Cell Therapy for Lung Disease

The following sections are included:

• Contents

• List of Contributors

• Foreword

• Preface

An increasing number of studies suggest that cell therapy approaches may be powerful tools for repair of injured or diseased lungs as well as for understanding mechanisms involved in both lung development and lung repair. This rapidly progressing field encompasses a number of disciplines and conceptual approaches including the study of endogenous stem and progenitor cells resident in the lung, and investigations utilizing exogenously administered cells for the repair of injured lung. Moreover, the field has undergone several conceptual shifts over recent years. For example, the initial focus on engraftment of exogenously administered cells as airway or alveolar epithelium has been shifted to the current emphases on immunomodulation of inflammatory and immune pathways in the lung by stem cells, and on bioengineering approaches to grow functional lung tissue ex vivo for subsequent use in in vivo implantation for destructive lung diseases, such as emphysema. Furthermore, it has become apparent that the variety of candidate stem and progenitor cell types can have different actions in the lung. Each of these areas is the focus of a comprehensive chapter in this book. The goal of this introductory chapter is to provide an overview of the field to date.

Nature builds the lung from scratch and understanding the signalling cascades that control the temporal and spatial construction of the airways, parenchyma and blood vessels will provide clues about how man could learn to repair or even replace damaged lung tissue. We describe the normal development of the airways and the vasculature, then discuss interaction between these systems during development and present a brief section on functional development as the lung adapts to extra-uterine life, the most critical period of lung development. Reference is made to abnormal development because it can be viewed as a vicarious experiment. Lastly, we have summarised current information on lung regeneration and repair from a developmental perspective.

Together with their precursors and descendants, epithelial and mesenchymal cells interact throughout development to ultimately define the physiology and architecture of the mature and functional lung. It is hypothesised that during development, certain classes of both epithelial and mesenchymal stem and progenitor cells are retained in an undifferentiated state so they may be reactivated later in adult life to repair the damaged lung. Furthermore, it is hypothesised that several classes of molecules intimately associated with the developmental process are reutilised later during times of repair. These molecules include members of the fibroblast growth factor and transforming growth factor families, their receptors, agonist and antagonist signalling systems, transcriptional superfamily members and components of the extracellular matrix and basement membrane. How these various molecules orchestrate a morphogenetic signal coordinating both appropriate proliferation and differentiation of the epithelium and restructuring or maintenance of the supportive mesenchymal mass in order to effect repair of damaged tissue, however, remains unknown. This chapter will detail some of the cellular and molecular mechanisms interpreting epithelial–mesenchymal interactions from the moment of gastrulation to the establishment of the early branching lung anlage, currently described in the literature. As continual research is applied to this field, it is predicted these signalling pathways instructive to the formation and allocation of epithelial and mesenchymal stem and progenitor cells during development will be exploited to provide advanced interventional therapeutics for treating congenital respiratory diseases and respiratory damage following environmental insult (see Chapter 2).

The adult lung is a quiescent organ whose function is highly dependent upon epithelial integrity. Maintenance and restoration of the airway epithelium relies upon regulated activation of progenitor cells whose identity varies between tracheobronchial, bronchiolar, and alveolar zones. Defects in epithelial repair are associated with a broad spectrum of lung diseases that range from the inability to fight infections to hyperproliferative diseases such as pulmonary fibrosis and lung cancer. The present chapter summarizes our current understanding of the hierarchical organization, function, and regulation of the stem/progenitor cell hierarchies that maintain the lung epithelium. Much of our current knowledge relies upon the availability of methods that have been widely used to characterize stem cell hierarchies in other highly proliferative systems. These methods and their limitations are described in detail while emphasizing the differences between quiescent and highly proliferative organs. This chapter identifies significant knowledge gaps and identifies future directions that will help advance the field of lung regenerative medicine.

Adult stem cells posses the ability to undergo both self-renewal and differentiation into multiple lineages. In chronic lung injury these cells are activated in response to tissue damage, migrate to the site of injury and contribute to both structural and functional repair. In acute injury they reduce the pulmonary inflammatory response via a number of mechanisms to cause downregulation of pro-inflammatory cytokines and a reduction in pathological lung damage. However, there are significant concerns that they are at risk of undergoing malignant transformation. Murine bone marrow-derived stem cells have been shown to acquire chromosomal abnormalities in vitro that corresponds to fibrosarcoma formation in vivo, a finding that was not reproduced using human adult stem cells. Despite these concerns a number of clinical studies have already been undertaken and initial results, although varied, seem promising for their use in a clinical setting with a good short-term safety profile. There have been no reports to date of an increase in the number of malignancies, but further long-term safety data is needed.

Progress on lung stem cell bioprocessing and tissue engineering has been slow due to the lung's complex architecture and difficulties in differentiating stem cells toward the various pneumocytic lineages, in particular type II pneumocytes. Recent advances on the use of stem cells appear promising in providing a reliable supply of clinically relevant cell numbers of lung cells for potential therapeutic applications. The development of bioprocess technologies for the successful transfer of the current laboratory-based practice of stem cell and tissue culture to the clinic as therapeutics necessitates the application of engineering principles and practices to achieve control, reproducibility, automation, validation, safety of the process and the product to be suitable for clinical applications. Furthermore, the utilisation of bioreactors addresses issues of mass transport and provides a three-dimensional growth environment. A variety of cell sources are being investigated for their suitability in generating lung epithelial cells. This review will discuss the complexity of the lung structure, evaluate the various cell sources, and present an up-to-date assessment of the bioreactor technology available that can contribute to the successful differentiation of stem cells into airway epithelial cells.

Two animal models, alveolar regeneration in lung emphysema and compensatory lung growth after pneumonectomy, are well established and characterised models for lung regeneration. Several promising reagents for lung regeneration were reported. Bone marrow-derived cells and lung endogenous stem cells are both thought to be contributors in experimental lung regeneration. The degree of lung regeneration differs among animal species and among ages. Small animals, such as rodents, have a better capacity for lung regeneration because its somatic growth continues throughout their lifespan. In spite of these limitations, studies in animal models are essential to understanding the pathophysiology of lung regeneration and the development of novel therapeutic strategies for human intractable lung diseases.

Human fibrocytes exhibit mixed phenotypic characteristics of haematopoietic stem cells, monocytes and fibroblasts, and originate from a precursor of the monocyte lineage. They constitutively produce chemokines and growth factors that are known to modulate inflammatory reactions or promote angiogenesis and the deposition of extracellular matrix molecules. Upon exposure to transforming growth factor-β₁ and endothelin-1, fibrocytes produce large quantities of extracellular matrix components and acquire a contractile phenotype. Such differentiation of fibrocytes into myofibroblast-like cells occurs at the tissue sites during repair processes and has been found to contribute to wound healing in vivo. Fibrocytes and fibrocyte-derived myofibroblasts are also involved in the pathogenesis of lung disorders characterised by chronic inflammation and extensive remodelling of the bronchial wall, like asthma, or progressive fibrosis with destruction of the pulmonary architecture, like idiopathic pulmonary fibrosis. They participate in tumour-induced stromal reactions and may either promote or inhibit the metastatic progression of cancers. Prevention of excessive extracellular matrix deposition and detrimental tissue remodelling in pulmonary diseases may be achieved by inhibiting the accumulation of fibrocytes in the lungs. Moreover, in vitro expanded fibrocytes may serve as vehicles for the delivery of gene constructs to improve ineffective lung repair or be used in anti-cancer cell therapy.

In this chapter we discuss how to engineer 3D pulmonary tissue constructs in vitro using primary isolates of foetal mouse distal lung cells. When cultured in hydrogel-based 3D constructs, the mixed cell population, comprised epithelial, mesenchymal and endothelial cells, organised into alveolar forming unit (AFU)-like sacculated structures, which, in terms of morphology and cytodifferentiation, were reminiscent of native distal lung. By using a unique, serum-free medium supplemented with a cocktail of tissue-specific growth factors, we were able to induce concomitant alveolisation and neovascularisation when culturing the cells in the hydrogels, but not in scaffolds composed of synthetic polymers. Our data suggest that our in vitro model is capable of recapitulating the parallel morphogenesis of epithelial and endothelial pulmonary tissue components, which may occur through dynamic paracrine interactions. These results also stress the importance of the complex input from co-cultures, tissue-specific growth factors and integrin signalling for successful tissue engineering in vitro. In a mouse model in vivo, incorporation of the primary lung cell isolates into Matrigel plugs, implanted either subcutaneously (s.c.), or under the kidney capsule, leads to the formation of sacculated AFUs in close proximity to patent capillaries. Effective functional vascularisation, however, was only observed upon addition of angiogenic growth factors to the scaffolds and their controlled release over time. Use of a fluorescent cell tracker confirmed that the neovessels in the constructs comprised endothelial cells from both the host and the grafts. These data demonstrate that it is feasible to generate vascularised pulmonary tissue constructs in vivo with proper epithelial differentiation, and that the degree of vascularisation may be manipulated by incorporating the release of an angiogenic factor within the construct.

There has been considerable research on harnessing the unique properties of stem and progenitor cells to develop more effective therapeutic approaches for respiratory diseases with limited treatment options. Cell-based gene therapy, combined with genetic engineering to enhance regenerative cell function, can be a powerful strategy able to provide additional benefits, and may overcome many of the limitations of cell or gene therapy alone. This dual strategy not only allows the direct targeting of cells to the lung for regenerative interventions, but also provides a site-specific source to release therapeutic proteins to the lung microvasculature or alveoli. This review discusses relevant literature on the use of stem/progenitor cells-based gene therapy for the treatment of acute and chronic pulmonary disorders, including acute lung injury, pulmonary arterial hypertension, emphysema, cystic fibrosis, and cancer therapy for lung metastasis. Different types of stem and progenitor cells such as mesenchymal stem cells or endothelial progenitor cells, which have been used in these therapeutic approaches, will also be covered.

Cystic fibrosis (CF) remains a devastating and incurable disease. One of the first targets for lung gene therapy and studies of lung gene transfer, early promising results were achieved in vitro and in small animal models. However, studies in primate models and in patients were discouraging, despite a large number of clinical trials, and gene therapy approaches for CF have generally fallen out of favour. Newer approaches with cell-based therapy, utilizing either embryonic stem cells or extra embryonic-derived adult stem cells (bone marrow or cord blood), have been investigated recently and may provide viable future therapeutic options. In parallel, further understanding of the role of endogenous progenitor cells in CF lungs may also provide both mechanistic understanding and potential therapeutic approaches for CF lung disease. In this chapter, endogenous lung progenitors in CF and the potential use of cell therapy-based approaches for CF will be considered.

Pulmonary arterial hypertension (PAH) is a progressive and lethal condition characterised by elevated pulmonary pressures and right ventricular hypertrophy. Significant advances have been made in the treatment of this condition; however, the prognosis remains extremely poor for the majority of patients. Over the last decade a number of preclinical studies have explored the potential for cell therapy in PAH. These have included novel investigations using somatic and stem/progenitor cells, either alone or transfected with potentially therapeutic genes. The success of early preclinical work has recently prompted the first clinical trials of cell therapy for PAH. This chapter will review the basic pathophysiology of PAH, the current pharmacotherapy, and then examine the preclinical and clinical investigations of cell therapy for this disease.

Alveolar regeneration using developmentally active small molecules is an attractive therapeutic option for the treatment of diseases where lung development is disrupted, such as bronchopulmonary dysplasia or where alveolar surface area is lost, such as pulmonary emphysema. Understanding programmes of alveolar development, maintenance and regeneration will be critical in exploiting these novel therapeutic opportunities.

Demonstrating that a cell therapy can directly contribute to functional recovery of a diseased organ is made difficult by experimental methods that are liable to introduce false-positive data. In this chapter we analyze the methods used to prove that a transplanted cell has grafted into the target organ and highlight the potentials for producing misleading results. Furthermore, we discuss the advantages and pitfalls of supporting graft data by quantifying improvement of organ function. We conclude that a multi-tiered experimental approach is required to thoroughly dispel the notion that engraftment and functional improvement are artefactual. Because of the relative immaturity of the field of lung cell therapy, we draw many of our examples from tissues with related challenges, such as cardiomyocyte cell therapy.

The development of cell therapies in animal models and patients requires robust in vivo imaging techniques to track the administration, migration, integration, division and fate of administered stem cells and to monitor any morphological and physiological effects on the damaged tissue after therapy. This chapter outlines the well-established imaging modalities such as CT (computed tomography), MRI (magnetic resonance imaging), PET (positron emission tomography), SPECT (single photon emission computed tomography), and EBUS (endobronchial ultrasound) and discusses recent advances in biophotonic technologies including OCT (optical coherence tomography), DOI (diffuse optical imaging), and CLSM (confocal laser scanning microscopy) and their integration with endoscopy for in situ, in vivo cellular level imaging. Comparisons are made of sensitivity and spatial resolution for detecting stem cells in vivo, as well as imaging the lung in small animal models and patients. We also outline the challenges of labelling stem cells in vivo and the role of responsive agents to visualise the local physiological conditions.

The discovery of donor-derived cells incorporating and apparently differentiating into human pulmonary tissue following haematopoietic stem cell transplantation (HSCT) was first reported in 2003, and subsequent studies on this phenomenon using ever more sophisticated techniques have described a wide range of findings. Thus far, it has been suggested that epithelial, endothelium, and interstitial tissues of the lung may be chimeric following HSCT, but debate about the extent and even existence of such chimerism continues. Although this phenomenon has only been described in humans in the incidental setting of transplantation, the implication is that such a process occurs in the absence of such conditions, relying on endogenous circulating marrow-derived stem cells to repair or remodel the lung. Whether these reports represent some other source of chimerism (such as maternal–foetal) or even cellular fusion, the incorporation of donor-derived cells into the lung, should it represent not only antigenic but functional conversion to lung tissue and be inducible at sufficient frequency, would present broad therapeutic possibilities. Furthermore, the study of this phenomenon may yield insights into disease pathogenesis that present even further therapeutic avenues, beyond the use of stem cells themselves.

Lung cancer is the most common cause of death from cancer. It is estimated that every 15 minutes in the UK, one person dies of lung cancer¹ and in both the UK and USA more people die of it than any other type of cancer,^2,3 it being responsible for about one quarter of all deaths from cancer. Its high mortality rate is also reflected on a global scale, with lung cancer accounting for more than 1 million deaths per year.⁴ Lung cancer is usually sub-divided into three types: small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), and mesothelioma — a rare type of cancer that affects the pleura. Stem cells and cancer are inextricably linked; the perceived wisdom is that the process of carcinogenesis initially affects normal stem cells or their closely related progenitors, and then at some point, neoplastic stem cells are generated that propagate and ultimately maintain the process. Many, if not all cancers contain a population of self-renewing stem cells; the so-called cancer stem cells (CSCs) that are entirely responsible for sustaining the tumour as well as giving rise to proliferating but progressively differentiating cells that contribute to the cellular heterogeneity typical of many solid tumours. Thus tumours, like normal cell populations, may have a hierarchical structure. Adherents of the CSC hypothesis believe that the bulk of the tumour is therefore not the clinical problem, and so the identification of CSCs and the factors that regulate their behaviour are likely to have an enormous bearing on the way we treat neoplastic disease in the future. This chapter summarises 1) the histogenesis and molecular pathogenesis of lung tumours that probably take origin from normal pulmonary stem cells; 2) the evidence for the existence of CSCs in neoplastic lung tissue; and 3) illustrates some of the cellular pathways, often related to stem cell behaviour, that are frequently aberrant in lung cancer and may represent druggable targets.

The following sections are included:

• Index