DOC

Stem Cell Therapy for Cystic Fibrosis Current Status and Future

By Hector Wagner,2014-04-05 21:49
9 views 0
Stem Cell Therapy for Cystic Fibrosis Current Status and Future

    Stem Cell Therapy for Cystic Fibrosis: Current Status and Future Prospects

Donatella Piro; Joanna Rejman; Massimo Conese

    Expert Rev Resp Med. 2008;2(3):365-380. ?2008 Expert Reviews Ltd.

    Posted 07/24/2008

Abstract and Introduction

    Abstract

    Although cystic fibrosis (CF), an autosomal recessive disease caused by mutations in the gene encoding for the CF transmembrane conductance regulator (CFTR), seems a good candidate for gene therapy, 15 years of intense investigation and a number of clinical trials have not yet produced a viable clinical gene-therapy strategy. In addition, the duration of gene expression has been shown to be limited, only lasting 14 weeks.

    Therefore, alternative approaches involve the search for, and use of, stem cell populations. Bone marrow contains different stem cell types, including hematopoietic stem cells and multipotent mesenchymal stromal cells. Numerous studies have now demonstrated the ability of hematopoietic stem cells and mesenchymal stromal cells to home to the lung and differentiate into epithelial cells of both the conducting airways and the alveolar region. However, engraftment of bone marrow-derived stem cells into the airways is a very inefficient process. Detailed knowledge of the cellular and molecular determinants governing homing to the lung and transformation of marrow cells into lung epithelial cells would benefit this process. Despite a very low level of engraftment of donor cells into the nose and gut, significant CFTR mRNA expression and a measurable level of correction of the electrophysiological defect were observed after transplantation of wild-type marrow cells into CF mice. It is uncertain whether this effect is due to the presence of CFTR-expressing epithelial cells derived from donor cells or to the

    immunomodulatory role of transplanted cells. Finally, initial studies on the usefulness of umbilical cord blood and embryonic stem cells in the generation of airway epithelial cells will be discussed in this review.

    Introduction

    In cystic fibrosis (CF), two important requisites for a disease to be considered an ideal candidate for gene therapy are fulfilled: it is a monogenic disorder and the main affected organ is the lung, which is relatively easily accessible via topical administration. Although initially successful gene transfer in small-animal models generated enthusiasm, [1]later studies in primate models and in patients were discouraging. This has led to a

    search for alternative approaches, including, for example, the use of stem cell populations. CF is a potential model disease for stem cell therapy because of the persistent lung inflammation that leads to damage and remodeling, which could promote engraftment of stem cells. As we will discuss later, injury is an essential catalyst that enables the production of lung cells from bone marrow (BM) stem cells.

    Cystic fibrosis results from mutations in the CF transmembrane conductance regulator (CFTR) gene and affects one in 3300 live births. The CFTR gene encodes an ion channel

    that conducts chloride across the cell membrane and is regulated by protein kinase A in a [2]cAMP-dependent fashion. Besides its function as a chloride channel, CFTR possesses many other functions, including the regulation of other transport proteins, including the [3]epithelial sodium channel (ENaC).

    Although CF affects many organs and tissues, including the lung, pancreas, liver, intestines, sinuses and reproductive organs, lung disease represents the major cause of [4]morbidity and mortality in CF patients. The lung of CF patients is normal at birth but,

    soon after birth, an endobronchiolitis ensues with surprisingly few pathogenic bacterial species (Pseudomonas aeruginosa in most cases) and is associated with an intense [5-7]neutrophilic response localized to the peribronchial and endobronchial spaces. CF

    primarily affects the airways and submucosal glands with sparing of the interstitium and [8,9]alveolar spaces until late in the disease.

    Pathophysiology of CF Lung Disease & Target Cells For gene/cell Therapy Determination of the mechanisms linking defective CFTR function to CF lung disease is of great importance in developing rational therapies to treat CF. However, the mechanism by which CFTR mutations cause lung disease remains uncertain. Various hypotheses have been proposed, such as defective airway submucosal gland secretion, abnormal +airway surface fluid (ASL) composition or oxygenation, Na hyperabsorption due to

    ENaC inhibition by CFTR producing ASL dehydration (reduced ASL volume), loss of [10,11]CFTR regulation of other transport proteins and intrinsic hyperinflammation.

    In the lung, CFTR is expressed in several cell types: high levels have been found in [12]serous cells of submucosal glands, at the apical surface of ciliated cells in submucosal

    gland ducts and in the apical plasma membrane of all ciliated epithelial cells in the [13]superficial epithelium. In very recent reports, it was demonstrated that CFTR is

    expressed in human lung alveolar epithelial type II [14,15]cells. Based on CFTR expression and on the onset and progression of CF lung disease previously outlined, it is currently believed that target cells for gene replacement in CF are the ciliated epithelial cells that line the conducting airways and the serous cells [16,17]of the submucosal glands. The ciliated cells display the ion- and fluid-transport [18]defect of CF airways. On the other hand, CF submucosal glands fail to secrete mucus in response to agonists, which is attributed to loss of both CFTR-mediated anion and [19]fluid secretion. While intraluminally delivered gene transfer agents are likely to reach only the surface epithelial cells, it is doubtful that they will gain access to the serous cells of the submucosal glands. Access to the target cells through the basolateral side of the [20]epithelium via the bloodstream, either through the bronchial artery or via an [21]intravenous route, has been attempted experimentally; however, clinical application is doubtful because of the low efficiency and potentially strong immune response provoked by this administration. Presently, it is unknown whether correction of the ion-exchange defect of epithelial cells alone could result in clinical benefit to the patient.

The CFTR is also expressed by cells of the immune system, such as human and murine [22][23]alveolar macrophages and human neutrophils. Although it has been recognized that [24]each of these cell types displays a biochemical or functional defect linked to CFTR,

    the role of CFTR in the immune system and the consequences for the CF

    pathophysiology are still under discussion. Hence, the usefulness of 'targeting' these cells by means of gene and/or cellular therapy remains to be established.

    Gene Therapy of CF Lung Disease

    Since the cloning of the CFTR gene in 1989, more than 30 Phase I/II clinical trials have [16]been carried out using viral and nonviral gene-transfer agents. In these trials, the CFTR

    gene transfer to the airways has been achieved predominantly with nonviral (cationic liposomes) or viral (adenoviruses) vectors, providing proof of principle that the CFTR [25-27]gene could be transferred to the airway epithelium. However, it was also clear that

    both kinds of gene-therapy agents failed to produce therapeutic correction of the basic [16,26-29]defect and persistent expression in the human CF nose and lung. Moreover, CFTR

    expression faded away in a timeframe of a few days to 4 weeks, depending on the vector. Airway epithelial cells (AECs) have a relatively short lifetime, suggesting that re-administration of the vector will be required for a life-long disease, such as CF. Results from studies in experimental animals and clinical trials have shown that inflammation and antibody and T-cell responses can limit the duration of transgene expression, as well [30,31]as the therapeutic value of repeated administration of the viral vectors. Reported

    strategies aimed at overcoming these immunological hurdles of lung gene therapy include pharmacological treatments (immunosuppressant drugs and corticosteroids), induction of [31]tolerance and modification of the vector backbone, especially in the case of adenovirus.

    Nonviral vectors based on cationic liposome/plasmid DNA complexes are [32]nonimmunogenic but can elicit adverse side effects, such as inflammatory reactions.

    This unfavorable outcome might be related to the unmethylated CpG dinucleotide motifs present in bacterial DNA. The current strategy is aimed at the design of plasmids with a [33]minimal number of such motifs.

    Presently, two kinds of gene-transfer agents are being evaluated in CF patients: recombinant adeno-associated virus (AAV) and nanoparticles. While some clinical [34,35]efficacy has been demonstrated for both kinds of vectors, they still must face

    immunological responses that limit their re-administration (AAV) or the establishment of a stable formulation to be administered by aerosol (nanoparticles). A recent review has [36]addressed the use of these vectors.

    The development of lentivirus (LV)- or Sendai virus-derived vectors is another recent advance in the field of viral gene-transfer technology. Recent reviews have addressed [36,37]these developments. LV vectors appear to be promising vehicles for gene delivery into respiratory epithelial cells. They are able to integrate in the host genome, infect nondividing cells and mediate long-term persistence of transgene expression in the lung [38]of animal models. Various experimental observations indicate that LV vectors can

mediate long-term gene expression (up to 1 year) in the airways by transducing a [39-41]'progenitor cell' compartment, although the latter has not been characterized.

    Other challenges faced by CFTR gene delivery to the relevant cell targets are physical

    and biological extracellular barriers. The mucus layer, cilia, ASL and glycocalyx on the airway epithelium luminal surface prevent direct contact between the administered [42-44]vectors and the cell membrane. Compounds of the airway secretions interact with [45]gene vectors and act as inhibitory factors, even under normal healthy conditions. In CF

    airways, inspissated mucus and mucus plaques will strengthen the barrier to airway gene [46]transfer even further. Moreover, the receptors for virus entry are localized more

    frequently on the basolateral membrane than on the apical side of the respiratory [47,48]epithelium and they are not accessible because of the airway tight junctions.

    Pseudotyping with heterologous envelopes, modification of paracellular permeability and use of viscoelastic gels to slow mucociliary clearance are the strategies currently used to overcome the hurdles in viral vector-mediated CFTR gene transfer to the airway [49]epithelium. Physical methods have been developed that accelerate vector particles toward the target cells. Recently, a method known as magnetofection was developed [50]using magnetic force to enhance gene delivery by nonviral vectors.

    Stem Cells in the Lung

    Characteristics that define stem cells include their capacity for self renewal, production of daughter cells and extensive proliferative capacity. In general, stem cells turn over slowly and display minimal physiological differentiation. As early descendants of stem cells, transiently amplifying (TA) cells retain significant growth capacity while acquiring differentiated functions. TA cells eventually become incapable of proliferation and enter the terminally differentiated compartment. To conserve growth potential and to prevent genetic injury while vulnerable during mitosis, stem cells are thought to cycle slowly and to be recruited only as demanded by tissue turnover. Thus, much of the increase in cell number at steady state occurs in the TA population.

    Multipotent, long-lived cells (stem cells) have been identified throughout airways and give rise to both TA and terminally differentiated daughters. It is clear that local stem or TA cells contribute to the repopulation of the injured epithelium in different anatomical [51-54]regions of the lung, as shown in mouse studies. The complex architecture of the lung

    is indeed reflected by various stem cell niches present at different anatomical levels (Figure 1). The trachea and bronchi are lined by a pseudostratified, ciliated, columnar epithelium, with goblet (mucous-secreting) cells and submucosal (mucous- and liquid-secreting) glands also present. At this level, abundant experimental evidence points to [55]basal cells as the main compartment containing stem and progenitor cells (TA cells).

    These proximal airway progenitors are keratin 5/14 positive and are located at [56,57]submucosal gland-duct junctions or intercartilaginous boundaries.

Figure 1.

Stem cell niches in the lung. The pseudostratified epithelium of the upper (proximal)

    airways contains mostly ciliated cells, globet cells and basal cells. Basal cells probably represent a compartment enriched for stem cells and/or TA cells. Basal cells in the ducts of SMGs also show stem/progenitor cell characteristics. In the lower (distal) airways, where the epithelium is columnar, Clara cells predominate over ciliated cells. Clara cells in the bronchioles are probably the progenitors of other Clara and ciliated cells, and a variant Clara cell of the bronchiolaralveolar junction (BASC) appears to regenerate both

    the bronchiole and the proximal alveoar region. In the distal alveoli, there is evidence that type II cells show stem cell capacity to renew themselves and type I cells. BADJ: Bronchoalveolar duct junction; BASC: Bronchioalveolar stem cell; NEB: Neuroepithelial body; SMG: Submucosal gland.

    More distally in the bronchioles, the epithelium changes to a simple ciliated, columnar epithelium with submucosal glands, Clara (nonmucous, nonciliated and liquid-secreting) cells and fewer goblet cells than proximally, and then progresses with further branching to a nonciliated, simple, cuboidal epithelium lacking both goblet cells and submucosal glands, in the terminal bronchioles. In the proximal bronchioles, Clara cells are thought to [58]provide a stem cell or a TA cell function, in the context of neuroepithelial bodies.

    The alveoli have a simple squamous epithelium, comprised of alveolar type I and II epithelial cells. Although these two epithelial cell types are present in roughly equal number, type I cells cover approximately 95% of the alveolar surface. On the other hand, type II cells are small, cuboidal cells that exhibit many functions, including the synthesis and secretion of surfactant. In this region, alveolar type II cells are considered to be the [59-61]TA cells in lung parenchyma.

    Finally, an epithelial stem cell niche was recently identified at the junction between the conducting and respiratory epithelium (the bronchioalveolar duct junction in terminal [59]bronchioles). Specific cells in this zone (known as bronchioalveolar stem cells [BASCs]) coexpress secretoglobin 1A1, a marker of Clara cells also known as CC10 or Clara cell secretory protein, the type II cell marker surfactant protein (Sp)-C, CD34 and [62]stem cell antigen (Sca)-1. BASCs proliferate in response to naphthalene or bleomycin

    injury, and when purified cells were cultured appropriately, they demonstrated a high clonal growth capacity and differentiation potential to form both Clara cells and distal [62]lung epithelium, composed of cells expressing type I and II cell markers.

    It is unclear which cell types give rise to lung cancers, although new data indicate that mutations within different endogenous stem cells situated throughout airways can drive [54]cancer formation. Thus, it is necessary to learn whether lung tumors originate from stem cells and, if so, to identify the cell types involved. In addition to the findings of Kim et al., who demonstrated that BASCs could expand in response to oncogenic K-ras in [62]culture and in precursors of lung tumors in mice, one report showed that side-

    population (SP) cells from human lung cancer cell lines have higher potential for [63]invasiveness, as demonstrated by Matrigel? invasion assay. Moreover, these cells

    showed resistance to multiple chemotherapeutic drugs and displayed a higher level of human telomerase reverse transcriptase expression, suggesting that SP cells may represent a reservoir with unlimited proliferating potential for generating cancer cells. Seo and colleagues studied the gene-expression profile of human lung adenocarcinoma [64]A549 cells using an oligonucleotide array. They found that several genes related to

    chemoresistance and metastasis were upregulated in SP cells compared with non-SP cells. These studies allow us to reflect on the real advantage of using stem cells (in particular SP cells) in stem cell-mediated therapeutic approaches for lung diseases. In contrast to insights regarding candidate stem cells in the respiratory epithelium of adult lung, much less information is available regarding stem cells in the vascular compartment of the lung. SP cells have been isolated from the lung using their Hoechst-33342 dye efflux properties, similar to those of hematopoietic stem cells (HSCs) and other stem cell markers, indicative of epithelial and mesenchymal lineages. They comprise 0.030.87%

    of mouse lung cells and are Sca-1 antigen positive, lin negative, heterogeneous for CD45 [65-67]and express the vascular marker CD31. On the basis of the expression of the

    hematopoietic marker CD45, lung SP cells were further subdivided into hematopoietic +-(CD45) and nonhematopoietic (CD45) subpopulations. Nonhematopoietic SP cells

    express markers of epithelial and mesenchymal cells, and share some characteristics with [67,68]-+--airway stem cells. Cocultures of CD45CD31 and CD45CD31 form complex [68]vascular-like structures.

    Multipotent, mesenchymal stromal cells (MSCs) have been detected in adult tissues, [69]including the lung. A recent study demonstrated the isolation and characterization of nonhematopoietic MSC populations from the lower respiratory tract of human lung-[70]transplant recipients. In another recent work, Hennrick and coworkers demonstrate for the first time that tracheal aspirate fluid from premature, mechanically ventilated infants contains fibroblasts with cell markers and differentiation potential typically found in [71]MSCs.

    BM-derived Stem Cells & Their Homing to the Lung

    [72]Bone marrow contains multipotent MSCs, HSCs, SP cells and multipotent, adult stem [73]cells. They are able to differentiate into a select range of cell types, heavily influenced by the microenvironment. Among these, the HSCs are the best characterized of the adult [74][75]stem cells. MSCs, which can also be found in tissues other than BM, depending on

    the tissue environment, can generate chondrocytes, osteoblasts, adipocytes, myoblasts and endothelial cell [76,77]precursors. In addition, circulating endothelial precursor cells have been identified as [78]being derived from BM. Finally, a circulating population of hematopoietic origin that [79,80]displays fibroblast cell characteristics has been described. In this section, we will

    describe the contribution of HSCs and MSCs only to the restoration of the lung epithelium, since the role of circulating endothelial and fibroblast precursor cells is presently unclear in the context of CF lung disease.

Hematopoietic stem cells generate all of the blood cells and can reconstitute the BM after [81]depletion caused by disease or irradiation. However, HSCs are also capable of giving

    rise to various lineages of cells, notably and especially relevant for this discussion [82]epithelial cells. A change in stem cell differentiation from one cell type to another is known as transdifferentiation, and the multiplicity of stem cell differentiation options is known as developmental plasticity. More recent studies have raised questions regarding [83]the plasticity of HSCs. In some organs, such as the liver, transplanted HSCs fuse with host cells and transfer genetic material to them, thus giving the false appearance of [84,85]having transdifferentiated, with the generation of new cells in the host. Both

    transdifferentiation and fusion are likely to occur in the lung. Harris et al. made use of the

    Crelox recombinase system to examine whether fusion occurs between BM-derived [86]stem cells and host cells after BM transplantation. Even if low frequencies of BM-

    derived epithelia were detected in the liver, lung and GI tract, the donor-derived lung cells were found to contain only one copy of each sex chromosome. Recently, the same group investigated the frequency of fusion events by performing a BM transplantation in a murine model of lung inflammation. In male mice lacking the lung-specific protein Sp-+C, which were transplanted with female wild-type marrow, the frequency of Sp-C cells [87]containing the Y chromosome was 65%, indicating their origin by fusion. The type of

    lung injury might be fundamental for detection of the two kinds of events. The seminal work by Krause and colleagues demonstrated that, after transplantation of total BM or enriched HSCs into irradiated recipient animals, the engraftment of BM-[88]derived cells in the lung could be detected. The level of pneumocyte engraftment was

    significantly higher (up to 20%) than that in the columnar epithelium of the bronchi (2.32%) and in other epithelial cell compartments in which BM engraftment could be demonstrated (0.193.39% in gastrointestinal lining cells, bile ducts, skin and hair [89-91]follicles). Further work has shown that this and other studies overestimated the

    extent to which engraftment of airway and alveolar epithelium can be obtained by BM-derived cells. Current state-of-the-art studies indicate that only a very small proportion [44](i.e., <0.010.025%) of lung epithelial cells arise from BM-derived cells. Much of this

    earlier literature relied on a green fluorescent protein (GFP) reporter gene to track BM-

    derived [92-94]cells; however, transgene expression has been shown to be relatively insensitive for the identification of BM-derived epithelial cells. Further literature, including studies both [95-97][98-100]in murine and human lungs, was based on sex-mismatched transplantation and

    subsequent demonstration of donor-derived cells by fluorescent in situ hybridization

    (FISH) for the Y chromosome coupled with cell-specific immunohistochemistry. However, meticulous care must be taken with microscopic techniques and a rigorous and consistent study design is mandatory. Current standards in this field demand either confocal or single-cell analysis of BM-derived epithelial cells (by deconvolution microscopy) to rule out the possibility of overlay. The addition of hematopoietic antigens (e.g., CD45) to staining protocols is appropriate in many cases. Indeed, many of the cells putatively identified as donor-derived were either leukocytes or leukocytes overlapping epithelial cells. Phenotypic analysis with cell-specific markers is indicated to ensure that the appearance of epithelial cells derived from BM is not the result of microscopy artifact.

Finally, when technically feasible, isolation and functional characterization of donor-[101]derived engrafted epithelial cells should be performed.

    In all studies published to date, lung damage, either from radiation utilized for myeloablation or otherwise, is required to detect engraftment. In fact, lung irradiation has a dose-dependent effect on apparent lung epithelial engraftment by BM-derived cells in [95,102]mice. It has been suggested that the lung injury associated with radiation creates a milieu that induces marrow progenitor cells to adopt the gene-expression pattern of mature pneumocytes and bronchial epithelium. For this reason, different authors have investigated the effect of the recruitment of BM-derived cells following the damage [86,89-91,95]induced by total-body irradiation. The combination of irradiation with other

    damage has not produced consistent data. The use of NO or endotoxin did not result in 2[103]significant engraftment of BM-derived cells in the lung. On the other hand, the

    combination of radiation with intratracheal elastase increased the proportion of BM-[89]derived cells in the lung, including type I pneumocytes. The use of the cardiotoxin

    does not notably increase the engraftment of BM-derived type II pneumocytes compared [102]with radiation alone.

    Most studies have shown repopulation of alveoli following transplantation of HSCs or [90,95,104,105]MSCs in irradiated recipients. However, as discussed previously, alveoli are

    involved in lung disease only at the end stage. More interestingly, others have reported formation of epithelial cells of the conducting airways. MacPherson and colleagues injected the BM-derived SP cells from ROSA26 mice (constitutively expressing β-[106]galactosidase) into irradiated hosts before polidocanol treatment. They demonstrated

    that mice engrafted with SP cells have donor-derived cells present in the epithelial lining +of the trachea following damage and repair. Donor-derived cells (Y chromosome) were

    found at a frequency of 0.83%. Confocal microscopy analysis revealed that 55% of the cells expressing cytokeratins (CKs) were donor derived. Analysis of X-gal staining and allele-specific ROSA26 PCR indicated that these cells did not have the ability to contribute to the developing blastocyst, nor were they able to contribute to primary epithelial cultures grown at an airliquid interface or denuded tracheal xenografts.

    Clearly, the necessary signals/factors required to allow BM-derived SP cells to contribute to the formation of the epithelia are not present in these in vitro and ex vivo systems. Very [107]recent work by the same authors extended and confirmed these observations. Indeed,

    they show that whole BM donor cells also contributed to the tracheal epithelium following damage but, without damage, the number of donor cells was tenfold lower. In animals transplanted with SP cells, Y chromosome FISH was used to identify donor-derived cells and deconvolved imaging to confirm localization of these cells with the epithelial marker pan-CK. The majority (60.2%) of donor-derived cells express CK and some of these also express the CD45 hematopoietic marker.

    Gomperts and colleagues have used a mouse model of sex-mismatched tracheal [108]transplantation. This model is associated with tracheal ischemia, followed by reperfusion from neovascularization post-transplantation. The airway injury is associated with complete sloughing of the epithelium from the basement membrane with gradual re-epithelialization starting by day 3 post-transplantation. Full regeneration of the pseudo

    columnar epithelium occurs by day 21 post-transplantation. The authors were able to harvest a population of oriented progenitor cells expressing the epithelial marker CK5 and the chemokine receptor CXCR4 from the BM. These cells, upon passing into the circulation, provide a cellular pool able to repair damaged tracheal epithelium. Depletion of CXCL12 prevents precursor recruitment and appropriate epithelial repair and favors ++squamous metaplasia. These findings demonstrate that CK5CXCR4 cells have a crucial

    role in the re-epithelialization of tracheal transplants and that the CXCL12CXCR4 axis

    is involved in epithelial precursor mobilization and recruitment at sites of injury (Figure 2).

Figure 2.

    Engraftment of stem cells into the injured lung. Lung injury induced by stimuli (e.g.,

    bleomycin, elastase and irradiation) generates mediators that stimulate the BM to produce and release HSCs and MSCs. Mediators are probably secreted by resident macrophages, fibroblasts, endothelial cells and epithelial cells, and also regulate HSC and MSC homing to the damaged lung. Once homed, BM-derived stem cells are thought to give rise to alveolar cells and epithelial cells of the airways. The same chemokine/chemokine receptor axis (i.e., stromal cell-derived factor-1-1/CXCL12 and CXCR4) is probably important for the homing of UCB SCs. BM: Bone marrow; HSC: Hematopoietic stem cell; MSC: Mesenchymal stem cell; SC: Stem cell; UCB: Umbilical cord blood.

    In these studies, lung damage was brought about by physical or chemical means. For obvious reasons, such treatments may not be desirable for application in patients. Therefore, we set out to investigate the effect of epithelial damage caused by P.

    aeruginosa (a pathogenic bacterium widely occurring in CF patients) on the engraftment of BM-derived cells in airway epithelium. Controlled damage was achieved by instilling different sublethal doses of P. aeruginosa, followed by assessment of different

    parameters of tissue damage. Upon intravenous injection of GFP-positive BM-derived +cells into P. aeruginosa-infected mice, virtually no GFP cells were detected in the lungs +of the recipient mice. When the donor cells were injected intratracheally, GFP cells were

    found but their frequency was very low. Only when the BM cells were first purified to +obtain a cell suspension enriched in progenitor cells were significant numbers of GFP

    cells detected in the lung epithelium. In control mice not infected with P. aeruginosa, no +GFP cells were observed in the lungs. Pulmonary localization of the donor cells was confirmed by Y chromosome FISH analysis. All donor-derived Y-chromosome-positive cells were found to express CK, revealing their epithelial nature (Figure 3). The fractions +56of GFP cells expressing CK were 0.34 and 0.76% for the 10 and 10 cfu bacterial

    inoculum, respectively. When scored by Y chromosome positivity, these numbers were 0.60 and 1.12%, respectively (Rejman J, Conese M, Unpublished data).

Figure 3.

    Identification of donor-derived epithelial cells after inoculation of bone marrow (BM)-derived cells into the lung. A recipient female C57Bl/6 mouse was inoculated 6through the trachea with Pseudomonas aeruginosa at a dose of 10 cfu. Lineage minus

    cells, obtained from a male mouse, were injected intratracheally in mice infected with P.

    aeruginosa 2 days earlier. The mouse was sacrificed after 6 weeks and the lung sections +were screened for Y chromosome cells and cytokeratin expression. Image shows a Y ++chromosome cell (arrow) that is cytokeratin (red signal). Nuclei were stained in blue

    with 4',6-diamidino-2-phenylindole.

BM-transplantation studies in CF mice. In the CF lung, the mucus clogs the airways,

    causing breathing problems and making it easy for bacteria to grow. The resulting chronic infections and airway inflammation lead to progressive lung injury. Neutrophils are considered to be responsible for the onset and promotion of the inflammatory [24,109]response within the CF lung. Moreover, neutrophil products, such as proteases, can

    act both as growth factors for mucous cells and as secretagogues for mucins. Indeed, CF airways usually display hypertrophy and hyperplasia of submucosal glands. Airway remodeling is recognized to be the product of long-standing bronchial inflammation. Inflammatory mediators, such as IL-8, and growth factors have the ability to cause cell infiltration, collagen deposition, matrix restructuring, smooth muscle [110,111]hypertrophy/hyperplasia and increased bronchial vascularity. The role of

    neutrophil elastase and metalloproteinases in CF airway inflammation and remodeling [112]has been shown in studies with humanized airway xenografts and in CF [113,114]patients. The remodeling involves formation of new glands (tubulogenesis), matrix breakdown, proteolysis and reticular basement membrane (RBM) thickening, related to TGF-β concentration. 1

    The difficulty of extensive studies in young infants with CF and the lack of relevant animal models can explain the current lack of knowledge about remodeling in CF airways. Mouse models created by targeted mutations in the murine Cftr gene typically [115]exhibit severe digestive impairment but barely any lung disease, although some show [116,117]spontaneous leukocyte accumulation in the lung interstitium. In spite of these flaws,

    two groups have recently reported BM transplantation of CF mice with wild-type cells. Loi et al. determined whether transplantation of adult marrow cells containing the gene [96]for wild-type Cftr might result in CFTR expression in the lung epithelium. The authors

    transplanted two populations of BM-derived cells, cultured stromal marrow and total BM cells containing the wild-type Cftr gene, into transgenic Cftr-knockout (KO) mice.

    Administration of plastic adherent stromal cells to naive nonirradiated mice resulted in the engraftment of donor-derived AECs, although in small numbers only (~0.025%). Naphthalene-induced airway remodeling doubled the number of chimeric AECs expressing CFTR but not significantly. No donor-derived AECs were detected in irradiated mice treated with total marrow cells. Cftr mRNA and protein could only be detected in the lungs of Cftr-KO recipients treated with isolated adherent BM stromal

Report this document

For any questions or suggestions please email
cust-service@docsford.com