Chapter Eight - Lung Stem and Progenitor Cells in Tissue Homeostasis and Disease

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Abstract

The mammalian lung is a complex organ containing numerous putative stem/progenitor cell populations that contribute to region-specific tissue homeostasis and repair. In this review, we discuss recent advances in identifying and studying these cell populations in the context of lung homeostasis and disease. Genetically engineered mice now allow for lineage tracing of several lung stem and progenitor cell populations in vivo during different types of lung injury repair. Using specific sets of cell surface markers, these cells can also be isolated from murine and human lung and tested in 3D culture systems and in vivo transplant assays. The pathology of devastating lung diseases, including lung cancers, is likely in part due to dysregulation and dysfunction of lung stem cells. More precise characterization of stem cells with identification of new, unique markers; improvement in isolation and transplant techniques; and further development of functional assays will ultimately lead to new therapies for a host of human lung diseases. In particular, lung cancer biology may be greatly informed by findings in normal lung stem cell biology as evidence suggests that lung cancer is a disease that begins in, and may be driven by, neoplastic lung stem cells.

Introduction

The mammalian respiratory system is a highly complex three-dimensional organ historically described as containing over 40 different cell types, each with specialized functions to maintain adequate gas exchange and protect against environmental exposures. During development, the primordial lung undergoes branching morphogenesis to form the proximal conducting airways and distal gas-exchanging alveolar space (Morrisey & Hogan, 2010). The adult murine lung contains several distinct epithelial cell populations with unique anatomical positions and specialized functions (Fig. 8.1). The proximal airway includes the cartilaginous trachea, lined by pseudostratified columnar epithelial cells with submucosal glands interspersed. Noncartilaginous bronchioles, lined with simple columnar epithelium, branch from the trachea in an organized pattern. Secretory Clara cells also line the basement membrane of the airway with ciliated, neuroendocrine, and goblet cell populations (Bertoncello & McQualter, 2013). Lung cell-type terminology is undergoing a transition as the name Clara cell is being replaced by club cell; this review will use the historic term Clara cell. Neuroendocrine cells are present individually as well as in clusters termed neuroendocrine bodies that may play a role in sensing stimuli within the airway lumen (Van Lommel, 2001). Terminal bronchioles lead to the distal alveolar space containing surfactant-producing alveolar type II (AT2) cells and gas-exchanging alveolar type I (AT1) cells (Rock & Hogan, 2011).

Diverse experimental approaches have provided evidence that different populations of lung stem/progenitor cells reside in distinct niches and act in region-specific homeostasis and injury repair. Murine mouse models of injury have been utilized to study stem cells because of the low baseline levels of lung cell turnover during homeostasis and the increased rate of proliferation to replace ablated tissue following injury (Rawlins & Hogan, 2006). For example, bleomycin injures the alveolar epithelium, and naphthalene specifically injures the bronchiolar epithelium (Rawlins & Hogan, 2006). For more proximal airway injury, sulfur dioxide inhalation damages the tracheal epithelium (Borthwick, Shahbazian, Krantz, Dorin, & Randell, 2001), while ozone and nitrogen dioxide damage airway epithelial cells (Evans et al., 1976, Evans et al., 1986). Using these region-specific epithelial injury mouse models, it is possible to study cellular proliferation and epithelial regeneration. Lineage tracing is another valuable in vivo tool that has been used to study stem cell populations and their role in lung injury and repair without removing them from the lung (Raiser et al., 2008, Barkauskas et al., 2013, Rawlins, Okubo, et al., 2009, Rock et al., 2011, Tropea et al., 2012). Investigators have created mouse models to label stem cell populations of choice, which coupled with the injuries mentioned earlier, allow for detection of the lineage label, which will remain in both the progenitor population and progeny cells after injury repair.

Three-dimensional (3D) culture systems have emerged as an important method of characterizing lung stem cell properties including proliferation, differentiation, and self-renewal (Lee et al., 2012, McQualter et al., 2010, Rock et al., 2009). Fluorescence-activated cell sorting (FACS) can be used to isolate individual stem cell populations (Kim et al., 2005, Lee et al., 2013, McQualter et al., 2009, Rock et al., 2009, Summer et al., 2007, Teisanu et al., 2009, Zacharek et al., 2011), which can then be grown in clonal 3D assays in the presence of various microenvironmental factors such as Matrigel, nonepithelial cells, and growth factors to assess growth and differentiation properties. An ongoing challenge has been the ability to directly compare the functions of FACS-isolated lung stem cell populations with reparative cells in situ. Limited knowledge of markers distinguishing lung cell types in vivo has prevented precise concordance between the identity of lung cells with stem cell functions in vitro and in vivo. Transplantation assays have also been lacking in lung stem cell biology; currently there is no in vivo transplant assay for freshly sorted stem cells delivered to the lung. Recently, a kidney capsule transplantation model has been utilized as an alternative in vivo method of examining stem cell autonomous properties (Chapman et al., 2011). Subcutaneous injection of multipotent lung cells with Matrigel and the use of ex vivo decellularized lung models have also provided a new means to assess potential progenitor cell function (Longmire et al., 2012, Mou et al., 2012). The development of in vivo or in vitro assays to interrogate the function of stem cells at the single cell level, and the discovery of unique marker sets (rather than single markers) for each lung cell type are critical advances necessary to better identify lung stem cell populations and understand their relative contributions to tissue maintenance and repair.

The pathology of devastating lung diseases including lung cancers, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis (IPF), cystic fibrosis (CF), asthma, bronchopulmonary dysplasia (BPD), and others is likely caused, in part, by dysregulation and dysfunction of lung stem cells. Investigators continue to work to unlock the mystery of the lung stem cell landscape by first identifying the stem cell populations, examining their cell autonomous properties, and trying to better understand their microenvironmental interactions. While much progress has been made over recent years, important questions remain and the complete picture remains unclear. Lung stem cell studies offer a new avenue for developing treatment strategies for lung disease. In this review, we discuss the current knowledge of the lung stem field and the assays and tools used to dissect the complex biology of the lung in homeostasis and disease.

Section snippets

Endogenous Lung Stem and Progenitor Cells

Endogenous adult lung stem/progenitor cells are regenerative cell populations important for epithelial cell maintenance and injury repair. In multiple adult organs, tissue-specific stem cells have been identified as multipotent cells with the capacity for long-term self-renewal and the ability to give rise to at least two distinct differentiated lineages. Tissue-specific stem cells are typically quiescent in normal conditions and proliferate during injury repair (Kolios and Moodley, 2013,

The Others: Lung Mesenchymal Stromal Cells and Lung Endothelial Progenitor Cells

Evidence continues to support the idea that adult mesenchymal stromal cells (MSCs) are an important element of epithelial stem/progenitor niches. Critical for regional specification of embryonic lung epithelium, MSCs at the distal tip of the branching epithelium are known to secrete FGF10, a critical component of the signaling network involving Bmp, Wnt, and sonic hedgehog pathways that is necessary for coordinating differentiation in the developing lung (Morrisey & Hogan, 2010). FGF10-positive

Directing Differentiation: Embryonic Stem Cells and Induced Pluripotent Stem Cells

Pluripotent embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells) hold great promise for regeneration of injured tissue and repair of disease states. ES cells are isolated from the inner cell mass of preimplantation blastocysts and under well-defined culture conditions, they can be maintained indefinitely in an undifferentiated state with the ability to give rise to cells of all three embryonic germ layers (Odorico, Kaufman, & Thomson, 2001). Takahashi and Yamanaka

MSCs: Potential Role of Cell-Based Therapy in Lung Disease

MSCs have been recently examined as a potential cell-based therapy for lung disease. MSCs secrete growth factors and antimicrobial peptides, have immunomodulatory properties, and exhibit low immunogenicity (Lee, Fang, Krasnodembskaya, Howard, & Matthay, 2011). MSCs can respond, migrate, and facilitate repair of damaged tissue making them an attractive candidate for both prevention and treatment of lung disease. MSCs can be isolated from a variety of human tissues including bone marrow, adipose

Stem Cells in Lung Cancer

Lung cancer is the leading cause of cancer related deaths in the United States and worldwide (Ettinger et al., 2013, Herbst et al., 2008). In the United States alone, nearly 200,000 people will be diagnosed with lung cancer in 2013, and the predicted 5-year survival rate for all patients is a dismal 15% (Ettinger et al., 2013). Evidence suggests that lung cancer may originate from neoplastic lung stem/progenitor cell populations and that certain lung tumors contain cancer stem cells (CSCs).

Future Directions

Identification and characterization of the various stem and progenitor cell populations in the lung will allow for the study of the mechanisms through which these cells are maintained and stimulated. Because the lung is an essential organ, understanding these mechanisms and exploiting them therapeutically for lung regenerative medicine holds great promise for improving public health. Research will likely focus now on identification of unique cell surface markers that can be used to enrich for

Acknowledgments

We thank members of our laboratory for their discussions and critical comments on the chapter. Work in our laboratory is supported by the Post-Doctoral Fellowship, PF-12-151-01-DMC, from the American Cancer Society (CMF), the Ikaria Advancing Newborn Medicine Grant (KTL), American Medical Association Foundation SEED Grant (KTL), 5 T32HD7466-15 (KTL), RO1 HL090136, U01 HL100402 RFA-HL-09-004, American Cancer Society Research Scholar Grant RSG-08-082-01-MGO, the V Foundation for Cancer Research,

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