Lung Stem Cell
Scientific Summary

Introduction

 Over the past decade, stem cell research has become one of the focal points of modern therapeutics. Alongside therapeutic cloning, stem cell manipulation has become one of the “holy grail” quests in laboratories across the world. It would seem logical that stem cells, with the ability to differentiate into every cell in the human body – from white blood cells to solid organ components – would be the ultimate resource in regenerative medicine. The ability to “build” failed organs, depleted immune systems or patches for localized sites of injury would be a modern medical miracle. The fact that these ailments could be relieved by harvesting cells from the patient’s own body further bolsters the opportunities presented by stem cells by removing the necessity of matching a recipient to an immunological match.

      The lung is a complex organ with up to 40 differentiated cell types in the cartilaginous airway (trachea), distal bronchioles, and gas exchanging airspace (alveoli). Lined with functionally distinct epithelium, the lung is thought to contain many different and unique types of epithelial progenitor and stem cells. The lung itself is characterized as a network of cells derived from the one of the three germ layers, the embryonic mesoderm and provides vascular structure for the gas exchanging system necessary to oxygenate our body’s blood supply (Sumner, 2005). The pulmonary airway and solid lung display mesenchymal cross talk during development. In essence the lung begins as a vascular bed, 10 days into development (Akeson, 2000). The first step towards a functional pulmonary system is the formation mesenchymal blood islands, sacs of endothelial cells and hematopoietic stem cells which form luminized connections and begin the budding and branching of the pulmonary system (Griffiths, 2005).  

Lung Stem Cell Therapy

      Given a growing interest in the characterization of tissue specific stem cells for regenerative therapy, attempts have been made to identify and categorize lung stem cells. Scientists believe that pulmonary stem cells may hold the key to curing a plethora of pulmonary diseases, ranging from hyper-proliferative cells disorders such as adenocarcinoma (which 35% of US cancer patients suffer from every year) to newly identified targets of the Severe Acute Respiratory Syndrome (SARS) as well as pulmonary hypertension and fibrosis- chronic pulmonary inflammation. Resident stem cells contribute to tissue regeneration and represent a target population that may be studied and manipulated to functionally restore injured pulmonary tissue (Majka, 2005). Scientists believe that these cells may function to replace pulmonary tissue, epithelium, mesenchyme or vasculature. In many of these diseases, such as adenocarcinoma, lung stem cells serve as a reservoir of pre-cancerous cells with the ability to proliferate ad infinitum (Majka, 2005). The ability to ablate, or completely wipe out, resident cells and then regenerate them following cancer therapy may save millions of lives across the world.

       SARS treatment is an excellent example of the advancements that could be offered by the identification and manipulation of lung-resident cells. Severe Acute Respiratory Syndrome is characterized by a high mortality rate induced by total lung failure and is an atypical form of pneumonia. Scientists have recently discovered that SARS is caused by a novel coronavirus - a group of viruses that have a halo or crown-like (corona) appearance when viewed under a microscope. Recent studies have shown that receptors expressed by stem cells resident in the lungs are primary targets of the SARS virus (Ling, 2006). Perhaps, by blocking these receptors through administration of receptor-blocking reagents doctors may be given the opportunity to prevent the lethal effects of this disease. 

Lung Stem Cells – Past and Present

      Unfortunately, over the past few years much of the forward advancement in lung stem cell research has been shadowed by controversy and the field has suffered much for it.  Since 2000, declarations of the characterization of lung stem cell progenitors have been published and met with much disdain as results have been neither repeatable by different laboratories nor shown with enough evidence to remove doubt. In addition, the anatomical complexity of the lung, its slow cell cycling period and the lack of adequate regenerative models are all among the issues that have made the study and isolation of adult lung stem cells very difficult (Kotton, 2004). Due to these scientific dilemmas, scientists now aim to design a rigorous method for identifying stem cells and characterizing them based on the previously designed models for stem cell research.

      Kotton et al. published a seminal paper in 2001 reporting the ability of bone-marrow cells to serve as precursors of lung alveolar epithelium. The report demonstrates the ability of cultured bone marrow cells to serve as precursors of lung cells in recipient mice. PCR analysis showed the engraftment of bone marrow with genetic markers in the host’s tissue as well as immuno-staining results for specific markers of lung stem cells (Kotton, 2001). In 2005, Kotton et al. published a second paper negating the results found in the former publication. The fact that Kotton reformed the concepts of his original paper is an incredibly important moment in lung stem cell biology. The mistakes learned from his experiments illustrate the problems currently inherent in this young field.

      As soon as Kotton’s publication was released in 2001, hopes for tissue repair flourished. Almost as immediately, his reports of hematopoietic stem cell (HSC) repair of endothelial layers was debated. Using, Fluorescence Activated Cell Sorting (FACS) analysis, no proof of bone marrow engraftment could be found. In fact, not a single report of HSC epithelial cell reconstitution could be identified (Kotton, 2005). Upon further microscopy, it became evident that signals originally asserted to represent engraftment events were actually auto fluorescent signals and that no real co-localization of markers for host and donor tissue had occurred.

      In a follow-up publication, Kotton concluded that immuno-fluorescence observations alone do not determine engraftment. Further results showed no donor derived cells in host tissue (Kotton, 2005).  Kotton’s team posits that experimentation should incorporate histology, FACS, and lineage markers in a concerted manner. Furthermore, Kotton surmises that due to the varied methods researchers in different laboratories utilize during experimentation, a standardized model for stem cell research must be designed. This will encourage uniformity in the field and bolster the veracity experimental results, such as immunochemistry results, as they will be able to be easily repeated. Using transgenic mice helps researchers ascertain the validity of their results by providing a controlled environment for experimentation (Kotton, 2004).  

Promising Discoveries in Lung Stem Cell Research

      While Kotton’s work may not have given the desired outcome, much research in the lung stem cell field is quite promising. A population of cells resident in the lung known as side population (SP) cells shows the potential to be enriched for epithelial precursors as well as some hematopoietic cells. Side population cells derive their name from their unique fluorescent appearance upon FACS analysis due to the presence of specific genes that cause the cells to efflux dyes (Summer, 2005). The efflux causes them to appear as a population of cells outside the main body of cells, or on the side of the main body. SPs comprise a very small fraction of cells in the lung and are found at all developmental points. These cells express many hematopoietic stem cells markers including GATA-1/2 and PU.1 as well as CD45, a common hematopoietic stem cell marker (Majka, 2005). While these cells do not show the ability to engraft all tissue, such as true hematopoietic stem cells, they do however show mesenchymal and epithelial regenerative properties. Sumner et al. report that these SPs tentatively appear capable of reconstitution of the bone marrow and have CD45+ marker (HSC activity)  and CD45- marker (endothelial and muscle cell activity). The CD45+ population is heterogeneous and the CD45+ Lin- cells re-populate bone marrow while the CD45- led to smooth muscle formation and the formation vascular structures (Sumner, 2005). Importantly, these cells express angiogenic cells markers (indicating that they can form blood vessels for growth) such as FLT-1 and the Tie-2 receptor (Sumner, 2005). These studies, however, are all in their earliest stages and while they show great promise, great precaution must be taken to ensure that the strictest steps are taken to validate all results.

      In addition to the ability of side populations to contribute to the bone marrow, reports of donor lung cells contributing to lympho-hematopoietic precursors (LHSCs) (our body’s immune system) have also recently appeared. Using green fluorescence protein (GFP) tagging, Majka et al. have shown that upon transplantation into the bone marrow, donor lung cells contribute to 30% of leukocytes (Majka, 2005). According to this study, lung cells were generated all components of the immune system, including T-cells, B-cells, granulocytes and macrophages. The presence of the CD45 correlated with a higher GFP expression in the blood. Additionally, in agreement with previously mentioned work, Majka’s group found side populations of lung cells capable of forming endothelial and epithelial progenitors but also report that only whole lung cells give rise to LHSCs.

      Akeson et al. report that ability of mouse fetal lung mesenchymal (MFLM) cells to produce endothelial precursors and respond to the microenvironment around them for activation queues (Akeson, 2000). Upon extraction of MFLMs from the fetal lung and host transplantation, Akeson proves using PCR and Southern Blot experiments that MFLM cell posses phenotypic and functional qualities distinct from embryonic source and are capable of chimerizing (forming) endothelial structures in-vivo. Characteristically, these cells express CD34+, a common endothelial cell marker and ACE - angiotensin converting enzyme, another classical hallmark marker of endothelial precursors. Further proof of their lineage from the mesenchyme is the presence of a mesenchyme marker, vinactin, and angiogenic markers such as the vascular endothelial growth factor receptor (VEGF) and TIE-2 (Akeson, 2000).

      Certain pulmonary progenitor cells have been identified and these cells may be important for lung tissue regeneration. In proximal coducting airways: Clara cells, basal cells have been shown to function as progenitors. (Kotton, 2004) Embryonic stem cells (ESCs), capable of differentiation into every part of the human body (totipotence) differentiate into Clara cells – the most characteristic airway epithelial cell. Clara cells express specific surfactant proteins which allow researchers to easily identify them upon experimentation. Coreaux et al. aimed to test the ability of murine ES cells to differentiate into airway epithelial cells and give rise to airway epithelium, composed of basal cells, ciliated cells, intermediated cells and nonciliated clara cells (Coraux, 2005).

      In their study, Coreaux et al. show the necessary substrates for Clara cell differentiation as well as the growth factors required. These cells can were able to generate a functional tracehobronchial surface epithelium similar to the native mouse epithelium. Morphologically, ES derived bioengineered epithelium resembles the native epithelium by Clara cells, that gives rise to mature Cilia. The similarities included the bioengineered surface formation of tight junction membranes to prevent particulates entering the interstitial spaces as well as the secretory function of the Clara cells, confirmed by staining for Cc10 protein, found in human broncheoalveolar lavages. They serve as progenitors for bronchioles as well and are characterized by the expression of Clara Cell Surfactant Protein (CCSP)(Reynolds, 2000).

      Clara cells have a major role in tissue regeneration of lung. Pulmonary Neuro Endocrine Bodies (PNEs) act as a progenitor for the establishment of hyperplasia (cell overgrowth) in hypoxia (oxygen deprived) exposed lung for lung repair.  Clara cells are associated with PNEs by association with the environment in which PNE hyperplasia takes place; regions known as NEBs – neuroendothelial bodies. (Reynolds 2000) The Clara cells associated with the NEBs are morphologically and functionally distinct from typical Clara cells and function to synthesize and secrete proteins in the ECM. Distinct pollutants cause this subset to proliferate and become associated with NEB hyperplasia and hypertrophy. Additionally, and importantly, they serve as multipotent progenitors for renewal of airway epithelia after oxidant injury. Morphologically, in their renewal phase, they express lower amounts of protein and synthesize less (Reynolds 2000) Naphthalene injury models suggest that there are Clara cells responsible for the regeneration of injured lung tissue. This specific subset of Clara cell is immune to naphthalene toxicity (Ling, 2006). These cells have been found experimentally in lung side populations: SPs isolated from the lung have been shown to have a very similar same molecular phenotype as the naphthalene-immune Clara Cell. Pulmonary stem cells have been identified as CD34+ SCA1+ CD45- PE CAM- cells - the classical Clara Cell marker composition - which express CCSP (clara cell secretion protein). All these facts point to a large and important role for Clara cells in tissue regeneration in the lung (Reynolds, 2000).

      The promises offered by the identification of pulmonary stem cells for regenerative medicine are clearly very realistic ones. As mentioned earlier, specific diseases have already been implicated in which lung stem cells may directly play a large role, including SARS, pulmonary fibrosis and various forms of lung cancers. With current advances in the identification of native regenerative models, as well as the identification of various lung stem cell progenitors and their ability to contribute to systems outside of the pulmonary system, researchers have an enormous amount of work lined up before them. While early attempts to characterize lung stem progenitors met with failure, the lessons learned from those mistakes have given scientists rigorous experimental methods by which they can continue their work in a manner that removes experimental doubt and ambivalence. It is the hope of the writer of this paper that the history of lung stem cell research continues to provide important lessons and that the enormous advancements already made ultimately lead to therapeutic models that will help all of mankind.  

References 

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Akeson A, Wetzel B, Thompson F, Brooks S, Paradis H, Gendron R, Greenberg J (2000) Embryonic vasculogenesis by endothelial precursor cells derived from lung mesenchyme. Dev Dyn. 217:11-23. 

Chang JC, Summer R, Sun X, Fitzsimmons K, Fine A (2005) Evidence that bone marrow cells do not contribute to the alveolar epithelium. Am J Respir Cell Mol Biol. 33:335-42. 

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Acknowledgements 

This review was prepared by the following graduate students in the Stem Cell Biology Class, Graduate School of Biomedical Sciences, University of Medicine and Dentistry of New Jersey: 

Tara Gooen, Joel Schneider, and James Sturzione (in alphabetical order) 

Teaching Assistant: Kathy Trzaska