There has been increasing excitement over the last few years with the suggestion that exogenous stem cells may offer new treatment options for a wide range of diseases. Within respiratory medicine, these cells have been shown to have the ability to differentiate and function as both airway and lung parenchyma epithelial cells in both in vitro and increasingly in vivo experiments. The hypothesis is that these cells may actively seek out damaged tissue to assist in the local repair, and the hope is that their use will open up new cellular and genetic treatment modalities. Such is the promise of these cells that they are being rushed from the benchside to the bedside with the commencement of early clinical trials. However, important questions over their use remain and the field is presently littered with controversy and uncertainty. This review evaluates the progress made and the pitfalls encountered to date, and critically assesses the evidence for the use of stem cells in lung disease.

INTRODUCTION

Lung disease is common and debilitating, with chronic respiratory conditions responsible for a large and increasing global burden of disease. The destruction of the gas exchange surfaces of the lung, be it by fibrosis in idiopathic pulmonary fibrosis or damage to the alveoli walls in emphysema, causes increasing dyspnoea, morbidity and eventually death. Medical therapies for chronic lung diseases have restricted efficacy and lung transplantation is often the only effective treatment.

Although the endogenous repair and continual tissue replacement by local stem cells is effective in some organs, such as the skin, this mechanism is not as effective in the respiratory tract and is insufficient to prevent the many progressive respiratory diseases [1]. The majority of alveolar development is in a well-defined postnatal period and there is no significant restoration of the gas exchange surface if it is disrupted by disease following this period [1]. In view of this, groups have been investigating the possibility of enhancing stem cell repair by the use of exogenous stem cells.

In addition to the repair of chronic lung diseases, there is also hope that recovery and outcome could be improved by the use of stem cells in acute lung conditions [2]. ARDS (acute respiratory distress syndrome) could be one such candidate, where, despite present treatment, there remains significant morbidity and mortality [3].

Gene therapy for genetic diseases has so far been hampered by the lack of a suitable vector, but exogenous stem cell delivery also has the potential to open up new avenues of delivering genes. CF (cystic fibrosis) and α1-antitrypsin deficiency are caused by recessive gene disorders and could be possible diseases targeted for this therapy.

STEM CELLS

Stem cells divide asymmetrically and have the ability to both self-renew and produce more differentiated cell progenitors. They characteristically have an unlimited proliferative capacity and usually divide infrequently in the steady state (Figure 1). Stem cells are typically categorized into embryonic stem cells and adult stem cells, with varying differentiation abilities or potencies. Embryonic stem cells are derived from blastocysts in a developing embryo and have the ability to differentiate into all cells of the three germ layers (endoderm, mesoderm and ectoderm). This ability is referred to as pluripotency. Conversely, adult stem cells are found in adult tissues, commonly in discrete niches, and are thought to act to help repair damaged tissues by replenishing specialized cells. The differentiated progeny of these adult stem cells are classically thought to be restricted to a specific cell lineage appropriate to its location. As such, adult stem cells are either multipotent, with an ability to differentiate into a limited range of cells (e.g. adult haematopoietic stem cells), or unipotent, with an ability to generate only one type of cell (e.g. type 2 alveolar cell).

An overview of stem cell properties including self-renewal and potency

Figure 1
An overview of stem cell properties including self-renewal and potency
Figure 1
An overview of stem cell properties including self-renewal and potency

ENDOGENOUS RESPIRATORY STEM CELLS

The endogenous stem cells of the respiratory system are complex. It has become clear that different regions of the respiratory tract rely on different populations of cells for repair; however, the precise mechanism and processes have not yet been fully elucidated [4,5]. The endogenous stem cells are more easily understood by dividing the respiratory system into the large airways, small airways and lung parenchyma.

The trachea and primary bronchi contain ciliated cells, Clara-like cells, and basal, neuroendocrine and submucosal cells (Figure 2). Various experiments have been performed to help to identify the stem cell of the proximal airway. Experiments looking at the normal turnover of the airway epithelium have included the use of [3H]thymidine labelling [6], rat tracheal xenograft models and in vitro cell cultures of air–liquid interfaces [7]. The response of the epithelium to injury has also been assessed. In a naphthalene injury model, a Cre recombinase under the control of basal cell promoter [K14 (keratin 14)] was used to express β-galactosidase in K14-positive basal cells and their descendants. After naphthalene injury, patches of β-galactosidase-positive epithelium containing basal, Clara-like and ciliated cells were seen [8]. These studies suggest that the basal cells in the proximal airway are able to function as local endogenous stem cells both in the steady state and in response to injury. Nevertheless, a role for Clara cells as stem cells in the proximal airways has not been ruled out.

Schematic representation of the cell types involved in the respiratory tract

Figure 2
Schematic representation of the cell types involved in the respiratory tract

(a) Section of the tracheal epithelium stained for K14, with a ciliated epithelial cell (black arrow), a K14-positive basal cell (white arrow), a K14-positive submucosal gland duct cell (asterix) shown. Magnification, ×20. (b) Section of bronchiolar epithelium stained for CGRP (calcitonin gene-related peptide; red; pulmonary neuroendocrine cells) and CCSP (green; Clara cells). Magnification, ×40. (c) Section of lung parenchyma stained for TTF-1 (thyroid transcription factor-1; green; type 2 cells) with 4′,6-diamidino-2-phenylindole (DAPI; blue) nuclear counterstaining. The red colour denotes autofluorescence of the alveolar wall structures. Magnification, ×40.

Figure 2
Schematic representation of the cell types involved in the respiratory tract

(a) Section of the tracheal epithelium stained for K14, with a ciliated epithelial cell (black arrow), a K14-positive basal cell (white arrow), a K14-positive submucosal gland duct cell (asterix) shown. Magnification, ×20. (b) Section of bronchiolar epithelium stained for CGRP (calcitonin gene-related peptide; red; pulmonary neuroendocrine cells) and CCSP (green; Clara cells). Magnification, ×40. (c) Section of lung parenchyma stained for TTF-1 (thyroid transcription factor-1; green; type 2 cells) with 4′,6-diamidino-2-phenylindole (DAPI; blue) nuclear counterstaining. The red colour denotes autofluorescence of the alveolar wall structures. Magnification, ×40.

In the smaller airways, there are no basal cells and the epithelium is made up of Clara, ciliated and neuroendocrine cells. In this region, the evidence from similar studies suggests subpopulations of Clara cells, including variant Clara cells, as the cells involved in proximal small airway repair and BASCs (bronchioalveolar stem cells) at the bronchioalveolar junction as the cells involved in distal epithelial reconstitution after injury [8,9]. BASCs have been demonstrated to express both alveolar [SP-C (surfactant protein C)] and bronchiolar [CCSP (Clara-cell-specific protein)] markers. Isolation of these cells has demonstrated their stem cell properties and their ability to differentiate into Clara cells and type 2 pneumocytes in vitro. In vivo, they appear to have a role both in bronchiolar and alveolar repair and homoeostasis [9].

The alveolar epithelium consists of thin-walled type 1 pneumocytes involved predominantly with gas exchange, and cuboidal type 2 pneumocytes concerned with many of the metabolic functions in the airspace. [3H]Thymidine labelling studies have suggested the type 2 cell as the local regenerating stem cell able to produce type 1 cells [10]. Furthermore, histological studies in ARDS have shown that there is an early loss of type 1 cells following injury, and that repair of the epithelium is preceded by a proliferation of type 2 cells [11]. The relative contributions and relationship between type 2 cells and BASCs in alveolar homoeostasis and repair has not been fully elucidated. There are also resident lung side population cells, which appear to have both mesenchymal and epithelial potential [12,13]. Side population cells can be characterized by their ability to efflux Hoescht dye. Their role in endogenous repair is not fully understood.

STEM CELLS FOR CELLULAR THERAPY

Early promise: epithelial engraftment

The idea of enhancing repair by the use of exogenous stem cells has grown over the last decade. Adult stem cells are attractive for several reasons as they avoid many of the ethical and moral problems that surround the use of embryonic stem cells. Furthermore, they allow for the possibility of expansion of a patient's own cells before transplantation. This delivery of autologous adult stem cells would also help prevent immunological rejection by the host [14]. Bone marrow stem cells are the best defined adult stem cell population. They are easily accessible and well understood. The bone marrow contains HSCs (haematopoietic stem cells), which can differentiate into all the mature blood cells and endothelial cell progenitors, and MSCs (mesenchymal stem cells), which differentiate into the stromal supporting tissue, including fat, bone and cartilage [15].

The concept that adult stem cells were lineage-restricted limited their potential for use as a cellular therapy in lung disease. However, exciting work at the beginning of this century suggested that the lineage-specificity of adult stem cells was not as rigid as thought previously. An early study showed that a single labelled HSC was able to reconstitute the bone marrow of an irradiated mouse, but also appeared to contribute to several organs including the lung [16]. Immunohistochemistry was used to show cellular colocalization of markers of both the donor bone marrow cell (Y chromosome) and markers, suggesting a type 2 epithelial fate. Studies such as this developed the concept of adult stem cell plasticity, whereby adult stem cells were able to cross lineage barriers and adopt the functional phenotypes of other tissues (Figure 3).

Traditional compared with the plasticity view of adult stem cells

Figure 3
Traditional compared with the plasticity view of adult stem cells

The traditional view is that adult stem cells are restricted in lineage such that bone marrow stem cells are only able to produce differentiated cells of a haematopoeitic lineage. However, recent evidence suggests these adult stem cells may have more plasticity (plasticity view) than thought previously and an ability to cross lineage boundaries to produce differentiated cells corresponding to the different germ layers (mesoderm, ectoderm and endoderm).

Figure 3
Traditional compared with the plasticity view of adult stem cells

The traditional view is that adult stem cells are restricted in lineage such that bone marrow stem cells are only able to produce differentiated cells of a haematopoeitic lineage. However, recent evidence suggests these adult stem cells may have more plasticity (plasticity view) than thought previously and an ability to cross lineage boundaries to produce differentiated cells corresponding to the different germ layers (mesoderm, ectoderm and endoderm).

These experimental findings led to the notion that adult bone marrow stem cells could, under certain conditions, engraft in damaged respiratory tissue and ‘transdifferentiate’ into various epithelial cells, such as the type 2 pneumocyte. A host of publications ensued using animal models and similar techniques of infusions of labelled bone marrow stem cells into recipient mice ([1727], but see [27a]). The donor cells were distinguished by phenotypic [β-galactosidase or GFP (green fluorescent protein)] or genotypic (Y chromosome) differences to cells originating from the host (Figure 4). A proportion of these donor-derived cells was then shown to express markers of lung epithelial cells. These experiments showed great variability in the type of bone marrow stem cells used {e.g. single bone marrow cells [16,28], whole bone marrow [18,2123,26,29,30], HSCs [26], MSCs [17,19,24,25], side population cells [20] and MAPCs (multipotent adult progenitor cells) ([27], but see [27a])}. Furthermore, the recipient was exposed to varying conditions, including the use or type of lung injury (e.g. radiation [18,19,26], bleomycin [17,23-25,29], lipopolysaccharide [21], elastase [22] and paracetamol [30]) and the use or amount of irradiation pre-infusion of the transplanted cells. As such, significant variations in the results were achieved, with some groups showing no contribution of adult bone marrow cells to lung epithelial cells [28] and others showing up to 20% engraftment as type 2 cells [16]. Contributions to different lung cell types were also described, including type 2 and type 1 pneumocytes [17], tracheal epithelial cells [20], fibroblasts [2931] and endothelial cell progenitors [32].

Bone marrow stem cell transplantation into recipient mice

Figure 4
Bone marrow stem cell transplantation into recipient mice

Transplanted bone marrow stem cells are labelled (GFP and Y chromosome), allowing detection of donor-derived cells in the host. These donor cells are found to have morphological and histochemical features of respiratory epithelial cells. The histology section shows labelled GFP-positive bone marrow cells (brown) engrafted into irradiated lung parenchyma with morphological characteristics of pneumocytes. Magnification, ×20.

Figure 4
Bone marrow stem cell transplantation into recipient mice

Transplanted bone marrow stem cells are labelled (GFP and Y chromosome), allowing detection of donor-derived cells in the host. These donor cells are found to have morphological and histochemical features of respiratory epithelial cells. The histology section shows labelled GFP-positive bone marrow cells (brown) engrafted into irradiated lung parenchyma with morphological characteristics of pneumocytes. Magnification, ×20.

In addition to the experimental variability, the exact mechanism of the phenotypic conversion of bone marrow stem cells has also not been elucidated. Fusion of donor-derived and host epithelial cells was suggested as a mechanism for colocalization of histochemical markers. Embryonic cells and adult somatic cells had been shown to fuse in vitro causing a change in the phenotypic properties of cells [33]. If fusion is the underlying mechanism, this may not alter the validity of engraftment-type studies as it is the functional properties of the resulting cells that will be more relevant to potential clinical applications. The concern, however, is that these fused cells may have a higher potential for malignant transformation [34]. In vivo, fusion has been shown to be important in engraftment of some organs (e.g. the liver) [35]; however, investigations of cell ploidy in several experiments have suggested that engraftment occurs without fusion [34]. Furthermore, an elegant set of experiments using a Cre/lox system with β-galactosidase and GFP transgenic mice illustrated that this was not the mechanism in lung engraftment [36]. Other possible mechanisms to explain the presence of donor-derived cells in host tissue include the differentiation of a small population of undifferentiated cells in the bone marrow and the phenotypic conversion of the adult bone marrow stem cells [34]. It is likely that any such transdifferentiation is mediated by the immediate environment with soluble factors and cell–cell contact playing an important role.

Despite the uncertainty, similar results were obtained in human subjects that had undergone sex-mismatched transplants. Male patients who had received female lung transplants were shown to have a proportion of male lung epithelial cells on lung biopsy [3740]. Conversely, female patients with male bone marrow allograft transplants were similarly shown to have a degree of pulmonary chimaerism, with Y-chromosome-containing epithelial and endothelial pulmonary cells [4043]. As with the animal results, there was significant variability between the studies with regards to the reported level of chimaerism, which varied between 20% [37] and 0% [44]. A quantative analysis suggested that the greatest chimaerism was observed in areas of significant injury [37]. In the healthy upper respiratory tract, sampled by nasal brushings, there was no reported chimaerism [44]. This relationship with injury was also repeated in animal models whereby bone-marrow-derived engraftment was increased if the respiratory system was damaged before transplantation [17,2126,29,30]. The suggestion from these studies was that the adult stem cells in the bone marrow were actively recruited by, and homed to, injured tissues.

Soluble factors or chemokines are implicated in the homing of bone marrow stem cells to the damaged lung tissue. Damaged lung produces chemokines such as CXCL12 [SDF1α (stromal-derived factor 1α)] and SLC (secondary lymphoid chemokine), which are ligands for CXCR4 and CCR7 receptors found on certain bone-marrow-derived stem cells [23,45]. This relationship has been shown in vitro whereby injured, but not normal, lung caused a proliferation and migration of MSCs across a transwell membrane [25].

Present state: reassessment and controversy

Over the last few years, the enthusiasm and optimism for the use of bone-marrow-derived cells as a cellular therapy in lung disease has been replaced by an appreciation of the need for caution and reassessment. A study by the authors of one of the original engraftment articles used a lineage-specific reporter system, whereby GFP expression in donor-derived bone marrow cells was linked to a surfactant protein promoter [46]. Consequently, GFP-positive cells would only be seen if they were both donor-derived and functioned as a type 2 pneumocyte. In this experiment, flow cytometry, histology and molecular methods were unable to detect GFP-positive cells, suggesting the lack of lung epithelial engraftment. A similar negative result was reported in a related paper suggesting that colocalization of epithelial and donor cell markers was artifactual and primarily as a result of overlapping cells [47]. This error was evident and could be removed by the use of a deconvolution or confocal microscope. Furthermore, recent reports [48] have cast doubt on the characterisation of certain groups of transplanted cells (MAPCs) in earlier experiments showing plasticity.

Nevertheless, despite these negative results, further studies have suggested some engraftment of bone-marrow-derived cells as pulmonary epithelial cells, as determined using advanced techniques such as deconvolution/confocal microscopy [26], flow cytometry [23] or laser capture microdissection and PCR or real-time PCR [37].

The present consensus appears to be that some engraftment of bone marrow stem cells as pulmonary epithelial tissue does occur, but at much lower rates than was thought previously [49]. Indeed, even the authors of the original papers that suggested up to 20% bone marrow engraftment as lung epithelium have re-assessed their findings. They have now concluded that engraftment is more likely to be 0.01-0.1% [50,51] and that the method of detection of donor-derived epithelial cells used (immunohistochemical co-localization of epithelial markers and Y chromosome with fluorescent in situ hybridization) was likely to have caused an overestimation in the earlier studies [51]. This lower level of epithelial engraftment appears too small to be a viable option of cellular therapy [51,52], whereby transplanted adult stem cells alone could be used to repair damaged lung by engrafting as replacement epithelial cells. The variability in results is due partly to different experimental conditions with different donor cells, injury and the like, as described above. Nevertheless, it is clear that the method of detection of the donor-derived epithelial cells is crucial and that much of the variability can be attributed to the over- or under-estimation inherent in many of the techniques. In order to overcome these discrepancies, methods of detection of small numbers of engrafted cells will have to improve. This should include advanced methods of photomicroscopy (e.g. confocal) or sampling (e.g. single-cell laser capture) to help avoid errors such as those caused by overlapping cells. These techniques should also be combined with others, including flow cytometry, and PCR and real-time PCR for DNA/RNA analyses. Ideally, functional outcomes should also be included in future studies [49]. At present, there is a great variability in the donor cells used in experiments. This is likely to increase in the future with the realization of other sources of stem cells, including umbilical cord blood and adipose tissue, and the increase in research with embryonic stem cells. The donor cells will need to be very accurately defined and possibly centrally sourced to allow accurate comparisons of like studies.

Repair of injury

Despite the re-evaluation of the promise of adult stem cells in lung repair, there is evidence to suggest that transplanted cells post-lung injury may have some effect on the disease process. Systemic MSC administration following bleomycin-induced lung fibrosis resulted in a reduction in fibrosis and collagen deposition in murine models [24]. An animal model of emphysema, with intranasal elastase, was used to demonstrate an improvement in outcome when endogenous bone marrow stem cells were augmented by all-trans retinoic acid or G-CSF (granulocyte colony-stimulating factor) [22]. Finally, suppressing a normal bone marrow response with busulphan or radiation led to an increased mortality in mice subjected to lung insults, which could be reversed with an infusion of bone marrow stem cells [25,26]. These studies suggest that the endogenous bone marrow does contribute to the disease phenotype after lung injury and that this can be manipulated by bone marrow stem cell transplantations. It is likely, however, that this effect is mediated not by cellular replacement, but by the paracrine release and delivery of cytokines and growth factors, producing an environment conducive to repair. Although the exact mechanism of improvement in these studies has not been elucidated, the promise is so great that the use of autologous bone marrow stem cells has already reached clinical trials in cardiology [5355]. Transplanted cells have been infused acutely following myocardial infarction and in chronic ischaemic heart failure. At present, these trials are still being evaluated, but tend towards a mild improvement in heart function [56].

STEM CELLS FOR GENETIC THERAPY

The property of stem cell homing to damaged tissues and possible engraftment as organ-appropriate cells has potential uses beyond simple cellular replacement and repair. Genetic therapy is based on the idea of inserting a normal gene into the genome to replace an abnormal gene which causes disease. There has been great promise for the use of this technique in single gene disorders; however, progress has been hampered by methodological problems such as delivering the therapy to the correct location, overcoming the immunological and inflammatory response to the delivery vector, and achieving stable genetic transfer [57]. Stem cells are attractive vectors as they are able to self-renew. In addition, delivery may be optimized by the homing potential of these cells to sites of disease, and the possibility of altering adult stem cells ex vivo before their autologous redelivery will avoid many of the immunological problems associated with the gene therapy vector. However, there are those who suggest that production of a new protein, as the end result of genetic correction, will lead to its recognition as non-self by the immune system [58].

The proof-of-concept of achievable long-term genetic alteration using this technique was provided by an experiment whereby bone marrow stem cells were genetically altered with retroviral transduction to express GFP before transplantation [59]. These transplanted cells were able to engraft as lung epithelial cells and maintained their long-term GFP transgene expression for at least 11 months. A further example illustrating the potential of adult stem cells in genetic therapy was provided by bone marrow transplantation in the FAH (fumarylacetoacetate hydrolase)-deficient mouse [60]. These mice serve as a model of fatal hereditary tyrosinaemia type 1 and die of liver and renal impairment unless treated. Transplantation of labelled bone marrow cells in these mice caused hepatic engraftment as wild-type hepatocytes, which were functionally active and were able to prevent the metabolic liver disease. In this situation, genetic therapy was very effective as the transduced cells had a selective advantage in the FAH-deficient liver.

CF is one of the most common fatal inherited diseases and has no cure. It is caused by a defective CFTR (CF transmembrane conductance regulator) gene which may benefit from genetic therapies. Unfortunately, despite extensive genetic research using viral and non-viral vectors, no suitable genetic treatment has been described to date. The use of adult HSCs provides a possible mechanism of genetic therapy which may bypass some of the problems encountered so far. As a proof-of-principle, MSCs have been taken ex vivo from patients with CF and transduced with the functional CFTR gene. These corrected stem cells were then co-cultured with CFTR-deficient epithelial cells at an air–liquid interface. The MSCs were able to differentiate into airways epithelium and had a functional effect contributing to cAMP-stimulated chloride secretion [61]. This work then led to in vivo studies [52,62], whereby wild-type bone marrow stem cells were transplanted into CFTR-knockout mice. There was some encouragement from these results with evidence of CFTR mRNA and the demonstration of a small restoration of epithelial chloride current by in vivo potential difference analyses in the nasal and gut epithelium. Nevertheless, these changes were small and lung epithelial engraftment of these bone marrow cells was a very rare occurrence (0.025% [62]; <0.01% [52]) when an approx. 10% correction of defective CFTR is needed to allow normal chloride currents [63].

As well as replacing faulty genes, genetic therapy of the stem cell may also be used to enable increased targeted expression of growth factors and the like to help with repair. EPCs (endothelial progenitor cells) are derived from the bone marrow and are thought to function to repair and regenerate blood vessels. These bone-marrow-derived progenitor cells have been shown to engraft into areas of vascular injury and prevent monocrotaline-induce pulmonary arterial hypertension in the rat [32]. When these cells were genetically transduced with human eNOS (endothelial nitric oxide synthase), established disease could also be reversed and survival improved [32]. Such has been the success of these animal experiments, that a phase 1 safety trial [PHACeT (Pulmonary Hypertension: Assessment of Cell Therapy)] has recently begun. In this clinical trial, patients with refractory pulmonary arterial hypertension received autologous EPCs which had been transduced with eNOS ex vivo [49].

POSSIBLE NEGATIVE EFFECTS

In contrast with their apparent positive effect in epithelial repair in injury, bone marrow stem cells have also been implicated in worsening disease. Compared with epithelial engraftment, there has been more robust evidence for the contribution of bone-marrow-derived cells as fibroblasts in disease [2931]. There is an increasing appreciation in fibrosis that the effector cell, the fibroblast, may not just be of resident tissue origin [64]. There has been a description of circulating fibroblasts, termed fibrocytes, for many years [65]. These cells, which are thought to have a role in fibrotic lesions, also appear to have a bone marrow derivation and typically express cell-surface markers typical of HSCs (e.g. CD34 and CD45). In an allergic asthma animal model, labelled fibrocytes were shown to participate, as myofibroblasts, in bronchial subepithelial fibrosis, which is thought to be one of the mechanisms contributing to airway remodelling in asthma [31]. Similar evidence is being collected for a role of these circulating fibrocytes in the development of pulmonary vascular remodelling in chronic pulmonary hypertension. In this condition, the cells are thought to contribute both to the vascular fibrosis directly, by the production of collagen, and to have a paracrine effect, releasing cytokines that cause the activation of resident fibroblasts [66,67].

A significant role for the bone marrow has also been suggested for parenchymal fibrosis. Research using a bleomycin model of lung fibrosis has shown that up to 80% of the collagen-producing fibroblasts in the lung have a bone marrow origin [23]. A separate study showed that the addition of intravenous MSCs could also contribute to fibroblasts and myofibroblasts in lung fibrosis models [29]. The negative effect that these bone-marrow-derived cells may have on fibrotic disease was demonstrated in vivo, whereby removal of the chemotactic gradient, with antibodies to CXCL12, reduced the bone-marrow-derived component to lung fibroblasts and the overall fibrotic response after injury [45].

There is a concern that the defining characteristic of a stem cell, unlimited self-renewal, leaves this cell an attractive candidate for malignant change [68]. Evidence is available for the development of karyotype abnormalities of bone marrow stem cells during in vitro passage [69]. Furthermore, MSC delivery in a mouse model has been associated with sarcoma development in vivo [70,71]. Bone marrow stem cells have also been implicated in the development of a gastric carcinoma in mice chronically infected with Helicobacter felis [72]. In this model, the tumour appeared to arise from GFP-labelled bone marrow cells. Bone marrow stem cells have also been shown to contribute to the stromal desmoplastic reaction of tumours in several similar lineage-labelling experiments [73,74]. There is a belief that this tumour stroma is directly involved in determining the progression and metastatic ability of the tumour [75,76]. Furthermore, subcutaneous injection of MSCs with colonic tumour cells has been shown to increase proliferation and metastasis of a subcutaneous tumour model [77]. However, the situation appears more complicated as this effect is not uniform, with systemic MSCs leading to an improvement in a Kaposi's sarcoma model [78]. The effects of bone marrow stem cells on tumour stroma appear dependent on tumour model and location [79].

Genetic therapy has an inherent need for caution. A clinical trial several years ago in ten children with X-SCID (X-linked severe combined immunodeficiency) used retroviral transfer of the IL2 (interleukin 2) receptor common γ-chain into ex vivo bone marrow cells [80]. This technique was very successful for the condition; however, one child developed acute lymphoblastic leukaemia, although secondary, at least in part, to insertional mutagenesis.

ROLE OF EMBRYONIC STEM CELLS

Embryonic stem cells are pluripotent cells derived from the inner cell mass of the blastocyst that are able to differentiate into cells of all three germ layers. These cells have great potential for cellular and genetic repair and, being more primitive than adult stem cells, should engraft as other cell types with greater ease. Furthermore, these cells could be used for tissue engineering aiming to produce cells and tissues ex vivo for implantation. Despite this, there are several disadvantages to these cells. They have a greater tumorigenic potential and have greater inherent risks associated with immune rejection than autologous adult stem cells [14]. For the most part, use of these cells requires destruction of an embryo. This has met with severe ethical, moral and political challenges that have gone some way to retarding the field of research. As such, research has developed more slowly than with adult bone marrow stem cells.

Although in vivo respiratory findings are presently lacking, embryonic stem cells have been demonstrated in vitro to form differentiated airways epithelial tissue [81] and type 2 pneumocytes [82], which illustrate their potential use as a cellular or genetic therapy. However, these procedures have not been very efficient, generating only small percentages of type 2 pneumocytes [83]. Therapeutic use requires a pure population of type 2 cells for transplantation, as pluripotent cell transplantation carries the risk of teratoma formation [84]. A recent study has developed a method of deriving a pure population of functionally active type 2 pneumocytes directly from embryonic stem cells without the formation of an embryonic body [84]. These cells have the potential to provide a transplantable source of type 2 pneumocytes in the future for both cellular and genetic therapy.

CONCLUSIONS

The excitement at the beginning of the decade that stem cells would be able to home to injured tissue throughout the body leading to repair has tempered somewhat in the light of further evidence. Studies suggest that, although there may be some migration of adult bone marrow cells towards chemotactic gradients, the amount of engraftment as phenotypically appropriate functional epithelial cells is small. Despite this, there does appear to be some effect from the use of exogenous stem cells in disease models. However, at present, there are significant numbers of uncontrolled variables, including the type of stem cell used, the timing of use and the injury model. As such, results from animal models are also conflicting with these same stem cells also being labelled as contributors to some diseases.

Although the exact mechanisms are not wholly understood, this potential is already seeing its way to clinical trials in cardiology [5355]. These trials have focused mostly on the local delivery of autologous whole bone marrow. This involves the transplantation of a very heterogeneous group of cells. This is likely to be one of the causes of the discrepant results, which suggest both some improvement [54] and no improvement [53] in left ventricular ejection fraction several months after acute delivery of autologous bone marrow stem cells. In order to fully assess the positive and negative implications of bone marrow stem cells, it is be important to characterize the individual effects of fractionated, universally defined, subgroups of bone marrow cells in well-designed in vitro and in vivo animal experiments.

Stem cells do seem to have a role in disease. The objective will be to try and define more accurately the contributions of the individual stem cell groups and how they can best be augmented in specific diseases to provide an acceptable therapeutic index. This may involve increasing the contribution of stem cells in areas of disease by local delivery or the concomitant use of growth factors and cytokines. Other possible strategies include the ex vivo manipulation of stem cells to increase possible engraftment or contribution to repair or to reduce any adverse effects. It is likely that the use of stem cells as genetic vectors will require either an increased engraftment of these cells in the organ of interest, possibly by the techniques described above, or other strategies, such as the addition of local injury or a selective survival advantage. The potential of all stem cells is high, but a greater understanding is still needed [85].

Abbreviations

     
  • ARDS

    acute respiratory distress syndrome

  •  
  • BASC

    bronchioalveolar stem cell

  •  
  • CCSP

    Clara-cell-specific protein

  •  
  • CF

    cystic fibrosis

  •  
  • CFTR

    CF transmembrane conductance regulator

  •  
  • eNOS

    endothelial nitric oxide synthase

  •  
  • EPC

    endothelial progenitor cell

  •  
  • FAH

    fumarylacetoacetate hydrolase

  •  
  • GFP

    green fluorescent protein

  •  
  • HSC

    haematopoietic stem cell

  •  
  • K14

    ketatin 14

  •  
  • MAPC

    multipotent adult progenitor cell

  •  
  • MSC

    mesenchymal stem cell

M. R. L. is an MRC Clinical Training Fellow. S. M. J. is an MRC Clinician Scientist and holds a Johnson & Johnson Focus-Giving Fellowship.

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