Role of Stem Cells in the Pathogenesis of Chronic Obstructive Pulmonary Disease and Pulmonary Emphysema

Irene Coppolinoa , Paolo Ruggeria , Francesco Nuceraa , Mario Francesco Cannavoa , Ian Adcockb, Giuseppe Girbinoa , and Gaetano Caramoria

aDipartimento di Scienze Biomediche, Unita Operativa Complessa di Pneumologia, Odontoiatriche e delle Immagini Morfologiche e Funzionali (BIOMORF), Universita degli Studi di Messina, Messina, Italy;

bAirways Disease Section, National Heart and Lung Institute, Royal Brompton Hospital Biomedical Research Unit, Imperial College, London, UK

Author for correspondence:

Gaetano Caramori

Dipartimento di Scienze Biomediche,

Unita Operativa Complessa di Pneumologia, Odontoiatriche e delle Immagini Morfologiche e Funzionali (BIOMORF),

Universita degli Studi di Messina,

Messina 98122,

Italy.

Email: gcaramori@unime.it

KEYWORDS

Stem cell; COPD; pulmonary emphysema; fibrocytes; lung; endothelial progenitor cells; mesenchymal stem cells; adipose tissue-derived stem cells

ABSTRACT

There are only few human translational studies performed in the area of stem cell research in patients with chronic obstructive pulmonary disease (COPD) and/or pulmonary emphysema. Before progress to clinical trials with stem cells we strongly believe that more human translational studies are essential, otherwise, the clinical rationale would be solely based on limited in vitro and animal studies. In the future, stem cell therapy could be a treatment for this incurable disease. As of now, stem cell therapy is still to be considered as an area of active research, lacking any strong rationale for performing clinical trials in COPD. Although stem cells would be likely to represent a heterogeneous population of cells, the different cell subsets and their importance in the pathogenesis of the different clinical phenotypes need to be fully characterised before progressing to clinical trials. Moreover, the potential side effects of stem cell therapy are underestimated. We should not ignore that some of the most deadly neoplasms are arising from stem cells.

Introduction

Chronic obstructive pulmonary disease (COPD) is defined as a “preventable and treatable disease that is characterised by persistent respiratory symptoms and airflow limitation that is due to airway and/or alveolar abnormalities usually caused by significant exposure to noxious particles or gases” (1). COPD is currently a leading cause of morbidity and mortality worldwide affecting an estimated 174.5 million people in the world and accounting for more than three million deaths annually (2). From 1990 to 2015, the all-age prevalence of COPD increased by 44.2% and the age-standardised prevalence decreased by 14.7%. The mortality from COPD worldwide in 2015 increase of 11.6% compared with 1990, despite a decrease in the age-standardised death rate of 41.9% (2). The burden of COPD estimated in 63.9 million disability-adjusted life years (DALYs) represented 2.6% of entire global burden of disease (GBD) in 2015 and COPD ranked eighth among the 315 GBD causes in 2015. Age standardised DALY rates due to COPD decreased of 43.7% in 2015 compared to 1990, mostly in countries with high middle SDI (2). The burden of COPD is projected to increase in the coming decades due to continued exposure to COPD risk factors and the changing age structure of the world population (1).

The aetiology of COPD is due to complex interactions between environmental factors (particularly cigarette smoking) and genetic factors. Long-term cigarette smoking is currently the cause of more than 90% of COPD in Westernised countries, whereas other factors, such as burning biomass fuels for cooking and heating, may be important causes of COPD in developing countries (3). Only ~25% of chronic smokers develop symptomatic COPD by the age of 80 years, suggesting a genetic component, but the influence of single gene polymorphisms is weak and the only clearly established, albeit rare, genetic risk factor for COPD is 1-antitrypsin deficiency (1-AT). However, there are, so far, very few studies comparing cigarette-smoking associated COPD and other causes of disease, for this reason, our review of the literature will be limited to the cigarette-smoking associated COPD.

We review here the evidence for a role of stem cells in the pathogenesis of COPD and pulmonary emphysema, both during stable phase and exacerbations. We will also review the potential role of stem cells in the pathogenesis of complications of the COPD such as lung cancer and secondary pulmonary hypertension.

Stem cells have been identified in nearly all adult tissues, including human lungs (4) and are thought to contribute to tissue maintenance and repair. These are rare unspecialised cells that are often localised in specialised niches within tissue. Importantly, those cells exhibit self-renewal capacity and can evoke daughter progenitor or transit amplifying cells. A progenitor cell, like a stem cell, tends to differentiate into a specific type of cell. A stem cell, however, is already more specific and is pushed to differentiate into its target cell. Stem cells can replicate indefinitely, whereas progenitor cells can only divide a limited number of times. Both stem and progenitor cells may give rise to differentiated cells of the organ (5).

Stem cells do not only have the ability to self-renew but also give rise to subsequent generations with variable degrees of differentiation capacities. This offers significant potential for the generation of tissue that could potentially replace diseased and damaged areas in the human body.

As the lung is a complex organ composed of more than 40 different differentiated cell types, identification of endogenous progenitor cells has been challenging, and it seems clear that different progenitor cell populations are localised in different anatomic regions of the adult human lungs (see Figure 1 for an example of the lung epithelial stem cells).

Although symptomatic care for COPD has improved over the years, as of now no knowledge exists of any disease modifying drug applicable for the treatment of this disease and new options are desperately needed. As COPD is characterised by loss of lung tissue and remodelling of the airways, there is growing enthusiasm for using stem cells to regenerate alveolar tissue and remodel lower airways and thereby restore lung function in patients with COPD. Indeed, it could be argued that the destructive, inflammatory processes induced by smoking that ultimately lead to COPD, could result from a failure of the regenerative processes in the lungs. Thus, the homeodynamic process maintaining lung structure and function could be preserved by enhancing lung regeneration. Recent studies conducted in animals have revealed that human clonal lung stem cells may contribute to distal lung tissue regeneration (4), therefore, administration of stem cells deriving from exogenous sources may be an innovative way to treat COPD. The main question is whether we already know enough to start using stem cells to treat patients with COPD.

Role of stem cells in animal models

In animal models (proteases-treated and transgenic [tight-skin or Tsk] mice) of pulmonary emphysema, the transplantation of wild-type mouse bone marrow cells (bone marrow- derived stromal cells) into emphysematous mice decreases emphysematous lesions in chimeric mice (6–10). In one study, the complete reversal of emphysematous lesions was obtained and the transplanted lungs resembled those of age-controlled normal mice with non-significant engraftment of donor-derived cells in chimeric mice lungs (8).

Some, but not all, studies performed in animals and humans suggest that after transplantation bone marrow-derived cells can migrate to the lungs and create cellular chimerism in the lower airways’ epithelium. However, the number of these cells is very low (11–19). Interestingly in animal models of pulmonary emphysema, there is little or no engraftment of donor-derived cells in chimeric mice lungs (6–10). These data suggest that adult bone-marrow-derived stromal mesenchymal stem cells (MSCs) and/or bone marrow mononuclear cells can migrate to injured areas in lungs, regenerating the pulmonary parenchyma and repairing pulmonary emphysema (5, 20). However, the mechanisms con- trolling this process are unknown and no data is available yet on the effects of bone-marrow-derived and other stem cells in animal models of COPD.

Human translational studies in stem cell research in COPD and pulmonary emphysema

Role of the basal stem cells of the lower airways

The human bronchial basal cells express in vivo on their surface CD44 (cell-surface glycoprotein) (21), CD49f (integrin subunit alpha 6 or ITGA6) (22), CD151 (tetraspanin, a member of the transmembrane 4 superfamily), CD166 (activated leukocyte-cell adhesion molecule [ALCAM]) a surface marker of all the epithelial bronchial cells and of the epithelium of the ducts of the bronchial glands (23), the nerve growth factor receptor (NGFR) (24), the epidermal growth factor receptor (EGFR, a tyrosine kinase receptor that is enriched in human airway basal cells and is implicated in regulation of tissue repair) (25), and in their cytosol the cytoskeletal cytokeratin (CK) CK5 (22), CK14 (26, 22) and CK17 (27), tissue-factor (TF) and in their cytosol and/or nucleus the protein Trp-63 (p63) (24). The p63 is a transcription factor of the p53 family. The human p63 gene encodes at least eight different isoforms that are generated by transcription from two different promoters in combination with alternative splicing: there are transactivating isoforms (TAp63) that contain an NH2-terminal activation domain (TAD), and truncated isoforms (Np63) lacking the TAD. TAp63 and DNp63 share a DNA binding domain and an oligomerisation domain. Only TAp63 and Np63 share a sterile alpha domain and a transactivation inhibitory domain at carboxy-terminal region (28, 29). The transactivating isoforms may have functions like p53 in inducing cell-cycle arrest and apoptosis, whereas Np63 can act inhibiting the activity of p53. The basal cells of human bronchi and bronchioli constitutively express in their nuclei high levels of p63, and the Np63 isoforms are the main isoforms expressed (30, 31). It has been hypothesised that the main physiological role of p63 is to antagonise p53 functions to ensure the survival and renewal of basal cells by contrasting apoptosis and cell cycle arrest (30). Indeed, in the p63-null mice, the tracheal epithelium is devoid of basal cells (28) and, in vitro, knockdown of p63 in the human bronchial basal cells reduces significantly their proliferation and migration with impaired capacity to generate a pseudostratified-like bronchial epithelium (31).

Functionally, the basal cells of the human tracheobronchial epithelium are considered stem cells because both in vivo (in xenografts) and in vitro are able to reconstitute a fully differentiated mucociliary epithelium. For example, the human tracheal basal cells (CD44+/CK5+/p63+) (obtained from healthy tissue of lung transplants of three donors non- smokers) xenotransplanted in the denuded rat trachea after five weeks gave rise to morphologically well-differentiated epithelia and occasionally submucosal glands (21). Likewise, the transplantation of human bronchial basal cells in trachea and intrapulmonary airways of mice post-naphthalene injury gave rise to completely differentiated epithelium (32).

In vitro, both the basal cells (CD44+/CD166+/NGFR+) of the human tracheal epithelium and of the ducts cells of tracheal submucosal glands (CD166+) have the same efficiency in self-renew and differentiation (23). In vitro, a sub-set of basal cells CD151-/CK5+/CK14+/TF+ has a high mitotic index (22). Again, in vitro human bronchial basal cells (CD49f+/NGFR+/p63+) are capable of self-renewal and to generate ciliated and secretory cells (24).

In vitro the epidermal growth factor (EGF) stimulating the its receptors (EGFRs) on the bronchial basal cells (obtained from bronchial [third and fourth order] brushings of non-smokers and smokers) changes their differentiation with expression of squamous cell markers (CK14, CK6, involucrin) and mesenchymal cell markers, like vimentin, and impairs their ability to generate the junctional apical complex, necessary for epithelial permeability (25). Interestingly, tobacco smoking increases EGF expression in ciliated cells of the bronchial epithelium (25).

The human basal cells (CK5+), isolated by brushings in third and fourth order of bronchial generation from non-smokers, after exposure to cigarette smoke extract (CSE) in vitro have an increased expression of amphiregulin (AREG, an EGF-like growth factor), an increased proliferation and expression of Ki-67 (marker of the proliferating cells), and of the major secretory mucins (MUCs) MUC5AC, and MUC5B, that is prevented with a selective inhibitor for EGFR. The same basal cells, treated with AREG, have decreased expression of tight junctions and of the genes implicated in the ciliogenesis (33).

In vitro, human basal cells (CK5+/p63+), obtained from bronchial (third and fourth order) brushings of non-smokers and COPD patients overexpressing the trophoblast cell sur- face antigen 2 (TROP2) have an increased proliferation rate (34). TROP2 is a transmembrane glycoprotein highly expressed in many cancers, it has stem cell-like qualities because provides signals for cellular survival and proliferation (35).

The treatment in vitro of the human basal cells (CK5+) of bronchiolar epithelium (10th–12th generations), obtained by brushing from healthy non-smokers, with EGF or with CSE, up-regulates genes normally expressed in the large air- ways, as uroplakin 1B (UPK1B), transcobalamin 1 (TCN1), interleukin 1 receptor type 2 (IL1R2) whereas down-regu- lates the expression of genes typical of the small airways, like secretoglobin family 3A member 2 (SCGB3A2), lacto- transferrin (LTF), matrix Gla protein (MGP), folate receptor 1 (FOLR1), mimicking the phenotype observed in the bronchiolar epithelium of smokers (36).

In vitro, the basal cells (CK5+/CK14+/p63+) obtained from bronchial biopsies of stable COPD patients produce significantly less clone-forming cells and ciliated cells (acetylated tubulin ACT+), but more basal cells (CK5+) and MUC5B+ cells compared with control subjects (37).

Likewise, in vitro the basal stem cells (CD151+/CK5+/p63+), obtained by brushing of the small airway epithelium (from 10th to 12th bronchial order) from COPD patients, have reduced capacity to successfully differentiate in a normal mucociliary epithelium compared to smokers with normal lung function and to non-smokers: at the 28th day of culture, the number of secretory cells (secretoglobin 1A1, SCGB1A1+) was significantly lower in the cultures derived from basal cells of COPD patients compared with nonsmokers samples, whereas were found similar numbers of ciliated cells (-tubulin IV+) (38).

In vivo, it has compared the gene expression of basal cells (CD151+/CK5+/p63+) obtained from brushing of large airways from lifelong non-smoking subjects and smokers with normal lung function: 676 genes have been identified differentially expressed in bronchial basal cells with 662 genes significantly up-regulated in smokers with normal lung function (13 of these 662 genes are localised in chromosome 19q13.2). Among the most up-regulated were NF-B inhibitor beta (NFKBIB), egl-9 family hypoxia-inducible factor 2 (EGLN2), latent transforming growth factor beta binding protein 4 (LTBP4), and transforming growth factor beta 1 (TGFB1) (39).

Another study has compared the gene expression of basal cells obtained by bronchial (up to the sixth bronchial generation) vs. small airways (from 10th to12th bronchial generation) brushings of stable COPD and control subjects, both smokers with normal lung function and non-smokers. The small airway epithelium in smokers with normal lung function, and more in smokers with COPD, showed down-regulation of bronchiolar genes, as LTF, SCGB3A2, MGP, FOLR1, SRY-box 9 (SOX1), GATA binding protein 6 (GATA6), and others, and up-regulation of uroplakin 1B (UPK1B), carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), transcobalamin 1 (TCN1), and interleukin 1 receptor type 2 (IL1R2), leading to shift towards the transcriptomic pattern normally displayed in the epithelium of large bronchi (33).

In vivo, AREG was up-regulated in the basal cells CK6+(cytokeratin 6, expressed in squamous differentiation) obtained from bronchial epithelium (third and fourth order) by both smokers with normal lung function and COPD subjects (33).

It was shown that the expression of TROP2 in airway basal cells (CK5+/p63+), obtained in lung samples by lobectomy or pneumonectomy performed for medical reasons, was significantly increased in COPD patients and in smokers with normal lung function compared to lifelong non-smokers. The expression of TROP2 was significantly correlated with the expression of Ki-67+ in epithelium from COPDs (34).

The number of bronchial basal cells (CK5+/CK14+/p63+) obtained by endobronchial biopsies at the secondary bronchus, was significantly decreased in COPD patients versus control subjects. It has been identified a subset of non-COPD subjects, former and current smokers, with lower basal stem cells count and lower airflows, that may represent an early, pre-diagnostic stage of COPD, indicating that basal stem cells exhaustion is involved in COPD pathogenesis (37).

Role of the submucosal bronchial gland stem cells

The duct cells of the human submucosal bronchial glands express CK5, CK14, p63, and -smooth muscle actin (-SMA) (40). Human bronchial epithelial cells xenotransplanted in denuded rat trachea produce many cell clones with different differentiation capacity including glandular cells (41).

Despite duct glandular stem cells (CD166+/CD44-/NGFR-) have been isolated from human trachea (23) and similar cells (p63+, CK5+, CK14+) have been obtained from autopsy lung specimens of patients with obliterative bronchiolitis due to lung transplantation (42), there are no studies about these cells in patients with COPD and/or pulmonary emphysema.

Role of the bronchioalveolar stem cells

The murine bronchioalveolar stem cells (BASCs) express the alveolar type 2 cell marker prosurfactant protein C (SP-C), the club cell marker secretoglobin family 1A member 1 (SCGB1A1), the stem cells antigen-1 (Sca-1), and CD34 (43). Other authors have identified these murine BASCs as Sca-1+/CD45.2-/CD31- - (platelet and endothelial cell adhesion molecule 1, also known as PECAM1, normally found on endothelial cells, platelets, macrophages, granulocytes, and lymphocytes) (44).

Murine BASCs in vitro have capacity of multilineage differentiation and self-renewal (43). In mouse lungs these cells are found exclusively at the bronchioalveolar ductal junction, are quiescent in the normal lungs and proliferate in response to lung injury (45).

The human BASCs are also cadherin-1+ (CAM 120/80 or epithelial cadherin [E-cadherin] or uvomorulin), SPC+ and vimentin+ (46). In vitro when these cells are co-cultivated with human lung fibroblasts produce bronchioalveolar structures (46).

The human lungs show stem cells in niches in the distal airways with capacity in vivo, transplanted in damaged lung of mouse, of self-renew, and differentiation in structures of endodermal origin (epithelial cells) and mesodermal origin (vessels), generating human bronchioles, alveoli, vessels, and partially restoring the recipient parenchyma. These cells are CD117+ (proto-oncogene c-kit or tyrosine-protein kinase kit or mast/stem cell growth factor receptor [SCFR]; a receptor tyrosine kinase protein) but negative for haematopoietic cell lineage (CD34, CD45, CD133 [prominin 1, a transmembrane glycoprotein of haematopoietic cells]), mesenchymal cell antigen (CD44, CD90 [previously termed Thy-1 cell surface antigen, because expressed in T cells], CD105 [endoglin, expressed in human vascular endothelium, in bone marrow proerythroblasts and in activated monocytes]), mast-cell lineage (CD6, CD29 [integrin subunit beta 1], CD49) and epithelial cells lineage (p63, pan-CK, SP-C) markers (4). Other authors have found that these CD117+ stem cells in the early developing human lungs also are CD34+, VEGFR-2+ (vascular endothelial growth factor receptor 2, also termed KDR) and Tie-2+ (a transmembrane tyrosine-kinase receptor of the angiopoietins), indicating their endothelial lineage (47).

Recently, in human lungs obtained from subjects ongoing lung surgery for cancer or nodule have been isolated many different subsets of CD117+ cells: the mast cells (CD117high/ CD45+/tryptase+ [a serine protease produced purely in the mast cells]) are mainly located around the peribronchial interstitium and are the majority (85%) of CD117+ cells; the innate lymphoid cells (CD117low/CD45+/tryptase-) that represent the 13% of c-kit cells; the endothelial stem cells (CD117low/CD45-/CD31+/CD34+/tryptase-) located in the alveolar walls, represent the 1.6% of c-kit population; the resident stem cells (CD117low/CD45-/CD31-/CD34-/tryptase-) that represent less than 0.1% of the total cells (48). When stable COPD patients were compared with smokers with normal lung function and non-smoking subjects no significant differences of these cells were observed between these three group of subjects (48).

There are no published studies about the human BASCs in patients with pulmonary emphysema.

Role of the alveolar stem cells

In mouse models, the subset of alveolar epithelial type 2 cells (AT2) axin-2+ (a protein regulating the stability of beta-catenin in the Wnt signalling pathway [regulates selfrenewal and lineage-specific differentiation in stem cells]) (49) show features of stem cells (50–52) including self renewal capacity and the ability to differentiate to alveolar type 1 cells (AT1 cells) (51). After H1N1 influenza virus injury of mice lungs, there is a large increase of the number of AT2 and AT1 cells with alveolar regeneration (52).

The murine alveolar stem cells (AT2) are also epithelial cell adhesion molecule (EpCAM)+, SP-C+, CD45-, podoplanin- (a small membrane glycoprotein that belongs to mucin-type proteins, required for lung development, specifically for maturation of alveolar type I epithelial cells). After knock-down the enzyme telomerase in mice, the AT2 cells decreased in association with decreased alveolar numbers (53).

Human lungs have resident alveolar stem cells that express markers of human mesenchymal stem cells (hMSCs) (CD73+ [5’-nucleotidase ecto, expressed in lymphocytes B and T]/CD90/CD105+/vimentin+), surfactant proteins (SPA, SP-C, SP-D) of the AT2 cells, but are negative for CD117 and for haematopoietic or endothelial stem cells markers (CD31, CD34, CD45, VEGFR-2) and, in vitro, have clonogenic ability and can differentiate in AT2 cells. In normal human lung tissue, these SP-C+/CD90+ cells are located exclusively in the alveolar walls (54). These human AT2 stem cells are also transmembrane-4-L-six-family-1 (TM4SF1)+ (a member of the transmembrane 4 superfamily, also known as tetraspanin family, that regulates cell movement), anti-AT2+ specific HTII-280 antibody, EpCAM+ and represent ~29% of the human AT2 total population (52).

Human alveolar epithelial stem cells (CD90þ, prosurfactant protein C [pro-SP-C]) exposed in vitro to all-trans retinoic acid, differentiate in AT1 (aquaporin-5þ[AQP-5]) and AT2 cells (SP-A+) (55). The same result was obtained stimulating the human alveolar stem cells (CD90+/pro-SPC+) with Am80 (a synthetic retinoid) (56).

It was found that phosphoinositide-3-kinase (PI3K)-protein kinase B (Akt) pathway (pathway that promotes cellular survival in response to growth factors) is involved in the mechanism of the human alveolar stem cells differentiation. During in vitro treatment with all-trans retinoic acid of alveolar stem cells, which leads to their differentiation, Akt is not phosphorylated (55). PI3K-inhibitors (e.g. Wortmannin) that block the phosphorylation of Akt, induces the differentiation of these alveolar epithelial stem cells in AQP-5+ (AT1) and SP-A+ (AT2) cells. In addition, in a mouse model of elastase-induced pulmonary emphysema, lung Akt phosphorylation is enhanced and PI3K-inhibitors lead to differentiation of the alveolar stem cells (57).

Role of the circulating blood pluripotent haematopoietic stem cells

A decreased number of circulating CD34þ stem cells has been found in the blood of patients with moderate to severe stable COPD, which correlates with hypoxaemia, severity of airflow obstruction and peak oxygen uptake, and low body mass index (BMI) (58–60). However, these studies used non-smoking subjects as controls and not, as in the ideal situation, age-matched smokers with normal lung function (58–60).

Another study showed that circulating CD34+ stem cells were significantly decreased both in stable COPD patients and during COPD exacerbations compared to control smokers with normal lung function, whereas no significant difference was found between stable and exacerbated COPD patients. In addition, the number of CD34+ cells during COPD exacerbations was inversely correlated with the pulmonary artery systolic pressure (PAPs), the N-terminal pro-B-type natriuretic peptide serum levels, and with resting heart rate, and positively correlated with the left ventricular ejection fraction (61).

Stable COPD patients have a decreased number of circulating haematopoietic progenitor cells (HPCs) (defined as CD34+/CD45+/CD133+ or CD34+/CD45+ cells) compared to both smokers with normal lung function and non-smoking control subjects. Whereas were not significant differences between the control non-smokers and smokers with normal lung function, suggesting that the number of these cells is independent of the smoking habit. In addition, COPD patients with PAPs >31mmHg have greater number of CD34+/CD45+/CD133+ cells compared to those with PAPs >31mmHg (62).

Role of lung mesenchymal stem cells and bone marrow mesenchymal stem cells

The Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy has provided three criteria to identify the MSCs in vitro: adherence to plastic when maintained in standard culture conditions, expression of CD73, CD90, CD105, and lack of CD11b, CD14, CD19, CD34, CD45, HLA-DR, and the differentiation capacity into osteoblasts, chondroblasts, and adipocytes (63).

In vivo, hMSCs are found at low frequency and are defined by their capacity to differentiate into bone, cartilage, and adipose tissue, depending on the stimuli and culture conditions under which they are expanded (64). In addition, under selective conditions of growth may also differentiate in myogenic cells (65). Lung tissue (or residents) MSCs (LTMSC) express CD44, CD73, CD90, CD105, CD146 (melanoma cell adhesion molecule) and NG2 (chondroitin sulphate proteoglycan 4), and lack the expression of haematopoietic, endothelial and monocyte markers (CD45, CD34, CD14) (66). MSCs can be isolated from the bronchoalveolar lavage fluid (and are termed BAL-MSC) of early (within the first 12 post-operative months) lung transplant recipients, whereas are rarely recoverable in the BAL of healthy subjects. The immunophenotype of these cells is similar to those of the LT-MSC (CD73+/CD90+/CD105+/CD45-), but they have lower differentiation capacity (in particular lack adipogenic differentiation), increased expression of COL1 and -SMA and express 76 more mRNAs, including those involved in fibroblast activation (67). In vitro, transforming growth factor beta 1 (TGF-b1) stimulation of the BAL-MSC increases their expression of -SMA and COL1 (68). These findings suggest that LT-MSC could acquire a profibrotic phenotype in pathological conditions (67). These functional properties explain why lung recipients who develop a bronchiolitis obliterans syndrome (BOS) have an increased number of BAL-MSC and that the increase of these cells is a predictor of the future development of BOS (69).

The bone-marrow MSCs (BM-MSC) are isolated for their expression of CD29, CD44, CD73, CD90, CD105, and HLAABC, and the absence of CD31, CD34, CD45, HLA-DR (70). During multiple passages in vitro these MSC exhibit various surface receptors with different expression profiles, including C-X-C motif chemokine receptor 3 (CXCR3, also known as CD183), C-X-C motif chemokine receptor 4 (CXCR4, also known as CD184), C-X-C motif chemokine receptor 6 (CXCR6, also known as CD186), C-C motif chemokine receptor 9 (CCR9, also known as CD199), and C-C motif chemokine receptor 10 (CCR10), which may be involved in the migration of the MSC to the damaged tissues (70). The most studied chemokine-chemokine receptor axis in MSC is C-X-C motif chemokine 12 (CXCL12) (known as stromal cell-derived factor 1 (SDF1), a potent chemoattractant from bone marrow of the endothelial progenitor cells [EPCs] and mesenchymal cells)-CXCR4. But MSC also express adhesion molecules such as C-C motif chemokine ligand 19 (CCL19) and C-C motif chemokine ligand 21 (CCL21), that are specific ligands of C-C motif chemokine receptor 7 (also known as CCR7 or CD197, expressed in B- and T-lymphocyte cell), and may also involve in the migration of MSC (70, 71).

In vitro, human lung-MSC have functional properties similar to BM-MSC: including capacity of self-renewal and to differentiate into multiple mesodermal lineages: osteocytes, chondrocytes, adipocytes, and myofibroblasts. Also, these cells have the same inhibitory effect on T cell proliferation and this may contribute to a local anti-inflammatory capacity (49, 66, 72). Human lung-MSC are considered important regulators of tissue repair following injury (49), through production of cytokines and growth factors such as fibroblast growth factor-10 and hepatocyte growth factor (HGF) (46). Human BM-MSC (CD29+/CD44+/CD90+/CD105+), isolated from bone marrow aspirates from the iliac crest of a 40 years old male healthy volunteer, in co-culture with human endothelial cells, produce VEGF, inhibiting the apoptosis of the endothelial cells (73).

In mouse models of elastase-induced pulmonary emphysema, there is a significant increase of number of lung-MSC (CD44+/CD73+/CD90+) (74). In animal models of pulmonary emphysema, lung-MSC or BM-MSC administrated intravenous or intratracheal, contribute to repair of the lung damage increasing the levels of epidermal growth factor (EGF), HGF, VEGF, reducing airway inflammation through the down-regulation of cyclooxygenase-2 and the inhibition of release of proteases by inflammatory cells, induce the proliferation of AT1 and AT2 cells and improve lung function (72, 75–79).

The BM-MSC (CD73+/CD90+/CD105+/CD14-/CD34-/CD45-) obtained from stable COPD patients have reduced levels of CXCR4 mRNA, of its specific ligand CXCL12, of CCL19 and CCL21 compared to control smokers with normal lung function. These findings suggest that in stable COPD there could be an impaired migration capacity of the stem cells from the bone marrow (80). The human BM-MSC obtained from patients with stable COPD and pulmonary emphysema compared to control non-smoking subjects have similar immunophenotype (CD31+/CD34+/CD45+/CD73+/CD80+/CD90+/CD105+ HLA- DR+), proliferation, differentiation, migration capacity, but the BM-MSC from pulmonary emphysema patients have a significantly higher potential to differentiate in adipocytes than the BM-MSC isolated from the control subjects (81).

The human lung fibroblasts, isolated from the lung tissue of stable COPD patients undergoing surgery for carcinoma, express the LT-MSC-like markers CD44, CD90, CD105, CD166, HLA class I, but not CD34, CD45, CD133, VEGFR-2 and in vitro, after stimulation with TGF-1, have decreased differentiation capacity in myofibroblasts (these cells express the a-smooth muscle actin) compared to smokers with normal lung function and non-smoking controls (82). The human lung fibroblasts isolated from non-smoking controls, cultivated in vitro with osteoblastic, chondrogenic, and adipogenic culture media, differentiate into osteocytes (expressing alkaline phosphatase and osteocalcin), chondrocytes (expressing SRY-box 9 [SOX9], a transcription factor essential for cartilage development) (83), and adipocytes (expressing the nuclear transcription factor peroxisome proliferator-activated receptor gamma [PPAR-c], that regulate adipocytes differentiation (84) and the enzyme lipoprotein lipase). The capacity of these human lung fibroblasts to differentiate into osteocytes is almost absent in COPD patients and is significantly decreased in control smokers with normal lung function compared to non-smoking subjects, whereas no differences were observed between these 3 groups in their differentiation capacity into chondrocytes and adipocytes (82).

Role of adipose-derived stromal stem cells

Adipose tissue-derived MSC (ADMSC), as BM-MSC, are mesoderm-derived cells able to differentiate into multilineage connective tissue (including osteogenic, chondrogenic, adipogenic, and myogenic cells) but adipose tissue contains a much higher number of MSCs than bone marrow (85, 86), in fact, while BM-MSC constitute 0.0001–0.01% of all BM nucleated cells, the adipose tissue contains 100.000 MSCs/g of fat (87).

In vitro, human ADMSCs, as BM-MSCs, express CD29, CD44, CD73, CD90, CD105, and lack CD14, CD34, CD45, CD133, whereas vascular cell adhesion molecule 1 (VCAM-1 or CD106) is more expressed in BM-MSC (88). However, other authors have reported that 50% of ADMSC express CD34 and almost all express CD49e (a5 integrin) (89). In addition, ADMSCs have higher proliferative capability (88), retain differentiation potential for a longer period in culture and have increased immunomodulating capacity compared to BM-MSCs (90, 91), secreting various immunomodulating molecules including HGF, VEGF, and interleukin-10 (IL-10) (85).

Human ADMSCs, as murine ADMSCs, administered to mice with elastase-induced pulmonary emphysema, accumulate in their lungs for long time, express growth factors such as fibroblast growth factor-2 (FGF2) and HGF, reduce matrix metalloproteases secretion and have decreased emphysema (92–94). Human ADMSCs, pre-treated in vitro with pioglitazone (a PPAR- agonist) and infused intravenously in mice with elastase-induced and smoke-induced pulmonary emphysema improve alveolar regeneration and increase lung tissue levels of FGF2, HGF, and VEGF compared with the same untreated cells (95).

In an animal model of pulmonary emphysema induced by the treatment of mice with a VEGFR neutralising antibody intravenous infusion of the human ADMSCs attenuates cell death (measured by IHC quantification of active caspase-3) suggesting that in the lung human ADMSCs have an anti-apoptotic role (96).

Role of circulating endothelial stem cells

Bone-marrow-derived EPCs, capable in vitro cultures of transforming into mature, functional endothelial cells, can be isolated from peripheral blood mononuclear cells (97–99). These cells express on their surface both the endothelial cell markers CD31 and CD34 (haematopoietic progenitor cell antigen), but lack the expression of CD14 (monocyte differentiation antigen), CD41a (integrin alpha chain 2b, mainly expressed on megakaryocyte-platelet lineage), CD45 (protein tyrosine phosphatase receptor type C, originally called leukocyte common antigen [LCA]), CD133 (prominin 1 or AC133; mainly expressed haematopoietic stem cells and cancer cells), CD235a (glycophorin A;expressed on erythrocytes and erythroid precursors) (100, 101). However, this field remains controversial, because other authors, using flow cytometry, have found that also the CD31+/CD34+/CD45dim/CD133+/CD14-/CD41a-/CD235a-cells have angiogenic properties in vitro (102). To further confuse the field other studies have defined, the circulating EPCs as CD34+/CD45dim/VEGFR-2+ cells (103). These CD34+/CD133+/VEGFR-2+þ cells are also expressing other endothelial cell markers such as the von Willebrand factor (vWF) and the endothelial nitric oxide synthase (NOS3) (104).

In vitro, the human endothelial colony forming units (CFUs) cells usually express CD31, CD144 (also termed vascular endothelial-cadherin [VE-cadherin] or cadherin 5, type 2) VEGFR-2 and vWF, but lack the expression of CD3 (a pan-lymphocytic marker) and CD45 (105). These cells also express CD184 (CXCR4) (106).

Circulating EPCs seem to serve as a reserve pool of cells to replace dysfunctional/damaged endothelium in mature blood vessels (99) and reduced levels of circulating EPCs are associated with increased risk of death in patients with stable coronary artery disease (107). Furthermore, the subjects with higher Framingham risk score have lower endothelial CFUs (108). The patients with heart failure have lower number of circulating CD34+/VEGFR-2+ cells and a lower count of these cells is an independent predictor of increased mortality (109).

Currently, the contribution of the EPCs to the pathogenesis of COPD has not been fully understood because the studies did not use the same phenotypic definition of these cells (100). In addition, many drugs such as statins (110) and systemic glucocorticoids (111), hypoxaemia (112) and comorbidities (systemic arterial hypertension, diabetes mellitus, increased age, smoking, and plasma triglycerides) may influence the number and the function of circulating EPCs (113) and not all the studies reviewed below have provided all these details.

Previous studies have suggested a role for an increased apoptosis of the endothelial cells in the pulmonary capillaries of the alveolar septa in the pathogenesis of human pulmonary emphysema (114, 115). In addition, evidence for autoimmune-induced destruction of endothelial cells has been reported in the lung of COPD patients (116) providing a further rationale that endothelial cell loss is a driver of pulmonary emphysema. In animal models, circulating endothelial stem cells, characterised by the concomitant expression of CD34, CD133, and VEGFR-2, may contribute to the repair of lung damage (19). Thus, one can hypothesise that a decrease in the blood of EPCs may contribute to the pathogenesis of pulmonary emphysema and/or COPD in humans. Indeed, some studies (58, 60), but not all (117), have found a significant reduction of the number of circulating EPCs in patients with stable COPD. Conversely, this suggests that increased blood EPCs may prevent pulmonary emphysema.

Again, the number of CD34+/VEGFR2+ cells and their proliferative ability in vitro, were significantly decreased in stable COPD patients with PAH (>25 mmHg) compared to both COPD patients non-PAH and control subjects, whereas there were not significant differences between the COPD non-PAH patients and control subjects. Also, the number of these cells was negatively correlated with the pulmonary artery pressure, suggesting that the number of EPCs could be related to involvement of pulmonary arteries. All the subjects had no cardiovascular disease, diabetes mellitus, and did not use statins, whereas in COPD patients the levels of triglycerides and arterial hypertension were significantly higher than those in non-COPD subjects (118).

We have investigated by flow cytometry the number of total (CD34+) and endothelial stem (triple positive for CD34/CD133/VEGFR-2) cells in the peripheral venous blood of age-matched smokers with or without pulmonary emphysema and with or without COPD, all the subjects were in stable phase (119).

The presence and the severity of pulmonary emphysema were determined using high resolution computed tomography (HRCT) scans of the chest with density mask and the National Emphysema Treatment Trial (NETT) Research Group score (0–4). We found a significant correlation between the absolute number of circulating CD34+ cells and the absolute number of circulating endothelial stem cells. Also, there was a significant correlation between the percentage of circulating endothelial stem cells and the number of pack-years smoked, however, no significant correlation was found between total and endothelial stem cells number and HRCT score of pulmonary emphysema or lung function data. These data indicate that the number of circulating endothelial stem cells is not related to the presence and/or severity of the pulmonary emphysema or the presence or absence of COPD in stable phase (119).

Using immunohistochemistry another study has demonstrated an increased number of CD133+ cells in the pulmonary arteries of patients with mild to moderate COPD compared with a small number of smokers with normal lung function (120). CD133+ cells are localised to the endothelial surface and in the vessel wall and their number in the intima shows a direct relationship with the pulmonary artery wall thickness (120), indicating their potential for a role in the pathogenesis of pulmonary artery remodelling and pulmonary hypertension, however, more research is mandatory in this area before we can reach firm conclusions.

Another study showed that the number of CD34+/CD133+/VEGFR-2+ cells and of CD34+/VEGFR-2+ cells from stable COPD patients and from control subjects was similar, and that, in vitro, the endothelial CFUs was increased in COPD patients compared to controls. There were no differences between COPD patients and control subjects in cardiovascular comorbidities (121).

Again, the number of circulating CD34+/CD45dim/VEGFR-2+ cells and CD34+/CD45dim cells obtained from stable COPD patients was similar to that of control subjects; instead, the number of CD34+/CD45+/CD133+/VEGFR-2+ cells was significantly reduced in COPD patients compared to control subjects and was correlated with FEV1. The number of these cells was also significantly reduced in patients with percentage of emphysema >26, whereas there was no correlation between the levels of CD34+/CD45dim/VEGFR2+ cells and percentage of emphysema. However, both COPD patients and control subjects used statins (122).

Circulating CD34+/VEGFR2+ cells and, in particular CD34+/CD133+/VEGFR2+ cells are reduced in stable severe COPD patients compared to controls and the number of CD34+/CD133+/VEGFR2+ cells, but not of CD34+/VEGFR2+ cells, was significantly and inversely correlated with the severity of the emphysema. These results were adjusted for confounders such as smoking status, diabetes mellitus, arterial hypertension, BMI, and statin use (123).

The dynamic of blood CD34+/CD133+/VEGFR2+ cells had been measured in COPD patients and control subjects during thoracic surgery for lung cancer. The number of these cells in the blood is not affected by thoracic surgery in stable COPD patients. Whereas in control subjects their number increases 2 hours after starting surgery and returns to the preoperative levels 24 hours later. In addition, in vitro, the endothelial CFUs cells from control subjects increased 2 hours after the operation started, whereas that from COPD patients unchanged (124).

The blood CD34+/CD133+/VEGFR2+ cells of stable COPD patients show, in vitro, a reduced proliferation capacity and significantly impaired migratory capacity in response to CXCR12 compared to those isolated from non-smoking control subjects (125). Likewise, circulating CD34+/CD133+/VEGFR2+ cells are decreased, have impaired proliferative capacity and express lower levels of NOS3 in stable COPD patients compared to controls non-smoking subjects. However, there were subjects with systemic arterial hypertension and with coronary heart disease and other comorbidities and statin use were not mentioned (104).

Others found a lower number of circulating CD34+/CD45dim/CD133+ cells from stable COPD patients compared to control subjects and showed an increase of these cells in sputum of COPD patients, suggesting that the lowering of these cells in the blood could be due to an increased recruitment in the lungs. However, 25% of COPD patients had arterial hypertension and hyperlipidaemia, the 17% used statins and one had cardiovascular disease, whereas the control subjects had not these comorbidities (126).

Another study shows that blood CD34+/CD31+/CD133+/CD144- cells, of patients with stable COPD with pulmonary emphysema differentiate, in vitro, in significantly fewer endothelial CFUs cells compared to those obtained from the blood of COPD patients without emphysema, smokers with normal lung function and non-smoking controls. A significant inverse correlation between the number of endothelial CFUs cells and the degree of pulmonary emphysema was observed. Moreover, the EPCs from both group of COPD patients (emphysema and non-emphysema) showed significantly lower migration after human recombinant VEGF stimulation compared with those obtained from non-smoking controls. No patient was receiving statins, but there are subjects with systemic arterial hypertension and diabetes mellitus included in the study (127).

The endothelial CFUs from blood samples of stable COPD patients and of smokers with normal lung function show, in vitro, a slower growth rate and display significantly increased senescence-associated -galactosidase activity, increased expression of p21 and p16 and decreased expression of SIRT1 (sirtuin-1, an important regulator of genomic stability and inhibitor of endothelial senescence) compared to controls non-smokers. Only one COPD patient had diabetes mellitus and one arterial hypertension, whereas in all groups statins were used (105).

Role of circulating fibrocytes

Fibrocytes are blood stem cells which originate in the bone marrow and express CD11b (integrin subunit alpha M; expressed on the surface of monocytes/macrophages, granulocytes, activated lymphocytes, a subset of NK cells, a subset of dendritic cells), CD13 (alanyl aminopeptidase membrane, expressed on granulocytes, endothelial cells, epithelial cells, macrophages), CD14, CD34, CD45, CXCR4, and major histocompatibility complex class II, but lack the expression of lymphocyte markers such as CD3, CD4, CD8, CD19, and CD25; they secrete the type 1 and 3 collagens, fibronectin, and vimentin (128). A large proportion of the fibrocytes also express CCR2 (also known as CD192, C-C motif chemokine receptor 2; produced by endothelial cells, smooth muscle cells, and macrophages), CCR3 (also known as CD193, C-C motif chemokine receptor 3, expressed in eosinophils and lymphocytes), and some express CCR7 (129). In the normal subjects, the fibrocytes constitute approximately 0.5% of circulating leukocytes (128).

In animal models, as well as in patients with pulmonary fibrosis, these blood fibrocytes are able to differentiate in vitro into lung fibroblasts (130). Fibrocytes now are recognised as mesenchymal cells that arise from circulating monocyte precursors and participate in the innate response to injury where they have functions of antigen presentation and tissue remodelling (131). In the damaged tissue, the fibrocytes can adopt the phenotype of contractile myofibroblast, losing the CD34 and acquiring the expression of the -SMA (128).

They can induce the differentiation of the mesenchymal cells into myofibroblasts, can produce proinflammatory cytokines (such as IL-6 and IL8), could be involved in the stimulation of angiogenesis with production of matrix metalloproteinases and proangiogenic factors such as VEGF and platelet-derived growth factor (PDGF) (132, 133). The fibrocytes differentiation is reduced by interferon , tumour necrosis factor , IL-12, serum amyloid P (SAP), and is augmented by IL-4, IL-13, TGF-1 and endothelin-1 (ET-1) (134).

Two different populations of fibrocytes may be identified: the classical phenotype CD34+/CD45med/CD14+/collagen type I alpha 1 chain (COL1)+ and the myeloid-derived suppressor cell-like fibrocytes CD34-/CD45dim/CD14-/COL1+(135).

So far, there are few studies on the number of these fibrocytes in the peripheral blood of patients with COPD. The level of myeloid-derived suppressor cell-like fibrocytes (CD34-/CD45dim/CD14-/COL1+ cells), obtained by blood of stable COPD patients is significantly increased compared to control subjects, whereas the number of circulating fibrocytes (CD34+/CD45med/CD14+/COL1+) is similar between COPD patients and controls. Also, the number of fibrocytes is not related with lung function measured by spirometry (135).

There is only one study quantifying the number of fibrocytes in the lung tissue. The fibrocytes (CD34+/COL1+) are been found located in subepithelial region of the proximal airways (obtained from healthy areas of lung resection for cancer) and their number for mm2 is similar between COPD patients and control subjects (135).

Role of the satellite cells of the skeletal muscles

Satellite cells are undifferentiated myogenic precursor cells, localised beneath the basal lamina and the myofiber plasma membrane. These cells are heterogeneous, but most of them express the transcription factors involved in myogenesis and skeletal muscle differentiation termed paired box 3 (Pax3) and 7 (Pax7), myogenic factor 5 (Myf5), BARX homeobox 2 (Barx2), the molecules CD34, CXCR4 (136) and the adhesion molecules cadherin 15 (M-cadherin, adhesion molecule highly expressed in developing skeletal muscle), neural cell adhesion molecule (NCAM) (137). Of these, Pax7 is the best biomarker as it is expressed in all quiescent and proliferating human satellite cells (136).

Satellite cells are usually quiescent but may be re-activated with their proliferation and differentiation into myoblasts after severe muscle injuries (e.g. extensive physical activity or exposure to mycotoxins) cause (136).

There are various studies on the satellite cells in skeletal muscle of stable COPD patients. Many stable COPD patients have a dysfunction of their skeletal muscles, and it has been hypothesised that is caused by an impaired muscle regeneration (138). In the vastus lateralis muscle, the number of satellite cells (Pax7+) obtained by needle biopsy of stable COPD patients with mid-thigh muscle cross-sectional area (MTCSA) >70cm2, has been compared with stable COPD patients with MTCSA <70cm2 and with control non-smoking subjects without showing any significant differences between groups. However, the proportion of skeletal muscle fibres with central nuclei (considered newly formed myofibres) is increased in COPD patients with MTCSA >70cm2 (138).

Another study has confirmed that the number of Pax7+ in the biopsies from vastus lateralis muscle is not different at the baseline between stable COPD patients compared with control subjects. But after 8 weeks of high-intensity resistance training, these satellite cells tend (p=0.07) to increase in both groups at 24 hours remaining elevated at week 8 (139).

It has been hypothesised that the satellite cells may be senescent in stable COPD patients, in fact, the maximal telomere length of satellite cells (NCAM+/Pax7+) obtained from vastus lateralis muscle of control subjects is significantly increased compared to stable COPD patients with MTCSA >70cm2 and COPD patients with MTCSA <70cm2 (140).

In vitro, the satellite cells (Pax7+) obtained from vastus lateralis muscle of stable COPD patients show increased proliferation rates compared to those obtained from control subjects and a significant increase of Myf5 compared to those from controls (138).

In another study, the satellite cells (NCAM+) obtained from biopsy of vastus lateralis muscle of COPD patients in vitro show similar proliferation and differentiation capacity compared to those of control subjects, but build myotubes with reduced diameter with increased expression of proteins, such as myostatin and atrogin-1, that inhibit myogenesis and promote muscle atrophy (141).

In vitro, the mRNA expression of markers of autophagy [microtubule associated protein 1 light chain 3 alpha (LC3), sequestosome 1 (SQSTM1) and BCL2 interacting protein 3 (BNIP3)] is significantly increased in myoblasts from stable COPD patients compared with healthy subjects (142).

Role of the above structural and circulating lung stem cells in the pathogenesis of COPD exacerbations

Interestingly, the number of blood CD34+/VEGFR2+ cells is increased in many patients during a severe COPD exacerbation leading to hospitalisation compared with their phase of clinical stability (117). However, this may be explained by the blood leucocytosis seen during COPD exacerbations as the number of EPCs in stable phase correlated significantly with the number of total blood leucocytes (119). The functional significance of this observation is unknown. Cardiovascular morbidity and mortality are increased in patients with COPD exacerbations but remains to be demonstrated if COPD patients with no increase in their blood EPCs level during COPD exacerbations have an increased risk for cardiovascular morbidity and mortality.

Very small embryonic-like stem cells (VSELs) (CD34+/CD133+/CXCR4+/CD45-) (cells with very small diameter [~5m] are found in most human tissues including the bone marrow and are characterised by the expression of several transcription factors involved in the self-renewal of undifferentiated embryonic stem cells, such as the octamer binding transcription factor 4 (Oct-4) and the homeobox transcription factor Nanog [from “Tir Na Nog,” the mythologic Celtic land of the ever-young]) are pluripotent stem cells because can differentiate into osteoblasts, chondrocytes, adipocytes, and neurons (143, 144). These cells are increased in the blood of COPD patients during their severe exacerbations (with arterial oxygen saturation [SaO2] ≤92%), compared to a different cohort of exacerbating COPD patients (SaO2 >92%) and control subjects, suggesting that the hypoxaemia may induce the mobilisation of these cells (145).

The level of circulating fibrocytes (CD45+/COL1+) is increased in COPD patients during exacerbation compared to stable COPD and to control subjects (129). Interestingly, the exacerbating patients, two months after exacerbation, had a number of fibrocytes significantly reduced compared to the exacerbation phase, and the percentage of fibrocytes was negatively correlated to FEV1. The same Authors showed that the fibrocytes in vitro have significantly increased migration when exposed to enhanced concentrations of CXCL12, and the fibrocytes from blood of COPD exacerbation patients have higher migration capacity compared to control subjects. The migration is abolished with antagonist of CXCR4. These results suggest that during exacerbation the fibrocytes are recruited by the CXCR4/CXCL12 axis (129).

Pulmonary epithelial stem cells as a target of the carcinogenic process of lung carcinoma

Lung cancer and COPD are leading causes of morbidity and mortality worldwide. They share a common environmental risk factor in cigarette smoke exposure and a genetic predisposition represented by the incidence of these diseases in only a fraction of smokers. COPD is also a major independent risk factor for lung carcinoma, among long-term smokers. Smokers with COPD also have a higher risk of developing a specific histological subtype of non-small cell lung cancer termed squamous cell carcinoma (146). Despite, current data supporting a direct relationship between proximal airway basal progenitors and cells associated with carcinogenesis in murine models, the precise stem cell target of this process in the development of human squamous cell lung carcinoma remains unknown (146).

In murine models, a pulmonary stem cell population is found at the bronchioalveolar duct junction. These cells, termed BASCs, are resistant to bronchiolar and alveolar damage and proliferate during epithelial cell renewal in vivo and their transformed counterparts give rise to adenocarcinoma (45). BASCs can be functionally defined in vivo because of their resistance to chemical (naphthalene) injury, their infrequent proliferation relative to other progenitor cell types, and their co-expression of the bronchiolar and alveolar secretory cell markers club cell secretory protein and pro-surfactant protein C, respectively. Their immunophenotype is CD45-, CD31-, CD34-, Sca-llow, and autofluorescencelow (147). Interestingly, the protein kinase Ciota (PKCiota) is an oncogene required for maintenance of the transformed phenotype of NSCLC cells (148). In mouse, the genetic loss of Prkc dramatically inhibits Kras initiated hyperplasia and subsequent lung tumour formation in vivo. This effect correlates with a defect in the ability of Prkc-deficient BASCs to undergo Kras-mediated expansion and morphologic transformation in vitro and in vivo (148). This suggests that both squamous cell lung carcinoma and lung adenocarcinoma may originate from the same stem cells (146). This is an area which clearly requires more human translational research.

Stem cell therapy in patients with COPD and/or pulmonary emphysema

Recently, good evidence has been provided that new lung growth can occur in an adult human lung. After pneumonectomy serial computed tomographic scans of the chest has shown progressive enlargement of the remaining lung and an increase in tissue density and magnetic resonance imaging with the use of hyperpolarised helium-3 gas has shown overall acinar-airway dimensions consistent with an increase in the alveolar number rather than the enlargement of existing alveoli, despite that the alveoli in the growing lung are shallower than in normal lungs (149). Stem cell treatment is a type of intervention strategy that leads new cells into damaged tissue, in order to treat a disease or injury. Many potential stem cell therapies already exist, but most of them, outside of bone marrow stem cells transplantation, are yet at an experimental stage. Nevertheless, before stem cell therapy could be applied in a clinical setting, more research is deemed necessary to understand stem cell behaviour upon transplantation, as well as the mechanisms of stem cell interaction with the diseased microenvironment.

Findings that embryonic stem cells and stem cells deriving from adult human tissues (4) might be used for the repair and regeneration of diseased lung tissues, has stimulated extensive investigations with respect to whether these approaches could be used for lung diseases. Understanding the identity and roles of endogenous progenitor cells in the lungs as well as the possible roles as lung cancer stem cells has increased (4, 5). These are rapidly moving fields that hold promise for improved comprehension of lung biology and a potential therapeutic approach for many lung diseases. For example, although MSCs were initially hypothesised to be the panacea for regenerating tissues, MSCs appear to be more important in therapeutics to regulate the immune response invoked in settings such as tissue injury and autoimmunity (64).

Results of controlled clinical trials of stem cells transplantation

Tables 1 and 2 are summarised the design and the results, respectively, for the primary and secondary outcomes of the clinical trials of stem cell therapies performed in stable COPD patients whereas Table 3 are summarised for comparison the minimally clinical important differences (MCIDs) for these outcomes.

Human bone marrow mesenchymal stem cells

During a phase I, controlled trial of PROCHYMAL (ex vivo cultured adult hMSCs produced by Osiris Therapeutics; www.osiristx.com) in patients with acute myocardial infarction, an improvement, in FEV1 and forced vital capacity (FVC) has been observed through an as yet unknown mechanism (150). These data have stimulated a multicentre, double-blind, placebo-controlled phase II trial of PROCHYMAL for patients with moderate-severe COPD (FEV1/FVC, <0.70, 30% ≤FEV1 post-BD ≤70%), which was initiated in May 2008. The primary goal of the trial is to determine the safety of MSCs infusions in patients with COPD. The trial has recruited 62 patients in 6 participating US sites. No toxicities or significant adverse events were reported post-infusion. Notably, a significant decrease in the circulating inflammatory marker C-reactive protein (CRP), which is commonly increased in COPD patients, was noted in treated patients as was a trend towards improvement in quality of life indicators but without any significant improvement in lung function, in 6-minute walking distance (6MWD), Borg dyspnoea scale, and number of COPD exacerbations (www.osiristx.com). The trial ended in the third quarter of 2010 (151).

A phase I open label and prospective study, performed in the Netherlands, has enrolled 10 patients with severe pulmonary emphysema who undergone a lung volume reduction surgery (LVRS) in two separate occasions. At the time of first surgery, the bone marrow was collected, the mesenchymal cells were isolate and expanded in vitro; after 6–10 weeks, the patients received two autologous BM-MSC infusions at a distance of one week, followed by the second LVRS. In the first 48 hours and 3 weeks after the second MSC infusion, no symptoms or toxicity were observed. At 12-month follow up, the FEV1 had increased from baseline of 390mL (p=0.03; minimal clinically important difference [MCID] =100–140mL), the residual volume decreased by 540mL (p=0.053) and the body weight of all patients significantly increased of mean 4.6Kg (p=0.016). In addition, after BM-MSC infusion the immunohistochemical analysis of the resected lung tissue, has shown a threefold increased expression of the endothelial marker CD31 (p=0.016) in the alveolar septa (152).

Granulocyte colony-stimulating factor (G-CSF), a haematopoietic growth factor stimulates the proliferation and mobilisation of bone marrow haematopoietic cells to the peripheral blood, including an increase in myeloid progenitor cells and CD34+ stem cells which are possibly involved in the regeneration of damage tissues in the lungs (59, 153–156).

In an uncontrolled phase I clinical trial, performed in Brazil, G-CSF was administered immediately prior to bone marrow harvest and then bone marrow mononuclear cells were isolated and infused into a peripheral vein in patients with advanced COPD (stage IV with dyspnoea) (157). The 12-month follow-up showed an absence of significant adverse effects but no proliferation of CD34+ and CD133+ cells (157). The same four patients were followed for 3 years. The authors stated that this treatment with autologous bone marrow mononuclear cells is safe, however, one patient died 12 months after the administration and one died 27 months after. The other two patients showed improved FVC, whereas FEV1 remained stable (158).

Ten patients with severe pulmonary emphysema have been enrolled in phase I, prospective, patient-blinded, placebo-controlled study, performed in Brazil from December 2013 and October 2014. All patients were subjected to bronchoscopic lung volume reduction with placement of endobronchial valves (EBV), but immediately before, five patients received allogeneic BM-MSC administration in the subsegmental bronchi whereas the remaining five patients received saline solution only. In the MSC-EBV group, no patient experienced adverse events and the serum CRP levels were significantly reduced (p<.05) at days 30 and 90 compared to saline-EBV group, without significant differences in FEV1, FVC, total lung capacity (TLC), arterial oxygen partial pressure, arterial carbon dioxide partial pressure, single-breath carbon monoxide diffusing capacity (DLCO), BMI, and 6MWD. The body mass index, airflow obstruction, dyspnoea and exercise capacity (BODE) index and St. George’s Respiratory Questionnaire (SGRQ) score were significantly decreased compared to baseline (dimensionless unit of BODE index from 8 to 3; dimensionless unit of SGRQ from 68 to 30, MCID = reduction of 4 or more units) at days 90 in the patients of the MSC-EBV group (159).

Human adipose-derived stromal stem cells

A phase I, open label clinical study performed in Mexico, has enrolled 12 severe stable COPD patients to test the safety of intravenous administration of autologous stromal vascular fraction. This contains ADSMC [30–40%], vascular endothelial cells, smooth muscle cells, pericytes, various circulating cells such as leucocytes, and not require culture expansion (91) immediately after taking it from the adipose tissue. During the infusion and at 12th month of follow-up no adverse events were observed and the SGRQ score was decreased from 73 units at baseline to 45 at 3 months (p=0.005) ant to 44 at 6 months after this treatment (160).

There are no other published studies treating COPD/pulmonary emphysema patients with human adipose-derived stem cells, but it has been published the case report of the intravenously delivery of human autologous adipose-derived MSCs (CD44+/CD73+/CD90+/CD105+), in a patient with lung injury after long-term exposure to sulphur mustard with improvement of 6MWT, as well as SGRQ, and Borg Scale Dyspnoea Assessment, but not of the FEV1, and without adverse effects (161).

Ongoing clinical trials of stem cell therapies

Table 4 are summarised the ongoing clinical trials of stem cell therapies for the treatment of patients with COPD/pulmonary emphysema.

Because the adipose-derived MSCs are effective in animal models of pulmonary emphysema and are easy to isolate, most of these ongoing clinical trials are using these cells (162, 163).

New potential approaches to regenerative medicine

Lasers typically generate electromagnetic radiation which is relatively uniform in wavelength, phase and polarisation, and classical medical applications of lasers are considered “high energy” because of their intensity, which ranges from about 10 to 100 W. Low level lasers (LLLs) elicit effects through non-thermal means and may activate endogenous pulmonary stem cells and neoangiogenesis which is optimal for stem cell growth. LLLs are currently in clinical trials for acute myocardial infarction and stroke and have been proposed as regenerative photoceutical treatment for COPD (164).

Side effects of stem cell therapies

The analysis of serious adverse events (including deaths) in four clinical trials of COPD treatment with stem cells has found that the total (combination of non-fatal adverse events and deaths) serious adverse events are more common in cell therapy group compared to controls, with a 1.5 and 1.3-fold increased odds ratios for death and non-fatal serious adverse events respectively (165).

In the clinical trial with the highest number of patients (151), 27 patients treated with stem cells and 28 in the placebo-group experienced an adverse event in the 2 years of follow-up. Most of the adverse events are of mild or moderate intensity and was not reported any fatal adverse event. The most common adverse events are infections (bronchitis and pneumonia, urinary tract infection), respiratory disorders (dyspnoea, cough, and respiratory failure), cardiac disorders, gastrointestinal disorders, dizziness, and lethargy.

Ethical issues of the stem cell therapies

Stem cell treatment for COPD is associated with certain risks that have been clearly identified, primarily by using allogenic bone marrow transplantation. Graft versus host disease, tumour formation, inappropriate migration of cells to other areas of the body, and immune system rejection have been observed. Stem cell therapy for COPD is in its infancy, but even so, its development already brings great hope to many patients who have exhausted other forms of COPD treatment. For this reason, it is mandatory that all the medical institutions worldwide enrol patients with COPD into controlled clinical trials on experimental stem cell therapy. In the lay world, there are high expectations of flexible cells that, in theory, can develop into a lot of cell types, but laymen do not know that there are many types of stem cells. Moreover, it not clears to the public that experimental stem cell therapy requires extensive preparation and monitoring in the laboratory for these cells to develop the correct features. Importantly, greater evidence-based studies are essential along with a greater understanding of the stem cell growth conditions required to obtain optimal success/growth particularly as some of the most deadly neoplasms arise from stem cells (166).

That COPD patients decide to try this therapy is not surprising when patients have exhausted other forms of COPD treatment and only have, for example, in cases of very severe COPD, two more years to live and can read on the Internet about the possibilities available. There is a great need for government and supra-governmental action in this area to prevent people from taking such therapies until adequate controlled research is performed. However, the banning of stem cell therapy in some EU countries since 2007 has excluded controlled clinical experiments in some excellent academic centres.

Conclusions

There are only few human translational studies performed in the area of stem cell research in patients with COPD and/or pulmonary emphysema. Before progress to clinical trials with stem cells we strongly believe that more human translational studies are essential, otherwise, the clinical rationale would be solely based on limited in vitro and animal studies. In the future, stem cell therapy could be a treatment for this incurable disease. As of now, stem cell therapy is still to be considered as an area of active research, lacking any strong rationale for performing clinical trials in COPD.

Although stem cells would be likely to represent a heterogeneous population of cells, the different cell subsets and their importance in the pathogenesis of the different clinical phenotypes need to be fully characterised before progressing to clinical trials. Moreover, the potential side effects of stem cell therapy are underestimated. We should not ignore that some of the most deadly neoplasms are arising from stem cells.

Declaration of interest

The authors report no conflict of interest.

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