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SERIES EDITORIAL
Reflection of stem cell therapy: An epilogue to the ‘Stem cells and the lung’ review series Key words: cell therapy, lung bioengineering, lung cancer stem cell, lung cell therapy stem cell, lung stem cell. Abbreviations: ALDH, aldehyde dehydrogenase; COPD, chronic obstructive pulmonary disease; CSC, cancer stem cell; MSC, mesenchymal stem cells; NSCLC, non-small cell lung cancer. Glossary of genes and transcription factors: c-myc, v-cytomyelomatosis viral oncogene homolog; EGF, epidermal growth factor; FGF, fibroblast growth factor; Fox, Foxhead; GATA, transcription factor binding to GATA sequences in DNA; Gli/Kruppel, Gli gene is part of the Kruppel family of genes; HNF, hepatocyte nuclear family; HOX, homeobox transcription factors; KLF, Kruppel-like factor 4; MUC, mucin; Nkx2.1/TTF, Nk2 homoeobox 1/thyroid transcription factor; OCT 4, octamerbinding transcription factor 4; PAX, paired box; PDGF, platelet derived growth factor; RA, retinoic acid; Shh, Sonic hedgehog; SMAD-, SMA- and Mad-related protein; Sox, sex-determining region Y-binding; SPARC, secreted protein acidic and rich in cysteine; TG, thyroglobulin; TGF, transforming growth factor; VEGF, vascular endothelial growth factor; Wnt, wingless-related MMTV integration site.
The review series on ‘Stem cells and the lung’, published in Respirology throughout 2013, has been exciting and informative and has explored the state of the art in the fields of lung cell therapy, lung stem cells and regenerative medicine. The series was authored by leading experts and explored the advances as well as the challenges of cell therapy for lung diseases. In this epilogue, we have highlighted that lung diseases represent a major issue for world health. Chronic obstructive pulmonary disease (COPD) is among the top five causes of global mortality and morbidity, and together with asthma, lung cancer and acute respiratory distress syndrome constitute a major health burden globally.1 There are few effective treatments for lung disease, and this is compounded by our limited understanding of the complex pathways that mediate inflammation and repair. Epithelial and endothelial damage as well as disruption of the basement membrane leads to chronic tissue remodelling and fibrosis.1 Delineating the pathways that lead to impaired repair and determining why chronic lung disease evolves despite the removal of the offending agent such as cigarette smoke in COPD remains a challenge. The available therapeutics such as bronchodilators and immunosuppressives have limited efficacy and do not reverse or retard the progression of lung disease. These drugs do not restitute damaged tissue, and impaired replacement of epithelial and endothelial tissue is a major driver of scar formation and damaged lungs. Replacing damaged lung tissue © 2013 The Authors Respirology © 2013 Asian Pacific Society of Respirology
by either enhancing endogenous repair or utilizing exogenous repair is highly attractive. As such, there is a strong need to understand the location and characteristics of endogenous stem cells within the lung. Stem cells can differentiate into tissue from all three germ layers, while progenitor cells can differentiate down a restricted lineage.2 Therefore, an airway progenitor cell can differentiate into airway epithelial cells only. In the lung, the progenitor populations of cells have been isolated and are central to restitution of damaged tissue and normal repair. McQualter and Bertoncello et al. have described in great detail the location and markers of progenitor cells.3 The lung is the largest organ exposed to the environment and has a complex structure with the proximal lung (airways) designed to protect the distal lung (alveoli) from the numerous infective and non-infective agents in the environment. As such, the progenitor populations are unique in each location of the lung. Briefly, these studies suggest that regional stem and progenitor cells are responsible for the maintenance of specific epithelial cell lineages in the proximal and distal conducting airways and the alveolar bed.4 These are as follows: Trachea and upper air ways (1) Krt14pos submucosal gland duct cells (2) P63pos cytokeratin 5-positive (Krt5pos) and/or Krt14pos basal cells (3) Secretoglobin family 1A member1-positive (Scgb1a1pos) club (Clara) cells (4) Naphthalene-resistant (cytochrome P450 negative: CyP450neg) Scgb1a1pos and (5) Scgb3a2pos variant club (Clara) cells5 Bronchiolar and bronchoalveolar duct (1) Neuroendocrine cells in the bronchiolar airways (2) Club (Clara) cell specific protein-positive (CCSPpos)6 (3) Surfactant protein C-positive (SP-Cpos) bronchioalveolar stem cells7 Alveolar (1) Type II progenitor of type I cells in the alveolar bed8 However, the presence of a lung stem cell, a cell within the lung that could differentiate into any cell or lung tissue, remains elusive, and to date, no such cell Respirology (2014) 19, 5–8 doi: 10.1111/resp.12222
6 has been found. Therefore, the progenitor cells in each location are responsible for repair. However, if these progenitor populations are overwhelmed, then there is defective repair of epithelium and fibrosis results. Chronic lung disease may be due to progenitor cell exhaustion leading to diseases such as idiopathic pulmonary fibrosis and COPD. Therefore, cellular therapies may in the future constitute an important therapeutic arm. Cell therapies may have an immunomodulatory role, can replace injured lung tissue or compensate for defects in current cell activities. One population of immunomodulatory cells are mesenchymal stem cells (MSCs). These cells are fibroblast-like and are defined by specific markers as laid out by the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy Standards. An MSC has three key characteristics:9,10 • Be plastic adherent when cultured under standard conditions • Express the cell surface proteins CD105, CD90 and CD73, but in addition, not express CD45, CD34, CD14/CD11b, CD79a/CD19 and human leucocyte antigen-DR surface molecules • Have the capacity to differentiate into the mesenchymal lineages—bone, cartilage and fat Sinclair et al. have described in great detail MSCs resident within the lung (Lr-MSCs) as well as exogenous cells that may be attractive modalities for treating lung diseases.10 Lr-MSCs are long lived within the lung, are immunoprivileged and enhance the repair of overlying epithelium. In addition, Lr-MSC also suppresses inflammation. However, since the Lr-MSCs are fibroblastic, they contribute to fibrogenesis in disorders such as bronchiolitis obliterans. Despite this role, preclinical studies in animal models have demonstrated that these cells reduce inflammation, enhance repair and may reduce downstream collagen deposition post-injury. Mechanisms of immune suppression include inhibition of effector T-cell activation and/or expansion of regulatory T-cell numbers. Contact-dependent mechanisms have also been found to be important in the induction of Foxhead P3 and CD25—the characteristic markers of the induced T regulatory cell phenotype. Soluble factors have also been implicated in mediating immunomodulation of MSCs.11 These trophic factors include prostaglandin E2, nitric oxide, angiopoeitin, tumour necrosis (TSG-6) and Interleukin-1 receptor antagonist (IL1Ra).10 Recently, MSCs have been shown to release organelles in microvesicles or exosomes that donate mitochondria to damaged tissue and enhance repair by increasing the energetics of damaged tissue.12 Preclinical studies of MSCs have demonstrated rescue of animal models of asthma, fibrosis, pulmonary hypertension, COPD, reperfusion injury, acute lung injury and bronchopulmonary dysplasia. Zhu et al. further examined the role of adult stem cells in lung disease. In addition to the immunosuppressive actions of MSC, they increase alveolar clearance and have antimicrobial properties that make them useful in acute lung injury/acute respiratory distress syndrome.13 The timing and dosage of MSCs need to be addressed since MSC administration within Respirology (2014) 19, 5–8
Y Moodley and P Thompson
24 h of injury is most effective. In addition, the most effective dosage in human studies needs to be determined. In the consideration of MSC therapy, Mora and Rojas have discussed the issue of MSC function in chronic lung disease and the influence of ageing on these cells.14 This is significant since MSC therapy will be increasingly used in the elderly. The authors of this review propose that ageing MSCs have limited reparative ability. The mechanisms underlying this defect in bone marrow-derived MSCs in elderly people include: • A different morphology • Increased production of reactive oxygen species and oxidative damage 18, DNA-methylation changes affecting cell differentiation and, slower proliferation rate in culture.15–17 • Shorter telomeres and a larger proportion stain positive for senescence-associated betagalactosidase.15,18 In this review, Mora and Rojas also draw caution to the extracellular matrix of the ageing lung and how this would influence the function of injected MSCs for treatment. There is altered fibronectin and an agedependent increase of transforming growth factor-β1 and transforming growth factor-βR1 expression in lungs from older individuals that correlates with increased Smad3 mRNA and protein expression.14 Therefore, MSC therapy in elderly persons may end up promoting fibrosis. The mechanism of immunoprivilege of MSCs is attributed to the absence of expression of histocompatibility molecules. The cells do not express human leucocyte antigen class II and have weak expression of human leucocyte antigen class I thereby not activating the immune response.19 However, recent studies in small animal models of cardiac disease suggest that the immunogenicity of MSCs became apparent with an increased differentiation of these cells. Specifically, major histocompatibility complex expression was upregulated in terminally differentiated progeny of MSCs such as myocytes and endothelial cells.10 The differentiated cells were then lysed by the recipient’s complement- and/or cell-mediated systems. Notably, in a primate model the administration of several highdose allogeneic MSCs resulted in the production of alloantibodies in two of six animals.20 Therefore, the use of MSCs in human disease must be approached cautiously since they are usually administered to human leucocyte antigen mismatched individuals. Although one study has demonstrated immunological concerns with MSCs in human studies, the safety of MSCs has been demonstrated in COPD, bronchiolitis obliterans and idiopathic pulmonary fibrosis. Phase 2 and phase 3 studies of MSC therapy in human lung diseases will commence or are ongoing, and outcomes are awaited with interest.10 However, given the above concerns, the expectation of MSC therapy in lung diseases needs to be tempered. It is unlikely that MSC therapy would make dramatic changes such as reversing chronic pathology in the lung and abnormal lung function. In the best-case scenario, this therapeutic option may modulate © 2013 The Authors Respirology © 2013 Asian Pacific Society of Respirology
Stem cells and the lung: An epilogue
inflammation and perhaps reduce exacerbations as well as stabilize any decline in lung function. To replace damaged tissue, pluripotent stem cells would be required to differentiate into lung cells. In the review by Moodley, Thompson and Warburton, the authors explored strategies to differentiate pluripotent cells into lung cells.21 They examined the various pathways during lung development and highlighted the role of these pathways in obtaining lung from induced pluripotent stem cells or embryonic stem cells. Researchers initially aimed to obtain anterior endoderm, the embryonic origin of the lung. They used several factors including activin and winglessrelated MMTV integration site.22 Once anterior endoderm was obtained, researchers used several factors that recapitulated development such as fibroblast growth factor 10, fibroblast growth factor 7 and bone morphometric protein23. In order to anteriorize the endoderm, they found that transforming growth factor-β antagonism was sufficient to get cells from definitive endoderm to anterior lung endoderm characterized by Nk2 homoeobox 1, Foxhead A2 and sexdetermining region Y-binding 2 expressing cells. In addition, Smad-dependent bone morphometric protein 2/4 signalling was critical for lung specification. These authors then found that a combination of bone morphometric protein 7, Noggin, low winglessrelated MMTV integration site and intermediate levels of retinoic acid generated proximal airway progenitors (Nk2 homoeobox 1+, sex-determining region Y-binding +), while bone morphometric protein 4, fibroblast growth factor 2, fibroblast growth factor 7, wingless-related MMTV integration site 2/2a and wingless-related MMTV integration site 3 as well as low levels of retinoic acid generated multipotent distal lung progenitors (Nk2 homoeobox 1+, Sox9+) or (Nk2 homoeobox 1+, Foxhead p2+) cells.23 The proximal airway cells obtained by the addition of growth factors were further placed in an air–liquid interface as occurs in vivo and these cells formed a pseudostratified layer that was similar to the epithelial lining of a human upper airway.23 Notably, the approach of recapitulating the developmental pathway in pluripotent cells to create lung cells presents new possibilities, but certain limitations remain. We need to confirm that functional lung cells are produced from these methods. In addition, there needs to be an efficient method of differentiation that would produce large amounts of cells for clinical applications. Although the developmental pathways may be exploited to create lung cells, they also play an important part in pathological processes. It is now increasingly recognized that developmental pathways are important mechanisms in driving lung malignancy. The review by Alamgeer et al. describes the growing link between stem cells and lung cancer.16 Lung cancer is the commonest cause of cancer mortality and has two distinct pathological groups: non-smallcell lung cancer (NSCLC, which makes up 80% of cases) and small-cell lung cancer (making up 20% of all lung cancers). The most common forms of NSCLC are adenocarcinoma (30–50% of NSCLC) and squamous cell carcinoma (30% of NSCLC).16 The © 2013 The Authors Respirology © 2013 Asian Pacific Society of Respirology
7 cancer stem cell (CSC) hypothesis is based on the concept that cancers have a hierarchy with respect to self-renewal, and a cancer stem cell can give rise to highly proliferative progeny that comprise the bulk of tumours.16 This has been reported in haematological malignancies, breast cancer, brain tumours and colorectal cancer.17 There are various assays that may identify CSC including side-population phenotypes. Side-population phenotyping is based on the differential ability of the cancer cells to efflux Hoechst 33342 dye, as imparted by the ATP-binding cassette family of transporter proteins present on the cellular membrane.18 Aldehyde dehydrogenase (ALDH) is involved in early stem development and is formed by the oxidation of retinol to retinoic acid. ALDH activity is an important functional marker of normal and malignant stem/progenitor cells.16 Other markers are CD133 and CD44 both are found in several tumour types.24 Based on these markers, cancer cells isolated from tumours grow as spheres in non-adherent cultures and, form colonies. The gold standard for CSC tumourigenic potential is their ability to grow in vivo as serially transplantable tumours. Despite the evidence that CSC may exist, no universal lung CSC marker has been described. Notably in many lung cancers CD133 is not detected. Furthermore both ALDH-positive and ALDH negative cells are tumourigenic.16 These findings suggest that either other tumour-initiating cells exist independently of these markers. There is plasticity in the cells of tumours where NSCLC cell lines and primary lung adenocarcinoma samples, that non-side population, CD133-negative and ALDH-negative cells can generate side-population, CD133-positive and ALDHpositive cells under adherent culture conditions.25 Therefore, cellular plasticity may explain the heterogeneity of CSC. Targeting CSC may have a major impact of lung cancer therapy. Finally, the prospect of regenerating whole lung was reviewed by Wagner et al.26 In the rapidly expanding area of ex vivo bioengineering, the authors described the possibility of deriving a lung using an extracellular matrix scaffold. Seeding this scaffold with epithelial and endothelial cells can result in the generation of a new lung that could then be implanted into patients with diseases such as COPD or idiopathic pulmonary fibrosis.26 This could be accomplished by utilizing either biologically derived or fabricated threedimensional matrices or other artificial scaffolding seeded with autologous stem, progenitor or other cells obtained from the eventual transplant recipient. The use of autologous cells would remove the need for lifelong immunosuppressive drugs. Synthetic options cannot reproduce the three-dimensional complexity of the lung. Therefore, another approach is to use whole lungs in which all cells and cellular materials are removed leaving an intact three-dimensional scaffold that comprises the innate extracellular matrix proteins of the lung. The approach is called decellularization and preserves native airway and vascular structure and provides an acellular matrix for cell seeding and functional recellularization. Commonly used substances for decellurization include Triton X-100, sodium deoxycholate, sodium dodecyl Respirology (2014) 19, 5–8
8 sulphate, 3-[(3-cholamidopropyl) dimethylammonio]1-propanesulfonate, ethylenediaminetetracetic acid, antibiotics, deoxyribonuclease, ribonuclease and heparin.26 However there are problems associated with these detergents since they may damage critical epitopes on the extracellular matrix and lead to impaired adherence as well as cell function in the explant graft. In addition, there may be retention of nucleic acids and other proteins from the donor lung that may precipitate an inflammatory reaction.26 The field of ex vivo bioengineering has major hurdles to overcome, but the preliminary studies utilizing animal models are very encouraging. This series has clearly demonstrated that cellular therapy can form a major component of future therapies. These therapies may address fundamental issues in lung regeneration. There are many barriers to overcome, but there is a quiet optimism about the prospect for future developments. Yuben Moodley, MBChb, MD, PhD, FRACP,1,2,3 and Philip Thompson, MBBS, FRACP1,4 1 Lung Institute of Western Australia and Centre for Asthma, Allergy and Respiratory Research, University of Western Australia, 2School of Medicine and Pharmacology, Royal Perth Hospital, 3Department of Respiratory and Sleep Medicine, Royal Perth Hospital, and 4Department of Respiratory Medicine, Sir Charles Gairdner Hospital, Perth, Western Australia, Australia
REFERENCES 1 Moodley Y, Thompson PJ. The burden of lung disease: the need for stem cell therapy—a review series prologue. Respirology 2013; 18: 393–6. 2 Moodley Y, Manuelpillai U, Weiss DJ. Cellular therapies for lung disease: a distant horizon. Respirology 2011; 16: 223–37. 3 Bertoncello I, McQualter JL. Lung stem cells: do they exist? Respirology 2013; 18: 587–95. 4 Rock JR, Hogan BL. Epithelial progenitor cells in lung development, maintenance, repair, and disease. Annu. Rev. Cell Dev. Biol. 2011; 27: 493–512. 5 Rock JR, Barkauskas CE, Cronce MJ et al. Multiple stromal populations contribute to pulmonary fibrosis without evidence for epithelial to mesenchymal transition. Proc. Natl Acad. Sci. U. S. A. 2011; 108: E1475–83. 6 Hong KU, Reynolds SD, Giangreco A et al. Clara cell secretory protein-expressing cells of the airway neuroepithelial body microenvironment include a label-retaining subset and are critical for epithelial renewal after progenitor cell depletion. Am. J. Respir. Cell Mol. Biol. 2001; 24: 671–81. 7 Giangreco A, Reynolds SD, Stripp BR. Terminal bronchioles harbor a unique airway stem cell population that localizes to the bronchoalveolar duct junction. Am. J. Pathol. 2002; 161: 173–82.
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Y Moodley and P Thompson 8 Zacharek SJ, Fillmore CM, Lau AN et al. Lung stem cell selfrenewal relies on BMI1-dependent control of expression at imprinted loci. Cell Stem. Cell 2011; 9: 272–81. 9 Dominici M, Le Blanc K, Mueller I et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 2006; 8: 315–7. 10 Sinclair K, Yerkovich ST, Chambers DC. Mesenchymal stem cells and the lung. Respirology 2013; 18: 397–411. 11 Aggarwal S, Pittenger MF. Human mesenchymal stem cells modulate allogeneic immune cell responses. Blood 2005; 105: 1815–22. 12 Islam MN, Das SR, Emin MT et al. Mitochondrial transfer from bone-marrow-derived stromal cells to pulmonary alveoli protects against acute lung injury. Nat. Med. 2012; 18: 759–65. 13 Zhu YG, Hao Q, Monsel A et al. Adult stem cells for acute lung injury: remaining questions and concerns. Respirology 2013; 18: 744–56. 14 Mora AL, Rojas M. Adult stem cells for chronic lung diseases. Respirology 2013; 18: 1041–46. 15 Zhou S, Greenberger JS, Epperly MW et al. Age-related intrinsic changes in human bone-marrow-derived mesenchymal stem cells and their differentiation to osteoblasts. Aging Cell. 2008; 7: 335–43. 16 Alamgeer M, Peacock CD, Matsui W et al. Cancer stem cells in lung cancer: evidence and controversies. Respirology 2013; 18: 757–64. 17 Reya T, Morrison SJ, Clarke MF et al. Stem cells, cancer, and cancer stem cells. Nature 2001; 414: 105–11. 18 Eramo A, Lotti F, Sette G et al. Identification and expansion of the tumorigenic lung cancer stem cell population. Cell Death Differ. 2008; 15: 504–14. 19 Ciavarella S, Dominici M, Dammacco F et al. Mesenchymal stem cells: a new promise in anticancer therapy. Stem Cells Dev. 2011; 20: 1–10. 20 Beggs KJ, Lyubimov A, Borneman JN et al. Immunologic consequences of multiple, high-dose administration of allogeneic mesenchymal stem cells to baboons. Cell Transplant. 2006; 15: 711–21. 21 Moodley Y, Thompson P, Warburton D. Stem cells: a recapitulation of development. Respirology 2013; 18: 1167–76. 22 Mou H, Zhao R, Sherwood R et al. Generation of multipotent lung and airway progenitors from mouse ESCs and patientspecific cystic fibrosis iPSCs. Cell Stem. Cell 2012; 10: 385–97. 23 Wong AP, Bear CE, Chin S et al. Directed differentiation of human pluripotent stem cells into mature airway epithelia expressing functional CFTR protein. Nat. Biotechnol. 2012; 30: 876–82. 24 Bertolini G, Roz L, Perego P et al. Highly tumorigenic lung cancer CD133+ cells display stem-like features and are spared by cisplatin treatment. Proc. Natl. Acad. Sci. U. S. A. 2009; 106: 16281–6. 25 Woo T, Okudela K, Mitsui H et al. Prognostic value of CD133 expression in stage I lung adenocarcinomas. Int. J. Clin. Exp. Pathol. 2010; 4: 32–42. 26 Wagner DE, Bonvillain RW, Jensen T et al. Can stem cells be used to generate new lungs? Ex vivo lung bioengineering with decellularized whole lung scaffolds. Respirology 2013; 18: 895– 911.
© 2013 The Authors Respirology © 2013 Asian Pacific Society of Respirology
SERIES EDITORIAL
Reflection of stem cell therapy: An epilogue to the ‘Stem cells and the lung’ review series Key words: cell therapy, lung bioengineering, lung cancer stem cell, lung cell therapy stem cell, lung stem cell. Abbreviations: ALDH, aldehyde dehydrogenase; COPD, chronic obstructive pulmonary disease; CSC, cancer stem cell; MSC, mesenchymal stem cells; NSCLC, non-small cell lung cancer. Glossary of genes and transcription factors: c-myc, v-cytomyelomatosis viral oncogene homolog; EGF, epidermal growth factor; FGF, fibroblast growth factor; Fox, Foxhead; GATA, transcription factor binding to GATA sequences in DNA; Gli/Kruppel, Gli gene is part of the Kruppel family of genes; HNF, hepatocyte nuclear family; HOX, homeobox transcription factors; KLF, Kruppel-like factor 4; MUC, mucin; Nkx2.1/TTF, Nk2 homoeobox 1/thyroid transcription factor; OCT 4, octamerbinding transcription factor 4; PAX, paired box; PDGF, platelet derived growth factor; RA, retinoic acid; Shh, Sonic hedgehog; SMAD-, SMA- and Mad-related protein; Sox, sex-determining region Y-binding; SPARC, secreted protein acidic and rich in cysteine; TG, thyroglobulin; TGF, transforming growth factor; VEGF, vascular endothelial growth factor; Wnt, wingless-related MMTV integration site.
The review series on ‘Stem cells and the lung’, published in Respirology throughout 2013, has been exciting and informative and has explored the state of the art in the fields of lung cell therapy, lung stem cells and regenerative medicine. The series was authored by leading experts and explored the advances as well as the challenges of cell therapy for lung diseases. In this epilogue, we have highlighted that lung diseases represent a major issue for world health. Chronic obstructive pulmonary disease (COPD) is among the top five causes of global mortality and morbidity, and together with asthma, lung cancer and acute respiratory distress syndrome constitute a major health burden globally.1 There are few effective treatments for lung disease, and this is compounded by our limited understanding of the complex pathways that mediate inflammation and repair. Epithelial and endothelial damage as well as disruption of the basement membrane leads to chronic tissue remodelling and fibrosis.1 Delineating the pathways that lead to impaired repair and determining why chronic lung disease evolves despite the removal of the offending agent such as cigarette smoke in COPD remains a challenge. The available therapeutics such as bronchodilators and immunosuppressives have limited efficacy and do not reverse or retard the progression of lung disease. These drugs do not restitute damaged tissue, and impaired replacement of epithelial and endothelial tissue is a major driver of scar formation and damaged lungs. Replacing damaged lung tissue © 2013 The Authors Respirology © 2013 Asian Pacific Society of Respirology
by either enhancing endogenous repair or utilizing exogenous repair is highly attractive. As such, there is a strong need to understand the location and characteristics of endogenous stem cells within the lung. Stem cells can differentiate into tissue from all three germ layers, while progenitor cells can differentiate down a restricted lineage.2 Therefore, an airway progenitor cell can differentiate into airway epithelial cells only. In the lung, the progenitor populations of cells have been isolated and are central to restitution of damaged tissue and normal repair. McQualter and Bertoncello et al. have described in great detail the location and markers of progenitor cells.3 The lung is the largest organ exposed to the environment and has a complex structure with the proximal lung (airways) designed to protect the distal lung (alveoli) from the numerous infective and non-infective agents in the environment. As such, the progenitor populations are unique in each location of the lung. Briefly, these studies suggest that regional stem and progenitor cells are responsible for the maintenance of specific epithelial cell lineages in the proximal and distal conducting airways and the alveolar bed.4 These are as follows: Trachea and upper air ways (1) Krt14pos submucosal gland duct cells (2) P63pos cytokeratin 5-positive (Krt5pos) and/or Krt14pos basal cells (3) Secretoglobin family 1A member1-positive (Scgb1a1pos) club (Clara) cells (4) Naphthalene-resistant (cytochrome P450 negative: CyP450neg) Scgb1a1pos and (5) Scgb3a2pos variant club (Clara) cells5 Bronchiolar and bronchoalveolar duct (1) Neuroendocrine cells in the bronchiolar airways (2) Club (Clara) cell specific protein-positive (CCSPpos)6 (3) Surfactant protein C-positive (SP-Cpos) bronchioalveolar stem cells7 Alveolar (1) Type II progenitor of type I cells in the alveolar bed8 However, the presence of a lung stem cell, a cell within the lung that could differentiate into any cell or lung tissue, remains elusive, and to date, no such cell Respirology (2014) 19, 5–8 doi: 10.1111/resp.12222
6 has been found. Therefore, the progenitor cells in each location are responsible for repair. However, if these progenitor populations are overwhelmed, then there is defective repair of epithelium and fibrosis results. Chronic lung disease may be due to progenitor cell exhaustion leading to diseases such as idiopathic pulmonary fibrosis and COPD. Therefore, cellular therapies may in the future constitute an important therapeutic arm. Cell therapies may have an immunomodulatory role, can replace injured lung tissue or compensate for defects in current cell activities. One population of immunomodulatory cells are mesenchymal stem cells (MSCs). These cells are fibroblast-like and are defined by specific markers as laid out by the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy Standards. An MSC has three key characteristics:9,10 • Be plastic adherent when cultured under standard conditions • Express the cell surface proteins CD105, CD90 and CD73, but in addition, not express CD45, CD34, CD14/CD11b, CD79a/CD19 and human leucocyte antigen-DR surface molecules • Have the capacity to differentiate into the mesenchymal lineages—bone, cartilage and fat Sinclair et al. have described in great detail MSCs resident within the lung (Lr-MSCs) as well as exogenous cells that may be attractive modalities for treating lung diseases.10 Lr-MSCs are long lived within the lung, are immunoprivileged and enhance the repair of overlying epithelium. In addition, Lr-MSC also suppresses inflammation. However, since the Lr-MSCs are fibroblastic, they contribute to fibrogenesis in disorders such as bronchiolitis obliterans. Despite this role, preclinical studies in animal models have demonstrated that these cells reduce inflammation, enhance repair and may reduce downstream collagen deposition post-injury. Mechanisms of immune suppression include inhibition of effector T-cell activation and/or expansion of regulatory T-cell numbers. Contact-dependent mechanisms have also been found to be important in the induction of Foxhead P3 and CD25—the characteristic markers of the induced T regulatory cell phenotype. Soluble factors have also been implicated in mediating immunomodulation of MSCs.11 These trophic factors include prostaglandin E2, nitric oxide, angiopoeitin, tumour necrosis (TSG-6) and Interleukin-1 receptor antagonist (IL1Ra).10 Recently, MSCs have been shown to release organelles in microvesicles or exosomes that donate mitochondria to damaged tissue and enhance repair by increasing the energetics of damaged tissue.12 Preclinical studies of MSCs have demonstrated rescue of animal models of asthma, fibrosis, pulmonary hypertension, COPD, reperfusion injury, acute lung injury and bronchopulmonary dysplasia. Zhu et al. further examined the role of adult stem cells in lung disease. In addition to the immunosuppressive actions of MSC, they increase alveolar clearance and have antimicrobial properties that make them useful in acute lung injury/acute respiratory distress syndrome.13 The timing and dosage of MSCs need to be addressed since MSC administration within Respirology (2014) 19, 5–8
Y Moodley and P Thompson
24 h of injury is most effective. In addition, the most effective dosage in human studies needs to be determined. In the consideration of MSC therapy, Mora and Rojas have discussed the issue of MSC function in chronic lung disease and the influence of ageing on these cells.14 This is significant since MSC therapy will be increasingly used in the elderly. The authors of this review propose that ageing MSCs have limited reparative ability. The mechanisms underlying this defect in bone marrow-derived MSCs in elderly people include: • A different morphology • Increased production of reactive oxygen species and oxidative damage 18, DNA-methylation changes affecting cell differentiation and, slower proliferation rate in culture.15–17 • Shorter telomeres and a larger proportion stain positive for senescence-associated betagalactosidase.15,18 In this review, Mora and Rojas also draw caution to the extracellular matrix of the ageing lung and how this would influence the function of injected MSCs for treatment. There is altered fibronectin and an agedependent increase of transforming growth factor-β1 and transforming growth factor-βR1 expression in lungs from older individuals that correlates with increased Smad3 mRNA and protein expression.14 Therefore, MSC therapy in elderly persons may end up promoting fibrosis. The mechanism of immunoprivilege of MSCs is attributed to the absence of expression of histocompatibility molecules. The cells do not express human leucocyte antigen class II and have weak expression of human leucocyte antigen class I thereby not activating the immune response.19 However, recent studies in small animal models of cardiac disease suggest that the immunogenicity of MSCs became apparent with an increased differentiation of these cells. Specifically, major histocompatibility complex expression was upregulated in terminally differentiated progeny of MSCs such as myocytes and endothelial cells.10 The differentiated cells were then lysed by the recipient’s complement- and/or cell-mediated systems. Notably, in a primate model the administration of several highdose allogeneic MSCs resulted in the production of alloantibodies in two of six animals.20 Therefore, the use of MSCs in human disease must be approached cautiously since they are usually administered to human leucocyte antigen mismatched individuals. Although one study has demonstrated immunological concerns with MSCs in human studies, the safety of MSCs has been demonstrated in COPD, bronchiolitis obliterans and idiopathic pulmonary fibrosis. Phase 2 and phase 3 studies of MSC therapy in human lung diseases will commence or are ongoing, and outcomes are awaited with interest.10 However, given the above concerns, the expectation of MSC therapy in lung diseases needs to be tempered. It is unlikely that MSC therapy would make dramatic changes such as reversing chronic pathology in the lung and abnormal lung function. In the best-case scenario, this therapeutic option may modulate © 2013 The Authors Respirology © 2013 Asian Pacific Society of Respirology
Stem cells and the lung: An epilogue
inflammation and perhaps reduce exacerbations as well as stabilize any decline in lung function. To replace damaged tissue, pluripotent stem cells would be required to differentiate into lung cells. In the review by Moodley, Thompson and Warburton, the authors explored strategies to differentiate pluripotent cells into lung cells.21 They examined the various pathways during lung development and highlighted the role of these pathways in obtaining lung from induced pluripotent stem cells or embryonic stem cells. Researchers initially aimed to obtain anterior endoderm, the embryonic origin of the lung. They used several factors including activin and winglessrelated MMTV integration site.22 Once anterior endoderm was obtained, researchers used several factors that recapitulated development such as fibroblast growth factor 10, fibroblast growth factor 7 and bone morphometric protein23. In order to anteriorize the endoderm, they found that transforming growth factor-β antagonism was sufficient to get cells from definitive endoderm to anterior lung endoderm characterized by Nk2 homoeobox 1, Foxhead A2 and sexdetermining region Y-binding 2 expressing cells. In addition, Smad-dependent bone morphometric protein 2/4 signalling was critical for lung specification. These authors then found that a combination of bone morphometric protein 7, Noggin, low winglessrelated MMTV integration site and intermediate levels of retinoic acid generated proximal airway progenitors (Nk2 homoeobox 1+, sex-determining region Y-binding +), while bone morphometric protein 4, fibroblast growth factor 2, fibroblast growth factor 7, wingless-related MMTV integration site 2/2a and wingless-related MMTV integration site 3 as well as low levels of retinoic acid generated multipotent distal lung progenitors (Nk2 homoeobox 1+, Sox9+) or (Nk2 homoeobox 1+, Foxhead p2+) cells.23 The proximal airway cells obtained by the addition of growth factors were further placed in an air–liquid interface as occurs in vivo and these cells formed a pseudostratified layer that was similar to the epithelial lining of a human upper airway.23 Notably, the approach of recapitulating the developmental pathway in pluripotent cells to create lung cells presents new possibilities, but certain limitations remain. We need to confirm that functional lung cells are produced from these methods. In addition, there needs to be an efficient method of differentiation that would produce large amounts of cells for clinical applications. Although the developmental pathways may be exploited to create lung cells, they also play an important part in pathological processes. It is now increasingly recognized that developmental pathways are important mechanisms in driving lung malignancy. The review by Alamgeer et al. describes the growing link between stem cells and lung cancer.16 Lung cancer is the commonest cause of cancer mortality and has two distinct pathological groups: non-smallcell lung cancer (NSCLC, which makes up 80% of cases) and small-cell lung cancer (making up 20% of all lung cancers). The most common forms of NSCLC are adenocarcinoma (30–50% of NSCLC) and squamous cell carcinoma (30% of NSCLC).16 The © 2013 The Authors Respirology © 2013 Asian Pacific Society of Respirology
7 cancer stem cell (CSC) hypothesis is based on the concept that cancers have a hierarchy with respect to self-renewal, and a cancer stem cell can give rise to highly proliferative progeny that comprise the bulk of tumours.16 This has been reported in haematological malignancies, breast cancer, brain tumours and colorectal cancer.17 There are various assays that may identify CSC including side-population phenotypes. Side-population phenotyping is based on the differential ability of the cancer cells to efflux Hoechst 33342 dye, as imparted by the ATP-binding cassette family of transporter proteins present on the cellular membrane.18 Aldehyde dehydrogenase (ALDH) is involved in early stem development and is formed by the oxidation of retinol to retinoic acid. ALDH activity is an important functional marker of normal and malignant stem/progenitor cells.16 Other markers are CD133 and CD44 both are found in several tumour types.24 Based on these markers, cancer cells isolated from tumours grow as spheres in non-adherent cultures and, form colonies. The gold standard for CSC tumourigenic potential is their ability to grow in vivo as serially transplantable tumours. Despite the evidence that CSC may exist, no universal lung CSC marker has been described. Notably in many lung cancers CD133 is not detected. Furthermore both ALDH-positive and ALDH negative cells are tumourigenic.16 These findings suggest that either other tumour-initiating cells exist independently of these markers. There is plasticity in the cells of tumours where NSCLC cell lines and primary lung adenocarcinoma samples, that non-side population, CD133-negative and ALDH-negative cells can generate side-population, CD133-positive and ALDHpositive cells under adherent culture conditions.25 Therefore, cellular plasticity may explain the heterogeneity of CSC. Targeting CSC may have a major impact of lung cancer therapy. Finally, the prospect of regenerating whole lung was reviewed by Wagner et al.26 In the rapidly expanding area of ex vivo bioengineering, the authors described the possibility of deriving a lung using an extracellular matrix scaffold. Seeding this scaffold with epithelial and endothelial cells can result in the generation of a new lung that could then be implanted into patients with diseases such as COPD or idiopathic pulmonary fibrosis.26 This could be accomplished by utilizing either biologically derived or fabricated threedimensional matrices or other artificial scaffolding seeded with autologous stem, progenitor or other cells obtained from the eventual transplant recipient. The use of autologous cells would remove the need for lifelong immunosuppressive drugs. Synthetic options cannot reproduce the three-dimensional complexity of the lung. Therefore, another approach is to use whole lungs in which all cells and cellular materials are removed leaving an intact three-dimensional scaffold that comprises the innate extracellular matrix proteins of the lung. The approach is called decellularization and preserves native airway and vascular structure and provides an acellular matrix for cell seeding and functional recellularization. Commonly used substances for decellurization include Triton X-100, sodium deoxycholate, sodium dodecyl Respirology (2014) 19, 5–8
8 sulphate, 3-[(3-cholamidopropyl) dimethylammonio]1-propanesulfonate, ethylenediaminetetracetic acid, antibiotics, deoxyribonuclease, ribonuclease and heparin.26 However there are problems associated with these detergents since they may damage critical epitopes on the extracellular matrix and lead to impaired adherence as well as cell function in the explant graft. In addition, there may be retention of nucleic acids and other proteins from the donor lung that may precipitate an inflammatory reaction.26 The field of ex vivo bioengineering has major hurdles to overcome, but the preliminary studies utilizing animal models are very encouraging. This series has clearly demonstrated that cellular therapy can form a major component of future therapies. These therapies may address fundamental issues in lung regeneration. There are many barriers to overcome, but there is a quiet optimism about the prospect for future developments. Yuben Moodley, MBChb, MD, PhD, FRACP,1,2,3 and Philip Thompson, MBBS, FRACP1,4 1 Lung Institute of Western Australia and Centre for Asthma, Allergy and Respiratory Research, University of Western Australia, 2School of Medicine and Pharmacology, Royal Perth Hospital, 3Department of Respiratory and Sleep Medicine, Royal Perth Hospital, and 4Department of Respiratory Medicine, Sir Charles Gairdner Hospital, Perth, Western Australia, Australia
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© 2013 The Authors Respirology © 2013 Asian Pacific Society of Respirology
