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Stem Cells and Regenerative Medicine, Section on Experimental Medicine and Toxicology, Hammersmith Hospital, London, United Kingdom
Correspondence and requests for reprints should be addressed to Anne E. Bishop, Ph.D., Imperial College Faculty of Medicine, Hammersmith Hospital, Du Cane Road, London W12 ONN, UK. E-mail: a.e.bishop{at}imperial.ac.uk
From time to time, an area of research erupts into the public arena and is promulgated as the new great white hope of medicine. The field of stem cells is one of the latest examples, with the media speculation on its potential applications being fueled and politicized by associated ethical issues concerning the use of embryonic stem cells and the occasional appearance of that ominous word cloning. For the basic scientist, stem cells offer new tools, for example in the investigation of developmental and pathogenetic pathways. For the clinician, the capacity of stem cells both to proliferate and to form different types of cells makes them, in theory at least, ideal for replacing or repairing damaged or diseased tissue. Sometimes the promise of medical research fulfils expectations, but too often it falls short of the hype. Stem/progenitor cell therapies are emerging in various medical disciplines, but their predicted status as a panacea has not yet been attained; the jury is still out. For the respiratory system, stem cell research has moved more slowly than that of many other organs, mainly because of the lung's structural complexity, cellular heterogeneity, and the low turnover rate of its epithelia. However, major advances have been and continue to be made, including clarification of the events underlying embryonic lung development and the discovery of previously unknown regenerative pathways in the adult lung. Targeted activation of endogenous stem cell pools would exploit existing repair mechanisms and augment the body's innate regenerative capability. Stem cells could form the basis of cell therapies and also offer the potential to create pulmonary tissue constructs ex vivo for implantation into the lung to repair more extensive damage. In addition, stem cells can be used to create in vitro models of lung development and disease for further investigation and manipulation. The 23rd Transatlantic Airway Conference was aimed at evaluating the progress made in our understanding of the stem cell biology of the lung and how far we may be from establishing effective therapies based on that understanding. The 2008 Conference took advantage of the unique nature of this series of meetings in bringing together not only physicians and research scientists at the center of the respiratory stem cell field but also a broad range of experts from related disciplines, including tissue engineering, bioengineering, and biophysics, to discuss stem and progenitor cell biology in relation to lung repair.
LESSONS FROM DEVELOPMENT
It is not only the knowledge gained by developmental biologists that is furthering our understanding of the turnover and stem cell biology of the lung epithelium but also some of their investigative approaches. This observation was neatly illustrated by the elegant studies done by Emma Rawlins in her examination of the hypothesis that distal tip epithelial cells form a multipotent progenitor population. Id proteins are negative regulators of basic helix-loop-helix transcription factors, and a functional role for Id2 in particular has previously been suggested in the developmental regulation of lung epithelial cell proliferation (1). By applying conditional gene targeting with Id2-CreER knock-in alleles, Emma showed that Id2 expression, activated at Embryonic Day 11.5 by tamoxifen, can be detected in branching tip epithelial cells in murine lung at Embryonic Day 12.5 and, at 7 days post-natally, these cells express markers of both proximal (ciliated, neuroendocrine, Clara, and putative [bronchoalveolar] stem cells) and distal (type I and II pneumocytes) lung epithelial phenotypes. By applying tamoxifen activation later, at Embryonic Day 16.5, it was possible to show that the cell types produced by the Id2-positive progenitors change over time and thus, at that stage, are committed to giving rise only to pneumocyte progeny.
The airway glandular stem cell niche may contribute to diseases associated with aberrant mucous production in the airway, including cystic fibrosis (CF), and John Engelhardt described his investigations of the factors that control the early establishment of this niche. John described the creation of a transgenic ferret model. Ferret airways have identical cell types and a distribution of submucosal glands similar to those in humans, making them a better model for CF airway disease than those that are currently available. Stem cells are slow-cycling and, therefore, can be labeled with bromodeoxyuridine, followed by a "chase" period in which the label is diluted out from rapidly dividing (transit amplifying) cells, and are thus identified as label-retaining cells (LRCs). LRCs in the glandular niche were found to be resistant to naphthalene and to express Clara cell secretory protein, characteristics they have in common with cells described by Barry Stripp in the smaller airways. A family of High Mobility Group transcription factors called TCF/Lef-1 was shown to co-ordinate events leading to gland formation in the airway and a mesenchymal signal, Wnt3a, was shown to be necessary for Lef-1 expression in gland buds.
The conference then focused on translational embryology as Jim Wells described how better understanding of molecular mechanisms that control endodermal differentiation in the embryo could help in the development of stem cell–based therapeutics for human diseases affecting endodermal organs. He drew parallels between the embryology and regeneration of pulmonary epithelium and pancreas and showed how he is translating information from his embryonic studies to promote the differentiation of human embryonic stem cells (hESCs) into endoderm. The formation of definitive endoderm from hESCs was upregulated with activin A (protocol adapted from Reference 2) and the effects of Wnt and FGF on the establishment of organ domains was studied by examining anterior-posterior patterning; increasing their concentration promotes posterior fates while their inhibition directs to anterior fates. The role of Sox 17 in the development and regeneration of endoderm-derived organs was also explored and it was shown that, in addition to being a transcription factor for master regulator genes in endoderm development and function, it also acts as a Wnt antagonist and regulates separation of ventral organs.
DEFINING POTENTIAL ROUTES TO STEM/PROGENITOR CELL–BASED THERAPIES
Regeneration of airway epithelium is a characteristic of several airway diseases, including chronic obstructive pulmonary disease and cystic fibrosis, and the exact nature of the human epithelial stem cells behind this repair is not yet known. Using in vitro models and xenografts, Christelle Coraux presented evidence that such stem/progenitor cells can be found among both human fetal basal and suprabasal cell subsets in the tracheal epithelium, and aquaporin 3 (Aq3) was identified as a marker for fetal basal cells that can effect regeneration. The roles of a variety of factors in airway regeneration were also explored using these models and it was revealed, for example, that trefoil factor family 3 induces ciliogenesis and promotes airway epithelial ciliated cell differentiation and that it achieves this in part through an EGF-R–dependent pathway.
Barry Stripp discussed the molecular regulation of the airway stem cell hierarchy and how the pathways that are being revealed might be exploited for the development of novel cell and molecular interventions for correction of epithelial reparative disorders. He showed, for example, that manipulation of signaling downstream from β-catenin impacts on stem cell pool size through altering cellular differentiation. The differences between bronchiolar stem cells and those of classical hierarchies, like those of the gut (stem >> transit amplifying >> terminally differentiated) were highlighted. Thus, the vCE (variant CCSP-expressing) stem cells, that he has previously described, give rise to transit amplifying cells after injury but not in the steady state, and can, in turn, form terminally differentiated (ciliated) cells or facultative progenitors (Clara cells).
Since 2001, when the first descriptions emerged, the phenomenon of adult bone marrow-derived cells (BMDCs) in the lung has been a hot topic and the drive to translate stem cell therapies in respiratory medicine appears to be strongest in this area. However, the basic research has thrown up several key and, as yet, unanswered questions, some of which were addressed by Diane Krause. Using cell tracing, it was possible to show that a single marrow-derived stem cell can engraft not only as blood but also as mature airway epithelial cells. Regarding the mechanism by which BMDCs become epithelial, it was shown that this can happen without cell fusion, although fusion can and does occur. Evidence that engrafted BMDCs can function was shown by the restoration of functional type II pneumocytes, expressing surfactant protein C (SP-C) and its mRNA, in SP-C–null mice after bone marrow transplantation; in this model, around 50% of BM-derived epithelial cells showed evidence of fusion.
David Warburton explored the roles of endogenous and exogenous progenitor cells in lung repair. He described, for example, the existence of a proliferative, injury-resistant subpopulation of type II pneumocytes (AEC2s) in hypoxia-treated lungs and that AECs from GFP-transgenic mice can engraft into the alveolar epithelium of wild-type mice after intravenous injection. Rates of engraftment are increased after oxygen injury. A new potential source of pluripotent cells for lung repair was also introduced in the form of amniotic fluid stem cells (AFSCs) that possibly are sloughed off from the fetus. It was shown that they can differentiate to airway epithelium in murine embryonic lung explants and, when injected intratracheally into adult mice, migrate and can be traced all over the body, including in the lung. Although pluripotent, AFSCs do not form teratomas (unlike embryonic stem cells) and, thus, may have wider applications.
Side population (SP) cells from adult lung, identified and isolated on the basis of their ability to efflux Hoechst dye, have been described in pulmonary airways and shown to comprise CD45+ or CD45– populations (3). Alan Fine showed how the latter were split on the basis of CD31 expression and the CD45–/CD31– population found to express the phenotype of primitive mesenchymal cells, giving rise to a variety of differentiated mesenchymal cell types in vitro, including smooth muscle. The cells were found only at very low frequency in the lung but could be expanded in vitro with full phenotype retention. When injected into mice with elastase lung injury, CD45–/CD31– SP cells displayed prolonged engraftment.
Bone marrow–derived endothelial progenitor cells have been shown to facilitate angiogenesis, possibly through direct incorporation and/or paracrine action. Clinical studies were described by Duncan Stewart that were designed to test the safety and efficacy of delivering autologous, adherent mononuclear cells, transiently transfected with endothelial nitric oxide synthase (eNOS) for the treatment of pulmonary arterial hypertension—The Pulmonary Hypertension And Cell Therapy (PHACeT) Trial. An overlapping dose escalation protocol was applied with panels of three patients each receiving from 7 to 150 million cells over 3 days. Among the measurable outcomes was the finding that pulmonary vascular resistance was reduced by 3 days in patients receiving 7 million cells, an effect that may have been due to eNOS. In addition, in a pre-clinical model of acute respiratory distress syndrome (intratracheal installation of LPS), administration via the jugular vein of mesenchymal stem cells (MSCs) transfected with the gene for angiopoietin-1, an anti-inflammatory and endothelial-protective protein, gave significant improvement of both alveolar inflammation and permeability, over and above that obtained with untransfected MSCs.
LOOKING TO THE FUTURE
Although BMDCs were initially considered to be beneficial in lung injury, engrafting in and repairing pulmonary epithelium, there is now substantial evidence that they contribute to respiratory disease, for example in pulmonary fibrosis, where proliferating cells of bone marrow origin have been identified (4). Sam Janes provided further evidence for the potential adverse effects of BMDCs in the lung by injecting CD45/CD11b– adherent murine BMDCs, transfected with GFP, into mice with sublethal whole body 
-radiation. By 24 hours, cells were seen throughout the lung in capillaries and in alveolar spaces. The lungs of mice killed at Days 14 and 28 contained bone and cartilage tumors expressing GFP, occupying a large percentage of the parenchyma at the later time. Similar results were obtained with untransfected cells but no tumors were found when the experiments were repeated using human BMDCs. This observation may relate to the finding that murine BMDCs showed karyotypic change with only 3 to 4 passages, whereas the human cells were karyotypically stable up to 14 passages, highlighting the need to use stem cell types that are stable in cell culture. These findings demonstrate the clear need for caution in the development of clinical trials for BMDCs in respiratory medicine.
Helen Rippon covered the potential applications of a particular exogenous cell source, embryonic stem cells (ESCs), in lung repair. Various methods were described that have been developed to enhance differentiation of ESCs into pulmonary epithelial lineages. The methods included recapitulation of developmental signaling events, mimicking the in situ physical environment and reprogramming the stem cell nucleus. Whether ESC-derived pulmonary epithelial cells or engineered tissue will be used in the clinic remains to be seen, as there are safety issues, principally regarding the possibility of accidental implantation of undifferentiated cells, that are yet to be addressed. In the meantime, there is no doubt that, at the very least, ESCs could have a range of indirect therapeutic applications, such as in disease modeling, drug discovery, and toxicological screening.
The perspective of a potential end user was given by Richard Lubman, who focused attention on potential targets for future stem/progenitor cell–based therapies, rationalizing many of the aspirations of basic scientists. By finishing on the subject of lung tissue engineering, this review neatly dovetailed into Joan Nichols's presentation on the generation of three-dimensional lung tissue constructs in bioreactor systems. Joan reviewed the current status of the field, providing a timeline that highlighted the major steps that have been made in the process of creating implantable lung constructs. She emphasized the need both to identify additional cell sources with reproducible and reliable differentiation efficiencies to form the basis of engineered lung tissues and to develop biomaterials that are tailored to fit the requirements of engineered lung, taking into consideration characteristics that include durability, elasticity, degradability, and low immunogenicity.
The conference was wound up with two presentations on the harsh realities of getting new therapies into clinic. Chris Mason gave a succinct account of the phases of regenerative medicine translation and commercialization, bringing the basic scientists in the audience down to earth with a bump. The period of 1985 to 2002, or RegenMed 1.0, was focused on attaining research goals, while 2006 heralded the era of RegenMed 2.0, in which the focus has shifted to the translation of research into commercially successful products. A necessarily pragmatic view was taken on the development of "regen" therapies, encompassing areas such as the need for simpler but superior products, scalability of manufacture, and the inevitable high costs. David Rodman proposed radiation injury as a suitable predictive animal model of human disease that could be used as a proof-of-concept strategy for stem/progenitor cell–based therapies in respiratory medicine. A number of suggestions were made as to how to limit the translational period, including using allogeneic cells, focusing on a restricted area of lung and assessing short-term (i.e., weeks rather than months/years) measurable outcomes using an accessible (i.e., epithelium) cell source.
Respiratory diseases are a leading cause of morbidity and mortality worldwide but, despite the severe impact of lung disease on society, research funding in this area is disproportionably low. Many lung diseases remain treatable only with a transplant, but the number of donor organs available is far outstripped by demand. However, this conference showed that all is not gloom and many opportunities for future therapies are being provided by the burgeoning field of stem cells. As the basis of natural pathways for tissue maintenance and repair, stem cells are a key target for mediating repair in vivo. Some researchers are focusing on the targeted activation of endogenous stem cell pools that would exploit existing repair mechanisms and augment the body's innate regenerative capability. Others are exploring the potential of stem cells in the engineering of pulmonary tissues, for implantation into the lung or for use as in vitro models of lung development and disease for further investigation and manipulation. Stem cell–based approaches to the treatment of lung diseases come with caveats, of course; the cells may contribute to the pathogenesis of the condition or may even give rise to neoplasms. However, given the numbers dying from respiratory disease and the wealth of evidence for the participation, direct or otherwise, of stem cells in maintaining the integrity and function of the lung, it is both surprising and disappointing that so few attempts have been made to carry out even initial exploratory trials. It is not clear why pulmonary medicine lags behind other fields, notably cardiovascular medicine, in which stem cell treatment has been used for some years, but it is hoped that sufficient research has now been done to allow potential therapies to move more swiftly to clinic. After all, there are huge numbers of patients with end-stage pulmonary disease who are already offering to participate in clinical trials.
FOOTNOTES
Conflict of Interest Statement: A.E.B. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.
(Received in original form April 14, 2008; accepted in final form April 28, 2008)
REFERENCES
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