EP1960553A2

EP1960553A2 - Pulmonary stem cells, related methods and kits

Info

Publication number: EP1960553A2
Application number: EP06826023A
Authority: EP
Inventors: John Cheng-Po Yu; Alice Lin-Tsing Yu; Thai-Yen Ling
Original assignee: Academia Sinica
Current assignee: Academia Sinica
Priority date: 2005-10-17
Filing date: 2006-10-16
Publication date: 2008-08-27
Also published as: JP2009514509A; WO2007047581A2; AU2006304390A1; TW200732475A; WO2007047581A3

Abstract

Methods for the isolation, cultivation, identification, and utilization of pulmonary stem cells are provided. Pulmonary stem cells are derived from mammalian lung tissue, being resident therein. Isolation methods include the identification of putative stem cells by their emergence in primary cultures as discrete slow growing colonies. Confirmation of identity as stem cells is by stem cell markers, such as Oct-4, and by their ability to differentiate into a mature phenotype. Isolation and selective methods further include cell sorting on the basis of stem cell markers. Methods of cultivation whereby stem cell populations are expanded while maintaining the undifferentiated phenotype include the use of defined culture media, culture surface modifications, and co-cultivation with pulmonary stroma cells. Methods for the use of pulmonary stem cells are further provided, for studies of the biology of the cells, including viral infectivity, production of virus, and the testing of anti-infective agents.

Description

Pulmonary Stem Cells, Related Methods and Kits

FIELD OF THE INVENTION The present disclosure relates to stem cells, adult pulmonary stem cells in particular, and associated methods and kits of parts for identifying, isolating, cultivating the cells, and for modeling viral infection of such cells and developing treatments therefore.

BACKGROUND OF THE INVENTION Stem cells are found in a number of tissues and organs from the earliest stages of development (embryonic stem cells) to adulthood (adult stem cells). While embryonic stem cells have been isolated and studied separately from non stem cells, adult stem cells have generally been identified only within and as a part of a larger cell population that includes non-stem cells. So-called "adult" or "tissue- specific" stem cells are reported to be resident in various tissues, including skin, bone marrow, muscle, brain, and other tissues. These cells are basically those associated with an organism at any stage of development beyond early embryonic development, and include, for example those derived from an organism at a neonatal stage of development. These tissue-specific stem cells act as a repair system, continually replenishing ageing tissues with normal cells. The ability of stem cells to create new body tissue provides stem cells therapeutic potential, offering, for example, the possibility of generating new cells to replace diseased and damaged body tissues in conditions such as Parkinson's disease, diabetes, cancer, and Alzheimer's dementia. Given a growing interest in the characterization of stem cells of lung tissues for regenerative therapy, attempts have been made to identify and enrich lung stem cells. The lung is an extremely complex, conditionally renewing organ composed of at least 40 differentiated cell types/lineages and can be divided into proximal cartilaginous airways (trachea and bronchi), distal bronchioles (bronchioles, terminal bronchioles, and respiratory bronchioles), and gas-exchanging airspace (alveoli). The lung is lined with functionally and structurally distinct epithelium that probably contains different and unique types of adult epithelial stem/progenitor cells. Because the epithelial surface is constantly open to potential injury, stem/progenitor cells serve as a primary protective lining armed with rapid response mechanisms for epithelial repair. The candidates for the putative stem/progenitor cells that repair the injured lungs and contribute to local needs in times of tissue damage include the basal cells for mucosal gland development and renewal of the branched epithelium of the trachea (from PNAS), the Clara cells of the bronchiole and the type-2 pneumocytes of the alveolus.

Lung injury models with naphthalene have suggested that there are cytochrome P450 negative (CyP450^~ )-variant Clara cells residing within neuroepithelial bodies or the bronchoalveolar duct junction that are spared from the toxicity of naphthalene and are responsible for the subsequent bronchiolar regeneration. In addition, the nonhematopoietic side population cells isolated from the lung airway have been shown to have the same molecular phenotype as the CyP450^~ -variant Clara cells. Recently, pulmonary stem cells residing in the bronchoalveolar junction of adult lungs have been identified and characterized as CD34⁺ Sca-1⁺ CD45^" PE-CAM^" cells expressing both cytoplasmic Clara cell secretion protein (CCSP) and prosurfactant protein-C proteins, which are markers for Clara cells and type-2 pneumocytes, respectively. However, the developmental relationship among the above reported pulmonary stem and progenitor cells remains to be defined. Lung tissue is the target of numerous infective agents, some responsible of fatal diseases, such as severe acute respiratory syndrome-associated coronavirus (SARS-CoV). In 2003, a new atypical pneumonia, severe acute respiratory syndrome (SARS), spread across several countries with a high mortality rate resulting from acute lung failure. The SARS pathogen was then subsequently identified as a new variant coronavirus (SARS-CoV) based on its cytopathic effect on VeroEβ cells. A number of animal models have been used to study the pathogenesis of SARS-CoV infection. Although the monkey model mimics to certain degree the clinical course of SARS, the mouse model provides the first genetic evidence for angiotensin converting enzyme 2 (ACE-2) as a crucial SARS-CoV receptor in vivo. Although type-1 pneumocytes, and to a lesser extent type-2 pneumocytes, have been shown to be the target cells of SARS-CoV infection in monkey studies, the identity of mouse bronchiolar epithelial cells infected by SARS- CoV remains unclear. Patients with SARS-CoV present with fever and pneumonia that initially responds to antiviral therapy. Viral titers typically decline rapidly after day ten following initial infection. Despite this decline in viral load, twenty percent of patients clinically develop acute respiratory distress syndrome (ARDS) by week three, which is associated with high mortality. These SARS-infected lung tissues demonstrate diffuse alveolar damage with edema, hemorrhage, and mononuclear infiltration. ARDS treatments are targeted to control inflammatory response, but the severity and recovery of lung injury also depend on epithelial cell function. In fact, the predominant pathological finding is diffuse alveolar epithelial damage. The alveolar epithelium is also the site of alveolar fluid reabsorption and plays a major role in the development of lung fibrosis associated with ARDS.

A greater understanding of the biology of pulmonary stem cells, their susceptibility to disease and infection, their role in recovery from disease, infection, or insult to the lung, will be helpful in the development of drugs and strategies with which to treat pulmonary diseases.

SUMMARY

According to a first aspect of the invention, a method of isolating adult pulmonary stem cells is disclosed. The adult stem cells, resident in the lung, when placed in vitro, present as slowly dividing cells that form distinct individual colonies, and express marker Octamer-4 (Oct-4, a protein, a transcription factor of the POU domain family, a marker of stem cells). The fact that the stem cells are dividing, even if slowly, distinguishes them the majority of cells present in the primary culture that do not form discrete growing colonies, even if cells are growing or dividing rapidly. Discrete colonies such as these typically arise from a single cell that undergoes a series of divisions, as such, these cells may be termed "clonogenic", referring to their clonal origin and the relatedness of daughter cells, as a result of having arisen from a single common ancestral cell. A population of cells derived from a primary culture of a tissue sample may already be presumed to be genotypically identical by virtue of coming from one individual. Clonal cell populations that grow out or are expanded in vitro, are typically further identical biologically with respect to phenotypic expression of cell type and state of differentiation. An early step in isolating the pulmonary stem cells is that of visually identifying them as growing colonies under a microscope and then manually plucking or harvesting them from the primary culture for further culture or analysis. Such a method is applicable to primary cultures of mammalian cells, including cells from a human or murine source. Such harvested cells, isolated from the larger population can be identified as stem cells by criteria further described below. One particular identifying feature of adult stem cells is that they are able to undergo terminal differentiation into a mature pulmonary cell phenotype. These methods of isolating stem cells resident in formed tissue are exemplified by the disclosure here with regard to mammalian pulmonary cells, but are anticipated to be broadly applicable to stem cells resident in other tissues as well.

According to a second aspect of the invention, a method to identify stem cells in a tissue is disclosed. Embodiments of the method comprise identifying Oct- 4 expressing cells of the tissue, the identified Oct-4 expressing cells able to form colonies in vitro, the identified Oct-4 expressing cells, upon isolation in primary culture, able to differentiate into a mature phenotype. Isolated adult tissue cells further are identifiable by their expression of Oct-4 or in combination with other stem cell markers, such as stem cell markers representing expression of genes or proteins associated with the undifferentiated phenotype of stem cells. Gene expression, more specifically, the process by which DNA is transcribed to form mRNA, may be shown by such methods as RT-PCR (reverse transcriptase-polymerase chain reaction) and quantitative PCR methods. Quantitative PCR is a method that has a significant advantage over immunofluorescent methodology for some purposes simply because it is quantitative, and permits, for example, high resolution comparisons of gene expression among various types of cells. Such methods may, for example, show the expression of genes for markers such as Oct-4, SSEA-1 , Nanog, or Sca-1. Protein expression, more specifically, the translation of RNA to form protein, may be shown by a method such as immunofluorescence and flow cytometric sorting procedure (fluorescence activated cell sorter, or "FACS"); by such methods, for example, the expression of Oct-4, SSEA-1 , Nanog or Sca-1 may be shown. Markers associated with the stem cell phenotype may be species specific. Human pulmonary stem cells, for example, express SSEA-3, SEEA-4 and Sca-1 , and Nanog. Murine pulmonary stem cells, for example, express SSEA-1, SCA-1, and Nanog. In addition, other markers not necessarily related to stem cells in particular, such as CXCR-4, CD54, etc can be used in conjunction with above-mentioned stem cell-specific markers to identify adult stem cells during the multiparameter sorting analysis of FACS. According to a third aspect of the invention, a kit for the identification of pulmonary stem cells is disclosed. Such kits are tangible embodiments of methods, including materials and reagents, for identifying and characterizing adult pulmonary stem cells, including the collective reagents and materials packaged as a unit. Embodiments of a kit for identifying pulmonary stem cells comprise a first identifier for the identification of slowly dividing cells and a second identifier for identifying the expression of the marker Oct-4, the first and the second identifier to be used to identify slowly dividing Oct-4 expressing cells, the identified slowly dividing Oct-4 expressing cells able to form colonies in vitro and to differentiate into a mature phenotype. The collective weight of the evidence by each of these markers creates an ever increasing confidence in the correctness of identifying such cells are stem cells. Some embodiments of the kit may include further identifiers (e.g., other stem cell-specific markers like SSEA-1 , Nanog, or Sca-1 or in combination with other nonspecific markers like CXCR-4, CD54, etc.) appropriate for distinguishing stem cells from non-stem cells, as described herein. According to a fourth aspect of the invention, a method for cultivating adult pulmonary stem cells in vitro is disclosed. Embodiments of the method comprise providing pulmonary tissue, the tissue including in major part differentiated pulmonary cells, but also a small population of resident stem cells. In general the methods of cultivating include methods of isolating the stem cell population of interest, and iteratively enriching or purifying the cell population to favor stem cells and reducing or eliminating the presence of non-stem cells, and expanding the obtained stem cell population in vitro. Methods of cultivating pulmonary stem cells begin with isolating them from the source tissue that is typically but not limited to mammalian tissue; incubating the isolated cells in a suitable medium. Some embodiments of the method include applying to the isolated incubated cells a cell sorting procedure to positively select for cells based on a stem-cell characteristic, expression of Oct-4 for example, to obtain cell populations increasingly enriched in stem cells. At any point in the culture and expansion of stem cell populations they may be tested by methods described herein to confirm or to quantify their identity as undifferentiated stem cells. Embodiments of this aspect of the invention may be directed toward laboratory scale cultures, or to larger scale operations, where large volumes of cells are sought for studies of the basic biology of the cells, studies of viral infection dynamics, or studies involving anti-infective agents.

According to a fifth aspect of the invention, a method for identifying an infective agent able to infect pulmonary stem cells is disclosed. Embodiments of the method comprise contacting an infective agent with the above identified isolated pulmonary stem cell; detecting the level of infection of the isolated pulmonary stem cell by the infective agent infection; and comparing the detected level of infection with a predetermined threshold level, the threshold level indicative or diagnostic of the development of an infection of the cell. Embodiments of this aspect of the invention may be used to identify infective agents in samples suspected of having infective agents, but basically of unknown content. Other embodiments of the invention may be used in a quantitative manner, wherein the infective agent may be known, but its level or titer is unknown, or where its infective efficiency is unknown. In these embodiments, samples are typically subjected to multiple levels of dilution, and one or more identifying test are run with controls, and subjected to statistical analysis, as is well known in the art. According to a sixth aspect of the invention, a method for the production of an infective agent, in particular a virus, is disclosed. Embodiments of the method comprise mutually contacting a viral inoculum with a population of pulmonary stem cells in a culture; and collecting virus particles from the culture after a suitable period of incubation. In particular, mutually contacting a virus inoculum and a population of isolated pulmonary stem cell in a culture can be performed in a serum-free culture (defined medium) condition. The cell culture system may further include treated surfaces or the presence of co-cultured cells to support the growth of the stem cell population, or to stabilize the phenotype in an undifferentiated state. The viral inoculum may be any virus of interest, such as, by way of example, Hanta virus, SARS-CoV, other influenza viruses, for example, influenza A, H1 N1 , H1 N2, H2N2, and avian flu viruses, such as H5N1 viruses. A now reasonably current list of the subtypes of the causative agent species of avian flu includes H1 N1 , H1 N2, H2N2, H3N2, H3N8, H5N1, H5N2, H5N3, H5N8, H5N9, H7N1, H7N2, H7N3, H7N4, H7N7, H9N2, and H10N7; any of these agents, as well as those discovered at a later time may be included as embodiments of methods of this invention. Embodiments of this aspect of the invention may be directed toward production at research scale, or at a more industrial scale. The viral products may be used in research, or for therapeutic purposes, such as gene delivery vehicles, as in the development and production of vaccines, and for the development of anti-infective agents.

According to a seventh aspect of the invention, a method for identifying an anti-infection agent that interferes with infection, or the progression of infection, of a pulmonary stem cell by an infective agent is disclosed. An anti-infective agent is a compound that interferes with any aspect of an infective process. Embodiments of the method comprise mutually contacting a candidate anti-infective agent and a pulmonary stem cell population; contacting the pulmonary stem cell with a candidate infective agent; detecting the level of infection of the pulmonary stem cell by the infective agent; and comparing the detected level of infection with a predetermined threshold level, the threshold level indicative or diagnostic of development of infection of the cell. The order by which cells are exposed to an infective agent and an anti-infective agent may vary according to experimental or procedural specifics, as is well known in the art. By way of example, the infective agent can be any virus such as SARS-CoV, Hanta virus, or influenza viruses, such as influenza A, and H1N1, H1N2, H2N2, or avian flu viruses, such as H5N1 viruses. Anti-infective agents may by of any chemical classification, and range in size from small molecules to large molecules such as antibodies. Embodiments of this aspect of the invention may be directed toward relatively positive/negative identification or diagnostic purposes, or for more quantitative purposes, where levels of anti-infective efficacy are sought. Embodiments may be directed to research scale experiments, or to larger scales, as in large scale screening of anti-infective therapeutic candidate compounds.

According to an eighth aspect of the invention, a method for generating differentiated pulmonary cells from pulmonary stem cells in long term in vitro culture is disclosed. Adult pulmonary stem cells have the capacity to undergo terminal differentiation to alveolar type 2 and type 1 pneumocytes as established by experimental procedures exemplified in Example 4, indeed this capacity is a hallmark of the stem cells isolated by methods disclosed. Embodiments of the method comprise the generation of alveolar type 2 and type 1 pneumocytes, the functional cells for lung tissues to carry out biological function. Embodiments of this aspect of the invention may be directed toward simple replacement therapy, for development of artificial lung tissues, or screening of anti-infective therapeutic candidate compounds, where functional lung cells are sought. Embodiments may be directed to research scale experiments, or to larger scales, as may be appropriate for therapeutic purposes.

According to an ninth aspect of the invention, a method for maintaining adult pulmonary stem cells in their undifferentiated state in long term in vitro culture is disclosed. Adult pulmonary stem cells have the capacity to stabilize the phenotype in the undifferentiated state. This method is important for studies of the biology of these cells, as for example when employing them as models of viral infectivity, and using them as models to test the efficacy of anti-infective agents. As provided by Example 10, culturing of isolated adult pulmonary stem cells in the presence of irradiated pulmonary stroma cells allows the stem cells to maintain their undifferentiated phenotype, and expressing Oct-4 for several weeks. The, stroma cells, in contrast to the stem cells, express smooth muscle α-actin, CD44, and CD90, and can partially been induced to become adipocytes, thus appearing as some form of mesenchymal cells. Long term robustness of stem cell cultures with a stabilized phenotype may be particularly useful for (1) stem cell as research tool for basic biological study, (2) tool for modeling viral infection process, (2) tool for identifying, screening, developing anti-infective agents.

According to a tenth aspect of the invention, an in vitro stem cell population derived from pulmonary tissue is disclosed. This population is resident in pulmonary tissue at a very low incidence, but can be isolated from the dominant population of non-stem cell pneumocytes and expanded in an in vitro system. The cell population is identifiable in the form of slow growing colonies that emerge from primary cultures of pulmonary cells from mammalian donors such as humans and mice. As provided by embodiments of the invention, slow growing colonies are visually apparent and may be physically plucked from cultures and transferred to another culture, where the population of cells grows and expands in number. Such a population is highly enriched in the incidence of stem cells in contrast to the non-stem cell pneumocytes simply by their physical capture to the substantial exclusion of other types of cells. Such an isolated population, particularly after expansion, may be further purified or enriched in stem cell constituency through cell sorting procedures. These sorting procedures, as provided by exemplary embodiments of the invention, exert a positive selection on the basis of the expression by the cells of stem cell markers. The in vitro culture system, as further provided by this tenth aspect of the invention, includes a defined mammalian cell culture medium, the medium including at least one growth factor, at least one metabolic hormone, and at least one cell- available iron source. The growth factor may be epidermal growth factor (EGF), or it may be other growth factors such as fibroblast growth factor (FGF), or any other suitable growth factor. The metabolic hormone may be insulin, or any analogue of insulin, or any other suitable metabolic hormone. The cell-available source of iron may be transferrin, or any combination of iron salts that cells can utilize in culture. Embodiments of the in vitro culture system may be used in the primary culture, and they may also be used in further culture of the isolated population that serves to expand the population. Embodiments of the in vitro system may further include treatments or alterations of the unadorned plastic surface of the culture vessel. Such treatments may include, for example, a coating of collagen, or any other material that promotes attachment and growth of the stem cell population in vitro. The in vitro system may include other features that promote the growth of the population or stabilize the phenotype in an undifferentiated state. Irradiated pulmonary stroma cells in co-culture, for example, as disclosed herein, stabilize the undifferentiated phenotype. The cell population has features characteristic of stem cells, such as the expression of Oct-4, and the ability to differentiate to a mature phenotype in culture. The cell population may further express other markers of undifferentiated stem cells, such the expression of of SSEA-I, SSEA-3, SSEA-4, Sca-1 , or Nanog. Such a pulmonary stem cell population may be useful, as noted above, as a research tool, or, more specifically for the development of models and diagnostic tools related to viral infection and for the screening and characterization of anti-infective agents. Further, the disclosed stem cell population may be directed toward the development of treatments for lung injury and disease.

An aspect of the invention related to the provision of a population of pulmonary stem cells is the application of these methods of isolation and identification of stem cells to stem cell populations resident in formed organs and tissues other than lung, such as, merely by way of example, heart, intestine, or kidney. The applicants have made the inventive realization that Oct-4, while conventionally understood as a marker for embryonic stem cells, in fact, is a marker broadly associated with stem cells resident in other organs, and that such cells may have, in addition to the Oct-4 marker, other markers that are tissue- or organ- specific. Accordingly, the applicants anticipate that stem cells resident in other tissues, and specific-to that site, or differentiated at least to some degree, will grow out in a clonogenic manner, as do the pulmonary stem cells, simply by virtue of being stem cells. Such cells will be identifiable by fundamental stem cell marker such as Oct-4, and other markers, some of which may be specific to the tissue. These markers will be tools which can be used to select in favor of stem cells in a cell sorting procedure, so as to enrich the population in the stem cell dominance, and to generally favor for increased homogeneity with regard to phenotype.

Still further, in an aspect related to the provision of a population of stem cells is provided, so also may be cohorts of genotypically homogeneous populations. As described herein, populations that emerge from primary culture and which are then expanded into populations useful for research, diagnostic, screening, or therapeutic purposes are clonal, as such, in addition to being genotypically identical (as they are by virtue of being from the same donor individual), they are also phenotypically homogeneous, the phenotype reflecting tissue or organ specific features as well as differentiated state. Any given donor tissue or biopsy can generate multiple such clonal populations. Such lines may be considered sibling or cohort populations, genotypically identical, but varying to small degrees to whatever degree the colony founder cells vary among themselves. BRIEF DESCRIPTION OF FIGURES

The patent application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

The above-mentioned features and objects of the present disclosure will become more apparent with reference to the following description taken in conjunction with the accompanying drawings wherein like reference numerals denote like elements and in which: Fig. 1 shows immunohistochemical staining for BrdU (bromodeoxyuridine) of formalin fixed lung tissues of neonatal mouse, immediately after five day injections; BrdU retaining cells are shown as darker grey stains; BrdU non-retaining cells are shown as grey stains. Fig. 2 shows immunohistochemical staining chase for BrdU of formalin fixed lung tissues of neonatal mouse, for 4 weeks after BrdU labeling; BrdU retaining cells are shown as darker grey stains and are indicated by arrows; BrdU non- retaining cells are shown as grey stains.

Fig. 3 shows immunohistochemical staining 4 weeks after BrdU labeling of formalin fixed lung tissues of neonatal mouse in a serial section (5 to 10 microns in thickness of the section showed in fig. 2 with anti-pancytokeratin antibodies (AE1/3) to demonstrate the bronchiolar area (dark grey stains); and lightest grey stains in nuclei of all cells in the tissue section.

Fig. 4 shows immunohistochemical staining of formalin fixed lung tissues of neonatal mouse with antibodies directed against Oct-4; Oct-4 expressing cells are shown as very dark grey stains indicated by arrows; Oct-4 non-expressing cells are shown as grey stains.

Fig. 5 shows immunohistochemical staining of formalin fixed lung tissues of neonatal mouse in a serial section (5 to 10 microns in thickness of the section showed in fig. 4 with anti-pancytokeratin antibodies (AE1/3) to demonstrate the bronchiolar area (medium grey stains); and very dark grey stains in nuclei of all cells in the tissue section.

Fig. 6 shows immunohistochemical double staining for BrdU and Oct-4 of formalin fixed lung tissues of neonatal mouse, for 4 weeks after BrdU labeling; Panel A shows staining for BrdU wherein BrdU retaining cells are shown as grey and light grey stains indicated by arrows; panel B shows staining for Oct-4 wherein Oct-4 expressing cells are shown as grey and light grey stains indicated by arrows; panel C shows merged images of Panel A and B, wherein the stains are indicated by asterisks. Fig. 7 shows a phase-contrast photograph of a primary culture from lung tissue of neonatal mouse; a pulmonary epithelial colony is indicated by arrow. Fig. 8 shows a diagram reporting the results of a flow cytometric sorting of pulmonary cells isolated from lung tissues; on the x axis the relative numbers indicating the size of the cells are shown; on the y-axis the fluorescence intensity is shown. The R1 region indicates the size of cells as well as the relative fluorescence intensity after incubation of cells with 2-bromoacetamidoethyl sulfonamide, a fluorogenic supravital dye. The R1 region refers to the subpopulation of cells with high fluorescence intensity and large size of cells.

Fig. 9 shows a phase-contrast photograph of a primary culture from lung tissue of neonatal mouse after enrichment; large pulmonary epithelial colonies are indicated by arrows.

Fig. 10 shows a phase-contrast photograph of a primary pulmonary cell culture.

Fig. 11 shows immunohistochemical staining of the primary pulmonary cell culture of fig. 10 with polyclonal antibodies anti-Oct-4; Oct-4 expressing cells are shown as grey stains; panel A is an amplified image of the area defined by the white box in fig. 11, the amplified image showing Oct-4 expression in the nuclei of the cells.

Fig. 12 shows counter staining with DAPI of the immunostained primary pulmonary cell culture of fig. 10 merged with immunostaining pattern of fig. 10; cell nuclei are shown as light grey stains; Oct-4 expressing cells are shown as dark stains.

Fig. 13 shows a phase-contrast photograph of a primary pulmonary cell culture.

Fig. 14 shows immunohistochemical staining with antibodies anti-SSEA-l; SSEA-1 expressing cells are shown as grey stains.

Fig. 15 shows counter staining with DAPI of the immunostained primary pulmonary cell culture of figure 13 merged with immunostaining pattern of fig. 14; cell nuclei are shown as light grey stains; cytoplasm of SSEA-I expressing cells are shown as grey stains; panel A is an amplified image of the area defined by the white box in fig. 15, the amplified image showing SSEA-1 was found on the cell surface and in cytoplasm of the pulmonary colony cells shown in fig. 15. Fig. 16 shows a phase-contrast photograph of a primary pulmonary cell culture.

Fig. 17 shows immunohistochemical staining with antibodies with antibodies anti Sca-1; Sca-1 expressing cells are shown as grey stains. Fig. 18 shows counter staining with DAPI of the immunostained primary pulmonary cell culture of figure 16 merged with immunostaining pattern of fig. 17; cell nuclei are shown as light grey stains; cytoplasm of Sca-I expressing cells are shown as grey stains; panel A is an amplified image of the area defined by the white box in fig. 17, the amplified image showing Sca-1 was found on the cell surface and in cytoplasm of the pulmonary colony cells shown in fig. 17.

Fig. 19 shows (A) phase contrast photograph and (B) the respective immunostaining merged with DAPI of primary pulmonary cultures treated with specific antibodies directed against cytokeratin-7; nuclei of all cells are shown as light grey stains; cytokeratin-7 positive cells are shown as grey stains. Fig. 20 shows (A) phase contrast photograph and (B) the respective immunostaining merged with DAPI of primary pulmonary cultures treated with specific antibodies directed against Clara cell secretion protein ; nuclei of all cells are shown as light grey stains; Clara cell secretion protein positive cells are shown as grey stains. Fig. 21 shows (A) phase contrast photograph and (B) the respective cytochrome P450 enzyme staining; cytochrome P450 enzyme activities in the cells is shown by grey stains.

Fig. 22 shows the RT-PCR analysis for the transcription of mRNA for Oct-4 in the cells of pulmonary epithelial colonies; results of RT-PCR performed on mouse testis Sertoli cell (TM4) for negative control is shown on lane 1 ; results of RT-PCR performed on cells picked up from pulmonary epithelial colonies is shown on lane 2; results of RT-PCR performed on mouse embryonic germ cells for positive control is shown on lane 3; a marker for molecular weight is shown on the M lane; the position of glyceraldehyde-3-phosphate dehydrogenase (GADPH), used as internal standard, is indicated by an asterisk.

Fig. 23 shows the RT-PCR analysis for the transcription of mRNA for Sca- 1 in the cells of pulmonary epithelial colonies; results of RT-PCR performed on VeroEδ cells for negative control is shown on lane 1 ; results of RT-PCR performed on cells picked up from pulmonary epithelial colonies is shown on lane 2; results of RT-PCR performed on BW5147 cells for positive control is shown on lane 3; a marker for molecular weight is shown on the M lane; position of GADPH used as internal standard is indicated by an asterisk.

Fig. 24 shows a phase-contrast photographs of the cells after subculture for day 5.

Fig. 25 shows immunohistochemical staining merged with DAPI counter- staining of the primary pulmonary cell culture of fig. 24 with anti-surfactant protein C antibodies; cells expressing surfactant protein C are shown as grey stains; the nuclei of the cells are shown as very light stains.

Fig 26 shows a phase-contrast photograph of the cells after subculture for day 9.

Fig. 27 shows immunohistochemical staining merged with DAPl counter- staining of the primary pulmonary cell culture of fig. 26 with anti-aquaporin-5 antibodies; cells expressing aquaporin-5 are shown as grey stains; the nuclei of the cells are shown as very light stains.

Fig. 28 shows a phase contrast photographs for pulmonary epithelial cells infected with SARS-CoV for 8 h; the pulmonary epithelial colonies are indicated by an arrow.

Fig. 29 shows immunohistochemical staining of the primary pulmonary cell culture of fig. 28 with antibodies against nucleocapside protein of SARS-CoV; positive cells are shown as grey stains.

Fig. 30 shows counter staining with DAPI of the immunostained primary pulmonary cell culture of figure 29 merged with immunostaining pattern of fig. 29; nuclei of all cells are shown as light grey stains; SARS-CoV positive cells are shown as grey stains; panel A is an amplified image of the area defined by the white box in fig. 30, the amplified image showing that SARS-CoV was found on the cell surface and in cytoplasm of the pulmonary colony cells shown in fig. 30. Fig 31 shows a phase contrast photograph for pulmonary epithelial cells infected with SARS-CoV for 24 h; the pulmonary epithelial colonies are indicated by an arrow. Fig. 32 shows immunohistochemical staining of the primary pulmonary cell culture of fig. 31 with antibodies against nucleocapside protein of SARS-CoV; positive cells are shown as grey stains.

Fig. 33 shows counter staining with DAPI of the immunostained primary pulmonary cell culture of figure 32 merged with immunostaining pattern of fig. 32, nuclei of all cells are shown as light grey stains; SARS-CoV positive cells are shown as medium grey stains; panel A is an amplified image of the area defined by the white box in fig. 33, the amplified image showing that SARS-CoV was found on the cell surface and in cytoplasm of the pulmonary colony cells shown in fig. 33. Fig 34 shows a phase contrast photograph for pulmonary epithelial cells infected with Influenza A/WSN/33 virus for 8 h; the pulmonary epithelial colonies are indicated by an arrow.

Fig. 35 shows immunohistochemical staining of the primary pulmonary cell culture of fig. 34 with anti-influenza A virus specific antibodies; positive cells are shown as medium gray stains.

Fig. 36 shows counter staining with DAPI of the immunostained primary pulmonary cell culture of figure 35 merged with immunostaining pattern of fig. 35; nuclei of cells are shown as blue stains; influenza A positive cells are shown as medium grey stains. Fig. 37 shows an electron micrograph of a primary pulmonary cell culture after 16 hours post-infection of SARS-CoV; swollen Golgi vesicles in the cells cytoplasm are indicated by arrows.

Fig. 38 shows a higher magnification of the electron micrograph of fig. 37 wherein the swollen Golgi vesicles in the cells, cytoplasm are indicated by arrows; SARS Co-V are shown as black particles inside the Golgi vesicles.

Fig. 39 shows a higher magnification of the electron micrograph of fig. 37 wherein SARS Co-V are shown as black particles; a SARS Co-V particle with spike proteins attached to the plasma membrane to enter via coated pit mediated is shown by an arrow. Fig. 40 shows a phase contrast photograph for pulmonary epithelial cells wherein pulmonary epithelial colonies are indicated by an arrow. Fig. 41 shows immunohistochemical staining of the primary pulmonary cell culture of fig. 40 with anti-ACE2 specific monoclonal antibody to characterize putative receptors for SARS-CoV in pulmonary epithelial colonies; positive cells are stained and show as white against a dark background. Fig. 42 shows counter staining with DAPI of the immunostained primary pulmonary cell culture of figure 41 merged with immunostaining pattern of fig. 41 ; nuclei of all cells are shown as blue stains; ACE-2 positive cells are stained and show as white and grey against a dark background; panel A is an amplified image of the area defined by the white box in fig. 42, the amplified image showing that ACE-2 was found on the cell surface and in cytoplasm of the pulmonary colony cells shown in fig. 42.

Fig. 43 shows a diagram reporting the bioactivity of SARS-CoV particles produced from a pulmonary primary culture; on the x-axis the post-infection time where the SARS-CoV particles are produced is reported; on the y-axis the titration of the SARS-CoV particles expressed as plaque numbers in outcome of a plaque assay is reported.

Fig. 44 shows a phase contrast photography of VeroEδ cells after infection with SARS-CoV particles produced by a pulmonary primary culture; cells showing cytopathic effects formation are indicated by arrows. Fig. 45 shows immunostaining for SARS-CoV used with antibodies directed against the nucleocapside of SARS-CoV; positive cells are shown as bright stains.

Fig. 46 shows identification of BrdU label-retaining and Oct-4 expression cells in the mouse lung. A: immunohistochemical staining for BrdU (bromodeoxyuridine) of formalin fixed lung tissues of neonatal mouse, immediately after five day injections and for 4 weeks after BrdU labeling; BrdU retaining cells are shown as darker clump stains; BrdU non-retaining cells are shown as very dark grey stains. B: immunohistochemical staining 4 weeks after BrdU labeling of formalin fixed lung tissues of neonatal mouse in a serial section (5 to 10 in thickness of the section showed in A with anti-pancytokeratin antibodies (AE1/3) to demonstrate the bronchiolar area (medium grey stains); and very dark grey stains in nuclei of all cells in the tissue section. C: immunohistochemical staining of formalin fixed lung tissues of neonatal mouse with antibodies directed against Oct-4; Oct-4 expressing cells are shown as circled (with dots) stains indicated by arrows; Oct-4 non- expressing cells are shown as blue stains. D: immunohistochemical double staining for BrdU and Oct-4 of formalin fixed lung tissues of neonatal mouse, for 4 weeks after BrdU labeling; panel BrdU shows staining for BrdU wherein BrdU retaining cells are shown as medium gray stains indicated by arrows; panel Oct-4 shows staining for Oct-4 wherein Oct-4 expressing cells are shown as bright stains indicated by arrows; panel BrdU/Oct-4 shows merged images of Panel BrdU and Oct-4, wherein the stains are indicated by asterisks. Fig. 47 shows the flow chart of a method to enrich pulmonary epithelial cells in vitro by a novel culture system.

Fig. 48 shows the immunofluorescence labeling of the stem cell markers in the pulmonary clonogenic stem cells. Top panels show phase-contrast photographs of primary pulmonary cell cultures. A: the immunohistochemical staining of the primary pulmonary cell culture with polyclonal antibodies anti-Oct-4; Oct-4 expressing cells are shown as medium grey outline and light grey center stains; the amplified image showing Oct-4 expression in the nuclei of the cells. B-D: immunohistochemical stainings with antibodies anti-SSEA-l, Sca-1, and Nanog. All counter staining with DAPI of the immunostained primary pulmonary cell culture merged with immunostaining patterns are shown in the bottom panels; cell nuclei are shown as light grey stains; stem cell marker- expressing cells are shown as medium grey stains.

Fig. 49 shows the characteristics of pulmonary clonogenic stem cells. A: Primary cells cultures were examined using specific antibodies directed against cytokeratin-7 (CK-7). B: To investigate the expression of putative receptors for SARS-CoV in pulmonary epithelial colonies, anti- ACE2 specific antibodies were applied. C: The cells were also examined for CCSP. D and E: Other two antibodies were applied to against to peroxiredoxin-2 (Prx-2) and peroxiredoxin-6 (Prx-6), respectively. Both phase-contrast photographs and the respective immunostaining merged with DAPI were shown. F: The CyP450 enzyme activities were examined , and shown.

Fig. 50 shows the expression of stem cell markers in the pulmonary stem cells by RT-PCR and quantitative -PCR. A: The RT-PCR analysis for the cells of individually plucked pulmonary colonies. For Sca-1 , the following cells were prepared and analyzed: lane 1, A549 cell line (negative control); lane 2, the cells plucked from pulmonary epithelial colonies; and lane 3, BW5147 cells (positive control). For Oct-4, lane 1 was TM4 cell line (negative control); lane 2 was the cells picked up from pulmonary epithelial colonies, and lane 3 was mouse R1 embryonic line (positive control). GAPDH was used as internal standard for both reactions. B and C are graphic presentations for quantitative RT-PCR experiment. B: Two primer sets were added in each PCR reaction to quantify GAPDH and Oct-4 simultaneously. Two fluorescence profiles will be acquired for each sample including J1 , 46c and R1 ES cells, pulmonary colony cells (colony), mouse embryonic fibroblasts (MEF) and negative control (H₂O). The fast raising curve (left) is for GAPDH, and the slower one (right) for Oct-4 respectively. The bold dashed horizontal line in the graph represents the threshold for the Ct determination. C:The values of Oct-4 expression for J1 ES cells, pulmonary colony and MEF were then normalized with respect to GAPDH and shown in a bar chart. D: Q-PCR analysis of Oct-4 expression in pulmonary colony cells with respect to J1, 46c and R1 embryonic stem cells; in contrast, mouse embryonic fibroblasts (MEF) were used as negative control. The results represent the average of three independent experiments with standard deviations. Fig. 51 shows the differentiation potential of pulmonary stem cell colonies.

The expression of alveolar cell markers, surfactant protein-C (SPC, a type-2 pneumocyte marker) and aquaporin-5 (Aqp-5, a type-1 pneumocyte marker), were examined of the cells after clone transfer at day 5 and day 9 or 11 , respectively. In primary cell culture, cell (Oct-4+) in the pulmonary epithelial colonies were picked and subcultured onto irradiated 5-day-old primary lung cell cultures or collagen-l coated plate with conditioned media. On the right, the picked colony cells were subcultured onto irradiated 5-days primary culture cells and the spherical colonies were observed on day 5. The colonies were stained Oct-4 positive. The spherical colonies maintain Oct-4 expression but with lower expression level in day 11. On the left, the picked cells were subcultured onto collagen-l coated plates and on day 5, the cells were stained positive with SPC protein. On day 9, the phase-contrast photograph of the cells indicated that morphology of individual cells changed and cells were flattened and the cells were stained positive with aquaporin-5 protein. Fig. 52 shows the identification of the pulmonary stem cells as the primary target for H5N1 infection. Confluent primary pulmonary cultures were infected with H5N1 in P3 facility and at 2, 2.5, and 3 day post-infection (p.i.), cultures were processed for immunostaining. Phase contrast photographs for pulmonary primary cultures were shown in panel A, D, and G. Immunostaining of infected cells within colony were shown (green) using antibodies specifically directed against H5 and counter-stained with DAPI.

Fig. 53 shows the identification of the pulmonary stem cells as the primary target for influenza A virus infection. Confluent primary pulmonary cultures were infected with H1 N1 , H1 N2, and H2N2 in P4 facility and at 5 day post-infection, cultures were processed for immunostaining. Phase contrast photographs for pulmonary primary cultures were shown in panel A, D, and G. Immunostaining of infected cells within colony were shown (red) using antibodies directed against H1 and H2, respectively and counter-stained with DAPI. Fig. 54 shows electron micrographs of H5N1 infected cells. Electron micrographs of infected cells at 2-3 day post infection were shown to have replicated virus particles.

Fig. 55 shows the identification of the receptors in pulmonary stem cell colony for both human influenza A (green, α-2,6 linkage) and avian flu virus (red, α- 2,3 linkage). Phase contrast photographs for pulmonary primary culture and staining of the receptors in pulmonary stem cell colony with specific lectins for human influenza A, NeuδAc- α-2,6-Gal (white) and avian flu virus, NeuδAc- α-2,3-Gal (white), were shown.

Fig. 56 shows the co-localization of nuclear Oct-4 (medium gray solid circles) and cytoplasmic SARS-CoV nucleocapsid proteins (white donut-shaped, surrounding the nucleus) in the same cells. Confluent primary pulmonary cultures were infected with SARS-CoV at 0.5 MOI and at 8 h post-infection, cultures were processed for dual staining of Oct-4 and SARS coronavirus. Immunostaining of cells within colony were shown (green) using antibodies directed against Oct-4 and 20- 30% of cells in colony shown as white against dark using antibodies directed against nucleocapsid protein of SARS-CoV. The merged image of Oct-4 and SARS-CoV and counter-stained with DAPI were shown. Fig. 57 shows the characteristics of the stromal cells surrounding the pulmonary stem cell colonies. The primary pulmonary cultures were stained with anti-smooth muscle actin and CD44. In addition, the culture was induced for adipocyte differentiation and stained for adipocytes. DETAILED DESCRIPTION OF THE INVENTION

Identifying and Isolating Adult Pulmonary Stem Cells

According to a first aspect, methods for identifying adult pulmonary stem cells and isolating them for subsequent culture in vitro are provided. "Stem cells" refers to primal, undifferentiated cells that retain an ability to grow or to differentiate into other cell types. In particular, the stem cells herein disclosed are a rare subpopulation of pulmonary cells with the characteristics of stem cells, identifiable in lung tissue of mammals such as mice and humans. The term "tissue" refers to a group of cells along with their associated intercellular substances, the cells, of one or several types, serving a specific function within a multicellular organism, such as a connective tissue or an epithelium.

The identified pulmonary stem cells are slow cycling cells and express Oct- 4, a marker associated with embryonic stem cells. The phrase "slowly cycling cells" or "slowly dividing cells" refers to cells that stand out against a population of rapid- dividing cells, based on DNA dye-retaining assay. The slowly dividing cells grow as visible colonies (clonogenic cells) that can be plucked or harvested from the cell cultures and either analyzed in various ways, or subcultured in vitro, so as to expand the cell population. Cells derived in this manner from primary culture, are thus highly enriched in the type of cell that is growing, in comparison to the initial population of cells present in the primary culture as a whole. Procedures appropriate for identifying pulmonary stem cells in situ are described in Example 1, procedures for isolating such cells and initiating their primary culture are described in Example 2.

Stem Cells Markers and Characteristics

The term "marker" refers to a protein, metabolite, gene, other compound, or biological event which is indicative of a relevant biological condition of a biological material, wherein a "biological condition" is a state or state of being of or relating to biology or life and living processes, and "biological material" is any material able of self-replication under appropriate condition, such as viruses, eukaryotic or prokaryotic cells, unicellular or multicellular organism, and other material identifiable by a person skilled in the art. For marker Oct-4, the biological material is a mammalian cell, and the biological condition is primal undifferentiated state of these cells, wherein they retain the ability to grow or to differentiate into other cell types, which characterize the stem cells.

Markers, typically proteins, DNA sequences, metabolites or biological events, are detectable by procedures known to a person skilled in the art, which include use of "identifiers" specifically suitable to detect the marker. An "identifier" is a molecule, for example, a metabolite, a protein, such an antibody or a cellular protein, a nucleotide, such DNA or RNA oligonucleotides, that reflects the existence, presence, or fact of or otherwise detects a marker; exemplary identifiers are primary and secondary antibodies and oligonucleotides as described in the examples, exemplary procedures are immunostaining, immunofluorescence, and reverse transcriptase - ploymerase chain reaction (RT-PCR) methodologies, as described in the examples. The term "express" or "expression" refers to a process by which a marker manifests in a cell, for example when the marker results from a gene's coded information, "expression" refers to the process by which the information is converted into the marker, and when the marker is a biological event such as BrdU retention, the process by which the biological event is initiated.

The pulmonary stem cells can also express stem cell specific markers other than Oct-4 and/or markers associated with biological conditions such as specific cell type, cell lineages and/or cell status. The phrase "cell type" refers to a morphological or functional form of a cell, distinct from other cell types. The phrase "cell lineage" refers to the ancestry of a particular cell type, including ancestral cells and all of the subsequent cell divisions that occurred to produce the cell type. The phrase "cell status" refers to a state or condition of a cell at a given time. Exemplary combinations of markers expressed by the pulmonary stem cells are described in the examples with reference to human and mice pulmonary stem cells. The pulmonary stem cells form individual colonies in vitro and are able to differentiate into a mature phenotype. The term "colony" refers to a group of cells, the descendants of a single cell, the cells thus being "clonal" and thus substantially identical genotypically; such colonies typically grow in the form of a contiguous aggregation outward from the founder cell. The term "cell culture" refers to the in vitro (i.e., "in glass", outside of the body) propagation or cultivation of cells isolated from living organisms. The term "differentiate" refers to the process cells undergo as the cells mature into a distinct, or differentiated, cell type, having distinct characteristics and specific functions, which typically, but not uniformly, and are less likely to divide. The term "phenotype" refers to the total characteristics shown by a cell under a particular set of environmental factors, resulting from interaction between the genotype and the environment, wherein a mature phenotype is the phenotype displayed by the organism complete in natural growth or development. Exemplary mature phenotypes of cells from a lung tissue are the phenotypes of alveolar type 2 and type 1 pneumocytes .

In mice, the pulmonary stem cells can be identified in vivo as scattered cells located at bronchoalveolar junctions of lung tissues. In in vitro cultures, the murine pulmonary stem cells form individual colonies, and express stem cell specific antigens including Oct-4 as well as markers of epithelial and Clara cell lineages and peroxiredoxin Il (natural killer enhancing factor B). Isolated stem cells from mouse lungs in primary cultures express stem/progenitor markers Oct-4, Stage Specific Embryonic Antigen 1 (SSEA-I), Nanog, and Sca-1 as established by experimental procedures exemplified in Example 3. The murine pulmonary stem cells also express cytokeratin 7, a marker of epithelial cells, not detected in the surrounding cells of lung epithelium. Additionally, the murine pulmonary stem cells express Clara cell secretion protein (CCSP) and display cytochrome p450 activities, as well as peroxiredoxin Il and Vl (see Example 3).

The murine pulmonary stem cells do not express other lung epithelial markers such as cytokeratin 5/8, 18 and 19, nor surfactant protein C or aquaporin-5, markers for alveolar type 2 and type 1 pneumocytes respectively (15, 16, 17, 18) (Example 3). The murine pulmonary stem cells have the capacity to undergo terminal differentiation to alveolar type 2 and type 1 pneumocytes as established by experimental procedures exemplified in Example 4. In /π vitro primary culture, pulmonary stem cells from humans express stem cells specific antigens such as Oct-4⁺, SSEA-3\ SSEA-4⁺ and Sca-1 ⁺, as detected following procedures analogous to the ones exemplified in Example 3. Human pulmonary stem cells are also able to form colonies and to differentiate into mature phenotypes, as established by procedures analogous to the ones exemplified in Example 4. Any adjustment and/or modification in the experimental procedures herein described particular in Example 3 required by the use of human cells instead of murine cells and/or detection of markers other than the one mentioned in the examples is identifiable by a person skilled in the art upon reading of the present disclosure, in particular the Example section, and will not be described in further detail.

Identifying Adult Stem Cells

According to a further aspect a method to identify adult stem cells in a tissue typically, comprising stem cell and non-stem cell, is disclosed. The term "identify" refers to discovering or determining the existence, presence, or fact of or otherwise detect an indicated item. The phrase "non-stem cells" refers to the vast majority of cells, of any type and in any state, which do not have the characteristic of stem cells. The adult stem cells can be in particular identified by the method for identifying stem cells in a tissue herein disclosed and exemplified for pulmonary stem cells in Example 1.

Embodiments of the method described herein comprise identifying colonies of cells of the tissue that are slowly dividing, and express the stem cell marker Oct-4, wherein the slowly dividing Oct-4 expressing cells are able to form colonies in vitro, the identified slowly dividing Oct-4 expressing cells, upon isolation in primary culture, able to differentiate in a mature phenotype. Identifying the cells of the tissue which are slowly dividing, can be performed by detecting a marker associated with slowly dividing cells. For example, identifying the slowly dividing cells of the tissue can be performed by detecting the cells that retain agents such as BrdU (see procedures described in Example 1 for pulmonary tissue) for a time period that is based on the,- cellular turnover rate of the tissue investigated. For murine pulmonary tissues, accumulation of BrdU after injection can be monitored for a time period of from about 1 week to about 5 weeks, in particular 4 weeks (see Example 1). Other tissues may require a different time of observations which could be days or weeks. Identifying Oct-4 expressing cells can be performed by detecting the expression of marker Oct-4 in the cells using any suitable identifier and methods known by a person skilled in the art upon reading of the present disclosure. Exemplary identifiers, such as antibodies and oligonucleotides, and exemplary methods such as immunofluorescence, RT-PCR and quantitative real time PCR, are shown in the Examples 2 and 3. In some embodiments, identifying the cells which are slowly dividing and express marker Oct-4 can be performed by identifying slowly dividing cells and identifying the slowly dividing cells that express Oct-4 or other embryonic stem cell marker, as exemplified in Examples 1, 2, and 3.

Quantitative PCR is a method that is powerful simply because it is quantitative, and permits, for example, high resolution comparisons of gene expression between various types of cells. Methods such as immunofluorescense are very useful as an approach to detecting the presence or absence of a marker, and the integration of the visual information from a micrographic image of a cell, with diagnostic staining for the presence of a marker, and especially with staining for multiple markers, showing co-localization, is useful and powerful for what it does deliver, but it does not provide quantitative and comparative information of the type that quantitative PCR delivers.

A Kit for Identifying Stem Cells

An identifier of slowly dividing cells and an identifier of the above listed markers can be included in a kit of parts for the identification of stem cells in a tissue, particularly stem cells from lung tissue, as provided by embodiments of this invention.

In one embodiment, the kit of parts can comprise a first identifier for the identification of slowly dividing cells and a second identifier for the identification of the expression of Oct-4 marker, the first and the second identifier to be used in any the methods to identify stem cells herein disclosed. In particular, the first and the second identifier to be used in a method for identifying stem cells of a tissue, wherein the first identifier is used to identify slowly dividing cells of the tissue and the second identifier is used to identify the cells expressing Oct-4 marker of the tissue, wherein the identified slowly dividing cells expressing Oct-4 marker, which are able to form a colony in vitro and to differentiate into an mature phenotype are the stem cells of the tissue.

Embodiments of a kit can further include a third identifier for identification of tissue stem cells specific markers] such as SSEA-1 , Nanog, and Sca-1 for murine pulmonary stem cells and SSEA-3, SEEA-4, Nanog, and Sca-1 in human pulmonary stem cells. The kit can further include a fourth identifier of the mature cell phenotype.

The first, second, third and fourth identifiers can be provided in kit embodiments, with suitable instructions and other necessary reagents, in order to perform the methods herein disclosed. A kit will normally contain the identifier in composition included in separate containers. Instructions, for example written or audio instructions, on paper or electronic support such as tapes or CD-ROMs, for carrying out the assay, will usually be included in the kit. A kit can also contain, depending on the particular method used, other packaged reagents and materials (i.e. wash buffers and the like).

The identified slowly dividing Oct-4 expressing cells of the adult tissue can be then tested for the ability to form colonies and differentiate into a mature phenotype, by procedures and examples herein described and/or identifiable by a person skilled in the art upon reading of the present disclosure. To this extent, the tissue cells can be cultivated in vitro according to any suitable procedure to cultivate the tissue cell identifiable by a person skilled in the art upon reading of the present disclosure, based on the type of cells to be cultivated. Further details concerning the identification of the identifier additional component to be included in the compositions, and generally manufacturing and packaging of the kit, can be identified by the person skilled in the art upon reading of the present disclosure. Additional kits of parts for performing at least one of the other methods herein disclosed, the kit comprising suitable identifiers, compositions and reagents for performing such methods can be identified by a persons skilled in the art and will not be further described in details. Additional embodiments of the method to identify adult stem cells wherein the above steps are performed in a different order and/or by additional procedures can be identified by a person skilled in the art and will not be further described in details.

Cultivating Stem Cells

In some embodiments of the present invention, the adult stem cells, particularly pulmonary stem cells, can be cultivated according to a method for cultivating adult stem cells in vitro herein disclosed. As provided by embodiments of the invention, the source of pulmonary cells is typically from a mammal, examples of mammals including humans and rodents, examples of rodents including laboratory mice. The method can comprise: providing a tissue comprising tissue cells, isolating the tissue cells from the tissue. The term "isolate" generally refers to a procedure to separate the tissue cells from other tissue material, and more specifically refers to. the separation of stem cells or putative stem cells from non-stem cells, non-stem cells being various types of differentiated cells. The tissue cells can be isolated by treating the tissue with appropriate procedure to release the tissue cells such as the ones described in Example 2.

The released tissue cells can be then incubated in a suitable medium for a variable time which can be determined on the basis of the type of cells to be cultivated and the desired number of cell colonies to be formed. The term "incubate" refers to place the cells under favorable conditions to grow and develop. The term "medium" refers to a substance used to provide nutrients for cell growth, according to a technology to cultivate cells of mammal origin, in a laboratory or production- scale device {i.e., in vitro). The medium may be liquid (e.g., broth) or solid (e.g., agar) and may include particular components (the right amounts of amino acids, glucose, vitamins, salts, and other minerals). As provided by embodiments of this invention, these methods are broadly appropriate for cultivating cells of mammalian origin. More specifically, such cells may be, for example, of human or rodent origin; an exemplary rodent is the laboratory mouse, cells from a mouse may be said to be murine. For pulmonary stem cells, the culture time period can be from 5 to 15 days. The medium can be selected on the basis of the nature of the tissue and cells to be cultivated, and the incubation performed according to procedures identifiable by a person skilled in the art and preferably including procedures suitable to enrich the tissue cells, such as centrifugation and resuspension in appropriate medium (see Example 2).

Embodiments of the method further comprise applying a cell sorting procedure such as a flow cytometric sorting procedure (the cell sorting instrument is known as fluorescence activated cell sorter, or "FACS)" to the incubated tissue cells to obtain colonies in primary culture according to procedures herein described in Example 2 and as shown in fig. 8 for pulmonary tissue, wherein any adjustment or modification for tissue cells other than pulmonary tissue cells is identifiable by a person skilled in the art upon reading of the present disclosure. The term "sorting procedure" refers to a process utilized by a user (e.g., by researchers) to sort/separate different cells; automated means of cell sorting include "biochips" (utilizing controlled electrical fields to collect specific cell types onto electrodes in the biochip), fluorescence- activated cell sorter (FACs) machines, magnetic particles (e.g., attached to antibodies), efc. The colonies so obtained can then be screened to identify the colonies including Oct-4 expressing cells able to differentiate into a mature type, as herein described. Additional embodiments of the method to cultivate adult stem cells in vitro can be envisioned by a person skilled in the art upon reading of the present disclosure and in particular the Examples section and will not be further described in details.

Viral infection of stem cells

As disclosed herein, the identified pulmonary stem cells are the target- or host of various infective agents. The phrase "infective agent" refers to any biological material capable of self-replication or reproduction, such infective agents typically interfere with the normal functioning and/or survival of a host cell. Infective agents include bacteria, parasites, fungi, viruses, prions, and viroids. As used herein, the term "interfere with" refers to specifically controlling, influencing or otherwise affecting, typically in an adverse way, the item indicated thereafter, and can include regulation by activation, stimulation, inhibition, alteration or modification. For example, to interfere with the normal functioning and/or survival of a cell refers to specifically controlling, influencing or otherwise affecting the normal functioning and/or survival of the cell and can include regulation by activation, stimulation, inhibition, alteration or modification of the normal functioning and/or survival of the cell. In particular, as disclosed herein, the pulmonary stem cells are infected by viruses such as SARS-CoV, influenza, e.g. influenza A, as established by experimental procedures exemplified in Example 5. The pulmonary stem cells are also infected with various types of avian flu such as H11N2 and H3N2, as verifiable by procedures analogous to the ones exemplified in Example 5. As disclosed herein, the pulmonary stem cells are also selectively targeted by SARS-CoV as shown in Example 5, wherein SARS-CoV are shown to infect the pulmonary stem cells and not the cells surrounding the pulmonary colonies. Production of infective agents

In some embodiments, the pulmonary stem cells can be used for the production of infective agents, e.g. virus , such as Hanta virus, influenza viruses, including of H1N1, H1N2, H2N2, or H5N1, and avian flu viruses, such as SARS and SARS-CoV. In one particular embodiment, exposure of the pulmonary stem cells to SARS-CoV leads to selective productive infection of the putative stem cells and the replication and release of infectious SARS-CoV particles as exemplified in Examples 5 and 6. Accordingly, the method can comprise contacting an infective agent, such as a SARS virus particle with an isolated pulmonary stem cell in a culture; and collecting the SARS virus particles produced from the culture.

The term "contact" or "contacting" refers to placing the pulmonary stem cell and an infective agent or other biological agent, in a mutual spatial relationship such that a biological interaction between the infective agent, or biological agent and the pulmonary stem cell is feasible; the phrase "biological agent" refers to any material able to interfere with the biological condition of the pulmonary stem cells, which include protein, metabolites other compounds and biological material such as an infective agent; the phrase "biological interaction" refers to the process by which the biological agent interferes with the normal functioning and/or survival of the pulmonary stem cells. Contacting a SARS virus particle with an isolated pulmonary cell in a culture can be performed, by incubating a confluent primary culture with virus particles for a predetermined time, as exemplified in Examples 5 and 6, or by other methods identifiable by a person skilled in the art upon reading of the present disclosure.

The term "collect" or "collecting" refers to picking up or accumulating in a harvesting-like operation. Collecting infective agents such as SARS virus particles from the culture can be performed by isolating supernatants of the cultures at predetermined times after infection as exemplified in Examples 5 and 6, or by other methods identifiable by a person skilled in the art upon reading of the present disclosure. An infective agent, such as SARS virus, can be identified in the supernatants by methods available in the art such as titration and/or immunofluorescence. Titration can be performed by plaque assay carried out on cells, such as Vero cells immunofluorescence can be performed using antibodies specific for the viral particles conjugated with a fluorescence agent (see Examples 5 and 6). Analogous procedures can be performed for the production of other viruses such as influenza and avian flu viruses.

The amount of viral particles (the inoculum) incubated with the culture, the duration of incubation, and the time of collection of the viral particles produced are determined by the user based on the type of cells being cultivated, the type of viral particles being produced, the culture medium utilized, culture condition, the desired amount of viral particles to be produced and/or other factors affecting the production of the viral particles in vitro, identifiable by a person skilled in the art. Exemplary procedures are illustrated in the Examples, in particular Examples 5 and 6. Additional methods to perform the above mentioned steps can be envisioned by a person skilled in the art upon reading of the present disclosure, in particular the Examples section, and will not be further discussed in detail. Additional embodiments can be designed to produce infective agents other than SARS virus, in particular viruses such as influenza, avian flu, and other susceptible viruses, wherein any adjustment and/or modifications of the procedures herein described required by the production of infective agents other than SARS virus, are identifiable by a person skilled in the art upon reading of the present disclosure, in particular the Examples section, and will not be further discussed in details.

Identifying compounds that Interfere with infection In some embodiments, the pulmonary stem cells can be used to identify compounds able to interfere with infection of the pulmonary stem cells by an infective agent, for example SARS-CoV. To this end, one or more candidate infective-interfering compounds can be administered to the pulmonary stem cells in combination with the infective agent, e.g. SARS-CoV particles and the effect of the administration of the candidate compound on the ability of the infective agent to infect the cells, determined. As used herein, the term "interfere with an infection", refers to specifically controlling, influencing or otherwise affecting an infection, and can include regulation by activation, stimulation, inhibition, alteration or modification of the process that leads to the infection. The term "infection" refers to the growth of an infective agent within the cell.

The ability of the infective agent to infect an isolated pulmonary stem cell can be determined by detecting, upon mutual contact of the infective agent and the pulmonary stem cells, the expression of a marker or cellular feature associated with the infection of a cell by the infective agent. In some embodiments, wherein the infective agent is SARS, exemplary markers associated with SARS virus infection are the presence of vacuoles filled with viral particles in the cells, production of viral particles, and expression of a viral protein or nucleic acid associated with reproduction of the virus in the pulmonary stem cells. Other markers are identifiable by a person skilled in the art upon reading of the present disclosure.

Accordingly, in the exemplary embodiments wherein the infective agent is SARS virus, the method can comprise contacting a candidate compound with isolated pulmonary stem cell; contacting the isolated pulmonary stem cell with SARS virus; detecting a level of SARS virus infection of the isolated pulmonary stem cell, and comparing the detected level of infection with a predetermined threshold value, the threshold value indicative of development of SARS infection of the cell.

Contacting a candidate compound with the isolated pulmonary stem cell can be performed by administering the compound in a manner and according to procedures known in the art to enable a biological interaction between the compound and the isolated pulmonary stem cell. A candidate compound, as referred to herein, can be a natural compound or a synthetically derived compound and include nucleic acid, proteins, carbohydrates, lipids, and derivatives of any of the preceding thereof. Additionally, the candidate compound can have biological or chemical properties, such as totally or partially defined signal transduction regulatory properties. A candidate compound can be obtained by methods identifiable by a person skilled in the art. For example, a candidate compound can be derived by rational drug design or can be selected from libraries of natural synthetic or natural compounds, including chemical biochemical or combinatorial libraries. Exemplary candidate compounds are anti-idiotypic antibodies and/or catalytic antibodies, or fragments thereof.

Suitable procedures to contact a cell with a candidate compound in an effective manner can be accomplished by those skilled in the art based on variables such as, the conditions under which the compound is being administered, the type of cell being contacted and the chemical composition of the candidate compound (i.e., size, charge efc.) being administered. Contacting the isolated pulmonary stem cells with SARS virus can be performed by incubating SARS viral particles according to methods herein described and exemplified, and/or by additional procedures identifiable by a person skilled in the art upon reading of the present disclosure.

Detecting the levels of SARS infection of the isolated pulmonary stem cells can be performed by qualitatively and/or quantitatively determining the expression of one or more markers associated with SARS infection of the pulmonary stem cell, wherein such a quantitative and or qualitative determination results in a value, the value being a detectable property or aggregate of properties; exemplary values are the concentration of viral particles in supernatants of a cell culture or numbers of vacuoles including viral particles which are detectable in a cell. The threshold value of SARS infection can be predetermined measuring the expression of a marker associated with development of viral infection in a significant population of cells and assessing, for example, by a statistically significant experimental procedures, the value ranges associated to the occurrence of infection, and to non-occurrence of infection. In light of this assessment, by comparing the two value ranges, it will then be possible to set a threshold value such that the above the threshold are indicative of the development of infection, and the values below such a threshold are indicative of non-occurrence of infection.

The above steps can be repeated in a statistically significant number of procedures to identify the effective amount of the compound, if any. The phrase "effective amount" of a compound refers to at least the minimum amount of a compound that is necessary to minimally achieve, and more preferably, optimally achieve, the desired effect (i.e. interference with infection of pulmonary stem cells by an infective agent). An effective amount for use in a given method can be readily determined by one skilled in the art without undue experimentation, depending upon the particular circumstances encountered (e.g. concentrations, type of infective agent and number, etc.).

Additional methods to perform the above mentioned steps can be envisioned by a person skilled in the art upon reading of the present disclosure and will not be further discussed in detail. Embodiments wherein the infective agent is an infective agent other than SARS, in particular viruses, such as influenza and avian flu viruses, can be performed by procedures analogous to the procedures herein described fo SARS wherein any adjustment and/or modifications required by testing infective agents other than SARS virus, are identifiable by a person-skilled in the art upon reading of the present disclosure, in particular the Examples section, and will not be further described in details.

According to an additional aspect, the isolated pulmonary stem cells can be used to identify an infective agent able to infect pulmonary cells. The method can comprise contacting a candidate infective agent with an isolated pulmonary stem cell; detecting the level of infection of the isolated pulmonary stem cell by the infective agent; and comparing the detected level of infection with a predetermined threshold level, the threshold level indicative of development of infection of the cell.

The level of infection and the threshold level can be determined by quantitative or qualitative analysis of the expression of one or more markers associated with infection of a cell by the candidate infective agent. Exemplary procedures for performing quantitative or qualitative analysis of the expression of markers associated with infection of a cell are herein described with reference to SARS, influenza, avian flu and other susceptible viruses. Additional markers and methods to perform such a quantitative or qualitative analysis can be identified by a person skilled in the art based the nature and biology of the infective agent upon reading of the present disclosure and in particular of the Examples section.

Suitable procedure to contact a cell with an infective agent are exemplified in the present disclosure and in particular in the Examples section with reference to SARS and influenza viruses. Additional procedures to contact a cell with an infective agent on can be accomplished by those skilled in the art based on variables such as, type and biology of infective agent, conditions under which the infective is being administered, and the type of cell being contacted, upon reading of the present disclosure and in particular of the Examples section. In some embodiments, the isolated pulmonary stem cells or their derivatives (e.g. differentiated pneumocytes) can also be used in treatment of various conditions associated with ARDS. In particular, the isolated pulmonary stem cells or their derivatives can be used to identify method of treatment of ARDS or other lung diseases involving regeneration of damaged pulmonary tissue. In one embodiment, the isolated pulmonary stem cells can be delivered into lung tissues, by means of a bronchoscope or other means of aspirations to position cells to damaged tissues of individuals diagnosed with conditions associated with ARDS or other lung diseases. Additionally or in the alternative, pulmonary stem cells or their derivatives can grow on biodegradable scaffold or polymers prior to delivery. Stem cells or their derivatives grown as artificial lung tissues can be used to test for efficacy of therapeutic agents intended for treatment of these individuals. These artificial tissues can also be used to identify specific pathogen strains, which these individuals are suffering from. Method to perform delivery of isolated pulmonary stem cells or their derivatives into lung tissues or grow isolated pulmonary stem cells on scaffold are identifiable by a person skilled in the art upon reading of the present disclosure and will not be further discussed in details.

Experimental Examples The following experimental examples of embodiments of the invention are provided to describe the invention in further detail. These examples, which set forth a mode presently demonstrated for carrying out the invention, are intended to illustrate and not to limit the invention.

Example 1 : Localization of BrdU-retaininq OCT-4 expressing cells in mice lungs a. Localization of BrdU retaining cells in mice lungs (fig. 46)

Neonatal ICR mice were injected intraperitoneal^ with 50 mg/kg of 5- bromo-2'-deoxyuridine (BrdU) (Sigma) at 12.5 mg/ml in phosphate- buffered saline (PBS) twice a day for five days. Mice were then maintained without additional BrdU injection and sacrificed on day 0, weeks 1 , 2, 3 and 4 after injection. Lungs were removed, fixed in 10% formalin fixative and embedded in paraffin. The paraffin- embedded sections were then subjected to immunohistochemical analysis.

The paraffin-embedded sections were dewaxed and re-hydrated. BrdU staining was performed as described (21) with anti-BrdU-monoclonal antibodies (M 0744, Dakocytomation) at 1:100 and a peroxidase detection kit (Vector VIP Substrate Kit, Vector) with diaminobenzene (DAB) as substrate according to manufacturer's instructions. Results are shown in figs. 1 - 3, and 46A.

At day 0, actively dividing, cells including some alveolar cells, in the lung demonstrated nuclear staining for BrdU (figs 1 and 46 A-day 0). At week 2 and week 3 after labeling, the number of BrdU positive cells significantly declined. At week 4 after labeling, only a few cells among the epithelial cells still retained BrdU , labeling (figs 2 and 46-4w chase wherein the BrdU retaining cells are also indicated by brown color). The localization of the BrdU retaining cells in the bronchoalveolar area was confirmed by staining performed with monoclonal antibodies against pan- cytokeratin (fig. 46 B) (14). b. Localization of Oct-4 expressing cells in mice lungs (figs. 3 and 46)

Paraffin-embedded section of lung removed 4 weeks after labeling with BrdU were also tested for detection of Oct-4-expression, as described in (22), wherein antigen retrieval for tissue sections was carried out by heating in 10 mM sodium citrate buffer (pH 6.0) for 8 min, with the tissue sections then incubated for another 15 min in room temperature. The tissue sections were incubated overnight at 4°C with the primary antibodies directed against Oct-4 (sc-908l, Santa Cruz Biotechnology), followed by peroxidase detection. Sections were counterstained with Mayer's hematoxylin to mark unstained nuclei.

The results of this series of experiments are shown in figs. 4, 5, and 46 C. A small number of cells located in the same region where BrdU retaining cells were identified (figs. 3 and 50B) were also found to be positive for Oct-4 (figs. 4 and 46 C) wherein localization of the Oct-4 positive cells in the bronchoalveolar area was confirmed by immunostaining performed on the same section with anti- pancytokeratin antibodies (figs. 5 and 46 B).

Sections of mice lungs taken 4 weeks after labeling with BrdU were tested for double staining with anti-BrdU and Oct-4 antibodies. Tissue sections were treated as described in sections [0108-0112] above, wherein the following fluorescence-labeled secondary antibodies were used: Cy3-labeled F(ab')₂ goat anti- rabbit IgG (H+L) and FITC-labeled F(ab')² goat anti-mouse IgG (H+L) (Jackson lmmuno Research).

The results shown in figs. 6 and 46 D confirm the presence of BrdU retaining cells (see medium gray-stained cells) and Oct-4 expressing cells (see light- gray-stained cells) in mice lungs 4 weeks after labeling. Additionally, this set of experiments also demonstrates co-localization of Oct-4 expression with BrdU retention in mice lungs 4 weeks after labeling (see merged images of BrdU retaining cells and Oct-4 expressing cells shown in figs. 6B and 46 D wherein the merged images of BrdU retaining cells and Oct-4 expressing cells are indicated by asterisks). These results show the presence of slow cycling, Oct-4 expressing epithelial cells in the terminal bronchioles adjuvant to alveolar sacs in lung tissues. Furthermore, the number of Oct-4⁺ cells and BrdU LRC was determined. The analysis revealed that there were 21 + 8 dual Oct-4⁺ BrdU retaining cells in each randomly selected lung section that contained bronchoalveolar junctions. Single Oct-4^* cells or BrdU LRC were not encountered in the double staining. As estimated by histological grid analysis, there are -1.25 x 10⁶ nucleated cells per slide, suggesting the presence of a small population (-0.0016 + 0.0006%) of slow- cycling, Oct-4-expressing stem cells in the neonatal lungs.

Example 2: Isolation and Primary Cultures of Clonoqenic Oct-4 expressing lung cells

To better characterize the rare BrdU retaining Oct-4 expressing cells identified by a procedure as exemplified in Example 1, a procedure to cultivate those lung cells in vitro, was developed (Fig. 47).

Neonatal ICR mice were killed, and their lungs were removed and cut into small pieces. After washing in Hank's buffer containing penicillin (100 units/ml) and streptomycin (100 ug/ml), the tissues were treated with 0.1% protease type-XIV (Sigma) in Joklik's MEM (Sigma) at 4°C overnight. Afterward, tissues were transferred to 10% FCS_Joklik's MEM, pipetted several times to release pulmonary cells, and then filtered through a 100-um nylon cell strainer. The released cells were washed and resuspended in MCDB-201 medium containing insulin- transferrin- selenium supplements only (GIBCO). One neonatal mouse can yield -1.0 - 1.5 x 10⁶ nucleated cells in this enzyme digestion procedure, which represents -5% to 8% of the total number of cells in lung tissues. These cells were cultivated at a density of 3 x 10⁵ cells per milliliter in culture dishes coated with collagen I (10 ug/cm²; BD Bioscience). After 1 day of incubation, the primary cultures were washed with MCDB-201 medium to remove unattached cells, and fresh medium with insulin- transferrin-selenium supplement and epidermal growth factors (1 ng/ml; Invitrogen) was then added.

This cell culture medium is serum-free; the serum being functionally replaced by defined components, is termed a "defined medium". Defined cell culture components may include any one or more of a metabolic hormone, a growth factor, an iron in a cellularly-available or cell-deliverable form, as well as other components. Insulin is a metabolic hormone, and in other embodiments of the invention may be replaced by an analogue or variant of insulin, or another metabolic hormone with effects similar to those of insulin. Epidermal growth factor (EGF), in other embodiments, may be substituted with another growth factor, such as fibroblast growth factor (FGF) that has similar effects as those of epidermal growth factor. Transferrin represents an iron transport system, and in other embodiments may be replaced with another iron transport system of similar efficacy as transferrin. The lung tissue from one neonatal mouse can yield approximately 1.0 - 1.5 x 10⁶ cells in this digestion procedure. Cells were cultivated at a concentration of 3 x 10⁵ /mL per well in 12-well Petri dishes which were coated with type I collagen (10 μg/cm²). Pulmonary cells were isolated from lung tissue from neonatal mice and grown in MCDB-201 medium supplemented with insulin, transferrin, and epidermal growth factor.

After 7-10 days of culture, a phase-contrast photograph for primary culture of lung tissue from neonatal mouse was taken as shown in figs. 7 and 47. The phase contrast photograph shows small, morphologically recognizable colonies. Cells in the colonies shown in fig. 47, were densely packed, highly reflectile, and easily distinguishable from the surrounding spindle shaped cells under phase contrast microscopy.

Pulmonary cells obtained from lung tissues as reported in section (a) and (b) of this example, were incubated at 1 x 10⁷ cells/mL in MCDB-20I medium with 2- bromoacetamidoethyl sulfonamide (Dapoxyl) at a final concentration of 2.5μM. After incubation for 5 mm at 37°C, cells were centrifuged and re-suspended in MCDB-201 with 5% FCS (v/v).

Fluorescence- activated cell sorting was carried out with a FACSvantage SF machine (BD Biosciences), using an Enterprise Il laser to generate UV lines for excitation. The fluorescence was collected using a 505-nm long-pass (LP) filter. Target cells were sorted into 1 mL of MCDB-201 medium supplemented with insulin, transferrin and epidermal growth factor. Cells were cultivated at 1 - 1.5 x 10⁵/mL and many colonies appeared after 10 to 14 days in culture. These colonies were comprised of at least 50 cells to hundreds of cells in a single colony. The results illustrated in fig. 9 show many large pulmonary epithelial colonies appearing after enrichment (see arrows in fig. 9). In particular, primary culture of sorted cells, comprising 8 to 10 % of total cells, yielded numerous colonies ranging in size from few cells to a few hundreds cells after ten days of culture (fig. 9). One neonatal mouse yields approximately 1.0-1.5 x 10⁶ cells in our procedure. Using ≥50 cells as criteria to count for a colony, the number of colonies were analyzed statistically. The number of the colonies per 3 x 10⁵ sorted cells was estimated to be 106 ± 5. Accordingly, the frequency of these clonogenic cells was estimated to be approximately 0.002% of all nucleated cells isolated from lung tissues.

Example 3: Pulmonary clonogenic stem cells express Oct-4, SSEA-I, Nanoq, and SCA-1

Pulmonary clonogenic colonies in primary cultures obtained as reported in Example 2, were examined for stem/progenitor markers by immunofluorescence using the embryonic and stem cell antigens, Oct-4, SSEA-I, Nanog and Sca-1, and by RT-PCR using probes for Oct-4 and Sca-1 mRNA. a. Immunofluorescence and enzyme activity assays

The pulmonary clonogenic cells were fixed in methanokacetone (1:1) for 3 min at room temperature for Oct-4, and in 4% paraformaldehyde in PBS for 10 min at room temperature for other antigen determination. Afterwards, cells were then permeabilized in 0.1% Triton X-100 in blocking solution (3% BSA in PBS), washed three times, and left in blocking solution for 1 h. Cells were incubated at 4 ⁰C overnight with primary antibodies. Primary antibodies used were as followed: aquaporin-5 (AB3069); c-Kit (CBL1359); cytokeratin 5/8, 7, and 18 (MAB3228, 3226, and 3234, respectively); pan-cytokeratin(AE1/3); pro-surfactant C protein (AB3786); SSEA-1 (MAB4301) (all from Chemicon International); cytokeratin 19 (IF15, Oncogene Research Products); AEC-2 (MAB933); Sca-I (AF1226), (both from R & D System); CCSP (sc-9773, Santa Cruz Biotechnology), Nanog (gift from Shinya Yamanaka), and Oct-4.

The following fluorescence-labeled secondary antibodies were used (Jackson lmmuno Research): Cy3-labeled F(ab')₂ donkey anti-goat IgG (H+L); F(ab')² goat anti-rabbit IgG (H+L); F(ab')₂ goat anti-mouse IgG (H+L); F(ab')₂ goat anti-mouse IgM, ychain specific; F(ab')₂ goat anti-rat IgG (H+L); and FITC-labeled F(ab')₂ donkey anti-goat IgG (H+L); and F(ab')₂ goat anti-mouse IgG (H+L). Cell nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI). Primary cultures were also analyzed for enzymatic activity of alkaline phosphatase, according to standard protocols using an AP detection kit (SCR004, Chemicon International).

Immediately after culture, Oct 4 expression was too rare to be detected. However, following 7-10 days of culture, expression of Oct-4, Nanog, SSEA-1 and Sca-1 were specifically detected in these pulmonary colony cells but not in the surrounding spindle shaped cells in the cultures as shown in figs. 10 - 12 and 48 A for Oct-4, figs. 13 - 15 and 48 B for SSEA , fig. 52D for Nanog and figs. 16 - 18, and 48 C for Sca-1. In particular, Oct-4 and Nanog expressions were detected in the nuclei of these pulmonary colony cells. Primary pulmonary cultures were also examined using specific antibodies directed against cytokeratin-7 and Clara cell secretion protein. The cells fixed with paraformaldehyde were permeablized in 0.1% Triton X-100 containing 3% BSA in PBS. The primary antibodies for anti-cytokeratin-5/8, -7, -18, -19 and anti-Clara cell secretion protein antibodies were used to examine the pulmonary culture cells. Cells were counterstained with DAPI. Primary cultures were also analyzed for enzymatic activity of P450 as described in (16). Results are shown in figs. 19 and 49A (cytokeratin-7), fig. 20 and 49C (Clara cell secretion protein) and fig. 49F (cytochrome p450 enzyme), respectively. The results shown that only cytokeratin-7 was expressed only in the colony cells, however, other cytokeratin (e.g. cytokeratin- 5/8, 18 and 19)could not be detected in the primary culture. The Clara cell secretion protein and cytochrome p450 enzyme activities were also detected in the colony cells. They also expressed peroxiredoxin Il and Vl (figs. 54 D and E), plasma levels of (which have been shown to be upregulated in response to infection with the SARS virus (19)). Taken together, these findings suggest that these pulmonary colony cells exhibit phenotypes characteristics of stem/progenitor cells of the lung and also bear markers for epithelial and Clara cells. b. RT-PCR

The presence of Oct-4, Nanog, and Sca-1 mRNA in the primary culture of clonogenic lung cells was confirmed with RT-PCR of cells in the colonies individually plucked from culture described in Methodology section. Under a microscope, a 26- gauge needle was used to delineate the boundary of pulmonary epithelial colonies, and the colonies were gently plucked from the cultures by using a finely drawn Pasteur pipette. Three mouse ES cell lines, 46c, R1 , and J1 , were used for a positive control for Oct-4 expression, and the TM4 cell line (mouse testis Sertoli cells; American Type Culture Collection) and MEF (mouse embryonic fibroblast) were used for a negative control. For Sca-1 , the BW5147 cell line (T lymphocyte cell) was used for a positive control, and the A549 cell line (type-2 pneumocyte) was used for a negative control (both from American Type Culture Collection). For Nanog analysis, MEF was used for negative control and mouse ES cell line J1 was used for positive control. Total RNA was prepared with the RNeasy Micro Kit (Qiagen, Valencia,

CA)₁ and reverse transcription was carried out with random primers by using the Superscript first-strand synthesis system (Invitrogen) according to the manufacturer's instructions. For PCR, the forward and reverse primers were as follows: (i) δ'-ATGGCTGGACACCTGGCTTC- 3' and 5'- CCAGGTTCTCTTGTCTACCTC-3' for Oct-4 expression (38), (ii) 5'-

GGACACTTCTCACACTACAAAG- 3' and δ'-TAACACAGACTCCATCAGGGTAG-S' for Sca-1 expression (39), and (iii) δ'-ACCACAGTCCATGCCATCAC- 3' and 5'- TCCACCACCCTGTTGCTGTA-3' for GAPDH as the internal control in both reactions (38). The primers for Nanog RT-PCR were prepared as described by Shinya Yamanaka.

The results are shown in figs. 22, 23, and 5OA. The bands for Sca-1 and Oct-4 were consistent with the expected size of 160 bp and 1121 bp, respectively. The GADPH (glyceraldehyde-3-phosphate dehydrogenase) was used as internal standard for both reactions. c. Quantitation of Oct-4 expression

Because Oct-4 expression had never been reported in adult cells and tissues, quantitative RT-PCR was performed to evaluate the level of expression. Quantitative PCR is a method that is powerful simply because it is quantitative, and permits, for example, well resolved comparisons of gene expression between various types of cells. The presence of Oct-4 mRNA is confirmed by quantitative RT- PCR performed with the colony cells plucked from the cultures to evaluate the level of Oct-4 expression (figs. 3 A-C, and 50 B-D). The Oct-4 expression in these pulmonary colony cells is high, about 51%, 52%, and 88% of those expressed in ES cell lines 46c, R1, and J1, respectively (figs. 3 and 50 D).

One aspect of the significance of high levels of Oct-4 in the present stem cell population is that it contributes to the developing general view that expression of Oct-4, generally considered a marker of embryonic stem cells, may be more appropriately considered a characteristic of stem cells in general, even as found resident in neonatal or adult tissue, tissue which would be considered fully differentiated. Oct-4, for example has been demonstrated in cells of the mouse heart (Mendez-Ferrer, et a/., "ES-like cells in the adult murine heart", Abstracts of the International Society for Stem Cell Research, Toronto Canada, June 29, 2006). This developing broader view of the significance of Oct-4 presence, and specific demonstration herein of the presence of the Oct-4 marker and its utility as an identifying and selective tool (by cell sorting, for example) has further significance. It informs the presently described inventive realization that identifying and selecting cells on the basis of Oct-4, particularly when coupled with the identification and selection on the basis of other, more tissue specific markers, provides an approach to isolating populations of adult stem cells from organs and tissues broadly, not just pulmonary tissue specifically.

Example 4: Primary cell cultures of Oct-4 expressing are able to differentiate into alveolar pneumocytes

To address the differentiation potential of these pulmonary colony cells, individual colonies were plucked from the primary cultures and transferred to culture dishes either precoated with collagen I or preseeded with irradiated primary pulmonary cultured cells. The transferred colonies were then monitored for the expression of Oct-4, surfactant protein C, and aquaporin 5 protein. In the presence of preirradiated cultured pulmonary cells, the transferred colonies did not attach well and appeared as spherical aggregates in which Oct-4 expression was maintained in the majority of the colony cells up to day 5 (figs 24, 25, and 51 ); by day 11 the colony cells displayed diminished Oct-4 expression (fig. 51). No surfactant protein C or aquaporin 5 protein could be detected in the transferred colonies during the 11- day culture period. In contrast, the individually plucked colonies transferred into collagen l-coated plates attached well, and the cells continued to grow, migrated out, and appeared as thinly spread flattened cell clusters. Oct-4 expression decreased rapidly within 2 days, and by day 5 the expression of surfactant protein C was detected in the cytoplasm, especially in the perinuclear region of these flattened cells (fig. 51).

All of these features are consistent with the type-2 pneumocytes. By day 9, the colony continued to expand and to spread thinly such that the average diameter of individual colony cells was ~5-fold greater than that of the parental primary epithelial colony cells. Expression of surfactant protein C decreased, whereas expression of aquaporin 5 protein, a marker for type-1 pneumocytes, became pronounced (fig. 51). These observations suggest that Oct-4⁺ SSEA-I⁺ , Nanog⁺, Sca-1⁺ pulmonary epithelial colony cells have the potential to differentiate to type-2 and -1 pneumocytes in a sequential fashion. These results show that the cells in the pulmonary colonies have the capacity to undergo differentiation into phenotypes characteristics of the differentiated alveolar pneumocytes.

Example 5: Primary cell cultures of Oct-4 expressing cells are selected target of SARS CoV infection a. Primary culture of Brdu-retaining/Oct-4 expressing cells are target of SARS Co-V infection

In order to investigate whether primary pulmonary cell cultures might be susceptible to SARS-CoV infection, we expose these primary cultures to SARS-CoV infection. All experiments for SARS-CoV infection were carried out at a P4 facility.

Seven to ten day old confluent primary cultures of pulmonary cells with epithelial colonies were incubated with an inoculum of SCAR-CoV (strain Tw7) at MOI of 0.5 (25) for 8 h and 24 h at room temperature. Cells were washed once with PBS buffer and incubated in MCDB-201 medium containing supplements. At 8, 16, 24 and 48 h postinfection, the supernatant of cells was collected for titration. The cells were then washed three times with PBS buffer and fixed with ethanol:acetone (1:1) before immunofluorescence analysis. The SARS-CoV antigen was detected using a mouse monoclonal antibody (diluted 1:2000) generated against the recombinant SARS-CoV nucleocapsid protein for this study. The epitope of this monoclonal antibody was shown to be localized to the N-terminal of the nucleocapsid. Results are shown in fig. 28 - 30 (8 h incubation) and in figs. 31 - 33 (24 h incubation). At eight hours of incubation, approximately 30% of cells within the pulmonary colonies displayed strong immunofluorescence for SARS-CoV (fig. 30). The percentage of SARS-CoV positive pulmonary colony cells rose to approximately 60% at sixteen hours, and by 24 hours, nearly every cells in the pulmonary colonies was positive for SARS-CoV infection (fig. 33). The pulmonary colony cells began to detach and exhibited cytopathic changes by 48 h. In contrast, none of the cells surrounding the pulmonary colonies became infected at any time point examined (fig. 30 and 33). Afterwards, electron microscopy was performed to confirm the presence of actively replicating virus in the cytoplasm of the infected cells of the pulmonary colonies. For ultrastructural analysis, the lung cells were cultivated on an embedding/cell growing film (Aclar-fluoropolymer films, Structure Probe) which were coated with type I collagen. The culture conditions were described in the example 3 above. The pulmonary epithelial cells were infected with SARS-CoV at 0.5 MOI.

Sixteen-hours after infection, the fixation of pulmonary epithelial monolayer culture was first carried out in 2.0% glutaraldehyde and 4.0% paraformaldehyde in PBS for 2 h, post-fixed in 1 % osmium tetroxide for 1 h. The cells were dehydrated in graded ethanol, washed in propylene oxide and infiltrated in a 1:1 mixture of propylene oxide and Spurr's resin (Sigma-Aldrich). Cells were then embedded in Spurr's resin and polymerized for 24 hours at 70⁰C. Sections were cut and stained with aqueous uranyl acetate and lead citrate. The ultrathin sections of infected cells were observed through transmission electron microscopy (Hitachi H-7000). Results are shown in figs. 37 - 39. At sixteen hours after the infection, the cytoplasm of infected cells contained numerous swollen empty sacs around perinuclear region, (fig. 37). At higher magnification, many enlarged swollen vacuoles filled with virus particles were observed (fig. 38). On the outside of the cells, mature virus particles were observed and some of these extracellular virus particles were seen to associate with coated pits (fig. 39) (20). b. Evidence for viral replication in primary cell culture Oct-4 expressing cells The titers of virus were determined by serial dilutions and analyzed by the monolayer procedure of plaque assay, which was modified from Burleson (26). After Vero-E6 reached confluence on 6-well tissue culture plate, 0.5 ml of appropriate virus dilutions were prepared and applied onto cells. Followed the absorption for 30 min at 37°C, the un-adsorbed virus was removed and overlaid 1 % of agar with

DMEM and 2% FBS. Until the plaques formed about four days, the agar overlay was removed and the cells were stained with crystal violet solution (10%) for 10 min. Finally the plaques could be counted and the titer can be calculated by dilution fold and the sample volume used. For bioactivity assay, a confluent VeroE6 cell culture was mixed with the SARS-CoV particles which collected from the supernatant of primary culture cells at 2 MOI for 24 h at room temperature. The procedures for virus identification were described above.

In order to ascertain whether SARS-CoV replicated and produced infectious virus particles in the cells of pulmonary colonies, culture media were collected at different time points after infection and assayed for infectious SARS- CoV using VeroEδ cells. Results are shown in fig. 43 to 45.

As shown in fig. 43, infected pulmonary yielded culture media with the increasing SARS-CoV infectivity: the titers of these culture media after 8, 16, 24 and 48h postinfection for VeroEβ infection were respectively, 5, 30, 210 and 104 x 10⁴ plaques). VeroEδ cells infected 16 to 48 hours with conditioned media demonstrated cytopathic alterations and SARS-CoV nucleocapsid protein immunostaining in all cells in the culture dish (figs. 44-45). These experiments demonstrated that SARS- CoV maintained its replicative activities in the pulmonary colonies. c. Primary cell culture Oct-4 expressing cells are selected target of SARS Co-V infection

To investigate whether SARS-CoV targets differentiated pulmonary stem cells the following experiments were performed. First, immediately after cell isolation from lung tissues, pulmonary cell suspensions were incubated with incocula of SARS-CoV at a range of MOI from 0.5 to 10 and assayed for their ability to become infected by immunofluorescence staining for SARS-CoV nucleocapsid protein. No nucleocapsid staining above background fluorescence was observed, suggesting that differentiated lung cells were not infected by SARS-CoV. Next, cultures of pulmonary stem cells that had undergone differentiation in vitro into type 1 and type 2 pneumocytes (as discussed in example 4 above and shown in for fig. 51. ) were incubated with SARS-CoV at the same 0.5 to 10 MOI. These in vitro differentiated mature pneumocytes were found not to be susceptible to infection under the same conditions. c. Primary cell culture BrdU-retaining/Oct-4 expressing cells are not a selective target of influenza infection

Cultures containing pulmonary colony cells were infected with influenza A virus, at a MOI of 0.5 for 8 hours under the same conditions described in section a. of this example. The influenza A/WSN/33 virus was propagated and maintained as described (27) (generous gift from Dr. Shieh-Shin-Ru at Chang-Gung University). Infections with the influenza A virus were performed similarly to SARS-CoV infection at same MOI. Cells were incubated for 8, 16 and 24 h postinfection and examined for evidence of infection as determined by immunofluorescence using a viral nucleoprotein specific antibody from an influenza detection kit (IMAGEN™ Influenza virus A and B, DakoCytomation).

Results are shown in figs. 34 - 36. In contrast to SARS-CoV, influenza A virus infected all cells without preference for pulmonary colony cells (figs. 34 - 36) and cytopathic effects were observed in the cultured cells as early as at 24 hours after infection. Overall, these experiments demonstrated that pulmonary stem cells are selectively susceptible to SARS-CoV infection.

Example 6. Co-localization of Oct-4 expression and SARS-CoV nucleocapsid infection

Because the pulmonary stem cells express both Oct-4 and ACE-2, a direct demonstration of the infection of SARS-CoV on Oct-4-expressing cells was sought. As shown in fig. 56, 20-30% of the Oct-4- cells showed cytoplasmic immunostaining for SARS-CoV nucleocapsid protein at 8 h after infection. Confluent primary pulmonary cultures were infected with SARS-CoV at 0.5 MOI and at 8 h post-infection, cultures were processed for dual staining of Oct-4 and SARS-CoV. Immunostaining of cells within colony were shown (medium gray) using antibodies directed against Oct-4 and 20-30% of cells in colony shown in medium gray using antibodies directed against nucleocapsid protein of SARS-CoV. The merged image of Oct-4 and SARS-CoV, and counter-stained with DAPI were shown. This has demonstrated the co-localization of nuclear Oct-4 (green) and cytoplasmic SARS- CoV nucleocapsid proteins (light gray) in the same cells.

In summary, these experiments demonstrate that pulmonary stem cells are capable of forming colonies in vitro, and continuously express stem cell markers such as Oct 4, SSEA-1, Nanog and Sea 1 antigen, and can differentiate to form type 1 and type 2 pneumocytes upon clone transfer. In addition, pulmonary stem cells also express markers known to be expressed in epithelial and Clara cell lineages, which have been implicated in pulmonary repair and regeneration. Exposure to SARS-CoV leads to selective productive infection of the stem cells and the replication and release of infectious SARS-CoV particles. In contrast, the alveolar pneumocytes either in the initial cell suspensions prepared from the lung tissues or those differentiated in vitro from the primary colony cells were resistant to SARS-CoV infection. The finding that the above mentioned stem cells express peroxiredoxin Il and Vl may be of clinical significance. Peroxiredoxins were originally identified as intracellular proteins with multiple functions including enhancing natural killer cell activity, increasing resistance to oxidative stress, regulating transcription activator proteins, and providing antiviral activity against HIV. A recent proteomic analysis of plasma samples from patients with SARS demonstrated that plasma levels of peroxiredoxin II, are significantly elevated in patients with SARS (19).

These findings support the notion that pulmonary stem cells may be an important target for SARS-CoV infection. Loss of this pulmonary subpopulation may thus compromise the ability of lung tissue to recover from initial injury, and may help explain the late phase of clinical deterioration in SARS patients which is associated with significant morbidity and mortality.

Example 7: Pulmonary stem cells are preferentially susceptible to many other viral infections

This example describes a method for selecting and growing pulmonary stem cells in lung cell culture. This in vitro method is useful for selecting drugs with which to treat respiratory infection. These pulmonary clonogenic cells were preferentially infected by SARS-CoV, Hantavirus, and some of influenza A viruses (H1N1 , H1N2, H2N2, H5N1 ) (figs. 64 and 67), which may account for the deterioration of lung tissues and the apparent loss of capacity for lung repair upon some respiratory viral infections. This culture system could also be a good model system for drug selection for respiratory infection. . Electron micrographs of H5N1 infected cells were shown in fig. 54 and the receptors for were identified in fig. 55. Staining of the receptors in pulmonary stem cell colony using lectins SNA and MAL II, specific for human influenza A₁ NeuδAc- α-2,6-Gal (green) and avian flu virus, NeuδAc- α-2,3-Gal (red), were shown in fig. 55. Example 8: Differentiation potential of the epithelial colony cells

To address the self-renewal and differentiation potential of these pulmonary colony cells, individual colonies were plucked from the primary cultures and transferred to culture dishes either precoated with collagen I or preseeded with irradiated primary pulmonary cultured cells. The transferred colonies were then monitored for the expression of Oct-4, surfactant protein C, and aquaporin 5 protein. In the presence of preirradiated cultured pulmonary cells, the transferred colonies did not attach well and appeared as spherical aggregates in which Oct-4 expression was maintained in the majority of the colony cells up to day 5 (fig. 4A); by day 11 the colony cells displayed diminished Oct-4 expression (fig. 4B). No surfactant protein C or aquaporin 5 protein could be detected in the transferred colonies during the 11-day culture period. In contrast, the individually plucked colonies transferred into collagen l-coated plates attached well, and the cells continued to grow, migrated out, and appeared as thinly spread flattened cell clusters. Oct-4 expression decreased rapidly within 2 days, and by day 5 the expression of surfactant protein C was detected in the cytoplasm, especially in the perinuclear region of these flattened cells (fig. 4C). These features are consistent with the type-2 pneumocytes. By day 9, the colony continued to expand and to spread thinly such that the average diameter of individual colony cells was ~5-fold greater than that of the parental primary epithelial colony cells. Expression of surfactant protein C decreased, whereas expression of aquaporin 5 protein, a marker for type-1 pneumocytes, became pronounced (fig. 4D). These observations suggest that Oct-4⁺ SSEA-I⁺ Sca-1⁺ pulmonary epithelial colony cells have the potential to differentiate to type-2 and -1 pneumocytes in a sequential fashion. In addressing whether these in vitro differentiated type-2 and -1 pneumocytes are susceptible to SARS-CoV infection, individual epithelial colonies were plucked and transferred to culture dishes precoated with collagen I. The cultures were then exposed to SARS-CoV at moi of 0.5 and 10 on days 5 and 9 after colony transfer. There is no evidence of SARS- CoV infection or virus replication on these differentiated type-2 and -1 pneumocytes by either immunostaining or virus titering.

Example 9: A method for studying interactions of pulmonary stem cells with surrounding stroma.

A rare type of lung cell, present at an incidence of 0.004% among primary cell cultures of lung cells, with the characteristics of pulmonary stem /progenitor cells has recently been identified that interacts with its co-cultured surrounding mesenchymal stroma cells to maintain its phenotype as a stem cell. These cells provide a system useful for designing and selecting drugs to treat respiratory infection, as well as for study of interaction with surrounding stromal cells and the process of remodeling of lung cells that may follow respiratory infection, thereby repairing damage done by the infection.

An defined (serum-free) culture system is described that supports the growth of epithelial colonies in primary pulmonary cultures that are positive for transcription factor Oct-4. Epithelial-like colonies appear in serum-free conditions after 10 - 14 days of culture with low concentrations of epidermal growth factor (EGF) in the medium.

These morphologically unique colony cells can be cultured in vitro for months and maintain characteristics of stem cells through interaction with surrounding stroma cells. On the other hand, once free from surrounding cells, they undergo differentiation to become more mature pneumocytes sequentially. The morphology of these cells changes over the course of subculturing, and they begin to express surfactant protein- C, and then Aquaporin-C, features characteristic of differentiated epithelial cells.

Cells plucked from individual colonies and subcultured onto irradiated stroma cells continue to express Oct-4 for several weeks (fig. 51 ). Therefore, the surrounding cells, which express smooth muscle α-actin, CD44, and CD90 (fig. 57), and can partially been induced to become adipocytes, appear as some form of mesenchymal cells (fig. 51). Finally, these pulmonary colony cells are infectable not only by SARS-CoV, but also by Hantavirus and influenza viruses (e.g., H1N1 , H2N2 and H5N1), thus exhibiting a specific susceptibility of the colony cells toward virus infection. The results of such virus infections may account for the deterioration of lung tissues and the apparent loss of capacity for lung repair that follows some respiratory viral infections. These data demonstrate this culture system to be a tool broadly useful for (1) drug design and selection for respiratory infection, and (2) for studying interactions of pneumocytes with surrounding stroma, such interactions being likely to play an important role in lung remodeling. Stabilization of the phenotype by the presence of the stroma cells serves to enhance the robustness of the in vitro system as a biological research tool, and as a system for the study of infective and anti-infective agents. Stabilizing the phenotype allows for a greater degree of expansion of the cultures, allowing larger populations, more replicate cultures, and longer life span. Example 10: BrdU label-retaining cells (LRC) in the neonatal lung express Qct-4.

To address whether the Oct-4⁺ epithelial colony cells growing in the serum-free medium cultures arise de novo or from the Oct-4⁺ cells present in the neonatal lungs, a pilot immunohistochemistry study was performed, and the presence of a small number of Oct-4- expressing cells at the bronchoalveolar junction of the neonatal lung was observed. To further ascertain whether these small number of Oct-4-expressing cells in neonatal lung can be the "putative" lung stem cells, BrdU pulse-chase experiments was performed as described in Materials and Methods to label the long-term LRC. Immediately after five daily injections of BrdU, a large number of cells, including some alveolar cells (fig. 5A left), demonstrated nuclear staining for BrdU. The number of BrdUpositive cells in the lungs declined significantly with time such that by 4 weeks only a few cells still retained BrdU labeling (arrows in fig. 5A right). These LRC were found almost exclusively in the epithelial layer of terminal bronchioles adjacent to alveolar sacs, as revealed by staining with monoclonal antibodies against pancytokeratin (AE1_3), general markers for epithelial cells (fig. 5B). lmmunohistochemical staining for Oct-4 was then performed and the number of Oct--4⁺ cells and BrdU LRC determined . The analysis revealed that there were 21 + 8 dual Oct-4⁺ BrdU retaining cells in each randomly selected lung section that contained bronchoalveolar junctions (fig. 5D). Single Oct-4⁺ cells or BrdU LRC were not encountered. As estimated by histological grid analysis, there are -1.25 x 10⁶ nucleated cells per slide, suggesting the presence of a small population (-0.0016 + 0.0006%) of slow-cycling, Oct-4- expressing stem cells in the neonatal lungs. Example 11 : Pulmonary stem cells susceptibility to viral infections, and the role that interaction with stromal cells has in stabilizing the stem cell phenotype

This example describes a method for selecting and growing pulmonary stem cells lung cell in culture. This in vitro method is useful for selecting drugs with which to treat respiratory infection, and for studying interactions of such cells with surrounding stroma, such interaction having a role in lung remodeling.

An defined (serum-free) culture system is described that supports the growth of epithelial colonies in primary pulmonary cultures that are positive for transcription factor Oct-4. Epithelial-like colonies appear in serum-free conditions after 10 - 14 days of culture with low concentrations of epidermal growth factor (EGF) in the medium. The presence of Oct-4 mRNA is confirmed by quantitative RT-PCR performed with the colony cells plucked from the cultures to evaluate the level of Oct-4 expression. The Oct-4 expression in these pulmonary colony cells is high, about 51%, 52%, and 88% of those expressed in ES cell lines 46c, R1, and J1 , respectively. In addition to Oct-4, these cells also express other stem cell markers such as Nanog, SSEA-1 , and Sca-1 , but not c-Kit, CD34 or p63.

Cells plucked from individual colonies and subcultured onto irradiated stroma cells continue to express Oct-4 for days. Therefore, the surrounding cells, which express smooth muscle α-actin, CD44, and CD90, and can partially been induced to become adipocytes, appear as some form of mesenchymal cells. In addition, the presence of Oct-4+, long term BrdU label retaining cells at the bronchoalveolar junction provides a link between the Oct-4+ cells in vivo and in vitro and enforces their identity as putative lung stem cells. Thus, these primitive pulmonary cells represent a population of slow cycling, Oct-4+ expressing cells, scattering at bronchoalveolar junctions of lung tissues.

Finally, these pulmonary colony cells are infectable not only by SARS-CoV, but also by Hantavirus and influenza viruses (e.g., H1N1 , H2N2 and H5N1), thus exhibiting a specific susceptibility of the colony cells toward virus infection. The results of such virus infections may account for the deterioration of lung tissues and the apparent loss of capacity for lung repair that follows some respiratory viral infections. These data demonstrate this culture system to be a tool broadly useful for (1) drug design and selection for respiratory infection, and (2) for studying interactions of pneumocytes with surrounding stroma, such interactions being likely to play an important role in lung remodeling.

Stabilization of the phenotype by the presence of the stroma cells serves to enhance the robustness of the in vitro system as a biological research tool, and as a system for the study of infective and anti-infective agents. Stabilizing the phenotype allows for a greater degree of expansion of the cultures, allowing larger populations, more replicate cultures, and longer life span.

Details of Methodology Used in Development of Examples a. Pulmonary Primary Cell Culture

Neonatal ICR mice were killed, and their lungs were removed and cut into small pieces. After washing in Hank's buffer containing penicillin (100 units/ml) and streptomycin (100 ug/ml), the tissues were treated with 0.1 % protease type-XIV (Sigma) in Joklik's MEM (Sigma) at 4⁰C overnight. Afterward, tissues were transferred to 10% FCS_Joklik's MEM, pipetted several times to release pulmonary cells, and then filtered through a 100-_m nylon cell strainer. The released cells were washed and resuspended in MCDB-201 medium containing insulin- transferrin- selenium supplements only (GIBCO). One neonatal mouse can yield ~1.0 - 1.5 x 10⁶ nucleated cells in this enzyme digestion procedure, which represents -5% to 8% of the total number of cells in lung tissues. These cells were cultivated at a density of 3 x 10⁵ cells per milliliter in culture dishes coated with collagen I (10 ug/cm²; BD Bioscience). After 1 day of incubation, the primary cultures were washed with MCDB-201 medium to remove unattached cells, and fresh medium with insulin— transferrin-selenium supplement and epidermal growth factors (1 ng/ml; Invitrogen) was then added. b. Virus Infection and Analysis

All experiments for SARS-CoV infection were carried out at a P4 facility in the Institute of Preventive Medicine (Taipei, Taiwan). Confluent primary cultures of pulmonary cells were incubated with SARS-CoV (strain Tw7) (34) at various moi (0.5, 1 , 2, and 10) for 1 h at room temperature. Cells were washed once with PBS and incubated in MCDB-201 medium containing supplements. At 8, 16, 24, and 48 h postinfection, the supernatants of the cultures were collected for viral titration and the cells were washed with PBS and fixed in ethanol/acetone (1:1) for immunofluorescence analysis. The SARS-CoV was detected by using a mouse monoclonal antibody (1 :2,000) generated against the recombinant SARS-CoV nucleocapsid protein for this study. The epitope of this monoclonal antibody was localized to the N-terminal region of the nucleocapsid (M. D. K., unpublished data). Virus titers were determined by plaque-forming assay with modifications using Vera E6 cells. Briefly, serial dilutions of the harvested supernatant were added into confluent culture of Vero E6 cells. After incubation for 30 min at 37°C, the unabsorbed virus was washed off and the culture was overlaid with 1% agar in DMEM and 2% fetal bovine serum (FBS). After plaques were formed, the agar overlay was removed, the cells were stained with crystal violet solution (10%) for 10 min, and the plaques were counted. c. Electron Microscopy

Transmission electron microscopy was performed as described, with modification (35). The pulmonary epithelial cells were cultivated on collagen l-coated ACLAR- Fluoropolymer films (Structure Probe). The cells were infected with SARS- CoV at 0.5 moi as described. At 16 h postinfection, cells were fixed with 2% glutaraldehyde/ 4% paraformaldehyde/ PBS for 2 h, followed by 1% osmium tetroxide for 1 h, and then embedded in Spurr's resin. Ultrathin sections (60 nm) of the embedded cells were prepared, stained with 2% uranyl acetate and 1% lead citrate, and analyzedwith an H-7000 electron microscope (Hitachi, Tokyo), and results are shown in figs. 37 - 39.

At sixteen hours after the infection, the cytoplasm of infected cells contained numerous swollen empty sacs around perinuclear region, (fig. 37). At higher magnification, many enlarged swollen vacuoles filled with virus particles were observed (fig. 38). On the outside of the cells, mature virus particles were observed and some of these extracellular virus particles were seen to associate with coated pits (fig. 39).

As shown in fig. 43, infected pulmonary yielded culture media with the increasing SARS-CoV infectivity: the titers of these culture media after 8, 16, 24 and 5Oh postinfection for VeroEδ infection were respectively, 5, 30, 210 and 104 x 10⁴ plaques). VeroEδ cells infected 16 to 48 hours with conditioned media demonstrated cytopathic alterations and SARS-CoV nucleocapsid protein immunostaining in all cells in the culture dish (figs. 44-45). These experiments demonstrated that SARS- CoV maintained its replicative activities in the pulmonary colonies. d. Immunocytochemistry. Cells in primary cultures were fixed in methanol /acetone (1 :1 ) for 3 min at room temperature. For analysis of Sca-1 expression, cells were fixed in 4% paraformaldehyde/PBS for 10 min, permeabilized with 0.1% Triton X-100 in PBS for 5 min, and then blocked with 3% BSA/PBS for 30 min. Cells were incubated at 4°C with primary antibodies against the following antigens: peroxiredoxin 2 [a kind gift from J. H. Chen (National Defense Medical Center)]; peroxiredoxin 6 (ab16824;

Abeam); CD34 (clone RAM34; BD Biosciences); aquaporin 5 (AB3069), cytokeratin- 5_8 (MAB3228), cytokeratin-7 (MAB3226), and cytokeratin- 18 (MAB3234), p63 (MAB4135), pan-cytokeratin (clone AE1_3), surfactant protein C (AB3786), and SSEA-1 (MAB4301) (all from Chemicon); α-smooth muscle actin (clone 1A4; DAKO); cytokeratin 19 (IF15; Oncogene); c-Kit (MAB1356) and Sca-1 (AF1229) (both from R & D Systems); and ACE-2 (sc20998), CCSP (sc9773) and Oct-4 (sc9081) (all from Santa Cruz Biotechnology). After overnight incubation, cells were washed and incubated for 1 h at room temperature with the following respective Cy3-labeled secondary antibodies: donkey anti-goat IgG, goat anti-rabbit IgG, goat anti-mouse IgG, goat anti-mouse IgM (μ- chain-specific), and goat anti-rat IgG (Jackson ImmunoResearch). Cells were then counterstained with DAPI. Primary cultures were also analyzed for enzymatic activity of alkaline phosphatase according to standard protocols using an AP detection kit (Chemicon) and CyP450, as described in ref. 36. e. RT-PCR and Quantitative RT-PCR

Pulmonary epithelial colony cells were collected for analysis. Under a microscope, a 26-gauge needle was used to delineate the boundary of pulmonary epithelial colonies, and the colonies were gently plucked from the cultures by using a finely drawn Pasteur pipette. Three mouse ES cell lines, 46c (37), R1, and J1, were used for a positive control for Oct-4 expression, and the TM4 cell line (mouse testis Sertoli cells; American Type Culture Collection) and MEF (mouse embryonic fibroblast) were used for a negative control. For Sca-1 , the BW5147 cell line (T lymphocyte cell) was used for a positive control, and the A549 cell line (type-2 pneumocyte) was used for a negative control (both from American Type Culture Collection). For Nanog analysis, MEF was used for negative control and mouse ES cell line J1 was used for positive control. Total RNA was prepared with the RNeasy Micro Kit (Qiagen, Valencia,

CA), and reverse transcription was carried out with random primers by using the Superscript first-strand synthesis system (Invitrogen) according to the manufacturer's instructions. For PCR, the forward and reverse primers were as follows: (i) δ'-ATGGCTGGACACCTGGCTTC- 3' and 5'- CCAGGTTCTCTTGTCTACCTC-3' for Oct-4 expression (38), (N) 5'-

GGACACTTCTCACACTACAAAG- 3¹ and 5'-TAACACAGACTCCATCAGGGTAG-S' for Sca-1 expression (39), and (iii) δ'-ACCACAGTCCATGCCATCAC- 3' and 5'- TCCACCACCCTGTTGCTGTA-3' for GAPDH as the internal control in both reactions (38). The primers for Nanog RT-PCR were prepared as described by Shinya Yamanaka.

Quantitative PCR is a method that permits high resolution comparisons of gene expression among various types of cells. Quantitative RT-PCR was performed by using the ABI Prism 7000 sequence detection system (Applied Biosystems) following the manufacturer's instructions. The primer_probe sets for mouse Oct-4 (TaqMan gene expression assay no. Mm00658129_gH; Applied Biosystems) and mouse GAPDH (TaqMan gene expression assay no. Mm99999915_g1) were used. Quantitative RTPCR was carried out for 45 cycles, and raw data were analyzed by ABI Prism 7000 SDS software (Applied Biosystems). The cycle threshold, Ct, of each sample was generated with the default setting. The Oct-4 expression level of each sample was normalized to the expression level of GAPDH in, the same sample by the following formula: Oct-4/GAPDH = 2-<^ctofOct-⁴-^ctofGAPDH>. The Oct-4/GAPDH ratio of J1 cell line was set to 1.0, and the values of all others were recalculated accordingly. The result represents the average of three independent experiments, with standard deviations. f. In vitro Differentiation

To analyze differentiation potential, cells were plucked from individual colonies as described above and transferred to either new culture dishes free of the surrounding stromal cells, or new culture dishes with the irradiated primary culture cells as a feeder layer. The primary culture cells used for the feeder layer were grown to near confluence and preirradiated with 1,500 rad in a 137Cs source (Atomic Energy, Ottawa). In this assay, conditioned media were applied to both conditions. The conditioned media were harvested from confluent pulmonary primary cultures and filtered with 0.2-um filters. g. BrdU Labeling, Oct-4 Expression, and lmmunohistochemical Analysis.

Neonatal ICR mice were injected i.p. with 50mg/kg BrdU (Sigma) in PBS twice a day for 5 days. Mice were maintained without further BrdU injection and killed on day 0 or after 1 , 2, 3, or 4 weeks of chase for BrdU labeling. Lungs were removed, fixed in 10% formalin fixative, and embedded in paraffin. Afterward, 5-um sections of lung tissues were obtained and stained for BrdU. For Oct-4 expression, the general staining protocol included the following details: antigen retrieval was carried out by heating for 8 min in sodium citrate buffer (10 mM; pH 6.0), followed by a 15-min incubation at room temperature. Mouse monoclonal anti-BrdU (DAKO) and rabbit anti-Oct-4 antibodies (Santa Cruz Biotechnology) were added, respectively. Afterward, immunohistochemical analysis was performed with a peroxidase detection kit (Vector Laboratories) by using diaminobenzene as substrate according to the manufacturer's instructions. Anti-pan-cytokeratin antibodies (AE1_3 clone; Chemicon) and an alkaline phosphatase detection kit with Fast-Red as a substrate were used to delineate the location of bronchoalveolar junctions. All sections were counterstained with Mayer's hematoxylin. For double staining of immunofluorescence with anti-BrdU and Oct-4 primary antibodies, the FITC-conjugated goat anti-mouse and Cy3-conjugated goat anti-rabbit antibodies (Jackson ImmunoResearch) were used, respectively.

In summary, these experiments (Examples 6 and 7) demonstrate the existence of a rare subpopulation of slow cycling pulmonary stem cells at the bronchoalveolar junction of the neonatal lung. They are capable of forming colonies in vitro, and continuously express stem cell markers such as Oct 4, SSEA-1 and Sea 1 antigen, and can differentiate to form type 1 and type 2 pneumocytes upon clone transfer. In addition, stem cells also express markers known to be expressed in epithelial and Clara cell lineages, which have been implicated in pulmonary repair and regeneration.

Exposure to SARS-CoV leads to selective productive infection of the stem cells and the replication and release of infectious SARS-CoV particles. In contrast, the alveolar pneumocytes either in the initial cell suspensions prepared from the lung tissues or those differentiated in vitro from the primary colony cells were resistant to SARS-CoV infection.

The finding that the above mentioned stem cells express peroxiredoxin Il and Vl may be of clinical significance. Peroxiredoxins were originally identified as intracellular proteins with multiple functions including enhancing natural killer cell activity, increasing resistance to oxidative stress, regulating transcription activator proteins, and providing antiviral activity against HIV. A recent proteomic analysis of plasma samples from patients with SARS demonstrated that plasma levels of peroxiredoxin II, are significantly elevated in patients with SARS (19). These findings support the notion that pulmonary stem cells may be an important target for SARS-CoV infection. Loss of this pulmonary subpopulation may thus compromise the ability of lung tissue to recover from initial injury, and may help explain the late phase of clinical deterioration in SARS patients which is associated with significant morbidity and mortality.

Embodiments of the invention and equivalents thereof

An element in a claim is intended to invoke 35 U.S. C. § 112 paragraph 6 if and only if it explicitly includes the phrase "means for," "step for," or "steps for." The phrases "step of and "steps of," whether included in an element in a claim or in a preamble, are not intended to invoke 35 U. S. C. § 112 paragraph 6.

While the cell populations, methods, and kits have been described in terms of what are presently considered to be the practical and preferred embodiments, it is to be understood that the disclosure need not be limited to the disclosed embodiments. The disclosure is intended to cover various modifications and similar arrangements included within the spirit and scope of the claims, the scope of which should be accorded the broadest interpretation so as to encompass all such modifications and similar structures.

Various terms have been used in the description to convey an understanding of the invention; it will be understood that the meaning of these various terms extends to common linguistic or grammatical variations or forms thereof. It will also be understood that when terminology referring, for example, to equipment, hardware, or software, has used trade names, brand names, or common names, that these names are provided as contemporary examples, and the invention is not limited by such literal scope. Terminology that is introduced at a later date that may be reasonably understood as a derivative of a contemporary term or designating of a subset of objects embraced by a contemporary term will be understood as having been described by the now contemporary terminology. Further, it should be understood that the invention is not limited to the embodiments that have been set forth for purposes of exemplification, but is to be defined only by a fair reading of claims that are appended to the patent application, including the full range of equivalency to which each element thereof is entitled.

Claims

CLAIMSWe claim:

1. A method of isolating stem cells of a pulmonary tissue comprising: providing a sample of cells from pulmonary tissue, the sample including a population of stem cells; growing a primary culture of the pulmonary cells in vitro; plucking slow growing clonogenic ceils that emerge from the primary culture, thereby isolating them from the other cells in the culture; and identifying the isolated cells as stem celis by finding expression of at least one stem cell characteristic.

2. The method of claim 1 , wherein the pulmonary tissue and the stem cells therefrom are mammalian.

3. The method of claim 2, wherein the pulmonary tissue and the stem cells therefrom are murine.

4. The method of claim 2, wherein the pulmonary tissue and the stem ceils therefrom are human.

5. The method ofclaim 1 , wherein the stem cell characteristic is a capability to differentiate into a mature phenotype.

6. The method ofclaim 1 , wherein the stem cell characteristic is the expression of Oct-4,

7. A method of identifying pulmonary tissue-derived cells as adult pulmonary stem celts, the method comprising identifying slow growing clonogenic cells within a larger population of pulmonary cells in a primary culture as a population of presumptive stem cells: identifying expression of a stem ceil marker in the population of presumptive stem cells; and; showing the population of cells to have the ability to differentiate into a mature phenotype, thereby confirming the stem cell identity,

8. The method of claim 7, wherein the stem cell marker represents expression of a stem cell phenotype associated gene.

9. The method of claim 8, wherein gene expression is shown by any of RT-PCT or quantitative PCR.

10. The method of claim 7, wherein the stem cell phenotype associated gene is any of the genes for Oct-4 or Sca-1.

1 1. The method of claim 7, wherein the stem cell marker represents expression of a stem cell phenotype associated protein.

12. The method of claim 1 1, wherein protein expression is shown by immunofluorescence.

13. The method of claim 1 1 , wherein the stem cell phenotype associated protein is any of Oct-4, SSEA-I, or Sca-1.

14. The method of claim 7, wherein the pulmonary tissue and the stem cells are murine, and wherein the stem cell marker expressed is at least one of SSEA-I, Sca-1 , and Nanog.

15. The method of claim 7, wherein the pulmonary tissue and the stem ceils are human, and wherein the stem cell marker expressed is at least one of SSEA-3, SSEA-4, Sca-1 , and Nanog.

16. A kit for the identification of pulmonary stem ceils, the parts comprising a first identifier for the identification of slowly dividing cells; and a second identifier for the identification of the expression of the marker Oct-4; the first and the second identifier identifying slowly dividing Oct-4+ cells, the cells able to form colonies in vitro and to differentiate in a mature phenotype.

17. The kit of parts of claim 16, wherein the first identifier is BrdU and the second identifier is selected from the group consisting of antibodies and oligonucleotides,

18. The kit of parts of claim 16, wherein cells are murine and the kit further comprises a third identifier for the identification of an additional marker, the additional marker selected from the group consisting of SSEA-I, SCA-I , and Nanog.

19. The kit of parts of claim 16, wherein the cells are human and the kit further comprises a third identifier for the identification of an additional marker, the additional marker selected from the group consisting of SSEA-3, SEEA-4 and Sca-1 , and Nanog.

20. The kit of parts of claim 16, further comprising a fourth identifier for the identification of the mature phenotype.

21. A method for cu Itivating pulmonary stem cells comprising: providing a sample of cells from pulmonary tissue; growing a primary culture of the pulmonary cells in an in vitro system, the system comprising a culture medium; plucking slow growing colonies of cells that emerge from the primary culture, the slow growing colonies comprising pulmonary stem cells, thereby isolating them from the other cells in the culture; and culturing further in vitro the cells from the plucked colonies, to expand the stem cell population.

22. The method of claim 21 , further comprising: sorting with a cell sorter at least a portion the expanded population comprising stem cells, to obtain a post-sort cell population enriched in pulmonary stem cells; and culturing further in vitro the cells from the post-sort population of pulmonary stem cells to expand the cell population.

23. The method of claim 22, wherein the cell sorting is on the basis of expression of at least one stem cell specific marker.

24. The method of claim 23, wherein the cell sorting is on the basis of expression of Oct-4.

25. The method of claim 22, wherein lhe stem cell population being sorted is one that has been previously sorted, the sorting thereby being a repeat operation on the population of cells.

26. The method of claim 21, wherein the culture medium is a defined medium.

27. The method of claim 26, wherein the defined medium comprises any of a growth factor, a metabolic hormone, and a cell-deliverable iron source.

28. The method of claim 26, wherein the growth factor is epidermal growth factor, the metabolic hormone is insulin, and the deliverabfe iron source is transferrin.

29. The method of claim 21, wherein the in vitro system includes a treated culture surface to facilitate the growth of the pulmonary stem cells.

30. The method of claim 29, wherein the treated culture surface includes any of a coating with collagen or the presence of irradiated cultured pulmonary cells,

31. The method of claim 21 , wherein the in vitro system includes co-cultured irradiated cultured pulmonary stroma cells, the presence of the stroma cells stabilizing the undifferentiated phenotype ofthe stem cells.

32. Λ method for identifying the presence of an infective agent able to infect pulmonary stem cells, the infective agent in a sample, the method comprising: mutually contacting the sample and a population of pulmonary stem ceils; detecting the level of infection of the pulmonary stem cell population by the sample; and comparing the detected level of infection with a threshold level, the threshold level indicative of development of infection in the cell population.

33. The method of claim 32, wherein identifying the presence of the infective agent further comprises quantifying the level of the agent in the sample, quantifying the level including: diluting the sample to create a dilutional series of samples, mutually contacting the series of samples and a population of pulmonary stem cells in a series of cultures; detecting the level of infection in the series of cultures; and evaluating the relative levels of infection in the series of cultures to determine the level of the infective agent in the sample.

34. The method of claim 33, wherein identifying the presence of an infective agent further comprises identifying the species of the agent.

35. A method for the production of a virus able to infect pulmonary stem cells comprising: mutually contacting an inoculum of virus and a population of pulmonary stem cells in an in vitro system, the system including cell culture medium; and collecting virus particles from the cell culture medium at a later point in time.

36. The method of claim 36, wherein the cell culture medium is a defined medium,

37. The method of claim 36, wherein the in vitro system includes a treated culture surface to facilitate the growth of the pulmonary stem cells.

38. The method of claim 36, wherein the viral inoculum is any of a Hanta virus, an influenza virus, or SARS-CoV.

39. The method of claim 36, wherein the influenza virus is any of H lN l , H IN2, H2N2, or H5N I .

40. The method of claim 36, wherein the influenza virus is an avian flu virus.

41 The method of claim 40, wherein the avian flu virus is H5N 1.

42. A method for identifying an anti-infective agent, the agent capable of interfering with infection of a pulmonary stem cell by an infective agent, the method comprising: mutually contacting an inoculum of virus and a population of pulmonary stem cells in an in vitro system; detecting the level of infection in the pulmonary stem cell population by the infective agent: and comparing the detected level of infection with a threshold level, the threshold level indicative of development of infection of the ceil.

43. The method of claim 42, wherein the infective agent is any of Hanta virus, an influenza virus, or SARS-CoV.

44. The method of claim 43, wherein the influenza virus is any of H IN l, H 1 N2, H2N2, or H5N 1.

45. The method ofclaim 43, wherein the influenza virus is an avian flu virus.

46. The method ofclaim 45, wherein the avian flu virus is I iSNl .

47. A method of generating differentiated pulmonary cells in vitro from pulmonary stem cells in vitro comprising: culturing the pulmonary stem cells in the an in vitro system, the system absent conditions that stabilize the undifferentiated phenotype of stem cells.

48. A method of maintaining pulmonary stem cells in vitro in an undifferentiated state comprising: culturing the pulmonary stem cells in the presence of irradiated pulmonary stroma cells.

49. A stem cell population from a mammalian tissue comprising: the population emerging from a primary culture of tissue in an in vitro system and identifiable as slow growing colonies of cells, the cells of the colonies being isolated from the other cells from the tissue, the population expressing Oct-4 and able to differentiate into a mature phenotype in vitro.

50. The population of claim 49 wherein the mammalian tissue is pulmonary tissue.

51. The population of claim 49 wherein the tissue is murine pulmonary tissue, and the ceil population expresses at least one of SSEA-I, Sea- 1₁ and Nanog.

52. The population of claim 49 wherein the tissue is human, and the cell population expresses at least one of SSEA-3, SSEA-4, Sea- 1 , and Nanog,

53. The population of claim 49 wherein the isolated population of stem cells is of clonal origin,

54. The population of claim 49 wherein the isolated population of stem cells is expanded an in vitro system to form an expanded cell population,

55. The population of claim 49 wherein the cell population is enriched in stem cell homogeneity by a cell sorting procedure that selects in favor of cell expressing a stem cell marker,

56. The population of claim 56 wherein the stem ceil marker is Oct-4.

57. The population of claim 56 wherein the stem cell marker is any of SSEA-I, SSEA-3, SSEA-4, Sea- 1 , or Nanog.

58. The population of claim 49, wherein the primary culture and the expansion of the population is in an in vitro system comprising a defined medium.

59. The population of claim 59 wherein the defined medium of the in vitro system includes any of a growth factor, a metabolic hormone, and a cell-deliverable iron source.

60. The population of claim 60, wherein the growth factor is epidermal growth factor, the metabolic hormone is insulin, and the deliverable iron source is transferrin.

61. The population of claim 49, wherein the in vitro system includes a treated culture surface to facilitate the growth of the pulmonary stem cells.

62. The population of claim 62, wherein the treated culture surface includes a coating with collagen.

63. The population of claim 49, wherein the in vilro system includes co-cultured irradiated cultured pulmonary stroma cells, the presence of the stroma ceils stabilizing the undifferentiated phenotype of the stem celis.

64. A cohort of cultured stem cell populations from a pulmonary tissue sample comprising: a plurality of populations emerging from a primary culture of pulmonary tissue in an in vitro system and identifiable as slow growing colonies of cells each arising from a single founder cell, the cells of the colonies being isolated from the other pulmonary cells, the populations expressing Oct-4 and able to differentiate into a mature phenotype in vilro, the individual populations arising from separate colonies being maintained separately from each other, the populations being genotypically identical, the populations having a level of pheπotypic variation consistent with that present in the founder cells of the primary culture, the respective colonies being members of a cohort.