one or more distinct lung epithelial progenitor cell types. For example, the method comprises determining the amount of a plurality of distinct lung epithelial cell types and determining a compound that directs differentiation toward one or more of those cell types and, optionally does not direct differentiation toward one or more other cell types.

It will be apparent to the skilled artisan based on the foregoing that the present invention also provides a method for identifying a compound that reduces or prevents differentiation of a lung epithelial progenitor cell into one or more lung epithelial cell types, said method comprising: i) contacting a lung epithelial progenitor cell or progeny thereof or population of cells enriched for same as described herein according to any embodiment with a test compound and determining the amount of lung epithelial cells differentiated therefrom; ii) determining the amount of lung epithelial cells differentiated from lung epithelial progenitor cell or progeny thereof or population of cells enriched for same as described herein according to any embodiment in the absence of the compound, wherein a reduced amount of differentiated cells at (i) compared to (ii) indicates that the compound directs differentiation of the lung epithelial progenitor cell. Preferably, the method comprises determining the amount of one or more distinct lung epithelial progenitor cell types. For example, the method comprises determining the amount of a plurality of distinct lung epithelial cell types and determining a compound that prevents or reduces differentiation toward one or more of those cell types and, optionally directs differentiation toward one or more other cell types. Such a compound is useful, for example, for treating a condition associated with excessive production of a cell type of the lung, e.g., mucous cell metaplasia characteristic of asthma and/or COPD.

The present invention also provides a method for identifying a compound that reduces proliferation of a lung epithelial progenitor cell and/or progeny thereof, said method comprising: i) contacting a lung epithelial progenitor cell or progeny thereof or population of cells enriched for same as described herein according to any embodiment with a test compound and determining the amount of cell proliferation; ii) determining the amount of cell proliferation of a lung epithelial progenitor cell or progeny thereof or population of cells enriched for same as described herein according to any embodiment in the absence of the compound, wherein a reduced amount of proliferation at (i) compared to (ii) indicates that the compound reduces proliferation of lung epithelial progenitor cells and/or progeny thereof. Such a compound is useful for, for example, protecting a lung epithelial progenitor cell during chemotherapy (e.g., when dividing cells are killed), so as to protect the ability of the cell(s) to repopulate and/or repair lung tissue.

The present invention also provides a method for identifying a candidate compound for the treatment of cancer, preferably lung cancer, said method comprising: i) contacting a lung cancer cell or population thereof with a compound and determining the level of proliferation and/or cell death; and ii) contacting a lung epithelial progenitor cell or progeny thereof of the present invention with the compound and determining the level of proliferation and/or cell death, wherein a compound that reduces cellular proliferation or induces cell death at (i) and not at (ii) or that reduces cellular proliferation to a greater degree at (i) compared to (ii) or induces a greater level of cell death at (i) compared to (ii) is a candidate compound for the treatment of cancer, preferably lung cancer.

In one embodiment, the method comprises determining the dosage of a candidate compound that compound that reduces cellular proliferation or induces cell death at (i) and not at (ii) or that reduces cellular proliferation to a greater degree at (i) compared to (ii) or induces a greater level of cell death at (i) compared to (ii).

In one embodiment, any method described herein for identifying a compound additionally comprises isolating the identified compound.

The present invention also provides a method for culturing a lung epithelial progenitor cell and/or progeny thereof in an undifferentiated state, said method comprising maintaining said cell(s) for a time and under conditions sufficient for cell growth and/or proliferation, wherein said cell(s) is(are) maintained in the presence of FGF-IO and/or HGF. Preferably, the cell(s) is(are) maintained in the presence of FGF- 10 and HGF.

Alternatively, or in addition, the cell is maintained in the presence of a stromal cell characterized by expression of platelet derived growth factor receptor (PDGFR)α and/or PDGFRβ. The skilled artisan will be aware of methods for identifying and/or isolating such cells, e.g., using a compound that binds to the protein and/or by identifying a cell previously characterized as expressing the protein.

The present invention additionally provides a method for identifying a gene or protein differentially expressed by a lung epithelial progenitor cell, the method comprising: (i) determining the level of expression of a gene and/or protein in a lung epithelial progenitor cell of the invention; and

(ii) determining the level of expression of a gene and/or protein in a cell other than lung epithelial progenitor cell of the invention (e.g., in a mature lung cell), Wherein increased or reduced expression at (i) compared to (H) indicates that the gene or protein is differentially expressed in a lung epithelial progenitor cell.

The foregoing method is useful for identifying additional markers of a lung epithelial progenitor cell.

The present invention also provides a method for identifying a gene or protein differentially expressed by a lung cancer cell, the method comprising:

(i) determining the level of expression of a gene and/or protein in a lung cancer cell; and

(ii) determining the level of expression of a gene and/or protein in a lung epithelial progenitor cell and, optionally, another lung cell type, Wherein increased or reduced expression at (i) compared to (ii) indicates that the gene or protein is differentially expressed in a lung cancer cell.

In one example, the method of the foregoing embodiment is for identifying a therapeutic target for treating cancer without substantially having detrimental effects on a lung epithelial progenitor cell. In one example, the method of the foregoing embodiment is for identifying a target for diagnosing/prognosing cancer without substantially detecting a lung epithelial progenitor cell.

In one embodiment, a method for identifying a protein differentially expressed in a cell additionally comprises identifying and/or isolating and/or providing a compound that binds to the protein. In the case of a protein expressed at an increased level on a lung cancer cell, the compound may kill a cell to which it binds.

The present invention also provides a method for isolating a lung epithelial progenitor cell, said method comprising maintaining a population of cells from a lung in a matrix, which is in contact with both a culture medium and the gas environment surrounding the matrix/medium for a time and under conditions sufficient for the cell to proliferate and differentiate and detecting a population of epithelial cells of the lung, wherein detection of the population of epithelial cells of the lung is indicative of a lung epithelial progenitor cell.

Preferably, the culture medium comprises secreted factors from a stromal cell, e.g., a lung stromal cell or a bone marrow stromal cell. Preferably, the culture medium is in contact with stromal cells, however the matrix and cells contained therein are spatially separated from the stromal cells.

Preferably, the method additionally comprises isolating a population of cells from the lung of a subject prior to maintaining the population in a matrix. For example, the cells are isolated based on expression of a cell surface marker, e.g., a marker described herein or any other cell surface marker known in the art.

The present invention also provides a kit for isolating a lung epithelial progenitor cell of the invention. For example, the kit comprises a compound, such as an antibody, that binds to a protein individually or collectively selected from the group consisting of EpCam, CD104, CD49f and CD24. Optionally, the kit comprises an enzyme for dissociating lung tissue or cells. Optionally, the kit is packaged with instructions for use in a method as described herein according to any embodiment.

The present invention also provides for use of a compound, such as an antibody, that binds to a protein individually or collectively selected from the group consisting of EpCam, CD 104, CD49f and CD24 to isolate a lung epithelial progenitor cell as described herein according to any embodiment.

Brief description of the Drawings

Figure IA shows an embodiment of a lung epithelial colony-forming assay generated by the present inventors. Panel A) Schematic representation of lung epithelial colony-forming assay. Panel B) Dotplot showing gating strategy used to sort CD- 45^negCD31^negSca-l^pos cells used to develop the assay. Panel C) Isotype control for Sca- 1. Panel D) Phase contrast image of colony formation and (insert) higher magnification (x20) image showing (i) airway-like cystic and (ii) alveolar-like ductal morphology of lung epithelial colonies grown using the assay.

Figure IB is a series of photomicrographs showing time course images of colony formation showing the development of (i) airway colony-forming units (airway- CFU); (ii) alveolar-CFU; and (iii) mixed lung epithelial colonies.

Figure 2A shows epithelial and mesenchymal progenitors isolated on the basis of EpCam and Sca-1 expression require co-culture for the generation of lung epithelial colony-forming units. Panel A) Dotplot showing subsetting of CD45^negCD31^ne§ lung cells on the basis of EpCam and Sca-1. Panel B) Isotype controls. Panel C) Morphology of CD45^negCD31^negSca-l^posEpCam^neg cells cultured alone. Mesenchymal cells stain positive for oil-Red O and α smooth muscle actin (SMA). Panel D) Image showing the lack of growth from CD45^negCD3r^egSca-r^eg/loEpCam^pos cultured alone. Panel E) Co-culture of Sca-l^neg/loEpCam^pos and Sca-l^posEpCam^neg cells showing the generation of epithelial-CFUs when stromal and epithelial stem/progenitor cells are in co-culture. Panel F) Phase contrast image of co-culture of Sca-l^neg/loEpCam^pos cells isolated from RFP and eGFP transgenic mice with Sca-l^posEpCam^neg cells isolated from C57BL/6 mice. Panels G and H) Fluorescent images showing that epithelial CFUs arise from Sca-l^neg/loEpCam^pos cells and that colonies are monochromatic.

Figure 2B is a graphical representation showing cloning efficiency of CD45negCD31^negEpCam^pos cells with linear regression analysis.

Figure 3A is a series of photomicrographs showing generation of distinct epithelial colony subtypes. Bright-field images of lobular cystic airway- like colonies (Panel A), dense saccular alveolar- like colonies (Panel B), and colonies with mixed cell types (Panel C). Fluorescent confocal images of DAPI (blue) (Panels D-F), MUC5AC (green) (Panels G-I), proSP-C (red) (Panels J-L), and overlay of MUC5AC and proSP- C staining of representative colonies (Panels M-O).

Figure 3 B is a heat map showing relative real time RT-PCR gene expression levels in cells harvested from single airway, alveolar and mixed epithelial colonies. Values represent average ΔCt values of genes relative to 18s RNA control. ND = not detected (Ct<40).

Figure 4A shows growth factor regulation of lung epithelial colony formation. Morphology of colony formation in co-cultures of Sca-l^neg/loEpCam^pos and Sca- l^p°ΕpCam^neg cells supplemented with epithelial and mesenchymal growth factors known to be involved in lung development; Panel A) no factor control (N=3); Panel B) FGF-7 (N=3); Panel C) FGF-IO (N=3); Panel D) HGF (N=2); Panel E) BMP-4 (N=3); Panel F) PDGF-A (N=3); and Panel G) TGF-Bl (N=3).

Figure 4B is a graphical representation showing normalised CFU incidence as percentage of control (no factors). A statistically significant difference (**P<0.01, ***P<0.001) between Growth factor treated groups and Control (no factors) was determined using a One-way ANOVA with Tukey post test.

Figure 5 A shows morphology of epithelial colonies in stromal-free culture (S ca- r^eg/lo/1°EpCam^pos cells alone) cultures supplemented with FGF-IO and HGF. Figure 5B is a graphical representation of the colony-forming efficiency showing number of colonies generated as a percentage of the number of cells seeded.

Figure 6A shows subsetting which can be used to enrich for epithelial colony- forming potential is enriched in EpCam^posCD49f^pos cell fraction. Epithelial stem/progenitor cells are enriched in the top approcximately 1.5% of cells with the highest expression of EpCam (Panels A and B). Subsetting with additional markers revealed that the phenotype of epithelial stem/progenitor cells can be further refined on the basis of CD104^pos (Panels C and D), CD24^pos (Panels E and F) and CD24^low (Panel G). The population gated in Panel A (EpCam Vs CD49f), Panel C (EpCam Vs CD 104) and Panel E (EpCam Vs CD24) appears to be the same population of cells.

Figure 6B is a graphical representation showing cloning efficiency of EpCAM^k CD24^1D (circles) and CD24^k (squares) cells with linear regression analysis (CD24¹⁰; r² = 0.9481, I/slope = 22.45, n = 7).

Figure 7 shows the self renewal potential of EpCam^posCD49f ^os epithelial stem/progenitor cells. Panel A) Dotplot of primary sort strategy showing minor population of EpCam^posCD49fpos cells. Panel B) Cloning efficiency of EpCam^posCD49fpos cells after serial re-cloning. Panel C) Dotplot of re-cloned cells from passage 3 showing expansion of the EpCam^posCD49F^os cell population. Panel D) Differentiation of re-cloned cells from passage 3. Panel E) Incidence of CFUs (mean number of colonies + SEM, n > 3 colonies for each generation) from serial recloning of single colonies. Figure 8 A shows fluorescence activated cell sorting (FACS) analysis of human lung cells compared to mouse lung cells based on EpCam and CD49f expression. Cells are gated on viability using Propidium Iodide exclusion. CD45 ^neg CD31^neg cells (gated in top row of figure) are then plotted on EpCAM versus CD49f. Data representative of a population of cells comprising lung epithelial progenitor is highlighted by the box. Figure 8B shows fluorescence activated cell sorting (FACS) analysis of human lung cells compared to mouse lung cells (as indicated). Cells are gated on viability using Propidium Iodide exclusion. CD45 ^neg CD31^neg cells (gated in top row of figure) are then plotted on EpCAM versus CD24. The shift to the right in the mouse sample is due to increased autofluorescence in the mouse compared to the human samples. Figure 9 is a depiction showing evidence of an epithelial hierarchy. (Panel A)

Images of a primary colony that was enzymatically dissociated and the secondary colonies generated after subsequent reseeding of disaggregated cells. Without being bound by any theory or mode of action Panel B shows a proposed lineage hierarchy of different lung epithelial CFU subsets. Figure 10 is a series of photomicrographs showing that an anti-EpCam antibody is capable of reducing, inhibiting or preventing colony formation by lung epithelial progenitor cells.

Key to Sequence Listing SEQ ID NO: 1 sets forth a sequence of Homo sapiens EpCam. SEQ ID NO: 2 sets forth a sequence of Mus musculus EpCam. SEQ ID NO: 3 sets forth a sequence of Homo sapiens CD 104. SEQ ID NO: 4 sets forth a sequence of Mus musculus CD 104. SEQ ID NO: 5 sets forth a sequence of Homo sapiens C49f. SEQ ID NO: 6 sets forth a sequence of Mus musculus CD49f. SEQ ID NO: 7 sets forth a sequence of Homo sapiens CD24. SEQ ID NO: 8 sets forth a sequence of Mus musculus CD24. SEQ ID NO: 9 sets forth a sequence of Homo sapiens CD45. SEQ ID NO: 10 sets forth a sequence of Mus musculus CD45. SEQ ID NO: 11 sets forth a sequence of Homo sapiens CD31. SEQ ID NO: 12 sets forth a sequence of Mus musculus CD31.

Detailed Description of the Preferred Embodiments

General

This specification contains nucleotide and amino acid sequence information prepared using Patentln Version 3.4. Each nucleotide sequence is identified in the sequence listing by the numeric indicator <210> followed by the sequence identifier (e.g. <210>l, <210>2, <210>3, etc). The length and type of sequence (DNA, protein (PRT), etc), and source organism for each nucleotide sequence, are indicated by information provided in the numeric indicator fields <211>, <212> and <213>, respectively. Nucleotide sequences referred to in the specification are defined by the term "SEQ ID NO:", followed by the sequence identifier (e.g. SEQ ID NO: 1 refers to the sequence in the sequence listing designated as <400>l).

The designation of nucleotide residues referred to herein are those recommended by the IUPAC-IUB Biochemical Nomenclature Commission, wherein A represents Adenine, C represents Cytosine, G represents Guanine, T represents thymine, Y represents a pyrimidine residue, R represents a purine residue, M represents Adenine or Cytosine, K represents Guanine or Thymine, S represents Guanine or Cytosine, W represents Adenine or Thymine, H represents a nucleotide other than Guanine, B represents a nucleotide other than Adenine, V represents a nucleotide other than Thymine, D represents a nucleotide other than Cytosine and N represents any nucleotide residue.

Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter. Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.

Preferred features and/or characteristics of one embodiment of the present invention shall be taken to apply mutatis mutandis to any other embodiment(s) of the invention unless explicitly stated otherwise.

Selected Definitions

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein the singular forms of "a", "and" and "the" include plural referents unless the context clearly dictates otherwise.

The term "and/or", e.g., "X and/or Y" shall be understood to mean either "X and Y" or "X or Y" and shall be taken to provide explicit support for both meanings or for either meaning. As used herein, the term "CD24" shall be taken to mean, for example, a protein capable of binding to monoclonal antibody BA-I (Abramson et al, J Immunol. 126:83- 88, 1981) and/or described in Pirrucello et al., J Immunol. 756:3779-3784, 1986. For the purposes of nomenclature and not limitation a sequence of Homo sapiens CD24 is set forth in SEQ ID NO: 7 and the sequence of Mus musculus CD24 is set forth in SEQ ID NO: 8.

The term "CD31" (syn "PECAM-I" or "platelet-endothelial cell adhesion molecule- 1") shall be taken to include a protein described in Newman et al., Science

247: 1219-1222, 1990. For the purposes of nomenclature and not limitation a sequence of Homo sapiens CD31 is set forth in SEQ ID NO: 11 and the sequence of Mus musculus CD31 is set forth in SEQ ID NO: 12.

For the purposes of nomenclature and not limitation, the sequence of Homo sapiens "CD45" is set forth in SEQ ID NO: 9 and the sequence of Mus musculus CD45 is set forth in SEQ ID NO: 10.

As used herein, the term "CD49f ' or "α6 integrin" shall be taken to include protein identified as α6 integrin in Hogervorst et al., Eur J Biochem. 199: 425-433,

1991. For the purposes of nomenclature and not limitation a sequence of Homo sapiens CD49f is set forth in SEQ ID NO: 5 and the sequence of Mus musculus CD49f is set forth in SEQ ID NO: 6.

As used herein, the term "CD 104" or "β4 integrin" shall be taken to include proteins identified as β4 integrin in Tamura et al,., J. Cell Biol. Ill: 1593-1604, 1990. For the purposes of nomenclature and not limitation a sequence of Homo sapiens

CD 104 is set forth in SEQ ID NO: 3 and the sequence of Mus musculus CD 104 is set forth in SEQ ID NO: 4.

The term "cell surface marker" shall be taken to mean a protein that is expressed and located or displayed on the surface of a cell. In the context of the present invention, this term will be understood to include EpCam and/or CD24 and/or CD49f and/or CD 104 and/CD45 and/or CD31.

By "collectively" is meant that the invention encompasses any number or combination of the recited cells or groups of cells, and that, notwithstanding that such numbers or combinations of cells or groups of cells may not be specifically listed herein the accompanying claims may define such combinations or sub-combinations separately and divisibly from any other combination of cells or groups of cells.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

As used herein, the term "derived from" shall be taken to indicate that a particular integer or group of integers has originated from the species specified, but has not necessarily been obtained directly from the specified source. The term "effective amount" " shall be taken to mean a sufficient quantity of lung epithelial progenitor cells and/or progeny thereof and/or cells differentiated therefrom to increase or enhance the number of lung epithelial cells in a subject and/or increase the amount of lung epithelial tissue in a subject and/or to improve lung function in a subject compared to the same feature in the subject prior to administration and/or compared to a subject to which the cells have not been administered. With regard to lung function, this may be measured, for example, by spirometry (e.g., the test may measure Forced vital capacity (FVC), Forced expiratory volume (FEV), Forced expiratory flow 25% to 75%., Peak expiratory flow (PEF), Maximum voluntary ventilation (MVV), Slow vital capacity (SVC), total lung capacity (TLC)functional residual capacity (FRC). expiratory reserve volume (ERV)) or may be a gas diffusion test or may be an exercise stress test. The skilled artisan will be aware of suitable methods for determining lung function. An effective amount of lung epithelial progenitor cells and/or progeny thereof and/or cells differentiated therefrom need not necessarily provide a therapeutically or prophylactically beneficial effect. The skilled artisan will be aware that such an amount will vary depending on, for example, the lung epithelial progenitor cells and/or progeny thereof and/or cells differentiated therefrom and/or the particular subject and/or the type or severity of lung dysfunction (if present). Accordingly, this term is not to be construed to limit the invention to a specific quantity, e.g., weight or number of cells, rather the present invention encompasses any amount of the lung epithelial progenitor cells and/or progeny thereof and/or cells differentiated therefrom sufficient to increase or enhance the number of lung epithelial cells in a subject and/or increase the amount of lung epithelial tissue in a subject and/or to improve lung function in a subject.

As used herein, the term "therapeutically effective amount" shall be taken to mean a sufficient quantity of lung epithelial progenitor cells and/or progeny thereof and/or cells differentiated therefrom to reduce or inhibit one or more symptoms of a lung disease or symptoms caused by damaged or missing or non- functional lung tissue. For example, a therapeutically effective amount of sufficient quantity of lung epithelial progenitor cells and/or progeny thereof and/or cells differentiated therefrom may improve lung function to a level observed in a normal or health subject or population thereof.

As used herein, the term "prophylactically effective amount" shall be taken to mean a sufficient quantity of lung epithelial progenitor cells and/or progeny thereof and/or cells differentiated therefrom to prevent or inhibit or delay the onset of one or more detectable symptoms of lung disease or symptoms caused by damaged or missing or non- functional lung tissue. For example, a prophylactically effective amount of lung epithelial progenitor cells and/or progeny thereof and/or cells differentiated therefrom prevent or reduce or delay a decrease in lung function of a subject below that observed in a normal or healthy subject or population thereof.

As used herein, the term "EpCam" or "Epithelial Cell Adhesion Molecule" or CD326 or "CO17-1A" or "TROP 1" or EGP or EGP40 or "GA733 2" or KSA or Ly74 or M1S2 or M4S1 or MIC 18 or "MK 1" or "hEGP 2" or "Adenocarcinoma associated antigen" or "KS 1/4" or "tumor-associated calcium signal transducer 1 "shall be taken to mean, for example, a protein recognized by monoclonal antibody KS 1/4 (Varki et al, Cancer Res., 44: 681-685, 1985) and/or a protein described in described in Strnad et al, Cancer Res. 49:314-311, 1989 or a homolog or paralog thereof. For the purposes of nomenclature and not limitation a sequence of Homo sapiens EpCam is set forth in SEQ ID NO: 1 and the sequence of Mus musculus EpCam is set forth in SEQ ID NO: 2.

As used herein, the term "differentiate" shall be taken to mean the process by which an immature cell becomes a specialized adult cell, for example, a lung epithelial progenitor cell becomes a specialized Clara cell.

As used herein, the term "enriched" in the context of a cell population shall be taken to mean that the number or percentage of lung epithelial progenitor cells is greater than the number or percentage in a naturally occurring cell population. For example, a population enriched in lung epithelial progenitor cells and/or progenitors is made up of at least about 0.02% of said cells, or at least about 0.05% of said cells or at least about 0.1% of said cells or at least about 0.2% of said cells or at least about 0.5% of said cells or at least about 0.5% of said cells or at least about 0.8% of said cells or at least about 1% of said cells or at least about 2% of said cells or at least about 3% of said cells or at least about 4% of said cells or at least about 5% of said cells or at least about 10% of said cells or at least about 15% of said cells or at least about 20% of said cells or at least about 25% of said cells or at least about 30% of said cells or at least about 40% of said cells or at least about 50% of said cells or at least about 60% of said cells or at least about 70% of said cells or at least about 80% of said cells or at least about 85% of said cells or at least about 90% of said cells or at least about 95% of said cells or at least about 97% of said cells or at least about 98% of said cells or at least about 99% of said cells.

As used herein, the term "healthy individual" or "healthy subject" shall be taken to mean an individual or subject that does not suffer from a lung disease and is not developing a lung disease. Preferably, a healthy individual or subject does not smoke and/or is not regularly exposed to inhalable toxins or agents that cause lung damage.

By "individually" is meant that the invention encompasses the recited cell or groups of cells separately, and that, notwithstanding that individual cells or groups of cells may not be separately listed herein the accompanying claims may define such cell or groups of cells separately and divisibly from each other. The term "lung disease" shall be taken to include any disease or condition that reduces lung function in a subject and includes, for example, asthma, chronic bronchitis, emphysema, and cystic fibrosis, respiratory failure, pulmonary oedema, pulmonary embolism, and pulmonary hypertension (high blood pressure), pneumonia and tuberculosis (TB), lung cancer, stiffening and scarring of lungs (e.g., caused by caused by drugs, poisons, infections, or radiation), lung disorders from unusual atmospheric pressure (e.g., caused by a mechanical ventilator). Preferably, the lung condition is a chronic lung condition and/or a lung condition associated with inflammation in the lung, e.g., the lung condition is COPD or cystic fibrosis or pulmonary fibrosis or bronchiolitis or alveolitis or vasculitis or sarcoidosis.

The term "lung epithelial progenitor cell" shall be taken to mean a progenitor cells capable of differentiating into one or more distinct cell lineages found in lung epithelium in nature. For example, a lung epithelial progenitor cell may be unipotent and capable of differentiating into only one cell lineage of lung epithelium. Preferably, a lung epithelial progenitor cell is multipotent, or capable of differentiating into a plurality of distinct cell lineages of lung epithelium. In one example, a lung epithelial progenitor cell is also capable of self renewal and, as a consequence, the term "lung epithelial progenitor cell" shall be taken to include a lung epithelial stem cell. This progenitor cell may be found either in the lung or outside the lung, e.g., in isolated form or any other tissue or ex vivo.

Reference to "mammal" or "mammalian" should be understood to include reference to a mammal such as but not limited to human, primate, livestock (animal (e.g. sheep, cow, horse, donkey, pig), companion animal (e.g. dog, cat), laboratory test animal (e.g. mouse, rabbit, rat, guinea pig, hamster), captive wild animal (e.g. fox, deer). Preferably the mammal is a human or primate. Most preferably the mammal is a human. Reference to a "mutant or variant" of the subject cellular population should be understood as a reference to a cell which is derived from the cellular population but exhibits at least one difference at the phenotypic or functional level. For example, the mutant or variant may have altered the expression of its cell surface markers or some aspect of its functionality subsequently to initial isolation. Such changes can occur either spontaneously (as exemplified by the spontaneous upregulation or downregulation of cell surface markers which can occur subsequently to in vitro culture or spontaneous transformation) or as a result of a directed manipulation, such as would occur if a cell was deliberately transformed (for example, in order to effect the creation of a cell line) or transfected (for example to effect the expression of a particular gene or marker).

As used herein, the term "normal individual" or "normal subject" shall be taken to mean an individual or subject that does not have reduced lung function compared to a population of subjects not suffering from lung disease.

Reference to "phenotypic profile" should be understood as a reference to the presence or absence or level of the transcription of the genes encoding the subject markers and/or the cell surface expression of the expression product translated therefrom. It should be appreciated that although most cells falling within the scope of the claimed lung epithelial cellular populations will be characterized by the presence or absence or level of the subject marker as a cell surface anchored expression product, some cells falling within the defined populations may exhibit changes only at the transcriptome level, such as when the transcription of a given marker has been upregulated but may not yet have resulted in a cell surface anchored expression product. In general, cells which progress to a new differentiative stage will transiently exhibit gene expression changes which are not yet evident in the context of changes to levels of an expression product. However, these cells nevertheless fall within the scope of the claimed cellular populations. It should also be appreciated that although the lung epithelial cell populations of the present invention are characterized by the defined phenotypic profiles, these cells will express a range of other intracellular and/or cell surface markers which are not necessarily relevant in terms of phenotypically characterizing the cellular population of interest. Still further, to the extent that a given lung epithelial cellular population of the present invention comprises a range of subpopulations, these subpopulations may exhibit variations in the expression of intracellular or cell surface markers other than those of the profiles defined herein.

As used herein, the terms "preventing", "prevent" or "prevention" include administering a therapeutically effective amount of cell(s) described herein sufficient to stop or hinder the development of at least one symptom of the specified disease or condition.

By "progenitor cell" is meant that a cell that is not fully differentiated but requires further differentiation to achieve maturation. Such cells are often also sometimes referred to as "precursor" cells, "multipotent" cells, or "stem" cells. The subject progenitor cell may be one which exhibits multipotentiality, for example is a progenitor which can be induced to differentiate down either the proximal or distal lung pathways. However, despite this initial level of commitment, the subject cell is nevertheless still a "progenitor" on the basis that it is not fully differentiated. The use of the term "progenitor" should not be understood as a limitation on the maturity/immaturity of the subject cell relative to that which might be implied by the use of the terms "stem cell", "multipotent cell", "pluripotent cell" or other such term.

As used herein, the terms "treating", "treat" or "treatment" include administering a therapeutically effective amount of cell(s) described herein sufficient to reduce or eliminate at least one symptom of the specified disease or condition. By "non-tumorigenic" or "non-malignant" shall be taken to mean that a cell will not cause a tumor or other form of cancer if administered to a subject. Methods for determining tumorigenicity of a cell will be apparent to the skilled artisan and/or described herein.

As used herein, the term "undifferentiated state" shall be taken to mean that a lung epithelial progenitor cell has the ability to differentiate into a lung epithelial cell, e.g., an epithelial cell of the proximal lung and/or an epithelial cell of the distal lung.

Reference to a "high" or "hi" level of expression, e.g., in respect of EpCam expression includes the 5% of cells, preferably 4%, 3% or more preferably 2% of cells expressing the highest level of the recited marker in a population of cells, e.g., as determined using FACS analysis. For example, high expression of EpCam refers to the highest level of EpCam expression in up to 5%, 4%, 4% or preferably, 2% of CD45^" CD31^" cells. In one example, the cells are resolved as a cluster of CD45^" CD31^" lung cells with the highest expression of EpCam in a bivariate plot of EpCam versus CD49f and/or CD 104 where these cells are also CD49f and CD104⁺. Reference to a "low" or "lo" level of expression, e.g., in the context of CD24 expression shall be taken to mean the 60% or 55% or 50% or less of cells with lowest CD24 expression in a heterogeneous population of CD24^pos cells. The skilled artisan will appreciate that if the population of cells is enriched for CD24^lc, the percentage of cells may be 99% or 95% or 90% or 85% or 80% or 75% or 70% or 60% or 55% or 50% or less of cells with lowest CD24 expression.

As used herein, the term "positive expression" or "+" shall be taken to mean expression above the level of background, e.g., as detected using an isotype control compound, e.g., antibody.

As used herein, the term "negative expression" or "-"shall be taken to mean expression equal to or less than the level of background expression, e.g., as detected using an isotype control compound, e.g., antibody.

As used herein, the term "isotype control compound" shall be taken to mean an compound, preferably an antibody of the same isotype as that used to detect expression of a protein, however having no relevant specificity to a protein and conjugated to the same detectable moiety as the compound used to detect expression of the protein. Such a control aids in distinguishing non-specific "background" staining from specific staining

Cell Surface Markers Cell surface markers suitable for detecting a lung epithelial progenitor cell of the present invention are described herein as the exemplary sequences of those markers. Preferred markers have at least about 70% identity to the exemplary sequences listed hereinabove, preferably at least about 75% identity or at least about 80% identity or at least about 85% identity or at least about 90% identity or at least about 95% identity or at least about 96% or 97% or 98% or 99% identity. In determining whether or not two amino acid sequences fall within the defined percentage identity limits supra, those skilled in the art will be aware that it is possible to conduct a side -by-side comparison of the amino acid sequences. In such comparisons or alignments, differences will arise in the positioning of non-identical residues depending upon the algorithm used to perform the alignment. In the present context, references to percentage identities and similarities between two or more amino acid sequences shall be taken to refer to the number of identical and similar residues respectively, between said sequences as determined using any standard algorithm known to those skilled in the art. In particular, amino acid identities and similarities are calculated using software of the Computer Genetics Group, Inc., University Research Park, Madison, Wisconsin, United States of America, e.g., using the GAP program of Devereaux et al, Nucl. Acids Res. 12, 387-395, 1984, which utilizes the algorithm of Needleman and Wunsch, J. MoI. Biol. 48, 443-453, 1970. Alternatively, the CLUSTAL W algorithm of Thompson et al, Nucl. Acids Res. 22, 4673-4680, 1994, is used to obtain an alignment of multiple sequences, wherein it is necessary or desirable to maximize the number of identical/similar residues and to minimize the number and/or length of sequence gaps in the alignment. Amino acid sequence alignments can also be performed using a variety of other commercially available sequence analysis programs, such as, for example, the BLAST program available form the National Centre for Biotechnology Information (NCBI). Preferably, the cell surface marker is a human protein or a mouse protein, more preferably a human protein.

Amino acid sequence mutants of the polypeptides described herein are clearly contemplated, as are isoforms thereof having an amino acid sequence differing from that explicitly taught herein, provided that said mutant or isoform is expressed on a lung epithelial progenitor cell. Mutants include, for example, those having deletions, insertions or substitutions of residues within the amino acid sequence. A combination of deletion, insertion and substitution can also be present.

Compounds that Bind Cell Surface Markers The skilled artisan will appreciate from the description herein, that some embodiments of the invention utilize a compound that binds to a cell surface marker or protein as described herein in any embodiment. Such a compound may be used to detect or isolate a cell expressing a particular protein or a cell that does not express detectable levels or expresses low levels of a particular protein. Exemplary compounds include antibodies, peptides and small molecules.

Antibodies

In one embodiment, the compound that binds to a protein or cell surface marker as described in any embodiment herein is an antibody. As used herein, the term "antibody" refers to intact monoclonal or polyclonal antibodies, immunoglobulin (IgA, IgD, IgG, IgM, IgE) fractions, humanized antibodies, or recombinant single chain antibodies, as well as fragments thereof, such as, for example Fab, F(ab)2, and Fv fragments.

The skilled artisan will be aware of suitable commercial sources of antibodies for use in a method as described herein according to any embodiment. For example, anti-EpCam monoclonal and/or polyclonal antibodies are available from Santa Cruz Biotechnology (USA), Abeam Ltd (Cambridge, UK), Acris Antibodies GmbH (Germany) or BD Biosciences, Inc. (USA). Monoclonal and/or polyclonal antibodies against CDl 04 are commercially available, for example, from Abeam Ltd (Cambridge, UK), Acris Antibodies GmbH (Germany) or BD Biosciences, Inc. (USA). Monoclonal and/or polyclonal antibodies against CD49f are commercially available, for example, from Abeam Ltd (Cambridge, UK), Abnova Corporation (USA), Antigenix America Inc. (USA) or StemCell Technologies, Inc. (USA). Monoclonal and/or polyclonal antibodies against CD24 are commercially available, for example, from Abeam Ltd (Cambridge, UK), Acris Antibodies GmbH (Germany) or BD Biosciences, Inc. (USA). Monoclonal and/or polyclonal antibodies against CD45 are commercially available, for example, from Abeam Ltd (Cambridge, UK), ABR-Affϊnity Bioreagents Inc (USA) or BD Biosciences, Inc. (USA). Monoclonal and/or polyclonal antibodies against CD31 are commercially available, for example, from Abeam Ltd (Cambridge, UK), Abnova Corporation (USA), Acris Antibodies GmbH (Germany) or BD Biosciences, Inc. (USA).

Alternative, or in addition, antibodies are prepared by any of a variety of techniques known to those of ordinary skill in the art, and described, for example in, Harlow and Lane {In: Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988). In one such technique, an immunogen comprising the antigenic polypeptide (e.g., as set forth in any one of SEQ ID NOs: 1-12 or an immunogenic fragment thereof) or a cell expressing same is initially injected into any one of a wide variety of animals (e.g., mice, rats, rabbits, sheep, humans, dogs, pigs, chickens and goats). The immunogen is derived from a natural source, produced by recombinant expression means, or artificially generated, such as by chemical synthesis (e.g., BOC chemistry or FMOC chemistry). A peptide, polypeptide or protein immunogen may be joined to a carrier protein, such as bovine serum albumin or keyhole limpet hemocyanin. The immunogen and optionally a carrier for the protein is injected into the animal host, preferably according to a predetermined schedule incorporating one or more booster immunizations, and blood collected from said the animals periodically. Optionally, the immunogen is injected in the presence of an adjuvant, such as, for example Freund's complete or incomplete adjuvant, lysolecithin and dinitrophenol to enhance the subject's immune response to the immunogen. Monoclonal or polyclonal antibodies specific for the polypeptide are then purified from blood isolated from an animal by, for example, affinity chromatography using the immunogen coupled to a suitable solid support. Monoclonal antibodies specific for the antigenic polypeptide of interest are prepared, for example, using the technique of Kohler and Milstein, Eur. J. Immunol. 5:511-519, 1976, and improvements thereto. Briefly, these methods involve the preparation of immortal cell lines capable of producing antibodies having the desired specificity (i.e., reactivity with the polypeptide of interest). Such cell lines are produced, for example, from spleen cells obtained from an animal immunized as described supra. The spleen cells are immortalized by, for example, fusion with a myeloma cell fusion partner, preferably one that is syngenic with the immunized animal. A variety of fusion techniques are known in the art, for example, the spleen cells and myeloma cells are combined with a nonionic detergent or electrofused and then grown in a selective medium that supports the growth of hybrid cells, but not myeloma cells. A preferred selection technique uses HAT (hypoxanthine, aminopterin, and thymidine) selection. After a sufficient time, usually about 1 to 2 weeks, colonies of hybrids are observed. Single colonies are selected and growth media in which the cells have been grown is tested for the presence of an antibody having binding activity against the polypeptide (immunogen). Hybridomas having high reactivity and specificity are preferred.

Monoclonal antibodies are isolated from the supernatants of growing hybridoma colonies using methods such as, for example, affinity purification as described supra.

Various techniques are also known for enhancing antibody yield, such as injection of the hybridoma cell line into the peritoneal cavity of a suitable vertebrate host, such as a mouse. Monoclonal antibodies are then harvested from the ascites fluid or the blood of such an animal subject. Contaminants are removed from the antibodies by conventional techniques, such as chromatography, gel filtration, precipitation, and/or extraction.

It is preferable that an immunogen used in the production of an antibody is one which is sufficiently antigenic to stimulate the production of antibodies that will bind to the immunogen and is preferably, a high titer antibody. In one embodiment, an immunogen is an entire protein. In another embodiment, an immunogen consists of a peptide representing a fragment of a polypeptide.

Preferably an antibody raised to such an immunogen also recognizes the full- length protein from which the immunogen was derived, such as, for example, in its native state or having native conformation.

Alternatively, or in addition, an antibody raised against a peptide immunogen recognizes the full-length protein from which the immunogen was derived when the protein is denatured. By "denatured" is meant that conformational epitopes of the protein are disrupted under conditions that retain linear B cell epitopes of the protein.

As will be known to a skilled artisan linear epitopes and conformational epitopes may overlap.

Alternatively, a monoclonal antibody capable of binding to a protein or cell surface marker as described herein in any embodiment is produced using a method such as, for example, a human B-cell hybridoma technique (Kozbar et al, Immunol Today

4:72, 1983), a EBV -hybridoma technique to produce human monoclonal antibodies

(Cole et al. Monoclonal Antibodies in Cancer Therapy, 1985 Allen R. Bliss, Inc., pages

77-96), or screening of combinatorial antibody libraries (Huse et al, Science 246:1215,

1989). Such an antibody is then particularly useful in detecting the presence of a protein or cell surface marker, and/or isolating a cell as described herein according to any embodiment.

Peptides In another embodiment, the compound that binds to a cell surface marker or protein as described herein according to any embodiment is a peptide. For example, the peptide is derived from a ligand of a cell surface marker or protein as described herein according to any embodiment (e.g., from a ligand binding region of the protein or marker). For example, a ligand for EpCam is an ectodomain of EpCam, designated EpEX. A ligand for heterodimerized CD 104 is laminin 332. Ligands for heterodimerized CD49f include laminin and Kalinin. Ligands for CD24 include P- selectin. Ligands for CD31 include CD38.

Alternatively, a ligand is a peptide isolated from a random peptide library. To identify a suitable ligand, a random peptide library is generated and screened as described in U.S. Patent Application No. 5,733,731, 5,591,646 and 5,834,318. Generally, such libraries are generated from short random oligonucleotides that are expressed either in vitro or in vivo and displayed in such a way to facilitate screening of the library to identify a peptide that, is capable of specifically binding to a protein or peptide of interest. Methods of display include, phage display, retroviral display, bacterial surface display, bacterial flagellar display, bacterial spore display, yeast surface display, mammalian surface display, and methods of in vitro display including, mRNA display, ribosome display and covalent display.

A peptide that is capable of binding a protein or peptide of interest is identified by a number of methods known in the art, such as, for example, standard affinity purification methods as described, for example in Scopes (In: Protein purification: principles and practice, Third Edition, Springer Verlag, 1994) purification using FACS analysis as described in US Patent No 6,455,63, or purification using biosensor technology as described in Gilligan et al, Anal Chem, 74(9): 2041 - 2047, 2002.

Other compounds

A chemical small molecule library is also clearly contemplated for the identification of ligands that specifically bind to a protein or cell surface marker as described herein according to any embodiment. Chemical small molecule libraries are available commercially or alternatively may be generated using methods known in the art, such as, for example, those described in U.S. Patent No. 5,463,564.

Labeled compounds

In one embodiment, the compound that binds to a protein or cell surface marker as described herein according to any embodiment comprises one or more detectable markers to facilitate detection and/or isolation. For example, the probe or primer comprises a fluorescent label such as, for example, fluorescein (FITC), 5,6- carboxymethyl fluorescein, Texas red, nitrobenz-2-oxa-l,3- diazol-4-yl (NBD), coumarin, dansyl chloride, rhodamine, 4'-6-diamidino-2- phenylinodole (DAPI), and the cyanine dyes Cy3, Cy3.5, Cy5, Cy5.5 and Cy7, fluorescein (5-carboxyfluorescein- N-hydroxysuccinimide ester), rhodamine (5,6- tetramethyl rhodamine). The absorption and emission maxima, respectively, for these fluors are: FITC (490 nm; 520 nm), Cy3 (554 nm; 568 nm), Cy3.5 (581 nm; 588 nm), Cy5 (652 nm: 672 nm), Cy5.5 (682 nm; 703 nm) and Cy7 (755 nm; 778 nm).

Alternatively, or in addition, the compound that binds to a protein or cell surface marker as described herein according to any embodiment is labeled with, for example, a fluorescent semiconductor nanocrystal (as described, for example, in US 6,306,610).

Alternatively, or in addition, the compound that binds to a protein or cell surface marker as described herein according to any embodiment is labelled with, for example, a magnetic or paramagnetic compound, such as, iron, steel, nickel, cobalt, rare earth materials, neodymium-iron-boron, ferrous-chromium-cobalt, nickel-ferrous, cobalt- platinum, or strontium ferrite.

Isolation or Enrichment of Cells

One exemplary approach to enrich for the desired cells is magnetic bead cell sorting (MACS) or any other cell sorting method making use of magnetism, e..g, Dynabeads®. The conventional MACS procedure is described by Miltenyi et al. {Cytometry 77:231-238, 1990). In this procedure, cells are labelled with magnetic beads bound to an antibody or other compound that binds to a cell surface marker or protein and the cells are passed through a paramagnetic separation column or exposed to another form of magnetic field. The separation column is placed in a strong magnet, thereby creating a magnetic field within the column. Cells that are magnetically labeled are trapped in the column; cells that are not pass through. The trapped cells are then eluted from the column.

Cells of the invention can be enriched, for example, from a suitable bodily reservoir, such as lung or a part thereof or fluid therefrom, using MACS to separate cells expressing a suitable protein. The sample is incubated with immunomagnetic beads that bind to the protein. Following incubation, samples are washed and resuspended and passed through a magnetic field to remove cells bound to the immunomagnetic beads, a nd cells bound to the beads collected. These techniques are equally applicable to negative selection, e.g., removal of cells expressing an undesirable marker, e.g., CD45 and/or CD31, i.e., undesirable cells. Such a method involves contacting a population of cells with a magnetic particle labelled with a compound that binds to a cell surface marker expressed at detectable levels on the undesirable cell type(s). Following incubation, samples are washed and resuspended and passed through a magnetic field to remove cells bound to the immunomagnetic beads. The remaining cells depleted of the undesirable cell type(s) are then collected. In another embodiment, a compound that binds to a protein or cell surface marker is immobilized on a solid surface and a population of cells is contacted thereto. Following washing to remove unbound cells, cells bound to the compound can be recovered, e.g., eluted, thereby isolating or enriching for cells expressing the protein to which the compound binds. Alternatively, cells that do not bind to the compound can be recovered if desired.

In a preferred embodiment, cells are isolated or enriched using fluorescence activated cell sorting (FACS). FACS is a known method for separating particles, including cells, based on the fluorescent properties of the particles and described, for example, in Kamarch, Methods Enzymol, 757:150-165, 1987). Generally, this method involves contacting a population of cells with compounds capable of binding to one or more proteins or cell surface markers, wherein compounds that bind to distinct markers are labelled with different fluorescent moieties, e.g., fluorophores. The cells are entrained in the center of a narrow, rapidly flowing stream of liquid. The flow is arranged so that there is a separation between cells relative to their diameter. A vibrating mechanism causes the stream of cells to break into individual droplets. The system is adjusted so that there is a low probability of more than one cell being in a droplet. Just before the stream breaks into droplets the flow passes through a fluorescence measuring station where the fluorescent character of interest of each cell is measured, e.g., whether or not a labelled compound is bound thereto. An electrical charging ring is placed at the point where the stream breaks into droplets. A charge is placed on the ring based on the immediately prior fluorescence intensity measurement and the opposite charge is trapped on the droplet as it breaks from the stream. The charged droplets then fall through an electrostatic deflection system that diverts droplets into containers based upon their charge, e.g., into one container if a labelled compound is bound to the cell and another container if not. In some systems the charge is applied directly to the stream and the droplet breaking off retains charge of the same sign as the stream. The stream is then returned to neutral after the droplet separates. Samples

To the extent that the method of the present invention is performed in vitro, on an isolated tissue sample, rather than as an in vivo based screen, reference to "sample" or "biological sample" or "tissue sample" (these terms being used interchangeably) should be understood as a reference to any sample of biological material derived from an animal such as, but not limited to, cellular material (e.g. tissue aspirate), tissue biopsy specimens (e.g. lung), surgical specimens or biological fluids (e.g. BALF). The biological sample which is used according to the method of the present invention may be used directly or may require some form of treatment prior to use. For example, a biopsy or surgical sample may require homogenisation or other form of cellular dispersion prior to use. Furthermore, to the extent that the biological sample is not in liquid form, (if such form is required or desirable) it may require the addition of a reagent, such as a buffer, to mobilise the sample.

As hereinbefore described the sample (which includes reference to "cells") may have been freshly isolated from a subject or it may have been sourced from a non-fresh source, such as from a culture (for example, where cell numbers were expanded and/or the cells were cultured) or a frozen stock of cells, which had been isolated at some earlier time point either from a subject or from another source.

Cell Culture

Cells of the invention can be maintained under standard cell culture conditions. For example, the cells can be maintained in Dulbecco Minimal Essential Medium (DMEM) or any other appropriate cell culture medium. To maintain cells in a substantially undifferentiated state cells are preferably cultured in the presence of FGFlO (for example, 5-lOOng/ml, preferably 20-80ng/ml, more preferably 50ng/ml) and/or HGF (for example, 5-50ng/ml, preferably, 20-40ng/ml, preferably 30ng/ml). Other growth factors may also be included in the culture medium, e.g., FGF-7, BMP-4, PDGF-AA (e.g., about 50ng/ml; Millipore, Billerica, MA).

Cell cultures are preferably incubated at 37°C in a humidified incubator. Cell culture conditions can vary considerably for the cells of the present invention, however, in exemplified embodiments, the cells are maintained in an environment suitable for cell growth, e.g., comprising 5% O₂, 10% CO₂, 85% N₂ or comprising 10% CO₂ in air.

In another embodiment, cells are cultured on or within a matrix, e.g., an extracellular matrix, e.g., Matrigel™, laminin, collagen, Culturex®, etc. In other embodiments, the cells may be cultured in the presence of an extracellular matrix. Suitable procedures for proliferating cells in the presence of such matrices are described, for example, in U.S. Patent No. 7,297,539.

Other appropriate media include, for example, MCDB, Minimal Essential

Medium (MEM), IMDM, and RPMI. Cells of the invention can be maintained according to culture methods known in the art to enhance proliferation. Cells of the invention can be co-cultured with other cells, e.g., stromal cells, e.g., lung stromal cells or bone marrow stromal cells, e.g., expressing low levels of EpCam and/or expressing CD45.

Lung epithelial progenitor cells and progeny thereof of the present invention can also be differentiated into various lung epithelial cells. For example, culturing in the presence of FGFlO (in the absence of HGF) s useful for inducing differentiation into a proximal cell type, e.g., goblet cells, basal cells and pulmonary endocrine cells. Such cells are identified, for example, by detecting expression of MUC-I and/or MUC5AC.

Culturing the cells in the presence of BMP4 is useful for inducing differentiation into a distal lung cell, e.g., alveolar type I pneumocytes and/or alveolar type II pneumocytes.

Such cells are identified, for example, by detecting expression of SP-C. Culturing the cells in the presence of retinoids is also useful for inducing differentiation into type II pneumocytes.

Models of Lung Disease

To assess the ability of cells of the present invention to treat or prevent a lung disease, cells are preferably administered to an animal model of lung disease, e.g., for pre-clinical analysis. Several models of lung disease are known in the art and reviewed, for example, in Puchelle and Peault, Respir Res. 1: 125-128, 2000. For example, rat tracheas are denuded of their surface epithelium by repeated cycles of freezing and thawing, then seeded with cells of the present invention and implanted subacutanenously into immunodeficient nude mice (Shimizu et al, Am J Respir Cell MoI Biol. 7:30-41, 1992). The types of epithelial cells generated can then be determined using standard methods in the art. Similar methods have also been described using human lung cells in SCID mice, e.g., Delplanque, et al, J Cell Sci. 113:161-11%, 2000.

Administration of polidocanol and sulphur dioxide is useful for producing a model of tracheal and proximal lung injury (Borthwick et al, Am. J. Respir. Cell MoI Biol, 24: 662-670, 2001). Administration of cells of the invention following such treatment determines those cells capable of treating tracheal and/or proximal lung injury. Bleomycin administration results in ATI cell injury, while naphthalene administration results in Clara cell injury (Aso et ah, Lab. Invest., 35: 558-568, 1976; and Giangreco et ah, Am. J. Pathol, 161: 173-182, 2002). Accordingly, these models are useful for determining cells useful for the treatment of ATI cell injury and/or Clara cell injury.

Cellular Compositions

In one embodiment of the present invention cells of the present invention are administered in the form of a composition. Preferably, such a composition comprises a pharmaceutically acceptable carrier and/or excipient.

The terms "carrier" and "excipient" refer to compositions of matter that are conventionally used in the art to facilitate the storage, administration, and/or the biological activity of an active compound (see, e.g., Remington's Pharmaceutical Sciences, 16th Ed., Mac Publishing Company (1980). A carrier may also reduce any undesirable side effects of the active compound. A suitable carrier is, for example, stable, e.g., incapable of reacting with other ingredients in the carrier. In one example, the carrier does not produce significant local or systemic adverse effect in recipients at the dosages and concentrations employed for treatment.

Suitable carriers for this invention include those conventionally used, e.g., water, saline, aqueous dextrose, lactose, Ringer's solution, a buffered solution, hyaluronan and glycols are preferred liquid carriers, particularly (when isotonic) for solutions. Suitable pharmaceutical carriers and excipients include starch, cellulose, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, glycerol, propylene glycol, water, ethanol, and the like.

In another example, a carrier is a media composition, e.g., in which a cell is grown or suspended. Preferably, such a media composition does not induce any adverse effects in a subject to whom it is administered.

Preferred carriers and excipients do not adversely affect the viability of a cell and/or the ability of a cell to reduce, prevent or delay lung disease.

In one example, the carrier or excipient provides a buffering activity to maintain the cells and/or soluble factors at a suitable pH to thereby exert a biological activity, e.g., the carrier or excipient is phosphate buffered saline (PBS). PBS represents an attractive carrier or excipient because it interacts with cells and factors minimally and permits rapid release of the cells and factors, in such a case, the composition of the invention may be produced as a liquid for direct application to the blood stream or into a tissue or a region surrounding or adjacent to a tissue, e.g., by injection.

Cells and/or progeny cells thereof can also be incorporated or embedded within scaffolds that are recipient-compatible and which degrade into products that are not harmful to the recipient. These scaffolds provide support and protection for cells that are to be transplanted into the recipient subjects. Natural and/or synthetic biodegradable scaffolds are examples of such scaffolds.

A variety of different scaffolds may be used successfully in the practice of the invention. Preferred scaffolds include, but are not limited to biological, degradable scaffolds. Natural biodegradable scaffolds include collagen, fibronectin, and laminin scaffolds. Suitable synthetic material for a cell transplantation scaffold should be able to support extensive cell growth and cell function. Such scaffolds may also be resorbable. Suitable scaffolds include polyglycolic acid scaffolds, e.g., as described by Vacanti, et al. J. Ped. Surg. 23:3-9 1988; Cima, et al. Biotechnol Bioeng. 38:145 1991; Vacanti, et al. Plast. Reconstr. Surg. 88:753-9 1991; or synthetic polymers such as polyanhydrides, polyorthoesters, and polylactic acid.

In another example, the cells may be administered in a gel scaffold (such as Gelfoam from Upjohn Company.

The cellular compositions useful for the present invention may be administered alone or as admixtures with other cells. Cells that may be administered in conjunction with the compositions of the present invention include, but are not limited to, other multipotent or pluripotent cells or stem cells, or bone marrow cells. The cells of different types may be admixed with a composition of the invention immediately or shortly prior to administration, or they may be co-cultured together for a period of time prior to administration.

Preferably, the composition comprises an effective amount or a therapeutically or prophylactically effective amount of cells. For example, the composition comprises about 1x10⁵ cells/kg to about 1x10⁷ cells/kg or about 1x10⁶ cells/kg to about 5x10⁶ cells/kg. The exact amount of cells to be administered is dependent upon a variety of factors, including the age, weight, and sex of the patient, and the extent and severity of the lung dysfunction.

In some embodiments of the invention, it may not be necessary or desirable to immunosuppress a patient prior to initiation of therapy with cellular compositions. Accordingly, transplantation with allogeneic, or even xenogeneic cells or progeny thereof may be tolerated in some instances. However, in other instances it may be desirable or appropriate to pharmacologically immunosuppress a patient prior to initiating cell therapy. This may be accomplished through the use of systemic or local immunosuppressive agents, or it may be accomplished by delivering the cells in an encapsulated device. The cells may be encapsulated in a capsule that is permeable to nutrients and oxygen required by the cell and therapeutic factors the cell is yet impermeable to immune humoral factors and cells. Preferably the encapsulant is hypoallergenic, is easily and stably situated in a target tissue, and provides added protection to the implanted structure. These and other means for reducing or eliminating an immune response to the transplanted cells are known in the art. As an alternative, the cells may be genetically modified to reduce their immunogenicity.

In one embodiment, a cell as described herein according to any embodiment, is administered with one or more of the drugs presently used to alleviate and/or prevent symptoms of a lung disease or condition (e.g., a bronchodilator or antiinflammatory corticosteroid). Alternatively, the cells are administered with an agent which causes a reduction in the expression or activity of one or more of the pro-inflammatory cytokines IL-I, IL-2, IL- 6, IFN-γ and TNF-α (e.g., a specific inhibitor or antagonist agent or specific antibody), and/or the expression or activity of the pro-fibrotic cytokine TGF-β (e.g., a specific TGF-β inhibitor or antagonist agent or specific anti-TGF-β antibody). The cells may also be administered with synthetic or animal-derived lung surfactant (e.g., colfosceril palmitate-based products (such as Exosurf® marketed by GlaxoSmithKline pic, Brentford, Middlesex, United Kingdom) and surfactant extracted from bovine lung lavage fluid (such as Alveofact® marketed by Boehringer Ingelheim GmbH, Ingelheim, Germany)), to open unperfused areas of the lung to thereby aid access of the AECs to sites of injury.

Preferably, a cell or composition of the present invention is administered directly into or near a lung or the lungs of a subject in need thereof, preferably in or near a site of injury. Alternatively, or in addition, the cells are administered by way of systemic transfusion, bronchoscopic or intra-nasal instillation.

Methods of Screening

The skilled artisan will be aware from the foregoing that the present invention encompasses various methods for identifying and/or isolating compounds using a cell as described herein according to any embodiment. Suitable compounds for screening include, for example, antibodies, peptides or small molecules, e.g., as described herein according to any embodiment. This invention also provides for the provision of information concerning the identified or isolated compound. Accordingly, the screening methods are further modified by:

(i) optionally, determining the structure of the compound; and (ii) providing the compound or the name or structure of the compound such as, for example, in a paper form, machine-readable form, or computer-readable form.

Naturally, for compounds that are known albeit not previously tested for their function using a screen provided by the present invention, determination of the structure of the compound is implicit. This is because the skilled artisan will be aware of the name and/or structure of the compound at the time of performing the screen.

As used herein, the term "providing the compound" shall be taken to include any chemical or recombinant synthetic means for producing said compound or alternatively, the provision of a compound that has been previously synthesized by any person or means. This clearly includes isolating the compound. In a preferred embodiment, the compound or the name or structure of the compound is provided with an indication as to its use e.g., as determined by a screen described herein.

The screening assays can be further modified by: (i) optionally, determining the structure of the compound; (ii) optionally, providing the name or structure of the compound such as, for example, in a paper form, machine-readable form, or computer-readable form; and (iii) providing the compound.

In a preferred embodiment, the synthesized compound or the name or structure of the compound is provided with an indication as to its use e.g., as determined by a screen described herein.

In one embodiment, the compound is provided in a library of compounds, each of which or a subset of which may be separated from other members (i.e., physically isolated). In such cases, a compound is isolated from the library by its identification, which then permits a skilled person to produce that compound in isolation, e.g., in the absence of other members of the library.

As will be apparent to the skilled artisan, methods of screening may involve detecting levels of cell death, cell proliferation and/or cell survival. Such methods are known in the art.

In one embodiment, cell death is assayed, e.g., using a method for the detection of cellular components associated with cell death, such as, for example apoptosis.

Methods for detecting cell death in a cell are known in the art. For example, APOPTEST (available from Immunotech) stains cells early in apoptosis, and does not require fixation of the cell sample (Martin et ah, 1994). This method utilizes an annexin V antibody to detect cell membrane re-configuration that is characteristic of cells undergoing apoptosis. Apoptotic cells stained in this manner can then be sorted either by fluorescence activated cell sorting (FACS), ELISA or by adhesion and panning using immobilized annexin V antibodies. Alternatively, a terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end-labeling (TUNEL) assay is used to determine the level of cell death. The TUNEL assay uses the enzyme terminal deoxynucleotidyl transferase to label 3'-OH DNA ends, generated during apoptosis, with biotinylated nucleotides. The biotinylated nucleotides are then detected by using streptavidin conjugated to a detectable marker. Kits for TUNEL staining are available from, for example, Intergen Company, Purchase, NY. Alternatively, or in addition, an activated caspase, such as, for example, Caspase 3 is detected. Several caspases are effectors of apoptosis and, as a consequence, are only activated to significant levels in a cell undergoing programmed cell death. Kits for detection of an activated caspase are available from, for example, Promega Corporation, Madison WI, USA. Such assays are useful for both immunocytochemical or flow cytometric analysis of cell death.

In one example, the phenotype being assayed is cell survival. Cell survival may simply be detected by maintaining the cells for a sufficient time for a visible colony of cells to form. Clearly, this provides a simple method for high-throughput screening of compounds as compounds capable of inducing cell survival are easily recovered from the colony of cells.

Alternatively, cell viability or cell metabolism assay may be detected and/or assayed. By way of example, non- fluorescent resazurin is added to cells cultured in the presence of a peptide of the present invention. Viable cells reduce resazurin to red- fluorescent resorufm, easily detectable, using, for example microscopy or a fluorescent plate reader. This marker of cell viability is useful for a variety of different cell types, from bacteria to higher eukaryotes. Kits for analysis of cell viability are available, for example, from Molecular Probes, Eugene, OR, USA. Other assays for cell viability include for example, assays that detect WST-8 reduction to formazan salt in live cells (Alexis Biochemicals), staining of live cells with cell-permeable calcein acetoxymethyl (calcein AM) which is converted to fluorescent calcein by intracellular esterases, detection of XTT reduction to formazan salt (Intergen), MTS reduction to formazan salt (Promega Corporation). In yet another embodiment, the phenotype of interest is cellular proliferation. Methods for determining cellular proliferation are known in the art. For example, incorporation of ³ H-thymidine or ¹⁴C-thymidine into DNA as it is synthesized is an assay for DNA synthesis associated with cell division. In such an assay, a cell is incubated in the presence of labeled thymidine for a time sufficient for cell division to occur. Following washing to remove any unincorporated thymidine, the label (e.g. the radioactive label) is detected, e.g., using a scintilation counter. Assays for the detection of thymidine incorporation into a live cell are available from, for example, Amersham Pharmacia Biotech. In another embodiment, cellular proliferation is measured using a MTT assay. The yellow tetrazolium MTT (3-(4, 5- dimethylthiazolyl-2)-2, 5- diphenyltetrazolium bromide) is reduced by metabolically active cells, in part by the action of dehydrogenase enzymes, to generate reducing equivalents such as NADH and NADPH. The resulting intracellular purple formazan is then solubilized and quantified by spectrophotometric means. Assay kits for MTT assays are available from, for example, American Type Culture Collection.

Alternative assays for determining cellular proliferation, include, for example, measurement of DNA synthesis by BrdU incorporation (by ELISA or immunohistochemistry, kits available from Amersham Pharmacia Biotech), expression of proliferating cell nuclear antigen (PCNA) (by ELISA, FACS or immunohistochemistry, kits available from Oncogen Research Products) or a Hoechst cell proliferation assay that detects DNA synthesis (available from Trevigen Inc.).

Alternatively, the growth rate of the cell is determined, for example, manually, by, for example observing or measuring the size of a colony of cells over a period of time or, alternatively or in addition counting the number of cells over a period of time.

The invention is described further in the following non-limiting examples.

EXAMPLE 1

Materials and methods

1.1 Mice All mice were maintained on the C57BL/6 background. Mice expressing nuclear-localized enhanced GFP derived by ES cell insertion of the pCagg promoter (chicken β-actin) driving an enhanced GFP gene were obtained from Klaus Matthaei (Australian National University, Canberra, Australian Capital Territory, Australia). C57BL/6 RFP mice derived from ES cells expressing a pbActin-CMV-DsRed T3 transgene were obtained from Patrick Tam(Children's Medical Research Institute, Sydney, New South Wales, Australia).

1.2 Mouse lung cell suspensions

Single cell suspensions of lung cells were prepared by mincing and enzymatic digestion. Briefly, lungs were rinsed in sterile PBS following removal of trachea and major airways, finely minced with sterile scissors and incubated in about 3mg/mL Collagenase Type I (Roche, Basel, Switzerland) in PBS (about 310 mOsm) in a volume of about 3mL per lung for about 60 min at 37°C in a shaking incubator. The resulting cell suspension was further disaggregated by titration through an 18 then 21 gauge needle, diluted in PBS, filtered through a 40 micron nylon cell strainer (Falcon, Becton Dickinson, NJ) and washed twice in PBS supplemented with about 2% fetal or newborn calf serum (PBS-2% Se) by centrifugation (about 1,300 rpm; 5 min; 10⁰C).

Discontinuous density gradient centrifugation was employed to remove contaminating erythrocytes, neutrophils and cell debris. About 2OmL aliquots of lung cell suspension (2-3 lungs per gradient) were layered on about 10 mL Nycoprep 1.077A (1.077g/cm³; 265 mOsm; Nycomed Pharma, Oslo, Norway) (about 60Og; 20 min; room temperature, brake off) in 50 mL sterile polypropylene tubes. Low density cells at the PBS-Nycoprep interface were collected and washed twice in excess PBS- 2% Se and resuspended in PBS-2% Se and held on ice prior to further manipulation.

1.2 Mouse flow cytometric analysis and sorting

Low density lung cells were resuspended in PBS-2% Se (about 5 x 10⁷ cells/mL) in an optimally pre-titered cocktail of antibodies (e.g., anti-CD45, anti-CD31, anti-Seal, anti-EpCam, anti-CD49f, anti-CD 104, anti-CD24 and relevant isotype controls) and incubated for about 20 minutes on ice. Labeled cells were washed in excess PBS-2% Se and resuspended at about 5-10 xlO⁶ cells/mL and held on ice for flow cytometric analysis and sorting. Propidium iodide (PI; about 1 μg/mL), was used as a viability dye for exclusion of non- viable cells.

For proSP-C analysis, cells were fixed using a Fix 'n' Perm kit (Invitrogen).

Sorting was performed using a BD Influx cell sorter (Becton Dickinson) equipped with five lasers (488 nm 200 mW, 640 nm 40 mW, 561 nm 75 mW, 405 nm 50 mW, and 350 nm 100m W) using a 90-μmnozzle at 30 psi. Analysis was done using aBDLSRII bench top analyzer (Becton Dickinson) equipped with four lasers (488 nm 100 mW, 640 nm 30 mW, 561 nm 40 mW, and 405 nm 50 mW). Data were analyzed using Flow Jo (Tree Star Inc, Ashland, OR).

1.3 Mouse cell culture

For cell cultures, sorted cells were resuspended in Matrigel® (BD Biosciences, San Jose, CA), which was pre-diluted at a ratio of 1 : 1 with serum-supplemented media. About 90 μL of Matrigel® cell suspension was added to the top of a 24-well transwell filter insert (Millicell-CM, Millipore, Billerica, MA) in a 24-well flat-bottom tissue culture plate containing about 400 μL of serum-supplemented media (Fig. IA). For organotypic co-cultures, different cell subsets were mixed prior to being resuspended in Matrigel®. Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Carlsbad, CA) was supplemented with 10% newborn calf serum (Hyclone, Logan UT) for all cultures. Where specified, growth factors were added to the medium at concentrations determined to be optimum for cell culture; FGF-7 (about lOOng/ml; Millipore, Billerica, MA), FGF-10 (about 50ng/ml; R&D Systems, Minneapolis, MN), HGF (about 30ng/ml; R&D Systems, Minneapolis, MN), BMP-4 (about 100ng/ml; R&D Systems, Minneapolis, MN), TGF-betal (about lOng/ml; Peprotech, Rocky Hill, NJ), and PDGF-AA (about 50ng/ml; Millipore, Billerica, MA). Cell cultures were incubated at 37°C in a humidified triple mix incubator (about 5% O₂, 10% CO₂, 85% N₂) and re fed three times per week. All images are representative of cultures grown for 14-16 days unless otherwise specified. For bulk passaging, whole cultures were dissociated in 1 mg/mL Collagenase Type I plus 3 mg/mL Dispase (Roche) in PBS to generate a single-cell suspension. For clonal passaging, single colonies were picked and dissociated in the Collagenase/Dispase solution. Single cells were reseeded in stromal- free cultures supplemented with FGF-10 and HGF.

1.4 Immunohistochemistry Whole-mount cultures were fixed with 4% vol/vol paraformaldehyde and removed from inserts, washed in PBS, subjected to antigen retrieval by boiling in 10 niM citrate buffer for 20 min, and incubated in blocking buffer (1 h; 5% wt/vol BSA, 1% skim milk, 0.05% Triton X-100 in PBS). Cultures were then incubated overnight with antibodies against proSP-C (goat anti-proSP-C, clone C-19; Santa Cruz Biotechnology, or rabbit anti-proSP-C; Millipore), CCSP (goat anti-CCSP; Santa Cruz Biotechnology), MUC-I (rabbit anti-MUC-1; Abeam), and MUC5AC (biotin-labeled mouse anti-MUC5AC) and were then washed in PBS (0.05% Tween 20) and incubated with Alexafluor-488, -568, or -647-conjugated anti-goat, anti-rabbit, or streptavidin secondary reagents (Invitrogen) for 3 h and then washed. Nuclei were stained with DAPI, and sections were mounted in Vectashield (Vecta Laboratories). Images were acquired using a Leica SP confocal microscope and colored and overlaid using Adobe Photoshop (Adobe Systems). Bright- field images and whole-mount fluorescent images of cultures were taken using an Olympus SZXl 6 stereo dissecting microscope.

1.5 In Situ Immunohistochemistry Five-micrometer sections of adult mouse lungs fixed with paraformaldehyde

(1%) fixed and embedded in optimal cutting temperature medium were incubated in blocking buffer (1 h, 5% wt/vol BSA, 1% skim milk, 0.05% Triton X-100 in PBS). Sections were then incubated overnight with biotinylated anti-CD104 in combination with goat anti-CCSP (Santa Cruz Biotechnology) or rabbit anti-proSP-C antibody (Millipore), washed in PBS (0.05% Tween 20), and then subjected to tyramide signal amplification with Alexafluor 488-labeled tyramide (Invitrogen). Sections were washed and then incubated with donkey anti-goat or anti-rabbit conjugated to Alexafluor 568 (Invitrogen) for 1 h and then washed. Nuclei were stained with DAPI, followed by rinsing and mounting in Vectashield mounting medium (Vecta Laboratories). Photomicrographic images were acquired using a Leica SP confocal microscope. Images were colored and overlaid using Adobe Photoshop (Adobe Systems).

1.6 Real-Time RT-PCR

RNA from individual colonies or primary sorted cells was prepared using the RNAqueous MicroElute RNA isolation kit (Applied Biosystems), and cDNA was prepared using the high-capacity RNA-to-cDNA kit (Applied Biosystems). For realtime PCR, isolated cDNA was subjected to 40 cycles of amplification using Applied Biosystems TaqMan gene expression assays (Table 1) and 18s RNA endogenous control as per the manufacturer's instructions. Reactions resulting in a Ct of less than 40 indicated the presence of target cDNA in the sample, and data were expressed as the number of cycles that the reaction sample differed (ΔCt) from an 18s RNA control. Relative expression of the target gene was expressed as raw ΔCt values relative to the endogenous control.

Table 1: Applied Biosystems TaqMan gene expression assays

EXAMPLE 2 Proliferation and differentiation of mouse lung epithelial colony- forming units.

Given that current methodologies fail to accurately determine the proliferative and differentiative potential of lung epithelial cells, an organotypic epithelial colony- forming assay was developed for the prospective identification of lung epithelial stem and progenitor cells, which combined culture of cells within a semisolid extracellular matrix (Matrigel®) with culture at the air-liquid interface (Fig. 1 PanelA).

Heterogeneous lung cells (sorted on the basis of Sca-1 expression; Fig 1 Panel B) cultured in this way resulted in the generation of epithelial colony- forming units (CFU) with distinct morphological structures from the Sca-l^ne§/1° fraction (Fig 1 Panel D); either polarized cystic structures which resembled proximal-lung epithelial cells with lobular branching, termed airway-CFU (i) or smaller ductal structures which resembled distal lung epithelium, termed alveolar-CFU (ii). Temporal analysis of colony formation (Fig. IB) revealed the appearance of epithelial colonies after 1 week in culture, with airway-CFU (i) and alveolar-CFU (ii) establishing distinct proximal or distal morphology early on. In addition, a third type of colony was observed that developed as a cystic structure up until Dl 3 after which it developed the morphology of an alveolar-CFU (iii). These colonies have been termed mixed lung epithelial CFUs.

EXAMPLE 3

Paracrine signaling between mouse lung epithelial and mesenchymal progenitors - Co- culture

To further characterize epithelial CFUs, subpopulations of distal lung cells isolated by multiparameter flow cytometry were analyzed using antibodies against the pan-epithelial marker, EpCam versus Sca-1 antigen. These experiments showed that EpCam subset an abundant population (about 39%) of CD45^negCD31^negSca-l^nes/low adult lung cells, which were distinct from the Sca-l^pos cell fraction (about 21%). Interestingly, when this subsetting strategy was used, all epithelial colony-forming potential was abolished in the EpCam^negSca-l^pos cell fraction (Fig. 2 PanelC), whereas the ability to generate mesenchymal lineages was retained. These mesenchymal cells stained positive for Oil-Red O or alpha smooth muscle actin (SMA). The EpCam^pos cell fraction was devoid of mesenchymal cells (Fig. 2 Panel D). When EpCam^pos cells were co-cultured with EρCam^negSca-l^pos cells, the growth of epithelial CFUs was restored (Fig. 2 Panel E).

To determine which cell fraction epithelial CFUs were derived from, co-cultures were performed using EpCam^pos cells isolated from fluorescent RFP and eGFP transgenic mice with EpCam^negSca-l^pos cells isolated from C57BL/6 mice. Fluorescent microscopy showed that all cells within epithelial colonies displayed either red (RFP) or green (eGFP) fluorescence (Fig. 2 Panels G and H), whereas the surrounding mesenchymal cells were non-fluorescent (Fig. 2 Panel F). To establish whether or not epithelial-CFUs were generated by clonal proliferation of epithelial progenitors or by aggregation of cells, equal numbers of red (RFP) and green (eGFP) fluorescent EpCam^pos cells were cultured with non-fluorescent EpCam^negSca-l^pos cells. This resulted in the generation of only monochromatic epithelial CFUs, signifying their derivation from the proliferation of single progenitors rather than the aggregation of differentiated cells, which would have generated mixed color colonies (Fig. 2 Panels G and H). To determine the linearity of colony formation with respect to the number of cells plated, lung cells were seeded at different concentrations ranging from about 1x10⁶ - 4x10⁶ cells/mL. Linear regression analysis of concentration versus the number of colonies generated (Fig. 2B) showed that the number of cells seeded was directly proportional to the number of colonies (r2=0.94, slope=l/4688, N=7). Extrapolation of the regression line also showed that the cloning efficiency (equal to 1 /slope) of epithelial colony- forming units within the CD45negCD31^negEpCam^pos cell fraction was less than 0.05%, e.g., about 0.021%.

EXAMPLE 4 Multi-lineage differentiation of mouse lung epithelial colony- forming units -

Immunohistochemistrv

Morphological characterization of epithelial colonies grown in organotypic cultures demonstrates the generation of three distinct subtypes of epithelial colonies from the EpCAM¹" CD24^low cell fraction, including large airway-like lobular cystic colonies with a clearly defined lumen (46% of colonies, n = 150 of 326; Fig. 4 Panel A), small dense saccular colonies (35% of colonies, n = 114 of 326; Fig. 4 Panel B), and colonies of mixed phenotype with distinct budding (19% of colonies, n = 62 of 326; Fig. 4 Panel C). Immunofluorescent labeling of epithelial colonies in fixed wholemount organotypic cultures confirmed the presence of three distinct colony subtypes. In cystic colonies, cells expressed the polymeric mucin MUC5AC, which was secreted into the lumen but did not express the AEC II marker, pro SP-C (Fig. 4 Panels D, G, J, and M). Fluid could be seen circulating within the lumen of cystic colonies along with beating cilia in cell patches on the inner surface of the lumen. Given that MUC5AC is produced specifically by airway mucous-secreting epithelial cells, these data suggest that cystic colonies comprise cells of the airway lineage. In contrast, the majority of cells in the smaller saccular colonies expressed proSP-C but did not stain for MUC5AC (Fig. 4 Panels E, H, K, and N), suggesting that these colonies comprised alveolar AEC II epithelial cells and their progeny. Mixed colonies showed immunoreactivity for MUC5AC and proSP-C (Fig. 4 Panels F, I, L, and O), suggesting that both airway and alveolar epithelial lung cell lineages can be derived from a multipotent lung epithelial CFU. Interestingly, proSP-C staining in mixed colonies was only observed on cells at the peripheral tips of the colonies.

Real-time RT-PCR gene expression analysis of individual colonies (Fig. 4B) established that genes encoding airway lineage markers, including FoxJl (Foxjl; ciliated cells), P63 (Trp63; basal cells), MUC5AC (Muc5ac; goblet cells) and CFTR (Cftr; anion secretory cells) were exclusively detected in cystic and mixed CFUs. The alveolar markers, ABCA3 (Abca3), CEBP/alpha (Cebpa) and SP-C (Sftpc), as well as SP-A (Sftpal) were highly expressed in saccular and mixed colonies, but were not detected (SP-C and SP-A) or expressed at lower levels (ABCA3 and CEBP/alpha) in cystic colonies. The Clara cell marker, CCSP (Scgblal) was enriched in cystic and mixed colonies, but also detected in saccular colonies, and SP-B (Sftpb), MUC-I (Mucl), ENaC (Scnnlg) and Sox-2 (Sox2) were expressed by all epithelial colony subtypes. CGRP (Calca) was not detected in any of the epithelial colonies examined suggesting that neuroendocrine cells are part of a separate lineage. Taken together, the differential expression of airway versus alveolar lineage markers supports the concept that different epithelial colony subtypes are derived from either lineage-committed epithelial CFUs or multipotent lung epithelial CFUs.

EXAMPLE 5 Growth factor regulation of mouse lung epithelial progenitors - Similarities with development

Epithelial stem cells in the organotypic clonal assay showed similar requirement for epithelial-mesenchymal interactions to the developing lung. Therefore, to further assess whether the epithelial progenitors identified respond to similar growth factor signals, cultures were supplemented with various epithelial and mesenchymal growth factors. The number of epithelial CFUs generated was significantly increased in the presence of FGF-IO (p<0.001) and HGF (P<0.01) but unaffected with FGF-7 (Fig. 4B). In cultures supplemented with FGF-7 (Fig. 4A Panel B), and more so with FGF-IO (Fig. 4A Panel C), there was a bias towards airway-CFU morphology. However, this was not the case for HGF supplemented cultures (Fig. 4A Panel D), in which the relative abundance of airway versus alveolar-CFUs remained similar to those observed in control cultures. With the addition of BMP-4, there was a marked increase in the differentiation of lipofϊbroblastic cells in the surrounding stroma, which coincided with a significant (p<0.001) reduction in the total number of epithelial-CFUs (Fig. 4B) and a skewing towards alveolar-CFUs (Fig. 4A Panel E). Conversely, after TGF-betal and PDGF-AA treatment, cultures were dominated by long filamentous fibroblastic cells and a complete absence of epithelial CFUs was observed (Fig. 4 Panels E-G and 4B).

Experiments were then conducted to determine whether or not FGF- 10 and HGF could replace the requirement for mesenchymal support of epithelial CFUs. Results of these experiments are shown in Fig. 5 A and B. Figs 5 B shows that that addition of FGF-IO or HGF to stromal-free matrigel cultures OfEpCAM⁺ cells supports the generation of epithelial colonies. A supraadditive increase in both size and incidence of epithelial CFUs was observed when FGF-IO and HGF were used in combination, indicating that these growth factors acted synergistically to support the proliferation of epithelial CFUs. Under these conditions, epithelial CFUs proliferated robustly to generate simple spheroid colonies after 1 week in culture (Fig. 5B)

EXAMPLE 6

Subsetting of mouse lung epithelial progenitors

The resolution of epithelial progenitors within the heterogeneous EpCam^pos cell fraction was attempted by co-fractionation with CD49f. EpCam and CD49f shared a similar expression pattern in CD45^negCD31^neg lung cells. The level of EpCam expression directly correlates with the proliferative and differential potential of lung epithelial stem/progenitor cells. Lung epithelial stem/progenitor cells were defined by high EpCam expression in combination with the other markers.

Further subsetting with additional markers revealed that the phenotype of epithelial stem/progenitor cells could be refined on the basis of CD104^pos (Fig. 6 PanelC) and CD24^pos (Fig. 6 Panel E), which could then be further refined based on the relative level of CD24 expression with stem/progenitor cells being enriched in about 50% of cells that show a low expression of CD24. The population gated in Fig. 6 Panel A (EpCam Vs CD49f), Fig. 6 Panel C (EpCam Vs CD 104) and Fig.6 Panel E (EpCam Vs CD24) is the same population of cells showing that different gating strategies can be used to isolate the stem/progenitor cell population. Organotypic culture of the CD24^hi and CD24^low cell subsets showed that epithelial CFUs were resolved in the CD24^low cell fraction. Linear regression analysis (Fig. 6B) shows that the incidence of colony formation is directly proportional to the number of CD241ow cells plated and that the cloning efficiency of this population is -4.5%. In the adult mouse lung, heterogeneity in CD24 staining could not be observed at the level of immunofluorescent staining on tissue sections, thus precluding localization of the CD104^pos CD24^low cells. EXAMPLE 7 Self-renewal potential of mouse lung epithelial progenitors

Self-renewal is the hallmark property of stem cells. To examine the self-renewal properties of EpCam^posCD49f^?os cells, primary stromal-free cultures supplemented with FGF-IO and HGF were dispersed by enzymatic digestion, re-seeded and epithelial-CFU generation quantitated. The re-cloning of CFUs generated from primary sorted EpCam^posCD49f^?os cells (Fig. 7 Panel A) resulted in the propagation of more epithelial - CFUs (Fig. 7 Panel B). Weekly serial passaging over 5 weeks demonstrated that these target cells have the capacity for self-renewal. Furthermore, serial passaging of single colonies demonstrated the robust self-renewal of single CFUs over three generations (Fig. 7 Panel E).

After passage 3, dispersed cells were subjected to re-analysis of EpCam and CD49f expression by flow cytometry analysis. The EpCam^posCD49F^os cell population in passaged CFUs was enriched (Fig. 7 Panel C) compared to that of the primary lung cell suspension (Fig. 7 Panel A). A population of cells with reduced expression of EpCam and CD49f emerged. In addition, when organotypic co-cultures combining passaged cells (passage 3) with freshly isolated EpCam^negSca-l^pos cells were set up, mixed lung epithelial CFUs were generated displaying both airway and alveolar morphology (Fig. 7 Panel D).

EXAMPLE 8 Isolating human lung cells

The markers EpCam and CD49f were also used for isolating and sub-setting human lung cells. Similar to mice, an EpCam^posCD49F^os cell population was detected in the human distal lung. This population shared the phenotype of their mouse counterparts with respect to EpCam and CD49f, as well as forward and side scatter properties (Fig. 8A). In the human sample this population was found in greater abundance than in mice.

Additional phenotypic analysis of human lung samples showed that a population of cells characterized by being CD45^neg CD31^neg EpCAM^pos CD24^low (homologous to the mouse population) cell population exists within the adult human lung (Fig. 8B). EXAMPLE 9 Culturing human lung cells

A human EpCam^posCD49f^pos cell population lung cell population isolated substantially as described in Example 8 are cultured substantially as described for mouse lung cells in the Examples above. For example, primary stromal-free cultures are supplemented with FGF-IO and HGF substantially as described in the examples above. Following a suitable time for cell growth cells are dispersed by enzymatic digestion, re- seeded and epithelial-CFU generation quantitated. Alternatively, or in addition, cells are cultured in the presence of stromal cells, e.g., bone marrow stromal cells and/or in the presence of one or more different or additional growth factors. Alternatively, or in addition, cells are cultured in the presence of a lung fibroblast cell line and/or a lung stromal cell population expressing platelet-derived growth factor receptor (PDGFR)-α and/or PDGFR-β

EXAMPLE 10 Transplantation of lung epithelial progenitor cells into animal models of lung disease

10.1 Animal models

10.1.1 Bleomycin model

Bleomycin sulphate is dissolved in sterile saline and administered as a single dose by intratrachael administration. Control animals receive sterile saline alone. Mice receive saline or bleomycin essentially as described in Kremer et al., Respiration 66: 455-462, 1999)

Animals are randomly assigned to the following groups: bleomycin treated and receiving lung epithelial progenitor cells; bleomycin treated without receiving lung epithelial progenitor cells; saline treated and receiving lung epithelial progenitor cells; and saline treated without receiving lung epithelial progenitor cells. For models to be treated with human cells, SCID mice are used.

10.1.2 Naphthalene model

Naphthalene is dissolved in corn oil and administered to mice by intraperitoneal injection essentially as described by Plopper et al., J. Pharmacol. Exp. Ther., 261: 353- 363. Control mice receive corn oil alone. Animals are randomly assigned to the following groups: naphthalene treated and receiving lung epithelial progenitor cells; naphthalene treated without receiving lung epithelial progenitor cells; corn oil treated and receiving lung epithelial progenitor cells; and corn oil treated without receiving lung epithelial progenitor cells. For models to be treated with human cells, SCID mice are used.

10.2 Cells

Human or mouse lung epithelial progenitor cells are isolated essentially as described above. In some experiments, cells are cultured in the presence of a fluorescent cell marker, to permit the cells to be tracked. Exemplary markers include 5-chloromethylfluorescein diacetate (Invitrogen Corporation).

Cells are administered to mice after lung injury via an intratracheal, intravenous or intercostal route of administration.

10.3 Analysis of lung injury

Lung injury and cell repopulation is assessed be detecting presence of fluorescently labelled cells, analysis of bronchoalveolar lavage fluid, measurement of hydroxyproline concentration in the lung and routine light microscopic examination as follows.

10.3.1 BAL analysis

A canula is placed into the trachea and BAL obtained using saline essentially as described in Kremer et ah, supra. The total number of cells in the BAL is counted and a differential count is performed in 200 cells per animal and expressed as a percentage of total cells recovered.

Reduced cell numbers, particularly of inflammatory cells in animals treated with cells compared to those in which injury is induced but no cells administered is indicative of reduced lung damage.

10.3.2. Hydroxyproline concentration

One lung is ligated and cut at the hilum, freed of extraneous tissue and homogenized in phosphate buffered saline. An aliquot of homogenized lung is hydro lysed in 6N HCl for 24h. at 106⁰C, and analysed using an amino acid analyser, essentially as described in Lossos et ah, Life Sci., 67: 2873-2881. Reduced hydroxyproline levels in animals treated with cells compared to those in which lung injury is induced without cell treatment is indicative of reduced lung injury.

10.3.4 Morphological examination

One lung is fixed by intratracheal infusion of paraformaldehyde. Lungs are removed, embedded and cut at either 5μm or 50μm for three-dimensional analysis. Alternatively, or in addition serial sections are taken through three 0.3cm sections of an inflated lung fixed in paraformaldehyde. CFDA fluorescence is detected to identify fluorescent cells, or sections are stained with haematoxylin and eosin and modified Masson's trichrome for morphological analysis of fϊbrotic lung injury, essentially as described in Izbicki et al, Int. J. Exp. Path. 83: 111-119, 2002.

These analyses permit determination of the effect of lung epithelial progenitor cells on lung disease and/or determination of the repopulation ability of cells in normal or injured lung.

EXAMPLE 11

Analysis of endogenous lung epithelial progenitor cells

Lung injury is induced in animals using the protocols described in Example 10. At various time points before, during and after lung injury lungs are fixed in paraformaldehyde, removed embedded and sectioned as described in Example 10.

Serial sections of lung are then stained for the presence of EpCam and/or CD 104 and/or

CD49f and/or CD24. Cells positive for at least two of these markers are considered lung epithelial progenitor cells. These analyses permit determination of the response of endogenous lung epithelial progenitor cells to injury.

EXAMPLE 12

Hierarchical ordering of lung epithelial progenitor cells

Without being bound by any theory or mode of action, the evidence presented herein appears to suggest that an epithelial stem/progenitor cell hierarchy exists in the adult mouse lung. For example, dissociation and reseeding of primary mixed colonies in co-culture with EpCAM^neg Sca-l^pos stromal cells results in the generation of secondary mixed, airway and alveolar colonies, suggesting that these colonies are derived from a multi-potent progenitor. In contrast, cystic airway and saccular alveolar colonies fail to generate secondary colonies, suggesting that these progenitor cells have only limited proliferative potential. A depiction of an exemplary hierarchy is shown in Figure 9.

To further characterize this hierarchy lung epithelial progenitor cells are separated on the basis of mid or high levels of expression of EpCam and/or CD 104 and/or CD49f and/or CD24. Accordingly, populations having various combinations of high and medium expression of each of these markers are isolated. Each population is then permitted to differentiate and the potential of each cell to form lung epithelial progenitor cells (i.e., the number of progenitor cells present) and the potential to form a variety of distinct epithelial progenitor cells is assessed. These analyses permit identification of populations having higher number of progenitor cells and/or comprising progenitor cells having higher differentiation potential.

EXAMPLE 13

EpCam signalling in lung epithelial progenitor cells Lung epithelial progenitor cells from mouse and/or human are isolated and cultured essentially as described above. Cells are treated with an antagonistic EpCam antibody or transfected with an expression construct encoding EpCam. Survival, proliferation and differentiation ability of cells are then assessed using standard methods and/or methods described herein.

EXAMPLE 14 Screening for an inhibitor of colony formation by lung epithelial progenitor cells

CD45^neg CD31^neg EpCAM^pos CD24^low adult lung epithelial progenitor cells were sorted and mixed with freshly sorted CD45^neg CD31^neg Sca-l^pos stromal cells and resuspended in Matrigel® (BD Biosciences, San Jose, CA), which was pre-diluted at a ratio of 1 :1 with serum-supplemented media. About 90 μL of Matrigel® cell suspension was added to the top of a 24-well transwell filter insert (Millicell-CM,

Millipore, Billerica, MA) in a 24-well flat-bottom tissue culture plate containing about

400 μL of serum-supplemented media (Fig. 10) containing lOμg/ml of purified anti- EpCam antibody or without antibody. Figure 10 shows that anti-EpCam antibody is capable of reducing or preventing colony formation.

Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Carlsbad, CA) or Alpha Modified Eagle's medium (alphaMEM; Invitrogen, Carlsbad, CA) was supplemented with 10% newborn calf serum (Hyclone, Logan UT) for all cultures.

Claims

WE CLAIM:

1. An isolated lung epithelial progenitor cell expressing a protein individually or collectively selected from the group consisting of EpCam, CD 104, CD49f and CD24 or progeny thereof or a cell differentiated therefrom.

2. An isolated population of cells enriched for lung epithelial progenitor cells and/or progeny thereof and/or cells differentiated therefrom, wherein cells in said population express a protein individually or collectively selected from the group consisting of EpCam, CD 104, CD49f and CD24.

3. The cell or population according to claim 1 or 2, wherein the cell(s) express at least EpCam and/or CD 104.

4. The cell or population according to claim 1 or 2, wherein the cell(s) express a high level of EpCam.

5. The cell or population according to any one of claims 1 to 4, wherein the cell(s) express a low level of CD24.

6. The cell or population according to any one of claims 1 to 5, wherein the cell(s) express low levels, or do not express detectable levels of a marker of a hematopoietic cell and/or a marker of an endothelial cell.

7. The cell or population according to any one of claims 1 to 6, wherein the cell(s) have a phenotypic profile selected individually or collectively from the group consisting of: i) EpCam⁺ and CD49f; ii) EpCarn¹" and CD49f^f; iii) EpCam⁺ and CD24⁺; iv) EpCarn¹" and CD24⁺; v) EpCam⁺ and CD24^lG; vi) EpCam¹¹¹ and CD24^lG; vii) EpCam⁺ and CD 104⁺; viii) EpCam¹¹¹ and CD 104⁺: ix) EpCam⁺, CD104⁺ and CD49f; x) EpCam¹¹¹, CD 104⁺ and CD49f ; xi) EpCam⁺, CD 104⁺ and CD24⁺; xii) EpCanΛ CD 104⁺ and CD24⁺; xiii) EpCam⁺, CD 104⁺ and CD24^lG; xiv) EpCanΛ CD 104⁺ and CD24^lG; xv) EpCam⁺, CD104⁺, CD49f and CD24⁺; xvi) EpCanΛ CD 104⁺, CD49f and CD24⁺; xvii) EpCam⁺, CD 104⁺, CD49f and CD24^lG; xviii) EpCam¹¹¹, CDl 04⁺, CD49f and CD24^lG; xix) any one of i) to xviii) and CD45^"; and xx) any one of i) to xix) and CD31 ^".

8. The cell or population according to any one of claims 1 to 3, wherein the cell(s) have a phenotypic profile selected individually or collectively from the group consisting of: i) CD104⁺ and CD49f⁺; ii) CD104⁺ and CD24⁺; iii) CD104⁺ and CD24^lG; iv) CD 104⁺, CD49f and CD24^lG; v) CD 104⁺, CD49f and CD24⁺; vi) any one of i) to v) and CD45^"; and vii) any one of i) to vi) and CD31 ^"

9. An isolated population of cells enriched for lung epithelial progenitor cells and/or progeny thereof and/or cells differentiated therefrom, wherein cells in said population have a phenotypic profile EpCam⁺, CD104⁺, CD49f , CD24⁺, CD45^" and CD31 or EpCam¹¹¹, CD104⁺, CD49f , CD24⁺, CD45^" and CD31 or EpCam⁺, CD104⁺, CD49f , CD24^lG, CD45^" and CD31 or EpCam^hl, CD104⁺, CD49f , C24^lG, CD45^" and CD31^".

10. The cell or population according to any one of claims 1 to 9, wherein the cell(s) is (are) non-tumorigenic and/or is (are) not derived from or isolated from a tumor.

11. The cell or population according to any one of claims 1 to 10, wherein the cells(s) is (are) capable of differentiating into a lung epithelial cell.

12. The cell or population according to any one of claims 1 to 11, wherein the cell(s) is (are) capable of differentiating into a plurality of distinct lung epithelial cells.

13. The cell or population according to any claim 12, wherein the cell(s) is(are) capable of differentiating into one or more cells of the proximal lung and/or of the distal lung.

14. A method for isolating a lung epithelial progenitor cell or population enriched for said cells, said method comprising identifying a cell expressing a protein individually or collectively selected from the group consisting of EpCam, CD 104, CD49f and CD24 or population of said cells and isolating said cell(s).

15. The method according to claim 14 comprising contacting a population of cells comprising lung epithelial progenitor cells with a compound that binds to a protein individually or collectively selected from the group consisting of EpCam, CD 104, CD49f and CD24 for a time and under conditions sufficient for a compound-protein complex to form and isolating the complex and/or a cell on which the complex has formed, thereby isolating a lung epithelial progenitor cell or population enriched for said cells.

16. The method according to claim 14 or 15, wherein the cell(s) are isolated from a tissue sample from a subject's lung and/or from a secretion from a lung or sample comprising same.

17. The method according to claim 16, wherein the tissue sample or secretion is from non-tumorigenic tissue or from a region of a tissue that does not comprise a tumor and/or from a normal and/or healthy subject.

18. The method according to any one of claims 14 to 17 additionally comprising culturing the isolated cell(s) to produce progeny or additionally comprising differentiating the isolated cell(s) and/or progeny thereof to produce a lung epithelial cell or population thereof.

19. A composition comprising a lung epithelial progenitor cell and/or progeny and/or differentiated cell and/or population of cells according to any one of claims 1 to

13 and a pharmaceutically acceptable carrier and/or excipient.

20 A method of treating or preventing a disease comprising administering to a subject in need thereof a lung epithelial progenitor cell and/or progeny and/or differentiated cell and/or population of cells according to any one of claims 1 to 13 or a composition according to claim 19.

21. The method according to claim 20, wherein the subject suffers from, is developing or is at risk of developing a lung disease and/or has undergone surgery to remove at least part of a lung.

22. A method of improving lung function or producing lung tissue in a subject, said method comprising administering to the subject a lung epithelial progenitor cell and/or progeny and/or differentiated cell and/or population of cells according to any one of claims 1 to 13 or a composition according to claim 19.

23. A method for identifying a compound that directs differentiation of a lung epithelial progenitor cell, said method comprising: i) contacting a lung epithelial progenitor cell or progeny thereof or population of cells enriched for same according to any one of claims 1 to 13 with a test compound and determining the amount of lung epithelial cells differentiated therefrom; ii) determining the amount of lung epithelial cells differentiated from lung epithelial progenitor cell or progeny thereof or population of cells enriched for same according to any one of claims 1 to 13 in the absence of the compound, wherein an increased amount of differentiated cells at (i) compared to (ii) indicates that the compound directs differentiation of the lung epithelial progenitor cell.

24. A method for identifying a compound that reduces or prevents differentiation of a lung epithelial progenitor cell into one or more lung epithelial cell types, said method comprising: i) contacting a lung epithelial progenitor cell or progeny thereof or population of cells enriched for same according to any one of claims 1 to 13 with a test compound and determining the amount of lung epithelial cells differentiated therefrom; ii) determining the amount of lung epithelial cells differentiated from lung epithelial progenitor cell or progeny thereof or population of cells enriched for same according to any one of claims 1 to 13 in the absence of the compound, wherein a reduced amount of differentiated cells at (i) compared to (ii) indicates that the compound directs differentiation of the lung epithelial progenitor cell.

25. A method for identifying a compound that reduces proliferation of a lung epithelial progenitor cell and/or progeny thereof, said method comprising: i) contacting a lung epithelial progenitor cell or progeny thereof or population of cells enriched for same according to any one of claims 1 to 13 with a test compound and determining the amount of cell proliferation; ii) determining the amount of cell proliferation of a lung epithelial progenitor cell or progeny thereof or population of cells enriched for same according to any one of claims 1 to 13 in the absence of the compound, wherein a reduced amount of proliferation at (i) compared to (ii) indicates that the compound reduces proliferation of lung epithelial progenitor cells and/or progeny thereof.

26. A method for identifying a candidate compound for the treatment of cancer said method comprising: i) contacting a lung cancer cell or population thereof with a compound and determining the level of proliferation and/or cell death; and ii) contacting a lung epithelial progenitor cell or progeny thereof according to any one of claims 1 to 13 with the compound and determining the level of proliferation and/or cell death, wherein a compound that reduces cellular proliferation or induces cell death at (i) and not at (ii) or that reduces cellular proliferation to a greater degree at (i) compared to (ii) or induces a greater level of cell death at (i) compared to (ii) is a candidate compound for the treatment of cancer.

27. The method according to any one of claims 23 to 26 additionally comprising isolating the identified compound.

28. A method for culturing a lung epithelial progenitor cell and/or progeny thereof and/or a population of cells comprising same in an undifferentiated state, said method comprising maintaining said cell(s) for a time and under conditions sufficient for cell growth and/or proliferation, wherein said cell(s) is(are) maintained in the presence of FGF-IO and/or HGF.

29. A method for isolating a lung epithelial progenitor cell, said method comprising maintaining a population of cells from a lung in a matrix, which is in contact with both a culture medium and a gas environment surrounding the matrix/medium for a time and under conditions sufficient for the cell to proliferate and differentiate and detecting a population of epithelial cells of the lung, wherein detection of the population of epithelial cells of the lung is indicative of a lung epithelial progenitor cell.

30. The method according to claim 29, wherein the culture medium comprises secreted factors from a stromal cell.

31. The method according to claim 29 or 30, wherein the culture medium is in contact with stromal cells, however the matrix and cells contained therein are spatially separated from the stromal cells.

32. The method according to any one of claims 29 to 32, wherein the method additionally comprises isolating the population of cells from the lung of a subject prior to maintaining the population in a matrix.