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1 Department of Medicine, University of Vermont College of Medicine, Burlington, Vermont; 2 Division of Pulmonary Medicine, Allergy, and Immunology, Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania; 3 University of Pittsburgh, Pittsburgh, Pennsylvania; 4 Department of Pediatrics, Division of Pulmonary, Allergy/Critical Care Medicine, and Division of Hematology/Oncology, Blood and Marrow Transplant Program, University of Minnesota, Minneapolis, Minnesota; and 5 Center for Gene Therapy, Tulane University Health Sciences Center, New Orleans, Louisiana
Correspondence and requests for reprints should be addressed to Daniel J. Weiss, M.D., Ph.D., Associate Professor of Medicine, Department of Medicine, University of Vermont College of Medicine, 149 Beaumont Avenue, HSRF 226, Burlington, VT 05405. E-mail: daniel.weiss{at}uvm.edu
EXECUTIVE SUMMARY
The University of Vermont College of Medicine and the Vermont Lung Center, with support from the National Heart, Lung, and Blood Institute (NHLBI), the American Thoracic Society, the Alpha-1 Foundation, and the Pulmonary Fibrosis Foundation, convened a workshop, "Stem Cells and Cell Therapies in Lung Biology and Lung Diseases," to review our current understanding of the role of stem and progenitor cells in lung repair after injury and to review the current status of cell therapy approaches for lung diseases. These are rapidly expanding areas of study that both provide further insight into and challenge traditional views of mechanisms of lung repair after injury and pathogenesis of several lung diseases. The goals of the conference were to summarize the current state of the field, discuss and debate current controversies, and to identify future research directions and opportunities for both basic and translational research in cell-based therapies for lung diseases.
This workshop was a follow-up to an inaugural workshop held at the University of Vermont in 2005, "Adult Stem Cells, Lung Biology, and Lung Disease," sponsored by the NHLBI and the Cystic Fibrosis Foundation, together with the Vermont Lung Center and the University of Vermont College of Medicine (1). That workshop was instrumental in helping guide research and funding priorities.
Since the 2005 workshop, investigations of stem cells and cell therapies in lung biology and diseases have continued to rapidly expand. However, there have been several distinct changes in focus and direction, particularly with respect to cell-based therapy approaches. For example, engraftment of airway or alveolar epithelium by stem or progenitor cells originating from outside of the lung is now viewed to be a rarer occurrence than previously described and of unclear physiologic or therapeutic significance. In contrast, circulating endothelial progenitor cells (EPCs) can contribute to regeneration of diseased pulmonary vasculature and are being investigated in patients with pulmonary hypertension in a clinical trial being conducted at the University of Toronto and in one recently completed at Zhejiang University, China. Circulating EPCs may also play roles in both acute lung injury and in fibrotic lung diseases. Furthermore, increasing evidence suggests that circulating fibrocytes can contribute to the pathophysiology of fibrotic lung diseases and thus may be a potential therapeutic target.
In addition, novel areas of investigation have developed that include increasing exploration of three-dimensional culture systems and bioengineering approaches to generate functional lung tissue ex vivo and in vivo. Mesenchymal stem cells (MSCs) have been found to exert profound suppressive effects on immune cells and pathways and have demonstrated both safety and efficacy in phase 1 and 2 trials in immune-mediated diseases such as graft-versus-host disease (GVHD) and Crohn's disease. Recent publications and several abstracts presented at the workshop demonstrate that MSCs suppress lung injury and inflammation in several mouse models of inflammatory and immune-mediated lung diseases. These areas are predicted to be of intense investigation over the next several years.
Progress continues to be made in investigations of local (endogenous) stem and progenitor cells resident in the lungs. Further understanding of the identity and lineage expansion properties of previously identified endogenous progenitor populations, including variant Clara cells, bronchoalveolar stem cells (BASCs), and side population cells, suggests an increasingly complex network of cellular repair after injury. Most recently, embryonic origin Oct-4+ Clara cell secretory protein (CCSP+) cells have been identified in neonatal mouse lungs and have been postulated to play a progenitor role in adult lung. However, study of endogenous lung stem and progenitor cells is complicated by the role of specific microenvironmental niches in which these cells reside. Alteration of the niches with experimental protocols or removal of cells from the niches can change their identifying characteristics and biologic activities. One of the challenges facing the field is to devise lineage tracing and other study mechanisms to define, characterize, and explore potential therapeutic and/or pathologic properties of endogenous lung progenitor cells. Notably, the existence of lung cancer stem cells is an area of increasing focus and high interest but remains poorly understood. Another challenge is that most studies of endogenous progenitor cells have used mouse models. Correlative information in human lungs remains poorly defined. Comparably, most studies of exogenous cells in lung repair have used mouse models, with relatively limited data in patient models.
A continuing issue of confusion is that of terminology. Precise definitions and characterizations of specific cell populations, notably MSCs and EPCs, are not agreed upon. The terms "stem cell" and "progenitor cell" are still used with varying degrees of clarity and precision by different investigators and in recent publications. This continues to complicate comparison of different investigative approaches. A glossary of relevant working definitions applicable to lung is depicted in Table 1. This glossary does not necessarily reflect an overall consensus for the definition of each term and will undergo continuing revision as overall understanding of the cell types and mechanisms involved in lung repair continues to be elucidated. Nonetheless, it is a useful framework.
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The second and third sessions highlighted advances in cell therapy approaches for lung diseases and also presented data from clinical trials of cell therapies in other diseases. New and developing areas in bioengineering approaches for cell therapies of lung diseases were explored in the second session. Presentations by Dennis Discher (University of Pennsylvania) and Mingyao Liu (University of Toronto) explored the effects of physical environment and three-dimensional matrices on stem cells and lung regeneration. Dario Fauza (Boston Children's Hospital) demonstrated that amniotic fluid–derived MSCs could be used for tracheal repair. Bethany Moore (University of Michigan) and Kurt Stenmark (University of Colorado Health Sciences Center) reviewed current understanding of the role of fibrocytes in fibrotic lung disease and of EPCs in pulmonary vascular and fibrotic lung diseases, respectively. Finally, Johnny Huard (University of Pittsburgh) presented an update in stem cell trials for muscle diseases.
Immunomodulatory effects of stem cells were explored in the third session. Results of a phase 2 clinical trial of MSCs in Crohn's disease were presented by Jane Onken (Duke University School of Medicine) demonstrating both safety and efficacy in otherwise treatment-resistant patients. This was followed by exploration of MSC effects in inflammatory and immune-mediated lung diseases by Donald Phinney (Tulane University) and Daniel Weiss (University of Vermont). Ellen Burnham (University of Colorado Health Sciences Center) and Duncan Stewart (University of Toronto) presented an update on EPCs and initial results from the EPC trial in pulmonary hypertension being conducted at the University of Toronto. Keith March (Indiana University) presented an update on cardiac trials and Donald Fink (U.S. Food and Drug Administration [FDA]) presented a perspective from the FDA. After extensive discussion, both immunomodulation and bioengineering approaches using MSCs and other stem cell populations were identified as areas of high priority for expanding study.
In the final session, Alan Michelson (National Institutes of Health [NIH]) discussed a systems-based strategy to cell therapies, and Mike Rosenzweig and John Walsh, respective presidents of the Pulmonary Fibrosis and Alpha-1 Foundations, presented perspectives on stem cell research from the views of nonprofit respiratory disease foundations. The conference concluded with vigorous discussion on future research and funding priorities, led by Darwin Prockop (Tulane University). As in the 2005 conference, discussion was spirited as to how and when to proceed to further clinical investigation in addition to the trial occurring at the University of Toronto. It was agreed that strong emphasis must continue to be placed on animal models of human lung diseases, with a focus on studies that incorporate relevant functional outcome measures. Nonetheless, the safety and efficacy results obtained with MSCs in Crohn's disease and other inflammatory and immune-mediated diseases suggest a potential role in inflammatory and immune-mediated lung diseases even in the absence of a comprehensive understanding of the mechanisms by which the MSCs are acting. There was a growing consensus at the meeting to move toward early studies in humans 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 progenitor cells and of cell therapy approaches for lung diseases remains a timely and exciting area of study. Nonetheless, there are many areas in which our understanding of the processes and mechanisms remain poorly understood. Recommendations for areas of continued and future investigation are presented in Table 2. More extensive details on each session are presented below. The conference program, executive summaries for each speaker, and abstracts from the poster sessions are included in the online supplement.
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A comprehensive summary of relevant published literature since the 2005 workshop and through January 2008 is presented below. See the report from that workshop for a comprehensive review of the literature up to that point (1). Readers are also referred to a number of reviews of each of the below topics that have appeared over approximately the past 2 years (2–27).
Structural Engraftment and Functional Effects of Circulating or Exogenously Administered Stem or Progenitor Cells
Structural engraftment.
A number of publications over approximately the past 10 years have suggested that a variety of bone marrow–derived cells, including hematopoietic stem cells, MSCs, multipotent adult progenitor cells, and other populations, could structurally engraft as mature differentiated airway and alveolar epithelial cells. This literature has been predominantly based on studies in mice using techniques that evaluated histologic demonstration of donor-derived marrow cells in recipient lungs after systemic administration of male marrow cells to female recipients, usually but not always after myeloablation of the recipient mouse bone marrow (reviewed in Reference 1). Prior lung injury was usually required to observe engraftment, although lung injury did not always result in increased apparent engraftment (28). Furthermore, the myeloablative regimen used, usually total body irradiation, was also believed to contribute to lung injury and be required for evident engraftment (29, 30). A smaller body of literature in clinical bone marrow and lung transplantation also appeared to have demonstrated varying degrees of apparent chimerism in lungs of the transplant recipients (reviewed in Reference 1). However, more recent reports have called into question whether epithelial engraftment does in fact occur (31, 32). Several technical issues contributed to misinterpretation of results in the initial reports, including inadequate microscopic techniques in which donor-derived cells superimposed on resident airway or alveolar epithelial cells were not effectively discriminated. Exquisite care and sophisticated microscopic approaches, including confocal and deconvolution techniques, must be used to effectively demonstrate potential engraftment (1, 31, 32). Furthermore, a variety of leukocytes, notably airway and alveolar macrophages, reside in the lung. Many of the early reports did not used antibodies directed against CD45 or other leukocyte markers to exclude the possibility that cells of donor origin detected in airway or alveolar epithelium were donor-derived leukocytes rather than epithelial cells. Other tools, such as use of green fluorescence protein (GFP) as a marker of donor-derived marrow cells obtained from transgenic GFP mice in recipient mouse lungs can be subject to error in the presence of autofluorescent cells (33).
Nonetheless, recent reports suggest that engraftment of donor-derived airway and/or alveolar epithelium can occur after perturbation of airway or alveolar epithelium in lung injury models. This has been observed with MSCs of bone marrow or cord blood origin (34), side population cells (35, 36), plastic adherent marrow stromal cells (36–40), or full marrow transplantation after a myeloablative regimen (36, 41–44) (Figure 1). These studies have tended to use more sophisticated microscopic and other analytical techniques. Nonetheless, epithelial engraftment in general is rare, except under conditions discussed below. In parallel, recent studies also continue to demonstrate rare apparent engraftment of pulmonary interstitium and vasculature after total marrow transplant in a variety of injury models (43, 44).
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The types of marrow-derived, cord blood–derived, or fully differentiated nonpulmonary cells that might engraft as lung epithelium, interstitium, or pulmonary vasculature remain to be fully explored. In addition to existing studies of hemapoietic stem cells (HSCs), MSCs (of bone marrow and cord blood origin), EPCs, and fibrocytes, the possibility remains that there may be other cell populations that could be recruited to lung or localize to lung after a systemic or other route of administration (47, 48). A population of circulating bone marrow–derived CD45+/CXCR4+/cytokeratin+ cells has been described to participate in reepitheliazation of denuded tracheal xenografts (49). A population of cord blood cells termed "multiplineage progenitor cells" isolated from human cord blood can be induced in vitro to express pro-surfactant C and thus suggests derivation of type 2 alveolar epithelial cells. However, these cells have not yet been extensively characterized or described to result in lung engraftment in vivo (50). Other sources of stem or progenitor cells, such as adipose tissues, also have not yet been extensively characterized for ability to engraft as lung tissue (51–53). However, the ability to structurally engraft in adult lung may not solely be a property of stem or progenitor cells. Intratracheal administration of neonatal mouse lung fibroblasts resulted in apparent alveolar and interstitial engraftment and engraftment was higher in areas of elastase-induced lung injury (54). More recently, it has been demonstrated that intratracheal administration of type 2 alveolar epithelial cells results in rare engraftment in areas of injured lung after bleomycin administration to rats (55). Notably, bleomycin-injured rats that received the type 2 cells had less histologic injury and decreased hydroxyproline content. In another report, systemically administered skin fibroblasts, transduced ex vivo to express angiopoeitin-1, protected against subsequent lung injury produced by intratracheal endotoxin administration in rats (56). These results suggest that lung injuries might be amenable to a variety of cell therapy approaches.
For those reports in which more robust evidence of engraftment has been suggested, the potential role of fusion has not yet been fully elucidated. Bone marrow–derived cells can be induced to fuse with lung epithelial cells in vitro, but an initial in vivo investigation in a transgenic mouse model suggested that fusion did not occur (57, 58). A more recent report suggests that fusion of donor-derived marrow cells with type 2 alveolar epithelial cells can occur in mouse lungs, but that the Y chromosome may be lost from the resulting heterokaryon cells (59).
Mechanisms by which circulating or systemically administered stem or progenitor cells might be recruited to lung remain poorly understood. After systemic (i.e., venous) administration, many cells initially localize in lung, and recent studies continue to confirm that lung injury results in increased localization and/or retention of marrow-derived cells in lung (60–62). The timing of cell administration after lung injury can also influence recruitment and phenotypic conversion. Systemic administration of MSCs 4 hours after lung irradiation resulted in apparent engraftment of cells as epithelial and vascular endothelial cells (60). However, MSCs administered at later time points appeared to engraft as interstitial cells and participate in development of fibrosis (60, 63). Recipient immune responses also play significant yet poorly characterized roles in retention of cells in lung (64). Commonly used approaches of sex-mismatched transplantation or cell administration may also result in clearance of cells (59). The range and identity of chemotactic soluble mediators released by injured lung cells and the role of up-regulation of adhesion molecules with which circulating cells might interact remain poorly understood (reviewed in Reference 1 and References 65–67). As with engraftment, a number of factors, including age of donor or recipient, type of cell administered, route of administration, and so forth, all might affect recruitment to lung.
Comparably, the mechanisms by which stem or progenitor cells might be induced to acquire the phenotype of lung epithelial, interstitial, or vascular endothelial cells remain poorly understood. In vitro studies continue to demonstrate that soluble factors released from lung epithelial cells or from injured lung homogenates can induce expression of lung epithelial markers in several types of marrow-derived cells, possibly through activation of β-catenin signaling pathways (68–70). One novel mechanism of inducing phenotypic change might involve release of membrane-derived microvesicles, a recently appreciated means of intercellular communication that involves horizontal transfer of mRNA and proteins between cells (71, 72).
Endothelial progenitor cells.
In the past decade, circulating bone marrow–derived cells similar to embryonal angioblasts have been identified (73), termed "endothelial progenitor cells." These cells have the potential to proliferate and differentiate into mature endothelial cells (73, 74). Increasing evidence demonstrates that EPCs play a role in pathogenesis of a wide variety of lung diseases, including pulmonary hypertension, pulmonary fibrosis, airway diseases including both asthma and chronic obstructive pulmonary disease (COPD), acute lung injury, and lung cancer (66, 75–82). However, studies of EPCs in lung diseases have been hampered by lack of consensus regarding identification of these cells. Early investigations relied almost exclusively on the use of flow cytometry in conjunction with immunostaining to identify and enumerate these cells both in bone marrow as well as in the circulation. Furthermore, when similar techniques were used in different reports, a different group of markers was used. This has complicated comparative assessments for similar disease processes. As a result, the field is beginning to encourage the use of functional assays both in vitro and in vivo in conjunction with the use of flow cytometry and immunohistochemistry to not only enumerate EPCs but to better characterize their true ability to form functional endothelium. A critical area for future study is to develop a consensus-based approach to definition and use of EPCs with particular emphasis on functional capabilities of these cells.
A recent development in the characterization of EPCs has been the observation of two major types of these cells in human peripheral blood (83, 84). The first, termed "early EPCs," are characterized by early growth in vitro, CD34/CD31/CD14 positivity, the inability to form tubes in a matrigel tube-forming assay, and high levels of cytokine secretion. The other type of EPC, termed "late outgrowth EPCs" or "outgrowth endothelial cells," is characterized by CD31 positivity, a better ability to form tubes either with or without human umbilical vein endothelial cell coculture, and a low level of cytokine secretion. Each of these cell types may have a unique potential in lung microvascular repair, with early EPCs having a role as paracrine cells, and late EPCs functioning more specifically in restoring vascular structures of the lung. Intravenous infusion of each type of cell in an animal model has shown their ability to preferentially localize to lung (85). In addition, preliminary evidence exists that the two EPC types when infused together may have a synergistic role in restoring vascular structure and function (84). Further characterization of these two EPC types, including methods to enhance their numbers ex vivo, could have implications for the development of therapy specific to the phenotypic abnormalities of a given lung disease.
The number of circulating EPCs has been correlated with a variety of clinical variables in several lung disease states, demonstrating the potential utility of EPCs as biomarkers. Although increased circulating EPC numbers correlate with survival in acute lung injury/acute respiratory distress syndrome (ARDS) (Figure 2) and are associated with less residual lung damage in patients with pneumonia (76, 77), increased numbers do not necessarily correlate with better outcomes or more normal physiology in all lung diseases. For example, an increase in the number of circulating EPCs in patients with COPD was associated with more abnormal spirometry (78), although a different study showed that levels of circulating EPCs were inversely correlated with COPD disease severity (79). Increased numbers of circulating EPCs also portended worse survival among those with non–small cell lung cancer (81, 82). In asthma, numbers of circulating EPCs were increased compared with nonasthmatic controls, but this did not correlate with clinical outcomes (75).
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–mediated hypoxia response (89). Use of HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A) reductase inhibitors had been previously shown to have a beneficial effect on the mobilization of EPCs (90). Recent in vitro work demonstrates that this may be related to the effect of this class of drugs in the prevention of EPC apoptosis in response to noxious stimuli, including the effects of tumor necrosis factor (TNF)- and IL-1β, thereby enhancing EPC survival and differentiation (91). These factors and others may simultaneously exist in patients with lung disease, making it incumbent on investigators to perform larger clinical studies of EPCs in specific lung diseases, using well-defined patient populations to account for their effects on the number and function of EPCs. Goals of increasing numbers of EPCs, or developing methods to enhance their mobilization, may not be appropriate for all diseases that affect the lung, particularly for lung cancers (12, 81, 82). EPCs may have an effect on the development of lung tumor vasculature and homing to sites of lung metastases as well as in other cancers (92–98). However, enumerating EPCs could be useful as a marker of lung cancer severity or to follow responsiveness to therapy. For example, increased levels of circulating EPCs were found to correlate with worsened outcomes in patients with non–small cell lung cancers (81). Because neovascularization involves the recruitment of EPCs from the bone marrow, these cells are a logical target for antiangiogenesis therapy. Recently, an investigational drug, TK 1-2 (the kringle domain of tissue-type plasminogen activator), was demonstrated to be useful in blocking adhesion, differentiation, and migration of ex vivo human EPCs (96). When EPCs were coimplanted with A549 cells into nude mice, tumor growth and vascular density were increased. TK 1-2 treatment of these animals resulted in a decrease in tumor growth and vascularity, and a decrease in the expression and secretion of vascular endothelial growth factor (VEGF) by EPCs. These findings suggest that blocking EPCs could be an important therapy in the prevention of cancer progression. In addition, after systemic injection, EPCs localize to lung and also appear to home to metastatic tumors in lung through as yet poorly understood mechanisms (92, 93). This suggests that modification of EPCs to express suicide genes or other therapeutic molecules could potentially be used in cell-based therapy approaches for lung cancer (92, 93). Mechanisms controlling mobilization and homing of EPCs to lung remain poorly understood and are the subject for more intense investigation (66).
Earlier studies in mice and dogs have also demonstrated a role for exogenously administered EPCs in vasculogenesis and vascular repair in experimental models of pulmonary hypertension (99–102). Furthermore, EPCs can be transduced to express proangiogenic factors, such as endothelial nitric oxide synthetase (eNOS), or inhibitors of smooth muscle cell proliferation, such as calcitonin gene–related peptide, and appear to home to sites of endothelial damage and lung injury (61, 101). EPCs also preferentially localize to areas of injured lung after systemic administration (61). As such, a pilot trial of autologous EPC administration for primary pulmonary hypertension conducted at Zhejiang University, Hangzhou, China, demonstrated increased six-minute-walk capacity and improved hemodynamic variables, including mean pulmonary artery pressure, pulmonary vascular resistance, and cardiac output, 12 weeks after systemic administration of autologous EPCs with conventional therapy (n = 15) compared with patients receiving conventional therapy alone (n = 16) (103). Importantly, no adverse effects of EPC administration were noted, although long-term follow-up is pending. A therapeutic trial of administration of autologous EPCs transduced to express eNOS for patients with pulmonary hypertension, the Pulmonary Hypertension and Cell Therapy (PHACeT) trial, has been initiated at the University of Toronto. As of January 2008, the PHACeT trial has completed enrollment of the first dose panel, with three patients receiving a total of 7 million early-growth EPCs transfected to overexpress human eNOS (information courtesy of Duncan Stewart, M.D., F.R.C.P.C., principal investigator of the PHACeT trial). The cell delivery procedure was well tolerated and there were no safety concerns. Notably, the first three patients all showed a remarkable (nearly 50%) reduction in total pulmonary vascular resistance over the course of the 3-day delivery period, which might represent the effect of increase NO release by the engineered EPCs within the pulmonary microcirculation. The trial's Data Safety Monitoring Board has approved moving to panel 2, which calls for a total of 23 million cells, again in three divided doses over 3 days. The investigators expect that completion of the third dose panel (50 million cells) will provide sufficient support to move forward with the design of a randomized controlled trial that can assess potential efficacy of this cell therapy approach in pulmonary arterial hypertension (PAH).
Circulating fibrocytes.
Circulating fibrocytes were first described as a subset of circulating leukocytes that produced collagen and homed to sites of inflammation (104, 105). The identity and phenotypic characterization of circulating fibrocytes are more firmly established, and these cells are described by the cell surface markers CD34, CD45, CD13, and major histocompatibility class (MHC) II, and also express type 1 collagen and fibronectin (10, 13, 23). Circulating fibrocytes have been implicated in the pathogenesis of lung fibrosis in several mouse models including irradiation and bleomycin-induced lung injury (reviewed in References 10, 13, and 23). Circulating fibrocytes have also been implicated in the subepithelial fibrosis that can develop in severe asthma (106, 107) and in clinical bronchiolitis obliterans in lung transplant patients (108). Several chemokines, including stromal-derived factor (SDF)-1 and the CCR2 and CCR5 axes, have been implicated in recruitment of circulating fibrocytes to fibrotic lungs, but overall mechanisms of fibrocyte recruitment to lung are poorly understood (109–112). Similarly, the mechanisms by which fibrocytes are induced to undergo phenotypic transformation into fibroblasts and myofibroblasts and contribute to fibrogenesis in lung are poorly understood, although recently both haptoglobin and cysteinyl leukotrienes have been implicated (113, 114). A recent study suggests that levels of circulating fibrocytes are increased in patients with usual or nonspecific interstitial pneumonitis (Figure 3) (115). Although suggestive, this small study needs to be confirmed in larger cohorts. Dr. Bethany Moore presented unpublished data that fibrocytes isolated from peripheral blood of patients with idiopathic pulmonary fibrosis exhibit increased proliferative and collagen-producing capacities. Together, the available information strongly suggests a role of circulating fibrocytes in fibrotic lung diseases and offers potential pathways for therapeutic interventions.
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MSCs can be induced in vitro to express phenotypic markers of airway and/or alveolar epithelial cells and can engraft as, or fuse with, airway and/or alveolar epithelium, interstitium, or vascular endothelium in vivo after systemic administration (reviewed in References 1 and 19). As discussed above, engraftment is generally rare, although many variables remain to be explored. Nonetheless, there are an increasing number of studies demonstrating a functional role of MSCs in rodent models of acute lung inflammation and fibrosis in the absence of significant lung engraftment. Intratracheal administration or systemic administration of MSCs immediately after intratracheal bleomycin administration decreased subsequent lung collagen accumulation, fibrosis, and levels of matrix metalloproteinases (152). Secretion of IL-1 receptor antagonist by MSCs has been hypothesized to account for at least some of these effects (153). Intratracheal administration of MSCs 4 hours after intratracheal endotoxin administration decreased mortality, tissue inflammation, and concentration of proinflammatory mediators, such as TNF- and macrophage inflammatory protein (MIP)-1β, in bronchoalveolar lavage fluid compared with endotoxin-only treated mice (46) (Figure 5). Systemic MSC administration also decreased lung inflammation after endotoxin administration in mice (70, 153–155). Notably, transduction of the MSCs to express angiopoietin-1 further decreased endotoxin-mediated lung injury, presumably through abrogation of endotoxin-mediated endothelial injury (154, 155). However, systemic administration of skin fibroblasts transduced to express angiopoietin-1 also decreased acute endotoxin-induced lung injury, suggesting that a variety of cell types might be used for cell therapy approaches to acute lung injury (56). Coculture of MSCs with lung cells obtained from LPS-treated mice resulted in decreased proinflammatory cytokine release from the lung cells (70). In other lung injury models, intratracheal administration of bone marrow–derived MSCs decreased pulmonary hypertension and other manifestations of monocrotaline (MCT)-induced pulmonary vascular injury (156). Systemic administration of a heterogenous population of autologous adipose-derived stromal cells decreased manifestations of elastase-induced emphysema in rats (157). Hepatocyte growth factor secreted by the stromal cells was postulated as a potential mechanism of injury repair in this report.
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, a proinflammatory cytokine that enhances bone marrow hematopoeisis, increases secretion of G-CSF, M-CSF, IL-6, and IL-11. The culture system in which MSCs are maintained ex vivo influences release of cytokines and other inflammatory molecules (145–148). For example, culturing MSCs in a three-dimensional hyaluronan scaffolding increased release of SDF-1, and matrix metalloproteinase-3 (146) compared with standard tissue culturing, whereas culturing under hypoxic conditions in a synthetic fibrous matrix increased fibronectin expression (144). Matrix stiffness can also affect the differentiation of MSCs (Figure 7) (147).
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, IL-4, IL-17, and IFN-
, as well as several Toll-like receptors, including the endotoxin receptor TLR4 (161, 162). The IL-171 receptor in particular is expressed in high abundance (161, 163) and IL-17 has recently been described as a proliferative stimulus for MSCs (163). Stimulation with IFN- can alter expression of MHC and costimulatory molecules by MSCs. Nonetheless, effects on MSC secretion of soluble mediators by other cytokines and chemokines by microenvironment conditions found during lung injury have been less well explored. MSCs can also inhibit the proliferation and function of a broad range of immune cells, including T cells, B cells, natural killer (NK) cells, and dendritic cells (Figure 6) (20, 158–160, 160–184). Notably, MSCs inhibit T-lymphocyte proliferation, activation, and cytokine release in response to either alloantigens or to mitogenic stimuli through a dose-dependent direct suppressive effect on proliferation. The mechanisms of MSC inhibition of T-cell proliferation and function in vitro are only partly understood and both direct cell–cell contact as well as release of soluble mediators have been proposed (158–160, 164–180). Furthermore, MSCs constitutively express low levels of HLA class I molecules and do not express HLA class II molecules or the costimulatory molecules CD40, CD80, and CD86, which are essential for activation of T-lymphocyte–mediated immune responses (20, 158–160, 164, 180). As such, these properties render MSCs generally nonimmunogenic and have been the basis of several clinical reports and, more recently, clinical trials using both autologous and allogeneic MSCs for immune-mediated diseases, such as GVHD and Crohn's disease (181–188). As discussed by Jane Onken at this workshop, these trials have demonstrated both efficacy and safety of MSC administration in otherwise treatment-resistant patients with Crohn's disease and, importantly, no significant adverse effects have been observed. These results suggest that administration of MSCs may be a safe and feasible clinical approach for immune-mediated lung diseases.
MSCs are also increasingly described as vehicles for delivery of therapeutic genes and proteins (189–193). Notably, MSCs can home to tumors, through as yet unclear mechanisms, and serve as vehicles for delivery of chemotherapeutic and other antitumor agents (194–197). This has recently been described in mouse lung tumor models and may provide a viable therapy for lung cancers (198–201). In contrast, MSCs may also contribute to tumor stroma and influence behavior of cancer cells (194, 202–206). Whether marrow-derived cells contribute to development of epithelial cancers remains an active area of investigation (205, 206).
Some additional cautions with regard to systemic administration of MSCs have been raised. Most culture strategies use fetal or bovine calf serum. Despite washing of the cells before systemic administration, some bovine antigens may remain adherent to cell surfaces and trigger immune reactions as well as decrease potential engraftment in recipient mice or patients (207). Culture of MSCs in medium with lower calf serum content, use of heterologous species-specific serum or alternative serum substitutes such as platelet lysate, and removal of calf serum antigens before administration are proposed strategies to decrease these potential adverse effects (208–211). In addition, after intravenous administration, MSCs initially lodge in the lung vasculature before moving through the pulmonary capillary system and on to other organs. However, depending on the preparative regimens used, MSCs can clump and potentially lodge as emboli in lung capillaries (212, 214). Pretreatment of mice with the vasodilator sodium nitroprusside has been proposed as a mechanism of decreasing cell trapping of MSCs in pulmonary capillaries (213). Furthermore, development of lung sarcomas from systemically administered MSCs has been described (212, 214). Interestingly, this has been described with administration of mouse but not human MSCs and may reflect a greater propensity of mouse MSCs to acquire chromosomal abnormalities with serial passages in culture (214, 215). However, extensive culture of almost any mammalian cell in culture can lead to crisis, followed by immortalization, and then transformation to tumorigenic cells, as has been well documented for mouse fibroblasts (216). Murine MSCs that were extensively expanded in culture through many passages developed chromosomal instability and produced lung sarcomas in mice (212, 214). Human MSCs that were cultured for 4 to 7 months underwent similar changes (217, 218). It is anticipated that additional strategies to maximize therapeutic utility of MSCs while decreasing chance of any adverse effects will develop over the next several years.
Lung tissue bioengineering.
One growing area of investigation is the utilization of three-dimensional matrices or other artificial scaffolding for growth of functional lung tissue from stem cells ex vivo and in vivo. These approaches have been increasingly successfully used in regeneration of other tissues, including skin, vasculature, cartilage, and bone. Given the complex three-dimensional architecture of the lung, this is a daunting task; nonetheless, there has been significant progress in several areas. Notably, MSCs isolated from amniotic fluid, umbilical cord blood, or bone marrow can be seeded on biodegradable polyglycolic acid or other biosynthetic scaffolds and generate tracheal cartilage for use in repair of congenital tracheal defects and also in tendon tissue for use in congenital diaphragmatic defects (142, 219–226). Notably, the extracellular matrix properties of the cartilage depend on the source of the MSCs (142). In data presented by Dr. Fauza at this conference, preclinical studies in large-animal models (sheep) suggest safety and efficacy, and clinical trials in neonates with congenital tracheal or diaphragmatic defects are being planned.
Three-dimensional culture systems have also been used as matrices for ex vivo lung parenchymal development and for study of growth factors and mechanical forces on lung remodeling (227–233). Culture of fetal rat lung suspensions in a three-dimensional glycosaminoglycan (GAG) scaffold resulted in formation of alveolar-like structures in the scaffold (232). Fetal mouse cells cultured in three-dimensional hydrogels and in synthetic polymer scaffolds resulted in generation of alveolar-like units (228). Notably, stimulation of fetal mouse cells in polymer scaffolds with different isoforms of fibroblast growth factor stimulated different patterns of development, demonstrating the power of three-dimensional culture systems to evaluate lung development and repair (227). In vivo, a recent study demonstrated that fetal rat lung cells cultured in a biosynthetic gelatin matrix and subsequently injected into normal rat lungs induced formation of branching, sacculated epithelial structures reminiscent of lung parenchymal architecture (234) (Figure 8). However, there are few studies as yet evaluating whether stem or progenitor cells isolated from adult bone marrow, cord blood, or other sources can also comparably form airway or alveolar-like structures when cultivated in a three-dimensional matrix or other scaffolding material or, furthermore, whether stem or progenitor cells cultured in such fashion can be used for functional lung regeneration in vivo. A population of cells described as adult lung somatic progenitor cells isolated from adult sheep lungs cultured in synthetic polymer constructs resulted in expression of airway and alveolar epithelial markers by the cells (235). Structures resembling lung airways and parenchyma developed when impregnated constructs were implanted subcutaneously in nude mice or inserted into the wound cavity after wedge lung resection in sheep. Adipose-derived MSCs, cultured ex vivo in sheets of polyglycolic acid and then applied to wound edges after lung volume reduction surgery in rats, accelerated alveolar and vascular regeneration (157). Several abstracts presented at this workshop and also to be presented at the 2008 American Thoracic Society International Conference have begun to further explore these questions. Lung tissue bioengineering with stem cells is projected to be an area of intense investigation.
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However, significant terminology problems exist as to how endogenous stem cells and other progenitor cell types (in this context, progenitor cell being used to describe any cell with more robust proliferative potential, such as toxin-resistant cells, Clara cells, basal cells, etc.). This is a problem that not restricted to the lung: for example, similar terminology issues occur in intestine, particularly as progenitor cell types in different organs differ in their properties and regulation. The glossary depicted in Table 1 does not necessarily reflect an overall consensus for the definition of each term and will undergo continuing revision as overall understanding of the cell types and mechanisms involved in lung repair continue to be elucidated. Nonetheless, it is a useful framework.
Evidence indicating the existence of cellular components of an airway epithelial stem cell hierarchy comes from studies in mouse models in which selective ablation of epithelial cells is achieved through exposure to toxic chemicals or through cell type–specific expression of toxic genes in transgenic mice. Using these approaches, several populations of progenitor cells with some properties of tissue stem cells have been identified along the tracheobronchial tree (7, 14, 15) (Figure 9). In trachea and large airways, a subpopulation of basal epithelial cells that express cytokeratins 5 and 14 has been implicated (236–238). In mice, Clara cells exhibit characteristics of transit-amplifying cells after injury to ciliated airway epithelial cells. However, unlike transit-amplifying cells in tissues with higher rates of epithelial turnover, such as intestine, Clara cells exhibit a low proliferative frequency in the steady state, are broadly distributed throughout the bronchiolar epithelium, and contribute to the specialized tissue function. In more distal airways, toxin (i.e., naphthalene)-resistant variant Clara cells have been identified as having stem cell functions and have been termed as "bronchiolar stem cells" (239–241). Naphthalene-resistant cells are also located within discrete microenvironments within bronchioles that include the neuroepithelial body and bronchoalveolar duct junction (BADJ) (239, 240).
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Recent investigations have begun clarifying cell signaling and other regulatory mechanisms regulating putative lung progenitor populations (7). For example, tumorigenic insults, including deletion of mitogen-activated protein kinase, p18 deletion, and p27 oncogenic mutation, have been shown to induce an expansion of CCSP/pro-SPC dual-labeled BADJ cell number and also enhance lung tumorigenesis. However, the precise role of these and other pathways in endogenous lung stem and progenitor cells remains to be determined (247–249). Most recently, another population of putative progenitor cells expressing CCSP, stem cell antigen (SCA)-1, stage-specific embryonic antigen (SSEA)-1, and the ES cell marker Oct-4 have been identified in neonatal mice (250, 251). These cells were able to form epithelial colonies and differentiate into both type 1 and type 2 alveolar epithelial cells. Interestingly, these cells were susceptible to infection with the SARS (severe acute respiratory syndrome) virus, raising the possibility that endogenous lung progenitor cells may be specific disease targets. Endogenous progenitor cells may also be attractive candidates for targeting with gene transfer vectors that provide sustained expression. The possibility remains that other endogenous stem or progenitor populations exist, and there is much room for additional information on regulatory mechanisms and pathways as have been elucidated in other epithelial progenitor cell populations (252).
Less information is available about differences in endogenous stem and progenitor cells in different clinical lung diseases. Airway epithelium in patients with cystic fibrosis (CF) contains primitive cuboidal cells that express primitive cell markers, including thyroid transcription factor and cytokeratin 7 (242). Neuroepithelial cells also express CF transmembrane conductance regulator (CFTR), which appears to play a role in neuropeptide secretion (243, 244). CFTR knockout mice also contain fewer pulmonary neuroendocrine cells during embryonic development but increased numbers of these cells postnatally (245). This suggests that endogenous progenitor cell pathways in CF lungs may be altered, but this has not been extensively investigated.
In addition to the role of endogenous lung stem and progenitor cells in repair from lung injury, increasing information suggests that mature differentiated lung cells may transdifferentiate and change phenotype. Best described for epithelial–mesenchymal transition, recent investigations describe a wider range of reversible phenotypes in epithelial and mucus cells (253–258). These mechanisms may also play significant roles in injury or repair from injury.
Overall, there remains much to be learned about endogenous stem and lung progenitor cells, including clarification of human counterparts to the cells identified in mouse models. Furthermore, relatively little is known about the behavior of endogenous stem or progenitor cells in clinical lung disease models.
Lung cancer stem cells.
There is intense current interest in the connections between endogenous stem or progenitor cells and cancer stem cells. Cancer stem cells have been defined in transplantation assays as the critical cells from tumors that are capable of propagating disease and are hypothesized to be the cells that maintain tumor progression and disease resistance (259, 260). Although cancer stem cells have been described in breast and other solid cancers, the existence of a lung cancer stem cell or cells is less well established (12). BASCs have been suggested to have a role in development of lung cancer in mice, but their role in tumor maintanance has not been established, nor has the human correlation been demonstrated. A recent study has shown that CD133+ cells from cultured lung cancer cells and primary lung tumors exhibit the ability to propagate lung cancer when injected subcutaneously in mice (261). Side population CD45– Hoechst-effluxing cells have also recently been identified in several human lung cancer cell lines and exhibit tumorigenic properties when subcutaneously implanted into NOD SCID mice (262). Side population cells have also been identified in clinical lung cancer specimens (262). However, despite suggestive data from these two reports, further work is needed to clarify the connections between endogenous lung stem cells, the cells of origin of cancer, and cancer stem cells, and to determine their potential role as therapeutic targets. Recent studies have begun elucidating cell signaling and gene expression pathways including Wnt, hedgehog, and others that may play roles in transformation of endogenous progenitor cells into cancer cells (263–267). In addition, bone marrow–derived or circulating MSCs, HSCs, EPCs, and fibrocytes may contribute to development of lung and other malignancies in mouse models, in part by providing a supportive stroma for the cancers and/or by participating in tumor vascularization (91, 94–98, 118, 194, 202–206). However, MSCs and EPCs have been demonstrated to home to areas of tumor development, and engineered EPCs and MSCs, as well as HSCs, have been used to suppress tumor growth in mouse tumor models (92, 93, 194–200, 268). Cell-based therapies may thus be useful in lung cancer therapeutics.
Embryonic Stem Cells
Mouse ES cells can be induced in culture to express pro-surfactant B, a marker of type 2 alveolar epithelial cells (269, 270). Furthermore, exposure of mouse ES cells to dissociated fetal lung cells induces pseudoglandular formation and pro-SPC expression (271). In one notable study, mouse ES cells cultured at air–liquid interface formed a pseudostratified epithelium resembling mouse tracheal epithelium (272). These studies demonstrate the capacity of ES cells to acquire airway epithelial phenotype in vitro. Comparable studies with human ES cells remain limited due to current restrictions on human ES cell use, particularly in the United States. Nonetheless, it has recently been demonstrated that cells of one of the approved human ES lines in the United States can be induced in vitro to express phenotypic characteristics of type 2 alveolar epithelial cells (273) (Figure 10). Comparable studies in England also have demonstrated induction of pro-SPC expression in cultured human ES cells (274). A recent study in Australia compared mouse and human ES cells exposed to dissociated fetal lung cells and found several differences in patterns of lung gene expression (275). The ATS issued a statement in 2006 calling for expanded human ES cell research and it is anticipated that human ES cell research will expand in the United States over the next several years (2). A human deltaF508 embryonic stem cell line has been established in England and another described in Belgium (276, 277). These cells exhibit normal morphology and protein expression compared with other ES cell lines but have not been studied in detail. Recent descriptions of induction of pluripotency in adult skin fibroblasts and other adult cell types offer another possibility of generating lung cells from autologous somatic cells (278–281). In particular, it might be possible to generate induced pluripotent cell lines from lung patients as models of disease.
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The workshop was organized into four 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 summary of the oral presentations is provided below. The executive summaries 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 this session was to review the current state of knowledge of endogenous stem cell populations in the lung, their role in lung development, and the role of the niche and microenvironment on lung progenitor cells. The session began with Diane Krause (Yale University School of Medicine) who provided a general overview of "stem" cells, highlighting that although many cells that are referred to as stem cells can differentiate into multiple cell types, true stem cells are capable of extensive self-renewal, whereas progenitors, found in different specialized regions of the lung, are limited in this capacity. The control of differentiation is determined in part by the cell's response to its microenvironmental niche that contains cues such as O2 tension and extracellular matrix components. Dr. Krause discussed the different endogenous lung progenitors, including CK5+CK14+ basal cells of the submucosal glands in the proximal airway, Clara cells and Clara cell variants (CCSP+SPC+) found in bronchoalveolar duct junctions of conducting airways, and type II pneumocytes as alveolar progenitors. The capacity of extrapulmonary precursors, such as those derived from bone marrow, to become pulmonary epithelial cells was described using the established male into female bone marrow transplant mouse model system together with the histochemical analyses required to demonstrate engraftment and pulmonary differentiation of the donor cells. There are several multipotent bone marrow subpopulations that can engraft and become epithelial cells, probably by different mechanisms, but an abundance of evidence demonstrates that tissue damage, such as that induced by irradiation, is required for bone marrow–derived epithelial engraftment, and such cells appear to be functional as shown in CFTR–/– and SPC–/– mouse experiments in which deficient gene expression was partially restored in the lungs. Many mechanisms for the plasticity of adult stem cells were proposed, including differentiation, direct and indirect transdifferentiation, fusion, and combinations thereof. Discrepancies in the literature are, in part, due to different types and severity of lung injury induced to provide homing signals, different assays that are used to determine "stemness" (e.g., label retention, retroviruses, transgenic reporter systems, cell ablation techniques), and the fact that different laboratories use different markers to identify and isolate the precursor cells. These areas certainly need to be addressed in the future. Other issues that need to be addressed include definition of the proliferative and differentiative activity of stem/precursor cells in different regions of the adult lung, development of improved methods to transplant and track cells in vivo, improved isolation and validation of such cells, identification of critical niche components, which should lead to better engraftment and differentiation, determination of risks of exogenous cell administration, and development of safe, effective treatments that promote lung progenitor function.
Wellington Cardoso (Boston University School of Medicine) discussed the role of specific pathways in the developmental programs of lung progenitor cells as these early programs may be relevant to adult lung regeneration/repair. Identification of critical factors in these programs may be used to design and develop treatments for lung disease. The key will be to understand the signals provided by the niche and the context in which they need to be delivered. Mouse studies describing the identification of the early pulmonary progenitors in the embryo were reviewed. The transcription factor TitF1 (Nkx2.1) labels lung progenitors in the foregut endoderm before formation of the lung buds. The importance of TitF1 and other transcription factors (e.g., Foxa2) in lung epithelial development and lung homeostasis was discussed. Dr. Cardoso underscored the critical role of fibroblast growth factor receptor 2b (FGFR2b) signaling via FGF10 in expansion of early lung progenitors during formation of the lung primordium and presented data on targets of FGF10 (BmpR1A, Tacstd2 [tumor-associated calcium signal transducer 2], cathepsin H) during branching morphogenesis (282–285). He discussed the control of the bone morphogenetic protein (BMP) pathway by FGF10, which maintains epithelial survival, and also presented evidence that FGF10 can act as a proto-oncogene. Transgenic mice expressing FGF10 in the lung epithelium develop multiple pulmonary tumors (286, 287). FGF10 is a potent inducer of Tacstd2, which is expressed by epithelial progenitors in FGF10-dependent structures, including the lung, as well as in many epithelial cancers. Tacstd2 may play a role in injury repair processes because it is induced in bronchial epithelial cells exposed to Pseudomonas and during renal regeneration and repair (288, 290). The importance of retinoic acid (RA) in lung progenitor cell expansion, through inhibition of the transforming growth factor (TGF)-β pathway and induction of FGF10, and the potential role of RA in alveolarization and lung injury repair were reviewed (289–293). Lessons from lung development need to be integrated into regeneration strategies. The inductive signals that trigger specific differentiation programs need to be identified, as does the context (timing and positional cues). Further investigation is also needed to identify the physical properties of the cells and of the local microenvironment vis-à-vis mechanical forces and bioelectricity (i.e., the correct microenvironment needs to be re-created) (294). Strategies and technologies to characterize the molecular signature of developing and adult lung components need to be developed, and studies that integrate genetic, developmental, and bioengineering aspects of the lung need to be designed using biologically relevant model systems.
Dr. Rick Wetsel (University of Texas–Houston) described the derivation of type II alveolar epithelial cells from one of the human ES cell lines, H9. Embryonic body formation was not required in contrast to what has been previously documented by Ann Bishop's lab using mouse ES cells. Dr. Wetsel's protocol (273) entailed the use of matrigel plates, and G418 selection, after which 99.6% of cells were SPC+ and had lamellar bodies. Molecular analysis showed that these cells expressed CFTR, surfactant proteins A, B, and C, 1-antitrypsin and complement components C3 and C5. After further incubation, these cells expressed alveolar type I cell markers. Preliminary data were also shown demonstrating that human ES–derived alveolar type II cells could ameliorate bleomycin-induced lung injury in mice, indicating the potential therapeutic use of these cells.
Barry Stripp (Duke University Medical Center) reviewed the current state of knowledge of the hierarchy of stem cells and their progeny in adult tissues. Comparisons were made between bronchiolar stem cells and other more classical stem cell hierarchies, such as those of the hematopoietic system and the intestine (295). The classical hierarchy consists of infrequently cycling cells and their more rapidly dividing transit-amplifying (TA) progeny. TA cells vary between organs in their abundance, have differentiation potential equivalent to a stem cell, but cycle frequently and have finite proliferative capacity. In contrast, the lung exhibits a nonclassical hierarchy where TA cells (e.g., Clara cells) cycle infrequently and have a differentiated phenotype, and their proliferative capacity is unknown. The anatomic sites of niches for different stem/progenitor cells in the lungs were discussed (296). Comparisons were also made to stem cell niches of other tissues and the role of asymmetric cell division and adherens junction complexes in the maintenance of the stem cell pool (e.g., intestinal crypts, hair follicles, bone marrow, subventricular zone of the brain) (297). Dr. Stripp reviewed data demonstrating the existence of latent bronchiolar tissue–specific stem cells and their role in airway repair after ablation of Clara cells by administration of naphthalene. This allowed the identification of distal airway stem cells that were resistant to naphthalene that localized to two discrete microenvironments, the neuroepithelial body and the BADJ. Other experiments showed that β-catenin stabilization (by truncating the N-terminus of β-catenin) altered the phenotype of Clara cells and increased the proliferation potential of the airways for 2 to 3 days after naphthalene injury (B. Stripp, unpublished data). Many fundamental issues and ideas remain to be addressed, including the relationship between a developmental progenitor cell and an adult tissue stem cell, whether lung tissue stem cells are required postnatally, what the constituents of a stem cell niche are, and how the niche changes in the setting of chronic or acute lung disease. Strategies to replicate or replace the stem cell niche need to be developed as do cell-based therapeutic approaches. Another challenge will be to define the relationship between normal repair versus tissue remodeling.
Ivan Bertoncello (Australian Stem Cell Center) described how local microenvironments in the lung interact with endogenous lung stem cells to specify their fate and modulate their cell surface phenotype. Stem cells exist in a state of dynamic reciprocity with their microenvironment, such that reciprocal signaling between stem cells, the extracellular matrix, supporting stromal cells, and accessory cells mediated by soluble and insoluble factors regulates their regenerative potential in the steady state and in response to injury (298). Dr. Bertoncello emphasized that stem cells are defined operationally by what they do rather than by how they are decorated. The evidence so far shows that stem cells are multiply marked, and that common stem cell–associated markers, including Sca-1, CD34, and membrane transport proteins, which confer the Hoechst 33342 side population phenotype, are not restricted to a single cell lineage and are typically shared and variably expressed by cells of differing developmental potential, activation state, and maturational age. Whether these markers define putative bronchioalveolar stem/progenitor cells is not clear and needs to be validated
35 (red line) or <35 (green line). Patients with EPC CFU counts 
