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Adult Stem Cells, Lung Biology, and Lung DiseaseReport of a National Heart, Lung, and Blood Institute and Cystic Fibrosis Foundation WorkshopProceedings 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:
BACKGROUND Adult Marrow–derived Cells in Lung Biology and Disease
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 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. 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. 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. 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. 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. 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. Endogenous lung stem cells.
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 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. Session 2: Other Adult Stem Cell Sources 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. Session 3: Adult Marrow–derived Stem Cells in 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. 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 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 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. RECOMMENDATIONS: TECHNIQUES
Functional outcome measures. RECOMMENDATIONS: FUNCTIONAL OUTCOMES MEASURES
Standardized preclinical models of human lung disorders. RECOMMENDATIONS: PRECLINICAL MODELS OF HUMAN LUNG DISORDERS
Tools. RECOMMENDATIONS: TOOLS
Fundamental understanding of lung repair mechanisms. RECOMMENDATIONS: LUNG REPAIR MECHANISMS
Mechanisms of recruitment and phenotypic conversion of reparative cells. RECOMMENDATIONS: MECHANISMS OF RECRUITMENT AND PHENOTYPIC CONVERSION
Development of in vitro methods. RECOMMENDATIONS: IN VITRO METHODS
Consideration of core centers and facilities. RECOMMENDATIONS: CORE CENTERS AND FACILITIES
Educational programs. RECOMMENDATIONS: EDUCATIONAL PROGRAMS
Clinical/Translational Recommendations TABLE 1. CLINICAL STUDIES OF STEM CELLS IN LUNG DISEASES Pro
Con
RECOMMENDATIONS: CLINICAL STUDIES
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)
References
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 |
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