© 2005 American Thoracic Society DOI: 10.1165/rcmb.F289
Bioengineered Lung EpitheliumImplications for Basic and Applied Studies in Lung Tissue RegenerationCenter for Lung Tissue Regeneration University of Pittsburgh Pittsburgh, Pennsylvania Strategies for replacement of lung epithelium that either lacks intrinsic regenerative capacity or harbors functional defects due to inherited or environmental factors have enormous therapeutic potential. A number of recent developments make significant inroads into our understanding of what it might take to achieve this objective. The confluence of studies investigating mechanisms of lung development, repair following injury, and development of in vitro methods for the derivation of lung epithelium either from pluripotent embryonic stem cells or multipotent adult somatic cells, represent the initial building blocks that could pave the way toward cell-based therapies for lung disease. This Editorial will discuss the novel findings presented in the article by Coraux and colleagues in this issue of the Journal (pp. 87–92; 1) in the context of implications and applications of this technology. Cellular Sources for Cell-Based Therapies Cellular sources for cell-based therapies are likely to be those that represent the intrinsic long-term regenerative pool for the tissue in which they reside. A good analogy would be the bone marrow, in which replacement of the hematopoietic stem cell in myeloablated recipients results in long-term restoration of hematopoiesis, whereas replacement with lineage committed cells only results in short-term restoration of hematopoiesis (2). This example highlights the features of stem cells and their more differentiated transit-amplifying or progenitor cell progeny that are of importance in tissue maintenance. Unifying properties of stem cells are their relatively undifferentiated phenotype, capacity for generation of differentiated progeny, and unlimited proliferative capacity. However, relationships between embryonic and adult stem cells, and among adult stem cells that vary in the range of differentiated progeny that they can generate, remain gray areas in our definitions of this class of regenerative cell. In a simplistic manner, embryonic and adult stem cells share the common properties that their maintenance is dependent upon the provision of environmental cues. Embryonic stem cells only exist transiently during pre-implantation development, and rapidly differentiate into the three embryonic germ layers during gastrulation. Despite this, embryonic stem cell lines have been derived from the pre-implantation embryos of a range of mammalian species including mice and humans. Interestingly, these cells, unlike their in vivo counterparts, can be stably maintained in culture provided that the appropriate culture conditions are maintained. Presumably these culture conditions provide a stable environment for the maintenance of the pluripotentiality of embryonic stem cells that is lacking in vivo. In contrast, adult stem cells are generally considered to be maintained indefinitely due to the creation of niches that provide a defined set of environmental cues that sequester a subset of cells and allow for their long-term maintenance at an arrested stage in the pathway toward their differentiation. In this context, tissue-specific stem cells are those regenerative cells that are maintained within a protective niche within a tissue and can be stimulated to proliferate for the generation of progeny. Differentiating progeny of stem cells is frequently referred to as a transit-amplifying population due to their retention of limited proliferative capacity and establishment of a larger pool of differentiated progeny. Coraux and colleagues determined that mouse embryonic stem cells cultured on type I collagen have the capacity to generate differentiated derivatives that include subsets of cells possessing the molecular properties of airway epithelium as indicated by expression of the Clara cell 10 kD secretory protein (CC10 or CCSP). They go on to demonstrate that when these differentiated cells are re-plated onto permeable supports and cultured at the air–liquid interface, the surface epithelium exposed to the air interface includes cell types and morphologic characteristics of the fully differentiated tracheobronchial epithelium. These findings are in contrast to the findings of Ali and coworkers, who reported the derivation of type II pneumocytes from differentiating mouse ES cells when cultured as embryoid bodies (3). Further insight into the significance and implications of these findings are drawn from further consideration of the processes regulating cell phenotype and function in the developing and adult lung. Progenitor Cells and Lineage Relationships in the Developing Lung By analogy with pluripotent cells of the developing embryo, there is strong evidence to suggest that multipotent lung precursor cells are specified, either intrinsically or through spatial cues, before lung organogenesis in the primitive foregut endoderm (4). These early lung precursors have the capacity to respond dynamically to local environmental cues to effect lung development (5). As such, even though a primitive multipotent lung precursor cell must theoretically exist in the developing embryo, it is not a stable entity whose properties have been objectively defined. The adult lung, by contrast, is composed of over 40 cell types whose properties have been defined using a combination of morphologic, ultrastructural, and molecular criteria. The process of lung development results in lineage commitment with the resulting formation of distinct zones along the proximal to distal axis of the airway (6). These zones are minimally defined by the alveolar, bronchiolar, and tracheobronchial regions, each of which is lined by an epithelium of unique cellular composition and function. The finding by Coraux and colleagues that differentiating mouse ES cells can give rise to tracheobronchial epithelium when exposed to an air–liquid interface suggests that these culture conditions support the differentiation of pluripotent cells for the recapitulation of at least two stages of lung development; that involved in early specification of foregut endoderm, and that involved in specification of the tracheobronchial zone of the conducting airway. However, the question of whether this epithelium is capable of long-term self-maintenance has not been addressed. Establishment of a Self-Maintaining Adult Lung Epithelium Whereas the majority of the adult airway epithelium is composed of cells that fulfill differentiated functions, a limited number of cells with stable properties of tissue-specific stem cells are sequestered within specialized niches (7–9). Within the conducting airway, these microenvironments appear to vary in cellular composition according to location and define the aforementioned airway zones that harbor distinct, non-overlapping epithelial lineages (Ref. 10, and reviewed by Engelhardt [11]). Even though stem cell niches are presumably established during the process of lung development, mechanisms by which they contribute to the establishment of the stem cell pool are not clear. It is possible that these microenvironments provide unique cues that allow local lung precursors to withdraw from a differentiation program involving dynamic interactions between epithelium and mesenchyme that would otherwise lead to the coordinate differentiation of both compartments. If this were the case, stem cells would represent a stable counterpart to a developmental precursor cell existing at the time the niche was established. Alternatively, once stem cell niches are formed they may confer upon neighboring cells unique characteristics that distinguish them from their neighbors in a more directed fashion, leading to the generation of a local population of cells with the intrinsic property of "stemness." An equally important issue that is of particular relevance to our understanding of pathophysiologic mechanisms contributing to lung diseases in which the regenerative capacity of the epithelium is diminished or lost, is the question of what mechanisms contribute to the maintenance of stem cells through adulthood. Potential answers to this question come from elegant studies investigating mechanisms involved in the maintenance of germline stem cells in Drosophila, for which direct interactions between the stem and somatic cells within the niche have been shown to govern stem cell maintenance and pool size (12). If similar mechanisms exist within the lung, then the physical association of stem cells with "structural" cells of the niche may be sufficient for their maintenance, while migration of cells from the niche commits the cell to a program of differentiation. It is unclear from the study by Coraux and colleagues whether the tracheobronchial epithelium generated after ES cell differentiation harbors stem cell niches and their associated stem cells that would presumably be necessary for its long-term maintenance. Microenvironments that are believed to participate in the maintenance of tracheobronchial stem cells are thought to reside at the opening of submucosal glands and intercartilaginous regions (7, 13). If the product of directed ES cell differentiation is to be used as a therapeutic tool, the generation of culture conditions that both establish and maintain tissue-specific stem cell would be critical. This would presumably require culture conditions that either recapitulate the stem cell niche or allow differentiating ES cells to recreate both the niche and the stem cell. In a similar vein, the potential that differentiating ES cells may be used as a source of more distal airway derivatives is a possibility that is highlighted by the independent findings of Ali and colleagues and of Kubo and colleagues (3, 14). However, notably absent in either of these reports, and that of Coraux and colleagues, is evidence of differentiation into bronchiolar derivatives. Expression of the airway secretory marker CC10 was shown in Coraux's paper, yet this was in the context of epithelial cell differentiation into tracheobronchial epithelium, suggesting that the distinct lineage of proximal secretory cells rather than bronchiolar (Clara) cells are present within these cultures. These studies highlight the need for unique molecular markers, such as locally restricted secretory proteins (15), whose expression can be applied to the more definitive classification of secretory cell subsets of the conducting airway epithelium. A number of hurdles must be addressed before tissue engineering approaches such as that detailed in the article by Coraux and colleagues may be extended into the realm of cellular therapy for lung disease. A fundamental issue is that the bioengineered tracheobronchial epithelium derived from ES cells may have overshot the desired stage of epithelial cell differentiation that preserves critical tissue-specific stem cell populations necessary for long-term maintenance of the tissue. Ongoing studies defining cellular and molecular mechanisms of stem cell maintenance in vivo and the unique properties of stem cell niches that supply this supportive microenvironment are critical to achieve this goal. Methods must be developed for the efficient engraftment of exogenously supplied cells at the appropriate site within the diseased tissue. This may also require the "homing" of engrafted multipotent cells to niches that can maintain their "stemness" to ensure long-term engraftment. Finally, as with any allograft, acute rejection of the transplanted tissue must be overcome for effective restoration of tissue function. However, these studies are testaments to the rapidly evolving field of lung tissue regeneration and provide critical insights into future directions for basic and applied studies in lung epithelial cell biology. Footnotes Conflict of Interest Statement: B.R.S. has no declared conflicts of interest; S.D.R. has no declared conflicts of interest. References
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