Stem Cells and the Lung - technical
Adult stem cells have the capacity for self-renewal and terminal differentiation to one or more cell types. Lung stem cells include basal cells and mucus-secreting cells in the trachea, Clara cells in the bronchioles, and type II pneumocytes in the alveoli. Mesenchymal cells predominate in fetal lung development. In postnatal life the Clara cell is the most actively dividing cell in the tracheobronchial epithelium while type II pneumocytes are the progenitor cells for the alveolar epithelium. Under specific circumstances basal cells and pulmonary neuroendocrine cells may also proliferate and act as stem cells. All these putative stem cells have varied roles in normal lung repair and in disease.
Mesenchymal progenitor cells and multipotential adult progenitor cells found in bone marrow differentiate into cells of endodermal, mesodermal, and ectodermal origin including lung fibroblasts. The finding of such heterogeneous stem cell populations in bone marrow and their presence in lung tissue may lead to stem cell manipulative and regenerative therapies for conditions such as idiopathic pulmonary fibrosis and cystic fibrosis transmembrane regulator gene therapy for cystic fibrosis.
Adult stem cells differentiate from pluripotent cells to more committed, tissue-specific cells. The majority of cells in adult tissues are differentiated but a number of stem cells are present in most tissues. Stem cells have the capacity to proliferate for self-renewal, to produce a large number of daughter cells, and to replace damaged or injured cells. Proliferation and replication are common to all stem cells but the number of daughter cells varies. In bone marrow, where there is a constant need to replenish blood cells, stem cells are relatively plentiful. In more stable tissues minimal numbers of dormant stem cells exist. Unipotent stem cells such as skin cells are capable of proliferation and differentiation along a single pathway while hematopoietic stem cells, mesenchymal stem cells, and intestinal stem cells are pluripotent and give rise to several different cell types.
Once committed to a somatic cell lineage, these cells were considered incapable of differentiating to cells of a different embryonic lineage. However, the concept of uncommitted stem cells and stem cell plasticity has changed our understanding of stem cell commitment and introduced the theory that stem cells are affected by local environmental conditions. Some postulate fusion of marrow-derived cells with recipient cells to explain the alteration in differentiation markers rather than true stem cell plasticity.
The hematopoietic stem cell (HSC) is the most well characterized adult stem cell. Attempts to purify HSCs on the basis of cell surface markers such as CD34, CD38, and CD45, have defined other multipotential stem cell subsets present in bone marrow. Mesenchymal stem cells (MSCs) give rise to bone, muscle, neural cells, cartilage, and hematopoietic stromal cells; multipotential adult progenitor cells (MAPCs) generate cell lineages of mesodermal, endodermal, and ectodermal origin. The CD34 þ stem cells generate HSCs and blood vessel endothelial cells. The CD45þ ClqRpþ CD34þ / cells generate HSCs and liver stem cells while MSCs and MAPCs are CD45 . The finding of such heterogeneous stem cell populations in bone marrow has led to the burgeoning field of stem cell manipulation for regenerative medical therapies. While the potential therapeutic use of embryonic stem cells has raised enormous public controversy, there are far fewer ethical concerns surrounding the use of adult stem cells.
The clinical use of allogeneic (donor-derived) hematopoietic stem cell transplantation (HSCT) for acquired disorders such as aplastic anemia and the leukemias is well known. Allogeneic HSCT is also curative for congenital and hereditary disorders of hematopoietic origin including red cell disorders (thalassemia), lymphocyte disorders (the immune deficiency syndromes), neutrophil disorders, and macrophage/monocyte disorders such as the storage diseases and other metabolic or enzyme deficiencies. MAPCs proliferate extensively, seemingly without senescence, and are an ideal source for stem cell therapy. Clinical trials of allogeneic transplantation are now being assessed to determine if MSCs and MAPCs can be successfully transplanted to treat hereditary disorders such as osteogenesis imperfecta.
Autologous bone marrow MSCs expanded in culture and embedded in collagen have been successfully transplanted into osteoarthritic joints. CD34 þ cells and MSCs differentiating into cardiac myocytes have been incorporated into the heart as therapy for myocardial infarction. Studies of sex-mismatched allogeneic bone marrow recipients have revealed donor-derived endodermal tissue cells such as hepatocytes and cholangiocytes. Bone marrow stem cells can be readily mobilized into the circulation using chemotherapeutic agents and/or cytokines and peripheral blood stem cell (PBSC)-derived ectodermal and endodermal tissues have also been demonstrated in sex-mismatched transplantation recipients. The availability of PBSCs circumvents the need for bone marrow harvesting and allows for enrichment by apheresis techniques. Umbilical cord blood is also rich in HSCs and is ideally suited for banking.
Stem Cell Function in the Normal Lung
The lung is composed of a conducting airway and a gas-exchange system with many different cell types including ciliated cells, mucus cells, basal cells, Clara cells, type I and II pneumocytes, and pulmonary neuroendocrine cells. Lung development and repair of injury require a balance between proliferation and differentiation of epithelium. The study of lung stem cells is frustrated by slow cell turnover and difficulty with stem cell isolation; however, several endogenous lung progenitors have been identified. In fetal lung, signals from mesenchymal cells play an important role in normal branching morphogenesis and pulmonary epithelial differentiation. Late in fetal development and in postnatal life, there is lineage restriction between bronchial and alveolar epithelial cells.
The most distinctive cell type in the tracheobronchial epithelium, the ciliated cell, is a terminally differentiated cell largely incapable of cell division. Progenitor cells, such as Clara cells, must differentiate to the ciliated phenotype. The Clara cells of the lung are nonciliated secretory cells characterized by apical cytoplasmic projections and abundant endoplasmic reticulum and secretory granules. Found throughout the respiratory system, Clara cells are the most actively dividing cells in the prenatal and postnatal lung and are the progenitors of themselves and of the ciliated cells in the bronchioles. A subset of Clara cells is resistant to airway pollutants and shows multipotent cell differentiation. Clara cells produce the anti-inflammatory protein CC10 in abundance and numerous cytochrome enzymes to inactivate carcinogens. Under oncogenic stimulation, the Clara cell induces expression of the cyclindependent kinase inhibitors to reduce proliferation. This prevents tumorigenesis. However, if the oncogenic stimulus persists, this mechanism is insufficient and the lung epithelium is transformed. Basal cells and pulmonary neuroendocrine cells affect the growth and differentiation of lung epithelium and may be recruited to act as stem cells.
Type I and type II pneumocytes make up the alveolar epithelium. Type I cells are flat with cytoplasmic projections and a protuberant nucleus. They do not divide. Type II cells are cuboidal, metabolically active cells with abundant cytoplasmic organelles. They secrete surfactant, proliferate after injury, and act as progenitor cells for type I and type II pneumocytes by transdifferentiating to an intermediate morphology and then differentiating to the alveolar type I phenotype with cytoplasmic projections, prominent nucleus, and loss of lamellar bodies. Type II pneumocytes are the progenitors responsible for repair of injured distal lung. The neuroendocrine cells are frequent in the developing lung and play a major role in airway growth and development. In the adult lung they form o1% of epithelial cells usually present in neuroepithelial bodies. They are unlikely to be stem cells but secrete regulatory factors for epithelial cell renewal.
To characterize pulmonary stem cells further, gene promotor analyses of the surfactant protein genes and the Clara cell protein CC10 have identified the NKX2 homeodomain gene thyroid transcription factor-1 (TTF-1) as critical for lung formation and epithelial cell differentiation. Expression of TTF-1 is increased in lung injury and repair. The lung maintains regulatory pathways to counteract transformation and oncogenesis. Indeed most small cell carcinomas and adenocarcinomas express TTF-1. In addition, fibroblast growth factor (FGF) signaling regulates these putative alveolar stem cells and FGF genes may provide therapeutic targets to induce lung repair and regeneration. The murine models of Krause et al. demonstrated that male bone marrowderived cells were capable of differentiation into bronchiolar epithelium and type II pneumocytes following transplantation into lethally irradiated female mice. Furthermore, mesenchymal stem cells appeared to differentiate into distal alveolar epithelial cells in bleomycin-treated mice.
Stem Cells in Respiratory Diseases
Asthma and Allergy
Bronchial myofibroblasts represent an important therapeutic target for asthma because of their role in the genesis of subepithelial fibrosis in airway remodeling. CD34þ hematopoietic precursor cells of myeloid lineage have been found in acute allergic inflammation in allergic rhinitis, asthma, and eczema. Indeed, allergen exposure induces the accumulation of CD34þ fibroblasts that differentiate into myofibroblasts. Thus, hematopoiesis and migrational pathways of hematopoietic cells are targets for treatment with corticosteroids, leukotriene inhibitors, blockade of the eosinophil-active cytokine, interleukin-5, antihistamines and chemokine receptor antagonists, which may modulate progenitor cell influx.
Idiopathic Pulmonary Fibrosis
Idiopathic pulmonary fibrosis (IPF) is a devastating disease for which no effective therapy exists. There is inexorable airspace obliteration and the mean time from diagnosis to death is 5 years. Alveolar type II pneumocyte injury and apoptosis is an important early feature in the pathogenesis of IPF. On pathological examination, there are areas of active fibrogenesis with fibroblast foci and extracellular matrix deposition. The number and appearance of fibroblastic foci on lung biopsy specimens can predict length of survival. Fibroblasts are assumed to arise from intrapulmonary cells, but circulating blood cells resembling fibroblasts are recruited to areas of lung injury. These cells migrate to sites of wound healing and serve as a source of fibroblasts and myofibroblasts that normally participate in the repair process. Mice models of pulmonary fibrosis, using bleomycininduced lung injury and radiation fibrosis models, have shown that the collagen expression in fibrotic lung is due mainly to bone marrow-derived precursor cells strongly suggesting a direct role for these cells in the pathogenesis of fibrosis.
The influx of these bone marrow-derived cells into the lung suggests fibrosis is mediated by chemokines in a similar fashion to the recruitment of leukocytes in pulmonary inflammation. Fibroblasts express the chemokine receptors CXCR4 and CCR7 and migrate towards stromal cell-derived factor 1 and secondary lymphoid tissue chemokine. The source of these chemokines is likely to be local lung fibroblasts or endothelial cells as well as bone marrow-derived macrophages that precede the arrival of the fibrocytes into areas of fibrosis. Idiopathic pulmonary fibrosis is a disease of abnormal wound repair and remodeling rather than inflammation, and treatment should be directed at inhibiting the fibroproliferative response. Perhaps there is a role for allogeneic hemopoietic stem cell transplantation to change the phenotype of the marrow-derived fibroblasts.
Identifying lung stem cells would provide an ideal target for gene therapy providing the ability to transfer the cystic fibrosis transmembrane regulator (CFTR) gene once. Gene therapy by topical delivery to the airway of patients with cystic fibrosis has been disappointing since vectors are inefficient and mucous barriers prevent transfection of airway cells. Even if transfection does occur, the cells are terminally differentiated and recurrent treatment is required. Indeed the CFTR gene is predominantly expressed in the submucosal glands, which are largely inaccessible to topical gene therapy.
Gene therapy aimed at hemopoietic stem cells has been successful in children with other single gene disorders. It is speculated that damaged lung attracts hemopoietic stem cells and in cystic fibrosis, inflammation and tissue injury occur early. However, retroviral vectors insert themselves randomly and insertional mutagenesis remains a risk (two patients treated for adenosine deaminase deficiency have developed leukemia).
If only 10% of cells need to be corrected for the chloride defect in cystic fibrosis to be reversed, then the bone marrow-derived engraftment rate may be adequate. However, if the sodium defect requires correction, 100% of the cells need to be corrected and this is unlikely to occur from a bone marrowderived cell.
Lung cancer patients often have multiple lesions at different stages of tumorigenesis with both stem cells and differentiated cells escaping repair and control mechanisms. Either a single common transformed stem cell gives rise to different tumor histologies, or multiple cells within a field are transformed. Cytogenetic studies of lung cancers frequently reveal abnormalities in the region of the cellcycle-regulating genes such as the cyclin-dependent kinase inhibitors.
Autologous peripheral blood stem cell rescue to allow for increased chemotherapeutic intensity is used to treat numerous malignancies. HSCs are collected by peripheral blood apheresis following chemotherapy and cytokine stimulation. CD34þ cell selection reduces the chance of malignant contamination of the stored aliquot. In small cell lung cancer, such chemotherapeutic intensification with stem cell rescue is feasible despite grade 4 hematological toxicity.
Respiratory Complications of HSCT
Approximately 50% of patients having HSCT will develop respiratory complications with 40% mortality. Both chemotherapy and radiation predispose to idiopathic pneumonitis in which no infectious causative agent can be found. Diffuse alveolar hemorrhage, characterized by severe thrombocytopenia, is also of obscure etiology and may coexist with bacterial, viral, or fungal pneumonia. Pulmonary veno-occlusive disease (VOD) presents with dyspnea, hypoxemia, pulmonary hypertension, and right heart failure. Like hepatic VOD, its pathogenesis is unknown although conditioning agents and viral infections are implicated.
Patients receiving total body irradiation for bone marrow transplantation or lung irradiation for esophageal or lung cancer frequently develop pulmonary damage. Acute pneumonitis and progression to fibrosis is dependent on the radiation dose, fraction size, and volume of lung irradiated. Treatment of acute pneumonitis with corticosteroids may not prevent the fibrotic changes that occur 6–24 months later.
Normal lung defense mechanisms to prevent infection include alveolar macrophages as well as the influx of neutrophils to eradicate bacteria and fungi. Cell-mediated immunity relies on adequate numbers of lymphocytes and dendritic cells to act as antigenpresenting cells. Post-transplantation, patients have marked pancytopenia and severe combined immune deficiency and are prone to respiratory infections. Neutrophil recovery occurs in 2–3 weeks but donorderived macrophages take 3–4 weeks to repopulate the lungs and may not function normally for 2–3 months. Lymphocytes reach normal numbers by 3 months post-transplantation but a return to normal immunoglobulin levels and cell-mediated immune function may take 12 months. The presence of acute or chronic graft-versus-host disease further delays immune recovery. Useful investigations include chest radiography, bronchoscopy with bronchoalveolar lavage, lung biopsy, and polymerase chain reaction techniques to detect viral antigenemia.
Therapy for bacterial pneumonia is frequently empiric and must include antipseudomonal antibiotics. Empiric antifungal therapy with amphotericin B is added for prolonged fever. Cytomegalovirus (CMV) causes 40% of pneumonias in HSCT and may occur in as many as 20% of all transplant patients. Prophylactic therapy with acyclovir and gancyclovir is recommended where the donor or recipient is CMV seropositive prior to transplantation. CMV-negative blood products should be given where possible. The addition of CMV hyperimmune globulin is more controversial. Invasive pulmonary aspergillosis or candida pneumonia occur in 5–10% of patients and require treatment with amphotericin B. The routine use of prophylaxis has made infection with Pneumocystis carinii relatively rare. Following engraftment, cotrimoxazole prophylaxis should be re-instituted and continued until immune function has returned to normal.
The outcome of patients requiring transfer to the intensive care unit for progressive respiratory failure during transplantation is poor.
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Hemopoietic stem cells (HSCs) – capable of differentiation into all blood elements and characterized by the expression of the cell surface marker CD34
Mesenchymal stem cells (MSCs) – capable of differentiating into osteoblasts, chondroblasts, fibroblasts, and adipocytes
Multipotent adult progenitor cells (MAPCs) – differentiate into most somatic cell types including lung
Stem cell – an undifferentiated cell capable of longterm self-renewal and multilineage differentiation
Stem cell fusion – fusion of 2 different somatic cells to form a cell with 2 nuclei
Transdifferentiation/stem cell plasticity – committed stem cell capable of differentiation into cells of a different tissue