Adult Stem Cells, Lung Biology, and Lung Disease

Report of a National Heart, Lung, and Blood Institute and Cystic Fibrosis Foundation Workshop

Proceedings of the ATS, May 2006; 3: 193 - 207. Internet address www.atsjournals.org

Daniel J. Weiss , Mary Anne Berberich , Zea Borok , Dorothy B. Gail , Jay K. Kolls , Christopher Penland and Darwin J. Prockop

University of Vermont College of Medicine, Burlington, Vermont; Will Rogers Institute Pulmonary Research Center, University of Southern California, Los Angeles, California; Division of Lung Diseases, National Heart, Lung, and Blood Institute, National Institutes of Health; and the Cystic Fibrosis Foundation, Bethesda, Maryland; Children's Hospital of Pittsburgh Center for Gene Therapy, Pittsburgh, Pennsylvania; and Tulane University Health Sciences Center, New Orleans, Louisiana

This workshop, sponsored by the National Heart, Lung, and Blood Institute of the National Institutes of Health and the Cystic Fibrosis Foundation, was held in Burlington, Vermont, July 25 and 27, 2005.

EXECUTIVE SUMMARY

The National Heart, Lung, and Blood Institute (NHLBI) and the Cystic Fibrosis Foundation, along with the Vermont Lung Center and the University of Vermont College of Medicine, convened a workshop, "Adult Stem Cells, Lung Biology, and Lung Disease," to review our current understanding of the role of endogenous lung stem or progenitor cells in lung development and repair after injury and also to review experimental studies suggesting a role for adult bone marrow–derived cells in normal lung physiology and in lung diseases. This is a new area of study that is challenging traditional views of mechanisms of lung development and lung repair after injury. The goals of the conference were to summarize the current state of the field, to discuss and debate current controversies, and to identify future research directions and opportunities in cell-based therapy for lung diseases. These included consideration of initial clinical investigations, and identification of opportunities for both basic and translational research investigating the use and role of adult bone marrow–derived and other adult stem cells in lung biology and disease.

An important consideration of the conference was that of terminology. The terms "stem cell" and "progenitor cell" are defined and used with varying degrees of clarity and precision by different investigators and in publications. To this end, some attendees broached the use of a more generic term, "reparative cells," to indicate a population or populations of cells that participate in lung remodeling after injury. Although agreement was not reached on this issue, all participants agreed that future study to define the true nature of the cells involved in lung repair is a critical issue. For the purposes of this workshop report, the term "adult marrow–derived cell" will be used in a generic sense to indicate the as-yet undefined population or populations of bone marrow–derived cells that might participate in lung remodeling and repair after injury. Similarly, the term "endogenous stem cell" will be used, unless otherwise specified, to refer to the populations of cells resident within the lung that might participate in lung development and repair.

The first session, "Endogenous Lung Stem and Progenitor Cells," reviewed the current state of knowledge of endogenous stem cell populations in the lung and their potential to initiate or augment repair. Discussions emphasized the need for new methods and markers that will enable identification and phenotyping of stem cells. The participants agreed on the importance of lung disease–specific preclinical models and use of functional outcome measures to evaluate the potential of lung cell–based therapy. Discussions also encouraged fostering partnerships between basic and clinical researchers, and moving ahead to explore in parallel basic and clinical research issues in cell-based therapy for lung disease.

Session 2, "Other Adult Stem Cell Sources," reviewed the basic biology of stem and progenitor cells and also explored potential sources (other than bone marrow) of adult cells that might participate in lung repair after injury. It is clear that lung injury results in recruitment of several cell types to the lung and that these cells can have either a beneficial or a detrimental effect on the postinjury process. A major challenge for the future will be to characterize the different recruited cell populations with respect both to behavior as "stem cells" as well as lung remodeling. Significant emphasis must be placed on developing and standardizing state-of-the-art techniques to adequately and unambiguously assess outcome measures at the molecular, cellular, and functional levels.

Session 3, "Adult Marrow–derived Stem Cells in Lung Diseases," reviewed preclinical data on adult marrow–derived cells for lung disease, translational research progress, and potential clinical protocols to investigate the safety and efficacy of cell-based therapy for lung diseases. Discussion was spirited concerning how and when to proceed to clinical investigation. It was agreed that strong emphasis must be placed on animal models of human lung diseases, with a focus on studies that incorporate relevant functional outcome measures. At the same time, it was recommended that early studies in humans, where appropriate, be launched to determine safety and dosing parameters, and to design carefully controlled clinical trials that offer patients benefits at relatively little risk.

It was acknowledged by all participants that the role of endogenous lung stem cells and of adult marrow–derived cells in lung biology and their potential therapeutic role in lung diseases represent a timely and exciting area of study. Nonetheless, there are a number of current controversies, including questions regarding the accuracy of assessments of lung chimerism by adult bone marrow–derived cells. Importantly, a functional role of adult marrow–derived cells in lung repair and remodeling after injury has not been clarified. As such, the following recommendations were developed:

  • Support high-quality translational studies focused on working toward cell-based therapy for human lung diseases. Preclinical models will provide proof of concept; however, these must be relevant to the corresponding human lung disease. Disease-specific models, particularly large animal models, should be developed for lung diseases.
  • Incorporate rigorous techniques to unambiguously identify outcome measures into this research. Advanced techniques such as high-resolution computed tomography (HRCT) scanning and histomorphometry should be used where applicable. Preclinical models require clinically relevant functional outcome measures, for example, pulmonary physiology/mechanics, electrophysiology, and other techniques.
  • Proceed with design and implementation of initial exploratory safety investigations in patients with lung disease, where appropriate.
  • Use advanced histologic imaging techniques, for example, confocal microscopy, deconvolution microscopy, electron microscopy, laser capture dissection, and so on, to avoid being misled by inadequate photomicroscopy and immunohistochemical approaches. Imaging techniques must be used in combination with appropriate statistical and other analyses to maximize the utility of detecting rare events.
  • Encourage new research to elucidate molecular programs for the development of lung cell phenotypes, including mechanisms of epithelial/endothelial/mesenchymal interactions in development.
  • Explore other potential sources of stem cells such as adipose tissue and cord blood. Uncover mechanisms of recruitment of and phenotypic conversion of adult marrow–derived cells to lung epithelial and interstitial cells.
  • Identify additional cell surface markers that characterize lung cell populations for use in visualization and sorting techniques.
  • Actively foster interinstitutional, multidisciplinary research collaborations and consortiums as well as clinical/basic partnerships. Include a program of education on lung diseases and stem cell biology.
  • Develop in vitro assays to assess the potential of cells to engraft in the lung.
  • Disseminate information about and encourage the use of existing core services and facilities. A partial list includes NHLBI Production Assistance for Cellular Therapies, National Center for Research Resources stem cell facilities, Good Manufacturing Practice Vector Cores, and small animal lung mechanics and CT scanner facilities at several pulmonary centers.

BACKGROUND

Adult Marrow–derived Cells in Lung Biology and Disease
It has long been known that adult bone marrow cells serve as precursors for hematopoietic stem cells. However, a number of reports have suggested that several cell populations derived from bone marrow of adult rodents, potentially including hematopoietic stem cells (HSCs), stromal-derived mesenchymal stem cells (MSCs), and circulating fibrocytes, can localize to and acquire phenotypic and functional markers of mature muscle, brain, bone, liver, skin, and lung ( 1 – 8 ) ( Figure 1 ). Whether the bone marrow–derived cells implicated are truly "stem" cells has not always been rigorously demonstrated and some of these studies are controversial. In some cases, notably liver and muscle, the marrow-derived cells, frequently monocytic cells, can fuse with existing organ-specific cells rather than transdifferentiating into mature organ-specific cells ( 9 – 11 ). Nonetheless, even fusion of normal adult marrow–derived cells with diseased or defective differentiated adult tissue might conceivably be a therapeutic approach for correcting defective tissues.

Figure 1

Figure 1. Schematic of endogenous and adult bone marrow–derived stem/reparative cells that potentially participate in lung development, injury, repair, and regeneration. EPC = endothelial progenitor cell; HSC = hematopoietic stem cell; MSC = mesenchymal stem cell or multipotent stromal cell.

In the lung, a number of publications have purported to demonstrate that bone marrow–derived cells can acquire phenotypic and/or functional markers of airway and alveolar epithelium, vascular endothelium, and interstitial cell types in both mouse transplantation models and from clinical lung tissue obtained from lung and bone marrow transplant patients. In rodents and other animal models, relatively small numbers of adult marrow–derived cells transplanted to adult recipient animals or progeny derived from these cells have been suggested to localize to recipient lungs and to acquire phenotypic characteristics of airway or alveolar epithelial cells, interstitial cells, and vascular endothelial cells ( 1 , 2 , 7 , 12 – 30 ). In humans, lung specimens from some clinical bone marrow transplant recipients demonstrate chimerism for both epithelial and endothelial cells ( 31 , 32 ). Similarly, lung specimens from some lung transplant patients demonstrate chimerism of lung epithelium ( 33 , 34 ).

However, chimerism or lung engraftment with adult marrow–derived cells has not been found in all studies. Further, data from several of the mouse studies have been questioned in light of more rigorous techniques and significant controversy exists as to the degree of chimerism or engraftment of adult marrow–derived cells that might actually occur ( 35 – 39 ). These issues are further discussed below.

Many of these reports are based on in situ demonstration of adult bone marrow–derived cells in lungs, evidenced by detection of a marker for the marrow–derived cells. In most cases, this has involved detection of cells containing the Y chromosome by fluorescence in situ hybridization after sex-mismatched transplantation ( 40 ) ( Figure 2 ). In one report, marrow-derived cells noted in lung tissue were quantitatively identified with single-nucleotide polymorphism assays of laser-excised cells previously labeled with phenotypic antibodies ( 33 ). Further suggestion that adult marrow–derived cells acquire the phenotype of lung cells is provided by examination of morphologic appearance and/or by coexpression of markers including cytokeratin (epithelium), smooth muscle actin (myofibroblast), or prosurfactant protein B or C (type 2 alveolar epithelium) ( 12 – 30 ). These markers are not significantly expressed in populations of adult marrow–derived cells, suggesting that cells recruited to the lung have undergone phenotypic conversion. Fusion of adult marrow–derived cells with lung epithelial cells may occur under some circumstances in vitro , but this phenomenon has not been demonstrated to occur to a significant extent in vivo in the lung ( 41 , 42 ).

Figure 2a

Figure 2b

Figure 2. Fluorescence in situ hybridization (FISH) and immunohistochemical (IHC) approaches used to detect adult bone marrow–derived cells in lungs after sex-mismatched transplantation. ( a and b ) Detection of donor marrow cells using dual Y chromosome FISH with cytokeratin and CD45 IHC. Female C57 mice were irradiated and engrafted with donor cells from a male C57BL/6 mouse. After 1 mo, mice were exposed to NO 2 to induce lung injury. One month after lung injury, mice were killed and lungs were removed and processed for FISH and IHC. ( a ) Y chromosome–positive cells were found in pan-cytokeratin–positive airways ( white arrow ) and surrounding CD45-positive interstitial tissue ( yellow arrow ). In this image, the vessel walls ( pink arrow ) do not show Y chromosome–positive cells, although rare donor cells were found in vessel walls on other images. Inset : The asterisk shows an enlarged image demonstrating a Y chromosome–positive, cytokeratin-positive cell in the airway wall, which also exhibits CD45 staining. ( b ) Y chromosome–positive cells are found in walls of distal airways and alveolar septa in ·NO 2 -injured mouse lung. Although many cells exhibit dual staining with Y chromosome probe and CD45 ( white arrow ), a small number of cells exhibit Y chromosome probe without CD45 ( yellow arrow ). Inset : The asterisk shows an enlarged image of Y chromosome-labeled alveolar cells that do not stain for CD45. Original magnification: ( a and b ) x 400. Reprinted by permission from Reference 40 .

Notably, the relevant adult marrow–derived cell population(s) capable of acquiring the lung cell phenotype have not been fully identified or characterized. Possible candidates include HSCs and the tissue culture plastic-adherent cells derived from bone marrow variously referred to as mesenchymal stem cells, marrow stromal cells, multipotent mesenchymal stem cells, or multipotent stromal cells (MSCs) ( Figure 3 ) ( 43 ). Endothelial progenitor cells (EPCs) have also been implicated in both pulmonary vascular remodeling in mouse models of pulmonary hypertension ( Figure 4 ) and lung repair in both pneumonia and acute lung injury ( 30 , 44 , 45 ). It has been suggested that circulating fibroblast precursors or fibrocytes participate in lung remodeling in several lung diseases, including asthma and fibrosing injuries ( 46 – 50 ) ( Figure 5 ). Recent data suggest the existence of discrete adult marrow cell populations (e.g., those expressing CXCR4, cytokeratin, and CD45 or CD34 and stem cell antigen-1 [Sca-1]) that are specifically recruited to the lung ( 51 ). However, additional characterization of these and other potentially relevant populations is needed. Furthermore, several studies have demonstrated the development of cells with the lung epithelial phenotype from murine embryonic stem cells ( 52 – 54 ). Although not a focus of the workshop, knowledge gained from these studies might be used for studies involving adult marrow–derived cells. Several studies have also suggested that the lung itself contains a cell population comparable to Hoeschst dye–effluxing side population cells characterized in bone marrow ( 55 – 61 ). The role(s) of these cells is not clear but may include the ability to serve as hematopoietic precursors under certain conditions as well as to provide precursors for repopulating several lung cell types, including epithelium, airway smooth muscle, and vascular endothelium.

Figure 3

Figure 3. Detection of CFTR expression in female CFTR knockout mouse lungs after transplantation with male CFTR wildtype marrow stromal cells. Donor-derived (Y chromosome, red ), CFTR-positive ( green ), and cytokeratin-positive ( blue ) cells are demonstrated in airway walls of lungs assessed 1 wk after transplantation. Original magnification, x 1,000. Reprinted by permission from Reference 29 .

Figure 4

Figure 4. Representative confocal projection images of lung sections ( a ) perfused with fluorescent microspheres ( green ) suspended in agarose (i.e., fluorescence microangiography) and immunostained for alpha-smooth muscle actin ( red ). Normal filling of the microvasculature was observed in control rats ( a ), whereas rats treated with monocrotaline (MCT) showed a marked loss of microvascular perfusion and widespread precapillary occlusion 21 d ( b ) and 35 d ( d ) after MCT injection. In the prevention model, animals receiving endothelial-like progenitor cells (ELPCs) displayed improved microvascular perfusion and preserved continuity of the distal vasculature ( c ). In the reversal model, endothelial nitric oxide synthase (eNOS)-transduced ELPCs dramatically improved the appearance of the pulmonary microvasculature ( f ), whereas progenitor cells alone resulted in more modest increases in perfusion and little noticeable reduction in arteriolar muscularization ( e ). Calibration bar (for a f ), 100 µm. Reprinted by permission from Reference 30 .

Figure 5

Figure 5. Cultured lung fibroblasts isolated from GFP bone marrow chimeric mice treated with bleomycin were analyzed by fluorescence microscopy ( a f ). The cells showed typical fibroblast morphology, many being stellate or spindle-shaped ( A ). On average, 80% of these cells expressed GFP (green fluorescence in a , at an original magnification of x 100; inset , x 400). Cells were stained with both anti-GFP ( green ) and anti–collagenase type I (Col I; red ) antibodies in ( b d ). The same microscopic field was photographed with the green ( b ) or red ( c ) filter only, or both simultaneously ( d ). Colocalization of both GFP and Col I expression resulted in a yellow color in d . Inset in d shows the cells stained with anti-GFP antibody ( green ) and isotype-matched control IgG for Col I ( red ). Cells were also stained with both anti-GFP ( green ) and anti–alpha-smooth muscle actin ( alpha-SMA) ( red ) antibodies ( e ). Colocalization of GFP and -SMA should appear yellow, but the two alpha-SMA + cells in this field did not appear to express GFP ( e ). Finally, cells were also stained with anti-GFP ( green ) and anti–telomerase reverse transcriptase (TERT; red ) antibodies. Colocalization of GFP and TERT appeared yellow , and most of the cells in this field expressed both TERT and GFP ( f ). Original magnification ( b f ), x 200. A representative example of at least three independent experiments is shown. Reprinted by permission from Reference 22 .

A number of studies suggest a functional implication for recruitment of adult marrow–derived cells to the lung. In mouse models of fibrosis, emphysema, and inflammatory lung injury, both MSCs and fetal liver cells (the fetal bone marrow equivalent) appear to abrogate lung injury ( 17 , 20 , 21 , 23 , 25 ). In an important proof-of-concept demonstration, ex vivo transduction of MSCs isolated from patients with cystic fibrosis (CF) resulting in expression of normal CF transmembrane conductance regulator (CFTR) was able to partially correct defective CFTR-dependent chloride current when ex vivo –transduced cells were mixed in culture with primary airway epithelial cells obtained from patients with CF ( 62 ) ( Figure 6 ). Conversely, it has been suggested that adult marrow–derived cells participate in the generation of fibrotic injuries ( 18 , 22 ). Additional phenotypes of adult marrow–derived cells, including MSCs and EPCs, may contribute to the development of lung and other malignancies in some mouse models, in part by providing a supportive stroma for the cancers ( 63 – 67 ). However, MSCs suppressed tumor growth when transduced to express interferon and thus adult marrow–derived stem cells may be potentially useful in cancer therapeutics. It has been demonstrated that EPCs participate in pulmonary vascular remodeling in a mouse model of pulmonary hypertension ( 30 ). Increased levels of circulating EPCs also correlate with lung repair in both pneumonia and in acute lung injury, although a causal relationship has not been established ( 44 , 45 ). Increasingly, adult marrow–derived circulating fibrocytes have been implicated in smooth muscle and myofibroblast proliferation in asthma and lung fibrosis ( 46 – 50 ). Nonetheless, despite an accumulating number of studies, the roles of adult marrow–derived cells in lung remodeling remain largely unclear and controversial.

Figure 6

Figure 6. CFTR-corrected MSCs from a patient with CF retained their multipotency and responded to cAMP stimulation by secreting chloride to the apical side. ( a ) Schematic for CF MSC isolation, expansion, gene correction, and positive drug selection. ( b ) Reverse transcription–polymerase chain reaction (RT-PCR) to verify successful CFTR gene transfer. RT-PCR was performed to amplify wild-type CFTR transcripts but not delta-F508 mutant transcripts. The gene-corrected CF MSCs and positive control Calu-3 cells have wild-type CFTR transcription, whereas non–gene-corrected CF MSCs and the no-RT control show negative amplification. In the RT-PCR control for the TATA-box–binding protein (TBP), all the samples except the no-RT control show positive PCR products. ( c ) Phase-contrast microscopic view of CFTR gene–corrected MSCs from a patient with CF. ( d ) Photomicrograph of a representative fibroblast colony-forming unit (CFU-F) assay. Purple -stained foci are the MSC colonies. ( e ) Osteogenesis of CFTR gene-corrected MSCs from a patient with CF. After differentiation in osteogenic medium, cells had mineral deposits visualized in red by alizarin red staining. ( f ) Adipogenesis of CFTR gene-corrected MSCs from a patient with CF. After differentiation in adipogenic medium, cells had lipid droplet accumulation stained in red with oil red O. ( g and h ) CFTR gene–corrected MSCs from CF patients contributed to apical cAMP-stimulated Cl – secretion. CFTR gene corrected CF patient MSCs or non–gene-corrected CF patient MSCs were mixed with delta-F508 CF airway epithelial cells at various ratios as indicated. After 1 mo in culture at the air–liquid interface, chloride efflux assays were performed as described in M ETHODS ( 62 ). Two-way analysis of variance revealed that cocultures with CFTR gene–corrected MSCs from the patient with CF ( 62 ) had greater chloride secretion in response to 3-isobutyl-1-methylxanthine and forskolin stimulation than did cocultures with non–gene-corrected CF patient MSCs (n = 4, p < 0.05). Reprinted by permission from Reference 62 .

Recruitment of adult marrow–derived cells to the lung.
Preexisting injury increases recruitment of adult marrow–derived cells to the lung ( 14 , 15 , 17 , 18 , 20 – 23 , 25 , 26 ). This suggests that chemotactic signals released by injured or remodeling lung serve to attract marrow–derived cells. Similarly, expression of specific adhesion molecules or other proteins by injured or remodeling lung may be important for recruitment of adult marrow–derived cells.

A number of soluble factors released by airway epithelium mediate recruitment of mature circulating leukocytes from bone marrow to the lung. There is a substantial body of literature demonstrating that increased release of many of these factors from stimulated or injured lung epithelium plays a significant role in inflammatory cell recruitment to the lung ( 68 ). Soluble factors also stimulate HSC mobilization and targeted tissue migration. Prominent among these is stromal-derived factor (SDF-1, CXCL12), which interacts with the CXCR4 receptor on the HSC surface ( 69 – 71 ). Other chemokine and angiogenic factors are also known to mobilize and recruit both HSCs and EPCs ( 69 – 71 ). HSCs and EPCs express several cell surface adhesion molecules (CD44, vascular cell adhesion molecule-1, and intercellular adhesion molecule-1) that can interact with corresponding tissue selectins and matrix proteins to mediate tissue binding ( 72 ). Increased release of soluble factors as well as increased expression of tissue ligands to which HSCs and EPCs can bind occur in a variety of lung injuries. Presumably this increases recruitment of HSCs and EPCs to the lung, but there are few direct data demonstrating this phenomenon ( 30 , 73 ).

Adult marrow–derived circulating fibrocytes exhibit chemotaxis in response to a number of ligands released from injured lung including SDF-1, CC chemokine ligand (CCL)-2, and eotaxin ( 47 – 49 , 74 ). Furthermore, exposure of circulating fibrocytes to ligands up-regulated in fibrotic lung injury including transforming growth factor-ß 1 and endothelin-1 promote differentiation into a myofibroblast phenotype ( 47 ).

Adult marrow–derived human MSCs express cell surface adhesion molecules including vascular cell adhesion molecule-1 and CD44. In experimental bleomycin-induced lung injury in mice, increased expression of the CD44 ligand hyaluronan is postulated to provide a homing signal for CD44-expressing MSCs ( 17 ). Bleomycin also increases lung expression of SDF-1, suggesting a role of this signaling pathway in recruitment of adult marrow–derived cells to the lung ( 48 ). SDF-1 has also been implicated in the recruitment of MSCs to liver and heart ( 69 , 73 ). Human MSCs express receptors for several chemokines and cytokines, including CXCR4, that might play a role in recruitment of MSCs to the lung, but there is little available information about the role of chemokines, cytokines, or other soluble factors in the recruitment of MSCs to the lung.

Mechanisms of phenotypic conversion of adult marrow–derived cells to lung cells.
Adult MSCs can be induced to develop phenotypic characteristics of fibroblasts, osteoblasts, chondrocytes, or adipocytes by in vitro exposure to growth factors and cytokines ( 75 ). In one report, coculture of human MSCs with primary human airway epithelial cells in vitro resulted in the acquisition of phenotypic and functional epithelial characteristics by the MSCs ( 62 ). This suggests that release of soluble mediators by lung epithelial cells and/or direct cell–cell contact between epithelial and mesenchymal cells can influence phenotypic conversion of MSCs to lung epithelial cells. However, specific factors directing conversion to the lung epithelial phenotype have not been elucidated. Exposure of circulating fibrocytes to ligands up-regulated in fibrotic lung injury, including transforming growth factor-ß 1 and endothelin-1, promotes differentiation into a myofibroblast phenotype ( 47 ). Little additional information is available concerning the mechanisms by which adult marrow–derived cells may be induced to undergo phenotypic change, acquiring characteristics of lung epithelial, vascular endothelial, or interstitial cells.

Endogenous lung stem cells.
Endogenous tissue stem cells are undifferentiated cells that reside in tissues and participate in regeneration after injury. Such cells have been identified for blood, intestine, and skin and their contribution to tissue maintenance and repair has been characterized at both the cellular and molecular levels. On the basis of these studies, tissue stem cells are understood to be a rare population of cells that cycle infrequently, an activity that results in self-maintenance and generation of transit-amplifying progeny. Transit cells are generally thought to have a finite life span and proliferative potential but exhibit organ-specific distinctions in overall abundance and frequency of cell proliferation ( see below ). These cells, as their name suggests, are a transient cell type that gives rise to differentiated cells, which participate in tissue-specific functions and are postmitotic. The small intestine harbors a regenerative epithelial unit that epitomizes such a classical stem cell hierarchy. Therein, intermittent proliferation of tissue-specific stem cells provides a continuous supply of transit-amplifying cells that proliferate repeatedly while positioned within the mitotic compartment. Eventually, these cells expend their mitotic potential or outdistance critical growth factors, activate the terminal differentiation program, and generate postmitotic cells of the villus epithelium. Evidence indicating the existence of cellular components of an airway epithelial stem cell hierarchy comes from studies in animal models in which selective ablation of epithelial cells is achieved through exposure to toxic chemicals or through cell type–specific expression of toxin genes in transgenic mice. Within airways of the lung, nonciliated bronchiolar epithelial (Clara) cells exhibit characteristics of transit-amplifying cells after injury to terminally differentiated cells. However, unlike transit-amplifying cells of tissues harboring classical stem cell hierarchies, Clara cells exhibit a low proliferative frequency in the steady state, are broadly distributed throughout the bronchiolar epithelium, and contribute to the specialized tissue activated through expression of differentiated characteristics. The existence of bronchiolar stem cells has been suggested from studies in which regenerative foci are maintained in airways after chemical ablation of the Clara (transit-amplifying) cell population ( 76 – 79 ). These cells, termed variant CCSP (Clara cell secretory protein)–expressing cells ( 78 ), contribute to distinct regenerative units within the bronchiolar and terminal bronchiolar epithelium and may participate in the establishment and maintenance of the distinct anatomic regions within intrapulmonary airways. Similar strategies have been used to identify putative stem cells for other lung epithelial compartments and suggest that the tracheobronchial zone is repaired by a subpopulation of basal epithelial cells and that the alveolar epithelium is maintained by type II cells ( 80 – 86 ) ( Figure 7 ). Endogenous lung stem cells are also suggested to participate in the development of lung carcinoma ( 64 ), although the connection between tissue stem cells and cancer stem cells has not been established. Side population CD45 – Hoechst-effluxing cells have also been identified in the lung, but their role in lung repair and remodeling is unclear ( 55 – 61 ). Future studies are needed to define the intrinsic and extrinsic mechanisms regulating activity of endogenous lung stem cells and aspects of this signaling hierarchy that distinguish this hierarchy from classical stem cell hierarchies of nonendodermally derived tissues.

Figure 7

Figure 7. Endogenous regenerative microenvironments in the bronchiolar epithelium of the mouse. In situ hybridization for CCSP (Clara cell secretory protein) mRNA ( white autoradiographic grains ) was used to identify regions of regenerating epithelium after naphthalene-mediated progenitor cell depletion. Regenerative zones of neuroepithelial bodies were identified at branch points in the airways ( red ovals ) and at the bronchoalveolar duct junction ( green ovals ). Figure courtesy of Susan Reynolds and Barry Stripp (University of Pittsburgh).

SCIENTIFIC PRESENTATIONS

The workshop was organized into three oral presentation sessions and one poster presentation session. The goal of the presentations was to provide a state-of-the-art summary of existing information and to highlight questions to be addressed by future research directions. A brief summary of the oral presentations is provided below. The executive summary of each speaker's presentation and the poster abstracts are presented in the accompanying online supplement.

Session 1: Endogenous Lung Stem and Progenitor Cells
The goal of Session 1 was to review the current state of knowledge regarding identification of endogenous stem cell populations in the lung and their potential to augment repair and regeneration. Jeffrey Whitsett (University of Cincinnati, Cincinnati, OH) introduced the workshop by providing a general overview of potential uses of adult stem cells for the treatment of pulmonary diseases. Potential applications of cell-based therapies include correction of disease caused by defective gene expression, provision of replicating cells for repair/regeneration, use as disease modifiers, and correction of genetic defects by correcting cells of hematopoietic lineages. Specific areas that need to be addressed include but are not limited to identification of the cell types that are most capable of contributing to repair of the lung, definition of the differentiation and proliferation potentials of cells that may have engrafted in the lung, identification of endogenous stem cell populations in the lung, and characterization of signals (extrinsic and intrinsic) that regulate their activities. Transcriptional programs activated during organogenesis were reviewed to assess the relevance of these pathways to regeneration, repair, engraftment, and differentiation of stem cells in the lung.

Scott Randell (University of North Carolina at Chapel Hill, Chapel Hill, NC) discussed the role of large airway epithelial stem cells. Clarifying progenitor–progeny relationships in this region of the airways is important for understanding phenotypic changes that occur in disease (e.g., CF); the possible role of stem cell transformation in lung cancer; post–lung transplant bronchiolitis obliterans syndrome, which may be due to death of stem cells; and also the potential of stem cells as targets for gene therapy. However, identification of somatic stem cells in the pseudostratified epithelium of the large airways remains rudimentary because of its cellular complexity, low baseline proliferation, and also its cellular plasticity as evidenced by the multiplicity of cell types participating after injury. Nevertheless, clonal expansion of progenitor populations and identification of label-retaining cells in focal areas in mouse trachea that are thought to represent a stem cell niche suggest the presence of discrete somatic stem cells at this site. Available experimental models for defining the biologic potential of airway epithelial cells were reviewed and include assays of colony formation, repopulation of tracheal surface epithelium in injury models, and in vitro differentiation of isolated cells. Dr. Randell described an approach for the identification of putative stem cells based on the use of a transgenic mouse in which enhanced green fluorescent protein (EGFP) expression was driven by the basal cell–specific promoter. Using this approach, these investigators were able to isolate and identify a keratin-rich subpopulation of basal cells capable of clonal growth. Dr. Randell emphasized the need for identification of additional stem cell markers and purification strategies to be able to perform molecular characterization of putative stem cells/label-retaining cells as well as the need for more detailed label-retaining cell/lineage studies and characterization of stem cell niches.

Barry Stripp (University of Pittsburgh, Pittsburgh, PA) reviewed the current state of knowledge of stem cells of distal conducting airways, highlighting differences between bronchiolar stem cells and other, more classical (e.g., small intestinal) stem cell hierarchies. Many tissue-specific stem cells and their progeny are organized into a so-called classical hierarchy consisting of infrequently cycling cells and their more frequently dividing transient-amplifying (TA) progeny. TA cells vary between organs in their abundance, have limited proliferative potential, and give rise to differentiated cells. The classical stem cell hierarchy is epitomized by the small intestine. Unlike this situation, bronchiolar Clara cells exhibit TA characteristics after injury but are usually quiescent in the steady state. Investigation of mechanisms of airway repair after TA cell depletion has produced data demonstrating the existence of a bronchiolar tissue–specific stem cell. Ablation of Clara cells by administration of naphthalene allowed the identification of distal airway stem cells that were resistant to naphthalene and that localized to two discrete microenvironments, the neuroepithelial body and the bronchoalveolar duct junction ( Figure 7 ). A putative stem cell population, so-called variant CCSP–expressing cells, was inferred by expressing the herpes simplex virus thymidine kinase gene under the control of the CCSP promoter. Ablation of all CCSP-expressing cells led to failure to regenerate. Unlike the intestinal epithelium, the Wnt–ß-catenin signaling pathway appears to be activated late in the regeneration of naphthalene-injured airways. These findings demonstrate the existence of a nonclassical stem cell hierarchy based on the unique properties of the TA cell and the unique molecular regulation of stem and TA cells during epithelial renewal. These studies highlight the existence of regiospecific stem cells in the conducting airways and the complexity of analyzing the differentiation potential of stem cells at different sites. Future research directions emphasized by Dr. Stripp included the need for development of a distinct molecular profile (and therefore novel molecular markers) of stem and other cells within the stem cell hierarchy, establishment of in vitro methods for stem cell maintenance and differentiation, dissection of intrinsic versus extrinsic cues involved in molecular regulation of stem cell maintenance, and development of methods for assessment of in vitro and in vivo differentiation potential of isolated stem cells.

Susan Reynolds (University of Pittsburgh) presented ongoing work on prospective purification and functional analysis of lung side populations. The existence of distinct proximal and distal stem cells hierarchies (i.e., tracheobronchial, bronchiolar, and terminal bronchiolar) suggests that discrete regenerative units maintain the conducting airway epithelium. Characteristics of the proximal and distal epithelial compartments are potentially a reflection of distinctions in the properties of region-specific stem cells as well as the influence of the niche/microenvironment on the activity of these cells. Roles for both intrinsic and extrinsic influences complicate identification, purification, and testing of tissue-specific stem cells and development of in vivo and in vitro models for assessment of selected cell types. Development of such methods for prospective purification and functional testing is also complicated by limitations of known molecular markers for airway stem cells (e.g., cytokeratin-14 and CCSP). These include the fact that many are intracellular proteins and therefore not useful for selection of viable cells and are also expressed redundantly by cells at multiple levels of the stem cell hierarchy. Strategies employing markers useful in the selection of hematopoietic stem cells have indicated some utility of Sca-1 and c-Kit for enrichment of clonogenic, multipotent lung cells. Similarly, application of Hoechst efflux methods to the lung demonstrated that the trachea contains a so-called side population (SP), a subpopulation of cells identified by low Hoechst blue and red fluorescence. Similar populations were identified in airway (0.6%) and alveolar (0.19%) preparations. SP cells from all compartments tended to be small and agranular and exhibited low red and green autofluorescence. Fractionation of the tracheal SP into upper and lower portions (USP and LSP, respectively) demonstrated both molecular and functional heterogeneity within the SP band. The USP was enriched with cytokeratin-14 and vimentin-expressing cells and the LSP with vimentin-expressing cells. Clonogenic cells were detected within tracheal, airway, and alveolar SP cells, and clonogenic cells were particularly enriched within the tracheal LSP. Fractionation of tracheal (and airway) populations in this way represents a step toward molecular characterization of clonogenic cells within conducting airways. Dr. Reynolds also presented data showing that label-retaining cells are detected in both the epithelial and the mesenchymal compartment of the neuroepithelial body microenvironment and emphasized the importance of mesenchymal–epithelial interactions in maintaining the stem cell niche. Future initiatives are needed to identify cell surface markers for stem and TA cells, to elucidate mechanisms regulating stem cell niche communication, and to assess the impact of inappropriate signals on stem cell function.

Douglas Losordo (Tufts University School of Medicine, Boston, MA) discussed the role of adult stem cells in the repair of ischemic cardiac tissue. Myocardial infarction and cardiomyopathy both involve loss of cardiomyocytes as well as blood vessels, so both these components need to be considered in any strategy to improve cardiac function. Isolation of putative EPCs that can participate in angiogenesis changed the strategies for improving vascularization in ischemic heart disease. Dr. Losordo presented work demonstrating successful ex vivo expansion of EPCs as well as an increase in circulating EPCs in response to vascular endothelial growth factor. Bone marrow–derived EPCs were shown to contribute to vasculogenesis in mouse uterus. Transplantation of isolated ex vivo –expanded human EPCs into nude athymic rats via tail vein injection reduced the extent of myocardial fibrosis observed after acute myocardial ischemia, with preservation of left ventricular function. Limitations in applying this transplantation approach in humans include the possibility of deleterious immune reactions when using allogeneic cell donors, thereby requiring the acquisition of autologous cells for transplantation; the large number of cells required for transplantation; as well as safety concerns attributable to the use of fetal bovine serum as a medium supplement for ex vivo cell expansion. Therefore, the strategy of using autologous CD34 + cells for transplantation was evaluated. Preclinical animal studies demonstrated that injection of autologous granulocyte colony-stimulating factor (G-CSF)–mobilized CD34 + cells into rats after myocardial infarction improved capillary density and myocardial function. A pilot clinical trial of intramyocardial transplantation of autologous CD34 + cells for intractable angina in 24 patients has been completed. Patients with class 3 or 4 angina and no other therapeutic options were enrolled. In individual patients there was improvement in treadmill time and time to angina at 6-mo follow-up. No significant serious adverse events were reported related to injection. This pilot study suggests promise for transplantation of CD34 + cells to improve outcome after myocardial infarction.

Discussion summary.
There is a major need for the development of new technical methods and identification of specific molecular markers with which to identify stem cells to specify the genetic and regulatory factors required for maintaining "stemness." Similarly, new methods (e.g., laser capture microdissection coupled with microarray and proteomic analysis) will be required to accurately phenotype stem cells. It was suggested that parallel studies be undertaken, focusing simultaneously on basic mechanisms and clinical applications. In this regard, the importance of performing functional correlations to assess the outcome of any therapeutic maneuver was emphasized. The timing of therapeutic interventions was also discussed in the context of the need for an intact underlying lung structure to effect epithelial regeneration. Consideration also needs to be given to whether the efficacy of stem cells is related to an actual change in cellular phenotype versus benefits that result from the effects of transplanted or modified endogenous cells on the microenvironment. The concept of "reparative" cells versus stem cells for cell-based therapy was suggested. In this regard there was also some discussion of which disease would be the appropriate initial target for any clinical application, with some debate about whether space needed to be made for cells to engraft. An obvious key question that will complicate any therapeutic approach concerns which cell type should be used, for example, the different endogenous cells identified in the conducting airways of the lung (e.g., basal, neuroendocrine, and variant CCSP–expressing cells). Also, it is unclear which cells will be most effective. In the case of MSCs, there are indications that preparations rich in early progenitors from low-density cultures engraft in vivo more efficiently than do more mature cells from confluent cultures ( 87 ).

Session 2: Other Adult Stem Cell Sources
The goal of Session 2 was to review the basic biology of stem and progenitor cells, including those cell populations already suggested to participate in lung remodeling, as well as to identify other potential sources of adult stem cells for investigation.

The initial presentation, by Brigid Hogan (Duke University, Durham, NC), reviewed current definitions of stem and progenitor cells, including self-renewal capacity and ability to differentiate into multiple cell types. The role of factors influencing lineage development, particularly the role of specialized niches, was examined. Important parallels between embryonic lung development and repair of adult lungs after injury were examined. Future research directions emphasized by Dr. Hogan include the development and use of appropriately adequate and sensitive tools to trace lineage-specific development, particularly the use of conditional transgenic and knock-in mice, and an understanding of the cellular events underlying lineage development.

Peter Quesenberry (Roger Williams Medical Center, Providence, RI) presented further information on underlying stem cell biology and the relevance for plasticity and organ remodeling. A paradigm was presented in which stem/progenitor cells represent a continuum rather than static populations. He suggested that stem cells change during cell cycling, particularly with respect to expression of surface markers and the ability to repopulate other organs including the lung. The ability to manipulate stem cell phenotypic lability may be a powerful tool for regulating tissue restoration effected by adult stem cells.

Diane Krause (Yale University, New Haven, CT) examined factors implicated in the recruitment of adult marrow–derived stem cells to lung tissue in mice, particularly the role of radiation-induced lung injury. The relative radiation doses producing engraftment after transplantation, radiation-induced lung injury and apoptosis, and lung engraftment with adult marrow–derived stem cells were evaluated. Lung epithelial engraftment rarely occurs after sublethal radiation doses (less than 1,000 cGy), which do not induce significant lung injury and apoptosis. In contrast, a lethal dose of 1,000 cGy, which leads to significant levels of apoptosis of lung epithelial cells, consistently resulted in low-level lung epithelial engraftment of bone marrow–derived cells. Data were also presented demonstrating that transplantation of wild-type bone marrow to transgenic mice lacking functional CFTR resulted in low-level CFTR expression in intestinal epithelium. Importantly, transplanted mice demonstrated a partial functional correction of a defective transepithelial potential difference and chloride current measured in rectal mucosa. Future research directions emphasized by Dr. Krause included emphasis on mechanisms by which adult marrow–derived cells were recruited to lung, studies to highlight physiologic relevance, and mechanisms to enhance recruitment to achieve therapeutic benefit.

The current status of knowledge with respect to the role of circulating fibrocytes was presented by Robert Strieter (David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, CA). Circulating fibrocytes or mesenchymal precursor cells are increasingly implicated in the pathogenesis of pulmonary fibrosis and in subepithelial remodeling and fibrosis that can occur with severe asthma. The role of CXCL12 and GM-CSF in the recruitment of circulating fibrocytes to the lung and in the conversion to myofibroblasts was reviewed. New information was also presented suggesting that circulating fibrocytes may contribute to tissue adipocytes and play a role in metabolic syndrome. This presentation highlighted the importance of local niches in the phenotypic conversion of fibrocytes and other adult marrow–derived stem cell populations.

Bruce Bunnell (Tulane University, New Orleans, LA) presented novel information on the isolation of MSCs from fat in both mice and in nonhuman primates. The capability of these MSCs with respect to propagation and differentiation is comparable to that of MSCs isolated from bone marrow. Data were also presented on the isolation of stem cells from umbilical cord blood and from Wharton's jelly. Dr. Bunnell emphasized the importance of evaluating alternative sources of stem/progenitor cells as well as the need to consider larger animal preclinical models for the study of lung remodeling due to adult stem cells.

The final presentation in this session, by Alan Fine (Boston University, Boston, MA), highlighted an important aspect of existing and future studies. The techniques required to demonstrate lung remodeling by adult stem cells must be rigorous. Current techniques used to assess lung chimerism, particularly in situ immunohistochemical assessments, are subject to significant artifacts that can lead to overestimation of actual chimerism. State of the art techniques including deconvolution microscopy, laser capture microdissection, flow cytometry, and others must be rigorously used. Most important, functional data should be obtained wherever possible.

Discussion summary.
It is clear that there are many potential sources of stem and/or progenitor cells that might conceivably participate in lung remodeling during growth and repair after injury. A major challenge for the future will be to characterize the different putative cell populations both with respect to behavior as "stem cells" as well as with respect to lung remodeling. A significant emphasis must be placed on developing and standardizing state-of-the-art techniques to adequately and unambiguously assess the desired outcome measure, including functional outcomes.

Session 3: Adult Marrow–derived Stem Cells in Lung Diseases
The goal of Session 3 was to review preclinical data on adult marrow–derived cells in lung disease, to review translational research, and to begin exploring potential clinical protocols to investigate the safety and efficacy of these cell populations for lung diseases.

Sem Phan (University of Michigan, Ann Arbor, MI) presented data from studies exploring the use of bone marrow progenitor cells in a preclinical model of interstitial lung disease. The source of fibroblasts in fibrotic lung has been the focus of intense investigation, but the contribution of intrapulmonary versus extrapulmonary cells in the fibrosis disease process remains unclear. Phan and colleagues used a well-characterized murine model of bleomycin-induced fibrosis to examine the contribution of bone marrow–derived cells in the generation of the fibrotic lesion. To accomplish this, his laboratory generated chimeric GFP mice by using bone marrow obtained from transgenic GFP mice and transplanted into irradiated wild-type recipients. After demonstrating engraftment with peripheral blood chimerism in excess of 90% donor-derived cells, mice were subjected to bleomycin injury. Analysis of fibrotic lesions histologically showed large numbers of GFP-positive cells in fibrotic lesions and flow cytometry showed that these GFP-positive cells expressed type I collagen and telomerase reverse transcriptase, but not alpha-smooth muscle actin. These cells also expressed functional CXCR4 and CCR7 and could migrate in vitro to CCL-12 (SDF-1 alpha) and CCL-21 (secondary lymphoid-organ chemokine), ligands for these two receptors, respectively. These data support prior published data on human lungs from patients suffering lung injury after bone marrow transplantation, showing that bone marrow–derived cells contribute to fibrogenesis and that their recruitment is likely regulated by specific chemokine pathways.

Another pulmonary disorder characterized by fibrosis and scarring largely localized to lung arterioles and microvasculature is pulmonary hypertension. Duncan Stewart (University of Toronto, Toronto, ON, Canada) presented translational data on the use of adult bone marrow–derived cells to regenerate lung microvasculature. This group has previously described a population of bone marrow–derived cells termed endothelial-like progenitor cells (ELPCs), which are obtained from Fischer 344 rat bone marrow, separated by Ficoll, and differentiated in culture in growth medium supplemented with endothelial growth factors. These cells express endothelial markers such as von Willebrand factor, UEA-1 ( Ulex europaeus agglutinin-1) receptor, and Flk-1 and have been shown to engraft into areas of vascular injury caused by monocrotaline. When transfected ex vivo with an expression plasmid encoding endothelial nitric oxide synthase cDNA, differentiated ELPCs were able to improve survival and to reduce pulmonary pressures in rats with monocrotaline-induced pulmonary artery hypertension. Dr. Stewart than presented an outline of the Pulmonary Hypertension and Cell Therapy (PHACeT) trial, which is a phase 1 clinical safety study involving 18 patients with refractory pulmonary artery hypertension who will receive endothelial nitric oxide synthase–transfected autologous ELPCs delivered via pulmonary artery catheter. The study design involves dose escalation of up to 150 million cells administered in divided doses. The trial is to begin Spring 2006.

Dan Weiss (University of Vermont, Burlington, VT) and Pam Davis (Case Western Reserve University, Cleveland, OH) each made presentations describing the use of adult marrow–derived cells for the treatment of CF. By transplanting bone marrow–derived cells obtained from CFTR wild-type mice into CFTR knockout (KO) mice, Dr. Weiss and colleagues reported detection of CFTR message in the lungs of transplanted mice as well as detection of small numbers of CD45-negative, CFTR-positive cells in lung epithelium ( Figure 3 ). Although these results are encouraging, the levels of engraftment observed were small and, although not directly assessed, they are not believed to be sufficient to result in physiological correction of the transepithelial chloride current. Dr. Davis investigated the effect of transplantation of wild-type bone marrow into CFTR KO mice (transgenic for CFTR expression in the gut) to examine the effect of bone marrow–derived cells on the excessive mortality and lung inflammation observed in CFTR KO mice challenged with Pseudomonas aeruginosa encapsulated in agarose beads (a model to simulate the chronic inflammation seen in CF). Wild-type or CFTR KO mice that were transplanted with CFTR KO bone marrow had 100% mortality by Day 6 of the P. aeruginosa challenge, whereas CFTR KO mice receiving wild-type bone marrow had 50% survival, which was nearly identical to that observed in wild-type mice receiving wild-type bone marrow. Lung sections from the CFTR KO chimeric animals were analyzed extensively for epithelial chimerism and more than 97% of the chimeric cells detected were confined to a CD45 + phenotype and were not epithelial cells, suggesting that bone marrow–derived cells control pulmonary inflammatory responses in this model of lung inflammation. As CFTR has been reported to be expressed at the level of messenger RNA in macrophages and lymphocytes, CFTR protein expression in these cells may alter host immune responses to pathogens.

Stephen Rennard (University of Nebraska, Omaha, NE) closed the session by presenting translational data regarding the potential use of adult marrow–derived cells to treat chronic obstructive pulmonary disease (COPD)/emphysema. Using parabiotic mice that share a common circulatory system, the Rennard laboratory has shown that bone marrow–derived cells are recruited to the lung during elastase-induced emphysema. Many of these cells in the lung are hematopoietic, but a subset expresses cytokeratins and on ex vivo culture, up to 20% of the fibroblasts are bone marrow derived. In a follow-up study, Dr. Rennard and colleagues investigated the recruitment of bone marrow–derived cells to the lung after treatment of elastase-induced emphysema with all-trans retinoic acid with or without G-CSF as a stem cell–mobilizing agent. Combined treatment with all-trans retinoic acid and G-CSF resulted in enhanced recruitment of bone marrow–derived cells to the lung as well as a significant decrease in mean linear intercept, a measure of alveolar destruction. These data raise the possibility that cell transfer or mobilization of endogenous stem cells may be feasible in the setting of COPD. However, numerous unanswered questions remain, including whether specific subpopulations of cells contribute to repair versus injury. Dr. Rennard also presented data indicating that FEV 1 is an inadequate outcome measure in COPD, and suggested that validation of advanced imaging techniques such as CT imaging with texture analysis or functional magnetic resonance imaging using hyperpolarized helium (which can also be used to image regional diffusion) might be necessary to provide more reliable outcome measures in clinical trials for COPD.

Discussion summary.
There are a number of clinically relevant situations in which initial investigations of stem cell administration may provide invaluable information. However, it is acknowledged that, at present, this is a controversial topic. Some participants voiced the opinion that there is not enough basic science knowledge to move into clinical investigation. Others argued that there are large numbers of patients who have severely debilitating and life-threatening pulmonary diseases such as COPD who frequently run out of treatment options. Therefore it should be possible to begin early studies in humans to determine safety and dosing parameters, and to design carefully controlled clinical trials that offer the patients potential benefits at relatively little risk. At the same time it was agreed that strong emphasis must be placed on relevant animal models of human lung diseases, with a particular focus on developing clinically relevant functional outcome measures.

RECOMMENDATIONS: SUMMATION AND FUTURE DIRECTIONS

Workshop participants acknowleged that the role of adult stem cells in lung biology and their potential therapeutic role in lung diseases represent a timely and exciting area of study. Nonetheless, there are controversies with respect to assessment of both lung chimerism achieved by transplantation of adult bone marrow–derived stem cells and the functional role of lung remodeling effected by adult marrow–derived and other adult stem cell populations. As such, the following recommendations were developed.

General Recommendations
Terminology.
As is true in the current scientific literature, the term "stem cell" was used with varying degrees of rigor during the conference. The use of the term "stem" or "progenitor cell" provokes a broad range of response from different scientific disciplines. The definition of stem and progenitor cells is often loosely and erroneously applied. This can result in confusion and misconceptions about research goals and methodology. This may further be problematic given current public debate about the use of stem cells in biological research. The term "pluripotent embryonic stem cell" is used for mammalian embryonic stem (ES) cells that can both self-renew and differentiate into most cellular phenotypes (mouse ES cells cannot give rise to trophoblasts as can human ES cells). The term "stem cell" is used for cells with restricted repertoires of differentiation such as HSCs, neural stem cells, and intestinal epithelial cells.

Most of the attendees agreed that the primary interest of the conference was in cells that could repair damaged lung even if they did not meet the most rigorous definition of a stem or progenitor cell. As a central goal of much of the current research in this field is in repair and restoration of diseased lung, a suggestion was made to redefine the field in terms of reparative or regenerative investigations. This includes using terminology identifying "reparative cells" rather than "stem or progenitor cells" to indicate a population or populations of cells that participate in lung remodeling after injury. A further clarification is the distinction between endogenous reparative cells, that is, originating in the lung itself as opposed to exogenous reparative cells, which originate in bone marrow, fat, cord blood, or other sources and that subsequently are recruited to the lung to participate in remodeling after injury. The potential interaction between endogenous and exogenous reparative cells is an important area of investigation. Although agreement was not reached on these issues of terminology, all participants agreed that future study to define the true nature of the cells involved in lung repair is a critical issue.

Basic Science Investigations
In addition to clarifying and understanding the mechanisms and role of adult marrow–derived and endogenous lung stem cells in lung biology, an important goal is to develop a firm scientific basis for translational approaches to the repair and regeneration of diseased lung, using endogenous and exogenous reparative cells.

To fully explore and promote an understanding of the role of reparative cells in lung development, repair, and remodeling, a number of recommendations were developed.

Techniques.
There is continuing controversy over the adequacy of methods currently used to assess outcomes in studies of endogenous and exogenous reparative cells in lung biology and diseases. This is most relevant with respect to immunohistochemical and other approaches used to detect and characterize reparative cells in the lung. Results obtained from routine photomicroscopy of lung sections to detect markers delineated by fluorescence in situ hybridization, immunohistochemical, as well as other techniques can be misleading.

RECOMMENDATIONS: TECHNIQUES

  • Photomicroscopy should incorporate advanced imaging techniques, including confocal microscopy, deconvolution microscopy, electron microscopy, laser capture, and other techniques as applicable.
  • Techniques including flow cytometry, DNA and/or RNA analyses, and other comparable approaches should be done in parallel where feasible.

Functional outcome measures.
The functional role of endogenous and exogenous reparative cells is largely unknown in different lung disease states. Furthermore, in the few available studies in which a functional role is suggested, the mechanism(s) by which functional alterations occur are unknown. These questions are crucial to potential therapeutic applications of reparative cells.

RECOMMENDATIONS: FUNCTIONAL OUTCOMES MEASURES

  • Functional outcomes should be included to the extent feasible in all preclinical studies.
  • Functional outcomes should be tailored to each specific disease model investigated.
  • Functional outcomes must be sensitive (high throughput) and easily and reproducibly quantitated. Examples include the following:
    • Expression of a missing gene or gene product and/or measurement of a gene product function (i.e., chloride current after CFTR expression)
    • Restored pulmonary function (HRCT scans, histomorphometry, small animal pulmonary mechanics)

Standardized preclinical models of human lung disorders.
The relevance of standardized preclinical models of human lung disorders is to promote a greater understanding of the cellular and molecular pathophysiology of human disorders.

RECOMMENDATIONS: PRECLINICAL MODELS OF HUMAN LUNG DISORDERS

  • Several models are currently available, primarily in mice. The development of additional models that mimic features of an important human disorder is a desired goal.
  • A suggested but not inclusive list of available (mouse) and/or desired models includes:
    • Pulmonary hypoplasia of prematurity, or due to surfactant deficiency
    • Idiopathic pulmonary fibrosis
    • Adult onset emphysema/COPD
    • CF
    • Acute airway injury due to environmental factors
    • Acute respiratory distress syndrome
    • Primary pulmonary hypertension
    • Post–lung transplant bronchiolitis
    • Asthma

  • The development of larger animal models of clinical lung diseases is a desirable goal. Acknowledging the high costs and other practical considerations for larger animal work, particularly in nonhuman primates, the development of cores for appropriate large animal models of specific lung diseases would be advantageous.

Tools.
The development of more robust tools for identification and characterization of potential endogenous and exogenous reparative cell populations is a high-priority area. Of particular importance is the need to develop these tools in parallel in both rodent and in human cells.

RECOMMENDATIONS: TOOLS

  • Characterization of cell-specific markers for isolating and characterizing different reparative cell populations
  • Development of additional cell-specific promoters that track endogenous expression with reasonable fidelity
  • Development of more robust markers for histology—suitable for laser capture microscopy, confocal microscopy, and so on
  • Development of more robust markers specific for flow cytometry—fluorescent, protease-resistant surface markers
  • Development of lung-specific (monoclonal) antibody libraries
  • Development of genetic mouse models for investigation of functional pathways in lung development and repair. Examples include the following:
    • Conditional gene activation/deletion based on the use of CreERt2 or rtTA (doxycycline regulated)
    • Modified genes bred onto different genetic backgrounds, including mutants lacking specific immune cells
    • Conditional deletion of specific cell populations, for example, neuroendocrine, ciliated, type II

Fundamental understanding of lung repair mechanisms.
The cells that mediate lung repair and the mechanisms by which they do so remain poorly understood. Both endogenous and exogenous reparative cells likely participate. Understanding the roles of each and their interactions is a crucial area of investigation. Recommendations focus largely but not exclusively on the use of different models.

RECOMMENDATIONS: LUNG REPAIR MECHANISMS

  • Use of different proof of concept models. Although some experimental injury/repair systems, for example, using naphthalene, ozone, bleomycin, SO 2 , and so on, do not necessarily mimic human lung disorders, they are useful for testing specific hypotheses.
  • Investigations of different plasticity/dedifferentiation models, including label-retaining tissue stem cell models for endogenous reparative cells.
  • Increased use and investigation of lung regeneration models. Some suggested models include postpneumonectomy regeneration, septal proliferation after retinoic acid treatment, nutrition after neonatal deprivation, and so on.
  • Investigations of basic development and repair mechanisms including:
    • Mechanisms for normal lineage specification of epithelial, mesenchymal, and endothelial cells
    • Mechanisms of epithelial/endothelial/mesenchymal interactions
    • Cell–matrix interactions
    • Identification of stem cell niches; how are they formed and maintained?
    • Impact of lung remodeling on niches
    • Impact of ageing, disease, and immune system on regenerative capacity

Mechanisms of recruitment and phenotypic conversion of reparative cells.
Little is known of the mechanisms by which exogenous reparative cells are recruited to lung and/or induced to undergo conversion to functional lung cells.

RECOMMENDATIONS: MECHANISMS OF RECRUITMENT AND PHENOTYPIC CONVERSION

  • Development of appropriate in vivo model systems including transgenic mice and use of gene transfer techniques for conditional or targeted overexpression of candidate recruitment mediators
  • Demonstration of functional relevance of identified potential recruitment mechanisms

Development of in vitro methods.
In addition to the in vivo studies delineated above, development of robust in vitro model systems are crucial to understanding the role of reparative cells in lung biology and disease.

RECOMMENDATIONS: IN VITRO METHODS

  • Development of methods for in vitro expansion of lung mesenchymal and epithelial cells
  • Expansion and improvement of methods for testing reparative cell potential including but not limited to:
    • Culture with and without feeder cells
    • Culture with and without growth factors/serum/retinoids
    • Culture using different substrata
    • Combined culture with mesenchyme and grafting into permissive tissues such as under kidney capsule
    • Expansion of air–liquid interface culture studies
    • Use of tracheal heterotopic transplantation models—either denuded and inoculated with cells from different sources or repopulation by endogenous cells
    • Investigation of three-dimensional organoid cultures

Consideration of core centers and facilities.
The pros and cons of whether to establish core centers for training, cell culture and generation and provision of specific cell types, functional outcome assessments, histologic assessments, and other methodologic techniques were discussed and the following recommendations were made.

RECOMMENDATIONS: CORE CENTERS AND FACILITIES

  • Widespread dissemination about already existing core services and facilities. A partial list includes the following:
    • Tulane Mesenchymal Stem Cell Core (expected to regain full functional capacity in several months)
    • NHLBI Production Assistance for Cellular Therapies Program
    • Good Manufacturing Practice Vector Core Facilities
    • University of Vermont Small Animal Airway Mechanics and Small Animal CT Scanner Facilities

  • Active fostering of interinstitutional multidisciplinary collaborations and consortiums

Educational programs.
A highly desirable goal is to inform and educate the stem cell community about the potential role of stem and reparative cells in lung biology and disease. Conversely, the lung community needs to be educated about stem and reparative cell biology. This will lead to fruitful interactions and collaborations and, importantly, will result in less overlapping and duplicative research efforts.

RECOMMENDATIONS: EDUCATIONAL PROGRAMS

  • Educational programs at national meetings
  • Stem cell training courses with both didactic and practical components from different disciplines
  • Arranging sabbaticals/training for both fellows and faculty in laboratories of established stem cell investigators not necessarily in the lung field

Clinical/Translational Recommendations
There is a divergence of opinion on whether clinical investigation of reparative cells in lung biology and disease is warranted at present. Importantly, a representative from the Food and Drug Administration was present at the meeting to add the current Food and Drug Administration perspective on stem cell research. The pro and con points raised at the meeting are summarized in Table 1 . After vigorous discussion, the following recommendations for clinical and translational studies were made.

TABLE 1. CLINICAL STUDIES OF STEM CELLS IN LUNG DISEASES

Pro

  • There is a considerable safety record with clinical use of stem cells, that is, bone marrow transplantation
  • Initial exploratory trials, phase 1 or otherwise, can be safely designed and implemented with appropriately measurable outcome assessments
  • Pulmonary and critical care medicine has traditionally lagged behind other fields, for example, cardiology and hematology, in translational studies of potential new therapies, including use of reparative cells
  • Initial exploratory clinical trials are important in hypothesis generation for future basic and translational studies
  • The FDA will support carefully designed initial investigatory trials. These will be reviewed on an individual basis
  • A Canadian exploratory trial of the role of reparative cells in pulmonary hypertension, under the direction of Duncan Stewart, M.D., University of Toronto, will shortly begin. Comparable trials may likely begin shortly in Europe. The United States stands to fall behind
  • There are many patients with end-stage lung diseases who are already offering to participate in clinical trials and investigations. Although patients with end-stage lung diseases may not be the optimal population to study, initial investigations will provide important data about safety and will generate future hypotheses for study

Con

  • There is not enough evidence for a function of exogenous cells in lung repair to venture into clinical trials
  • Despite the track record of safety with bone marrow transplantation, there is not enough information about long-term effects of "plasticity." Cogent examples are the publications demonstrating development of gastric cancer from transplanted stem cells in a mouse model of chronic H. pylori infection and the potential role of adult stem cells in development of lung cancer
  • There are unresolved ethical issues
  • There is concern that unanticipated adverse outcomes of early clinical application could impact future progress in the field as happened with early gene therapy trials

RECOMMENDATIONS: CLINICAL STUDIES

  • Proceed with design and implementation of initial exploratory and safety investigations.
  • All appropriate ethical issues should be considered.
  • Clinical and translational investigations should include consideration of mechanisms of reparative cell actions and not be limited to simple outcome measurements.
  • Careful consideration should be given to sample collections (i.e., blood, bronchoalveolar lavage fluid, biopsies) from already existing clinical trials wherein analyses of these samples might provide insight into potential roles and mechanisms of lung remodeling by adult reparative cells.
  • A central coordinating center should be considered to take advantage and appropriately distribute clinical samples to ensure maximal usefulness.
  • The National Institutes of Health, Food and Drug Administration, and other relevant regulatory agencies should work closely together to define parameters for adequate cell product characterization as well as preclinical proof of concept and toxicologic testing to support the design and supervision of phase 1 clinical trials.

SUMMARY AND CONCLUSIONS

A continuing accumulation of data in both animal models and in clinical lung specimens suggests that exogenous reparative cells play a significant role in lung development and in lung repair and remodeling after injury. Nonetheless, these data are controversial both from the perspective of adequacy of experimental techniques to assess participation of reparative cells and from a need for better assessment of the specific functional role reparative cells might play in lung remodeling. It is hoped that the workshop recommendations will spark new research that will provide further understanding of the role of stem and reparative cells in lung biology and also provide a sound scientific basis for therapeutic use of adult reparative cells in lung diseases.

ACKNOWLEDGMENTS

The authors thank Robert Beall, Patricia Cowles, John Evans, Jean Hood, Charles Irvin, Jennifer Nachbur, Robert Prenovitz, Christine Ross, Carol Whittaker, Susan Reynolds, and Barry Stripp.

Conflict of Interest Statement : None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Participants:

Co-chairs— Darwin J. Prockop, M.D., Ph.D. ( Tulane University Health Science Center) and Daniel J. Weiss, M.D., Ph.D. ( University of Vermont College of Medicine)

  • Melissa A. Ashlock M.D. (Cystic Fibrosis Foundation Therapeutics, Inc.)
  • Jason H.T. Bates, Ph.D. (University of Vermont College of Medicine)
  • Robert J. Beall, Ph.D. (Cystic Fibrosis Foundation)
  • Mary Anne Berberich, Ph.D.(Nation Heart Lung and Blood Institute)
  • Zea Borok,, M.D. (University of Southern California)
  • Kenneth L. Brigham, M.D. (Emory University School of Medicine)
  • Steven L. Brody, M.D. (Washington University, St. Louis)
  • Bruce Bunnell, Ph.D. (Tulane University Health Science Center)
  • Wellington V. Cardoso, M.D. Ph.D. (Boston University)
  • Frances Carr, Ph.D. (University of Vermont)
  • Pamela B. Davis, M.D., Ph.D. (Case Western Reserve University)
  • Barbara Driscoll, Ph.D. (Saban Research Institute of Children's Hospital Los Angeles)
  • Marie E. Egan, M.D. (Yale University School of Medicine)
  • John F. Engelhardt, Ph.D. (University of Iowa)
  • Michael Epperly, Ph.D. (University of Pittsburgh School of Medicine)
  • John Evans Ph.D. (University of Vermont)
  • Alan Fine, M.D. (Boston University Medical Campus)
  • Donald W. Fink, Jr., Ph.D. ( Center for Biologics Evaluation and Research)
  • Dorothy B. Gail, Ph.D. (Nation Heart Lung and Blood Institute)
  • Brigid L. Hogan, Ph.D. (Duke University)
  • Charles Irvin, Ph.D. (University of Vermont College of Medicine)
  • Jay K. Kolls, M.D. (Children's Hospital of Pittsburgh)
  • Darrell N. Kotton, M..D. (Boston University Medical Campus)
  • Diane Krause M.D, Ph.D. (Yale University School of Medicine)
  • Douglas William Losordo, M.D. (St. Elizabeth Medical Center)
  • Carolyn Lutzko, Ph..D. (Children’s Hospital Los Angeles)
  • William J. Martin II, M.D. (University of Cincinnati)
  • Dr. Michael Mengel (Institut Fuer Pathologie Medizinische Hochschule Hannover)
  • Marc B. Moss, M.D. (Emory University)
  • Brooke T. Mossman, Ph.D. (University of Vermont College of Medicine)
  • Luis A. Ortiz, M.D. (University of Pittsburgh)
  • Polly E. Parsons, M.D. (University of Vermont College of Medicine)
  • Chris Penland, Ph.D. (Cystic Fibrosis Foundation)
  • Sem H. Phan, M.D. (University of Michigan)
  • Donald Phinney, Ph.D. (Tulane University)
  • Matthew E. Poynter, Ph.D. (University of Vermont)
  • Peter J. Quesenberry, M.D. (Roger Williams Hospital)
  • Scott H. Randell, Ph.D. (University of North Carolina,Chapel Hill)
  • Stephen I. Rennard, M.D. (University of Nebraska Medical Center)
  • Susan D. Reynolds, Ph.D. (University of Pittsburgh)
  • Richard M. Rose, M.D. (University of California, San Diego)
  • Steve D. Shapiro, M.D. (Brigham and Womens' Hospital)
  • Jeffrey L. Spees, Ph.D. (University of Vermont College of Medicine)
  • Duncan J. Stewart, MD, FRCPC (University of Toronto)
  • Robert M. Strieter, M.D. (David Geffen School of Medicine at UCLA)
  • Barry R. Stripp, Ph.D. (University of Pittsburgh)
  • Benjamin T. Suratt, M.D. (University of Vermont College of Medicine)
  • Jakub Tolar, M.D., Ph.D. (University of Minnesota Medical School)
  • Anna M. Van Heeckeren, D.V.M., M.S. (Case Western Reserve University)
  • Nobert F. Voelkel, M.D. (University of Colorado Health Science Center)
  • Guoshun Wang, D.V.M., Ph.D. (Louisiana State University)
  • David Warburton, M.D. (Children's Hospital LA Research Institute)
  • Jeffrey A Whitsett, M.D. (University of Cincinnati)
  • Mary C. Williams, Ph.D. (Boston University)
  • Lawrence N. Yager, Ph.D. ( National Center for Research Resources)

References

  1. Pereira RF, Halford KW, O'Hara MD, Leeper DB, Sokolov BP, Pollard MD, Bagasra O, Prockop DJ. Cultured adherent cells from marrow can serve as long-lasting precursor cells for bone, cartilage, and lung in irradiated mice. Proc Natl Acad Sci USA 1995;92:4857–4861.
  2. Jiang Y, Jahagirdar BN, Reinhardt RL, Schwartz RE, Keene CD, Ortiz-Gonzalez XR, Reyes M, Lenvik T, Lund T, Blackstad M, et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 2002;418:41–49.
  3. Wagers AJ, Christensen JL, Weissman IL. Cell fate determination from stem cells. Gene Ther 2002;9:606–612.
  4. Korbling M, Estrov Z. Adult stem cells for tissue repair: a new therapeutic concept? N Engl J Med 2003;349:570–582.
  5. Prockop DJ. Further proof of the plasticity of adult stem cells and their role in tissue repair. J Cell Biol 2003;160:807–809.
  6. Herzog EL, Chai L, Krause DS. Plasticity of marrow-derived stem cells. Blood 2003;102:3483–3493.
  7. Anjos-Afonso F, Siapati EK, Bonnet D. In vivo contribution of murine mesenchymal stem cells into multiple cell types under minimal damage conditions. J Cell Sci 2004;117:5655–5664.
  8. Neuringer IP, Randell SH. Stem cells and repair of lung injuries. Respir Res 2004;5:6.
  9. Aliotta JM, Passero M, Meharg J, Klinger J, Dooner MS, Pimentel J, Quesenberry PJ. Stem cells and pulmonary metamorphosis: new concepts in repair and regeneration. J Cell Physiol 2005;204:725–741.
  10. Wang X, Willenbring H, Akkari Y, Torimaru Y, Foster M, Al-Dhalimy M, Lagasse E, Finegold M, Olson S, Grompe M. Cell fusion is the principal source of bone-marrow–derived hepatocytes. Nature 2003;422:897–901.
  11. Vassilopoulos G, Wang PR, Russell DW. Transplanted bone marrow regenerates liver by cell fusion. Nature 2003;422:901–904.
  12. Camargo FD, Green R, Capetanaki Y, Jackson KA, Goodell MA. Single hematopoietic stem cells generate skeletal muscle through myeloid intermediates. Nat Med 2003;9:1520–1527.
  13. Krause DS, Theise ND, Collector MI, Henegariu O, Hwang S, Gardner R, Neutzel S, Sharkis SJ. Multi-organ, multi-lineage engraftment by a single bone marrow–derived stem cell. Cell 2001;105:369–377.
  14. Kotton DN, Ma BY, Cardoso WV, Sanderson EA, Summer RS, Williams MC, Fine A. Bone marrow–derived cells as progenitors of lung alveolar epithelium. Development 2001;128:5181–5188.
  15. Theise ND, Henegariu O, Grove J, Jagirdar J, Kao PN, Crawford JM, Badve S, Saxena R, Krause DS. Radiation pneumonitis in mice: a severe injury model for pneumocyte engraftment from bone marrow. Exp Hematol 2002;30:1333–1338.
  16. Grove JE, Lutzko C, Priller J, Henegariu O, Theise ND, Kohn DB, Krause DS. Marrow–derived cells as vehicles for delivery of gene therapy to pulmonary epithelium. Am J Respir Cell Mol Biol 2002;27:645–651.
  17. Ortiz LA, Gambelli F, McBride C, Gaupp D, Baddoo M, Kaminski N, Phinney DG. Mesenchymal stem cell engraftment in lung is enhanced in response to bleomycin exposure and ameliorates its fibrotic effects. Proc Natl Acad Sci USA 2003;100:8407–8411.
  18. Epperly MW, Guo H, Gretton JE, Greenberger JS. Bone marrow origin of myofibroblasts in irradiation pulmonary fibrosis. Am J Respir Cell Mol Biol 2003;29:213–224.
  19. Abe S, Lauby G, Boyer C, Rennard SI, Sharp JG. Transplanted BM and BM side population cells contribute progeny to the lung and liver in irradiated mice. Cytotherapy 2003;5:523–533.
  20. Ishizawa K, Kubo H, Yamada M, Kobayashi S, Numasaki M, Ueda S, Suzuki T, Sasaki H. Bone marrow–derived cells contribute to lung regeneration after elastase-induced pulmonary emphysema. FEBS Lett 2004;556:249–252.
  21. Yamada M, Kubo H, Kobayashi S, Ishizawa K, Numasaki M, Ueda S, Suzuki T, Sasaki H. Bone marrow–derived progenitor cells are important for lung repair after lipopolysaccharide-induced lung injury. J Immunol 2004;172:1266–1272.
  22. Hashimoto N, Jin H, Liu T, Chensue SW, Phan SH. Bone marrow–derived progenitor cells in pulmonary fibrosis. J Clin Invest 2004;113:243–252.
  23. Abe S, Boyer C, Liu X, Wen FQ, Kobayashi T, Fang Q, Wang X, Hashimoto M, Sharp JG, Rennard SI. Cells derived from the circulation contribute to the repair of lung injury. Am J Respir Crit Care Med 2004;170:1158–1163.
  24. Dooner M, Cerny J, Colvin G, Demers D, Pimentel J, Greer D, Abedi M, McAuliffe C, Quesenberry P. Homing and conversion of murine hematopoietic stem cells to lung. Blood Cells Mol Dis 2004;32:47–51.
  25. Rojas M, Xu J, Woods CR, Mora AL, Spears W, Roman J, Brigham KL. Bone marrow–derived mesenchymal stem cells in repair of the injured lung. Am J Respir Cell Mol Biol 2005;33:145–152.
  26. Beckett T, Loi R, Prenovitz R, Poynter M, Goncz KK, Suratt BT, Weiss DJ. Acute lung injury with endotoxin or NO 2 does not enhance development of airway epithelium from bone marrow. Mol Ther 2005;12:680–686.
  27. Macpherson H, Keir P, Webb S, Samuel K, Boyle S, Bickmore W, Forrester L, Dorin J. Bone marrow–derived SP cells can contribute to the respiratory tract of mice in vivo . J Cell Sci 2005;118:2441–2450.
  28. Schoeberlein A, Holzgreve W, Dudler L, Hahn S, Surbek DV. Tissue-specific engraftment after in utero transplantation of allogeneic mesenchymal stem cells into sheep fetuses. Am J Obstet Gynecol 2005;192:1044–1052.
  29. Loi R, Beckett T, Goncz KK, Suratt BT, Weiss DJ. Limited restoration of defective cystic fibrosis lung epithelium in vivo with adult marrow derived cells. Am J Respir Crit Care Med 2006;173:171–179.
  30. Zhao YD, Courtman DW, Deng Y, Kugathasan L, Zhang Q, Stewart DJ. Rescue of monocrotaline-induced pulmonary arterial hypertension using bone marrow–derived endothelial-like progenitor cells: efficacy of combined cell and eNOS gene therapy in established disease. Circ Res 2005;96:442–450.
  31. Suratt BT, Cool CD, Serls AE, Chen L, Varella-Garcia M, Shpall EJ, Brown KK, Worthen GS. Human pulmonary chimerism after hematopoietic stem cell transplantation. Am J Respir Crit Care Med 2003;168:318–322.
  32. Mattsson J, Jansson M, Wernerson A, Hassan M. Lung epithelial cells and type II pneumocytes of donor origin after allogeneic hematopoietic stem cell transplantation. Transplantation 2004;78:154–157.
  33. Kleeberger W, Versmold A, Rothamel T, Glockner S, Bredt M, Haverich A, Lehmann U, Kreipe H. Increased chimerism of bronchial and alveolar epithelium in human lung allografts undergoing chronic injury. Am J Pathol 2003;162:1487–1494.
  34. Spencer H, Rampling D, Aurora P, Bonnet D, Hart SL, Jaffe A. Transbronchial biopsies provide longitudinal evidence for epithelial chimerism in children following sex mismatched lung transplantation. Thorax 2005;60:60–62.
  35. Bittmann I, Dose T, Baretton GB, Muller C, Schwaiblmair M, Kur F, Lohrs U. Cellular chimerism of the lung after transplantation: an interphase cytogenetic study. Am J Clin Pathol 2001;115:525–533.
  36. Davies JC, Potter M, Bush A, Rosenthal M, Geddes DM, Alton EW. Bone marrow stem cells do not repopulate the healthy upper respiratory tract. Pediatr Pulmonol 2002;34:251–256.
  37. Zander DS, Baz MA, Cogle CR, Visner GA, Theise ND, Crawford JM. Bone marrow–derived stem-cell repopulation contributes minimally to the type II pneumocyte pool in transplanted human lungs. Transplantation 2005;80:206–212.
  38. Chang JC, Summer R, Sun X, Fitzsimmons K, Fine A. Evidence that bone marrow cells do not contribute to the alveolar epithelium. Am J Respir Cell Mol Biol 2005;33:335–342.
  39. Kotton DN, Fabian AJ, Mulligan RC. Failure of bone marrow to reconstitute lung epithelium. Am J Respir Cell Mol Biol 2005;33:328–334.
  40. Trotman W, Beckett T, Goncz KK, Beatty BG, Weiss DJ. Dual Y chromosome painting and in situ cell-specific immunofluorescence staining in lung tissue: an improved method of identifying donor marrow cells in lung following bone marrow transplantation. Histochem Cell Biol 2004;121:73–79.
  41. Spees JL, Olson SD, Ylostalo J, Lynch PJ, Smith J, Perry A, Peister A, Wang MY, Prockop DJ. Differentiation, cell fusion, and nuclear fusion during ex vivo repair of epithelium by human adult stem cells from bone marrow stroma. Proc Natl Acad Sci USA 2003;100:2397–2402.
  42. Harris RG, Herzog EL, Bruscia EM, Grove JE, Van Arnam JS, Krause DS. Lack of a fusion requirement for development of bone marrow–derived epithelia. Science 2004;305:90–93.
  43. Horwitz EM, Le Blanc K, Dominici M, Mueller I, Slaper-Cortenbach I, Marini FC, Deans RJ, Krause DS, Keating A. Clarification of the nomenclature for MSC: the International Society for Cellular Therapy position statement. Cytotherapy 2005;7:393–395.
  44. Yamada M, Kubo H, Ishizawa K, Kobayashi S, Shinkawa M, Sasaki H. Increased circulating endothelial progenitor cells in patients with bacterial pneumonia: evidence that bone marrow derived cells contribute to lung repair. Thorax 2005;60:410–413.
  45. Burnham EL, Taylor WR, Quyyumi AA, Rojas M, Brigham KL, Moss M. Increased circulating endothelial progenitor cells are associated with survival in acute lung injury. Am J Respir Crit Care Med 2005;172:854–860.
  46. Direkze NC, Forbes SJ, Brittan M, Hunt T, Jeffery R, Preston SL, Poulsom R, Hodivala-Dilke K, Alison MR, Wright NA. Multiple organ engraftment by bone-marrow–derived myofibroblasts and fibroblasts in bone-marrow–transplanted mice. Stem Cells 2003;21:514–520.
  47. Schmidt M, Sun G, Stacey MA, Mori L, Mattoli S. Identification of circulating fibrocytes as precursors of bronchial myofibroblasts in asthma. J Immunol 2003;171:380–389.
  48. Phillips RJ, Burdick MD, Hong K, Lutz MA, Murray LA, Xue YY, Belperio JA, Keane MP, Strieter RM. Circulating fibrocytes traffic to the lungs in response to CXCL12 and mediate fibrosis. J Clin Invest 2004;114:438–446.
  49. Moore BB, Kolodsick JE, Thannickal VJ, Cooke K, Moore TA, Hogaboam C, Wilke CA, Toews GB. CCR2-mediated recruitment of fibrocytes to the alveolar space after fibrotic injury. Am J Pathol 2005;166:675–684.
  50. Ishii G, Sangai T, Sugiyama K, Ito T, Hasebe T, Endoh Y, Magae J, Ochiai A. In vivo characterization of bone marrow–derived fibroblasts recruited into fibrotic lesions. Stem Cells 2005;23:699–706.
  51. Gomperts BN, Belperio JA, Burdick MD, Streiter RM. Circulating progenitor cells traffic via CXCR4/CXCL12 in response to airway epithelial injury. J Immunol 2006;176:1916–1927.
  52. Ali NN, Edgar AJ, Samadikuchaksaraei A, Timson CM, Romanska HM, Polak JM, Bishop AE. Derivation of type II alveolar epithelial cells from murine embryonic stem cells. Tissue Eng 2002;8:541–550.
  53. Rippon HJ, Ali NN, Polak JM, Bishop AE. Initial observations on the effect of medium composition on the differentiation of murine embryonic stem cells to alveolar type II cells. Cloning Stem Cells 2004;6:49–56.
  54. Coraux C, Nawrocki-Raby B, Hinnrasky J, Kileztky C, Gaillard D, Dani C, Puchelle E. Embryonic stem cells generate airway epithelial tissue. Am J Respir Cell Mol Biol 2005;32:87–92.
  55. Abe S, Lauby G, Boyer C, Rennard SI, Sharp JG. Transplanted BM and BM side population cells contribute progeny to the lung and liver in irradiated mice. Cytotherapy 2003;5:523–533.
  56. Summer R, Kotton DN, Sun X, Ma B, Fitzsimmons K, Fine A. Side population cells and Bcrp1 expression in lung. Am J Physiol Lung Cell Mol Physiol 2003;285:L97–104.
  57. Giangreco A, Shen H, Reynolds SD, Stripp BR. Molecular phenotype of airway side population cells. Am J Physiol Lung Cell Mol Physiol 2004;286:L624–L630.
  58. Abe S, Lauby G, Boyer C, Manouilova L, Rennard SI, Sharp JG. Lung cells transplanted to irradiated recipients generate lymphohematopoietic progeny. Am J Respir Cell Mol Biol 2004;30:491–499.
  59. Summer R, Kotton DN, Liang S, Fitzsimmons K, Sun X, Fine A. Embryonic lung side population cells are hematopoietic and vascular precursors. Am J Respir Cell Mol Biol 2005;33:32–40.
  60. Majka SM, Beutz MA, Hagen M, Izzo AA, Voelkel N, Helm KM. Identification of novel resident pulmonary stem cells: form and function of the lung side population. Stem Cells 2005;23:1073–1081.
  61. Liang SX, Summer R, Sun X, Fine A. Gene expression profiling and localization of Hoechst-effluxing CD45 – and CD45 + cells in the embryonic mouse lung. Physiol Genomics 2005;23:172–181.
  62. Wang G, Bunnell BA, Painter RG, Quiniones BC, Tom S, Lanson NA Jr, Spees JL, Bertucci D, Peister A, Weiss DJ, et al. Adult stem cells from bone marrow stroma differentiate into airway epithelial cells: potential therapy for cystic fibrosis. Proc Natl Acad Sci USA 2005;102:186–191.
  63. Houghton J, Stoicov C, Nomura S, Rogers AB, Carlson J, Li H, Cai X, Fox JG, Goldenring JR, Wang TC. Gastric cancer originating from bone marrow–derived cells. Science 2004;306:1568–1571.
  64. Kim CF, Jackson EL, Woolfenden AE, Lawrence S, Babar I, Vogel S, Crowley D, Bronson RT, Jacks T. Identification of bronchioalveolar stem cells in normal lung and lung cancer. Cell 2005;121:823–835.
  65. Studeny M, Marini FC, Dembinski JL, Zompetta C, Cabreira-Hansen M, Bekele BN, Champlin RE, Andreeff M. Mesenchymal stem cells: potential precursors for tumor stroma and targeted-delivery vehicles for anticancer agents. J Natl Cancer Inst 2004;96:1593–1603.
  66. Hilbe W, Dirnhofer S, Oberwasserlechner F, Schmid T, Gunsilius E, Hilbe G, Woll E, Kahler CM. CD133 positive endothelial progenitor cells contribute to the tumor vasculature in non–small cell lung cancer. J Clin Pathol 2004;57:965–969.
  67. Shinde Patil VR, Friedrich EB, Wolley AE, Gerszten RE, Allport JR, Weissleder R. Bone marrow–derived lin – c-kit + Sca-1 + stem cells do not contribute to vasculogenesis in Lewis lung carcinoma. Neoplasia 2005;7:234–240.
  68. Strieter RM, Belperio JA, Keane MP. Cytokines in innate host defense in the lung. J Clin Invest 2002;109:699–705.
  69. Hattori K, Heissig B, Tashiro K, Honjo T, Tateno M, Shieh JH, Hackett NR, Quitoriano MS, Crystal RG, Rafii S, et al. Plasma elevation of stromal cell–derived factor-1 induces mobilization of mature and immature hematopoietic progenitor and stem cells. Blood 2001;97:3354–3360.
  70. Rafii S, Heissig B, Hattori K. Efficient mobilization and recruitment of marrow–derived endothelial and hematopoietic stem cells by adenoviral vectors expressing angiogenic factors. Gene Ther 2002;9:631–641.
  71. Zielske SP, Braun SE. Cytokines: value-added products in hematopoietic stem cell gene therapy. Mol Ther 2004;10:211–219.
  72. Frenette PS, Subbarao S, Mazo IB, von Andrian UH, Wagner DD. Endothelial selectins and vascular cell adhesion molecule-1 promote hematopoietic progenitor homing to bone marrow. Proc Natl Acad Sci USA 1998;95:14423–14428.
  73. Kobayashi H, Tahara M, Worgall S, Rafii S, Crystal RG. Mobilization of hematopoietic stem cells and progenitor cells to lung by intratracheal administration of an adenovirus encoding stromal cell–derived factor-1. Mol Ther 2003;7:S112.
  74. Dorman SC, Babirad I, Post J, Watson RM, Foley R, Jones GL, O'Byrne PM. Progenitor egress from the bone marrow after allergen challenge: role of stromal cell–derived factor 1 alpha- and eotaxin. J Allergy Clin Immunol 2005;115:501–507.
  75. Phinney D, Kopen G, Isaacson RL, Prockop D. Plastic adherent stromal cells from the bone marrow of commonly used strains of inbred mice: variations in yield, growth, and differentiation. J Cell Biochem 1999;72:570–585.
  76. Stripp BR, Maxson K, Mera R, Singh G. Plasticity of airway cell proliferation and gene expression after acute naphthalene injury. Am J Physiol 1995;269:L791–L799.
  77. Reynolds SD, Giangreco A, Power JH, Stripp BR. Neuroepithelial bodies of pulmonary airways serve as a reservoir of progenitor cells capable of epithelial regeneration. Am J Pathol 2000;156:269–278.
  78. Hong KU, Reynolds SD, Giangreco A, Hurley CM, Stripp BR. Clara cell secretory protein–expressing cells of the airway neuroepithelial body microenvironment include a label-retaining subset and are critical for epithelial renewal after progenitor cell depletion. Am J Respir Cell Mol Biol 2001;24:671–681.
  79. Giangreco A, Reynolds SD, Stripp BR. Terminal bronchioles harbor a unique airway stem cell population that localizes to the bronchoalveolar duct junction. Am J Pathol 2002;161:173–182.
  80. Borthwick DW, Shahbazian M, Krantz QT, Dorin JR, Randell SH. Evidence for stem-cell niches in the tracheal epithelium. Am J Respir Cell Mol Biol 2001;24:662–670.
  81. Schoch KG, Lori A, Burns KA, Eldred T, Olsen JC, Randell SH. A subset of mouse tracheal epithelial basal cells generates large colonies in vitro . Am J Physiol Lung Cell Mol Physiol 2004;286:L631–L642.
  82. Engelhardt JF, Schlossberg H, Yankaskas JR, Dudus L. Progenitor cells of the adult human airway involved in submucosal gland development. Development 1995;121:2031–2046.
  83. Engelhardt JF. Stem cell niches in the mouse airway. Am J Respir Cell Mol Biol 2001;24:649–652.
  84. Otto WR. Lung epithelial stem cells. J Pathol 2002;197:527–535.
  85. Hong KU, Reynolds SD, Watkins S, Fuchs E, Stripp BR. Basal cells are a multipotent progenitor capable of renewing the bronchial epithelium. Am J Pathol 2004;164:577–588.
  86. Bishop AE. Pulmonary epithelial stem cells. Cell Prolif 2004;37:89–96.
  87. Lee RH, Hsu SC, Munoz J, Jung JS, Lee NR, Pochampally R, Prockop DJ. A subset of human rapidly-self renewing marrow stromal cells (MSCs) preferentially engraft in mice. Blood 2006;107:2153–2161.

Correspondence and requests for reprints should be addressed to Daniel J. Weiss, M.D., Ph.D., Pulmonary and Critical Care, University of Vermont College of Medicine, Burlington, VT 05405. E-mail: dweiss@uvm.edu



Twitter iconTwitterExternal link Disclaimer         Facebook iconFacebookimage of external link icon         YouTube iconYouTubeimage of external link icon         Google+ iconGoogle+image of external link icon