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Pulmonary and Critical Care, University of Vermont College of Medicine, Burlington, Vermont
ABSTRACT
Rationale: Recent literature suggests that adult bone marrow–derived cells can localize to lung and acquire immunophenotypic characteristics of lung epithelial cells. We speculated this might be a potential therapeutic approach for correcting defective lung epithelium in cystic fibrosis.
Objective: To determine whether adult bone marrow–derived cells containing normal cystic fibrosis transmembrane conductance regulator protein (CFTR) could repopulate lung epithelium in transgenic mice deficient in that protein.
Methods: Stromal marrow cells or total marrow obtained from adult male wild-type mice were transplanted into adult female Cftr knockout mice. To increase marrow cell recruitment naphthalene was used to induce airway epithelial injury in recipient mice.
Measurements and Main Results: At 1 wk, 1 mo, and 3 mo after transplantation, Cftr mRNA was detected in lung homogenates of recipient mice by reverse transcription–polymerase chain reaction. Cftr mRNA was not found in either donor marrow cells or mature circulating leukocytes. In situ examination of recipient mouse lungs demonstrated rare (0.025%) chimeric airway epithelial cells, some of which (0.01%) expressed CFTR protein. Naphthalene-induced airway remodeling nonsignificantly increased the number of chimeric airway epithelial cells expressing Cftr.
Conclusions: These results demonstrate that adult marrow cells can be recruited to airway epithelium and induced to express Cftr in mice otherwise lacking this protein. However, the number of observed chimeric epithelial cells is small and new strategies for enhancing airway epithelial remodeling by adult bone marrow–derived cells will be necessary for correction of defective CFTR-dependent chloride transport.
Key Words: cystic fibrosis cystic fibrosis transmembrane conductance regulator protein epithelium lung stem cell
Although controversial, a growing number of studies suggest that adult bone marrow–derived stem cells exhibit plasticity and can transdifferentiate into a variety of organ-specific cells (1–4). In certain instances, fusion of hematopoietic stem cells or of differentiated marrow-derived myeloid cells with organ-specific cells such as hepatocytes and skeletal muscle fibers can also occur (5, 6). Other populations of cells isolated from adult bone marrow including mesenchymal stem cells and multipotent adult progenitor cells have been demonstrated in vitro and to a limited extent in vivo to acquire phenotypic and in some cases functional characteristics of organ-specific cells (7, 8).
The complex structure of the lung is made up of many different cell types in which limited regenerative capacity has been attributed predominantly to endogenous lung stem or progenitor cells (9–11). Nonetheless, reports have demonstrated that adult bone marrow–derived stem cells can localize to lung and acquire phenotypic characteristics of lung epithelial, interstitial, and vascular endothelial cells (12, 13). As in other organs, injury increases marrow-derived stem cell recruitment to lung and functional participation of marrow-derived cells in lung remodeling after injury has been described (14–21). This can be deleterious, with marrow-derived cells participating in the formation of fibrotic lesions (13, 16, 20). However, amelioration of both fibrotic and inflammatory lung injury by marrow-derived cells has been demonstrated (15, 18, 19). Fusion of marrow-derived cells with lung epithelial cells, while inducible in tissue culture, does not appear to occur with significant frequency in vivo (22, 23).
Clinical manifestations of cystic fibrosis (CF), a devastating genetic disease for which there remains no cure, result from a defect of a multifunctional protein, the CF transmembrane conductance regulator protein (CFTR). In airway epithelium, defective CFTR contributes to alterations in airway surface fluid, mucus, and antibacterial defenses leading to chronic airway occlusion and infection (24). Since identification of the CFTR gene in 1989, CF has been a target of gene therapy efforts although no viable approach has yet proven successful (24). We therefore determined whether transplantation of adult marrow cells containing the gene for wild-type Cftr might result in functional Cftr expression in lung epithelium. To accomplish this, we transplanted two different populations of adult bone marrow–derived cells containing the wild-type Cftr gene into transgenic Cftr knockout (KO) mice: cultured stromal marrow cells and total marrow cells. We were subsequently able to detect Cftr mRNA and protein in the lungs of the Cftr KO recipient mice by reverse transcription–polymerase chain reaction (RT-PCR) and immunohistochemistry, respectively. However, the total number of chimeric lung epithelial cells exhibiting Cftr expression was small and unlikely to affect overall CFTR-dependent chloride transport and other functions in airway epithelium. Nonetheless, if strategies for increasing airway epithelial repopulation with adult bone marrow–derived stem cells can be found, this approach may be a potential therapeutic option for CF lung disease.
METHODS
Animals
Adult male (6–12 wk) C57BL/6 mice and transgenic C57BL/6 mice constitutively expressing green fluorescent protein (GFP) were used as donors (25). Adult female (8–12 wk) Cftr KO mice [Cftrtm1Unc-Tg(FABPCFTR)1Jaw/J] were used as recipients (26). All studies were subject to Institutional Animal Care and Use Committee review at the University of Vermont (Burlington, VT) and conformed to institutional and American Association for Accreditation of Laboratory Animal Care standards for humane treatment of laboratory animals.
Bone Marrow Harvest, Culture, and Transplantation
Total marrow was obtained from adult male GFP-expressing mice as previously described (12, 13). Plastic-adherent stromal cells isolated from C57BL/6 mice were cultured for 7 to 10 d in Dulbecco's modified Eagle's culture medium with 15% fetal bovine serum and then administered by tail vein injection to nonirradiated female Cftr KO mice (106 stromal cells/mouse) (13, 27). For transplantation of total marrow cells, as the Cftr KO mice are on a mixed strain background (C57BL/6, 129, FVB/NJ), CD3-positive T cells were depleted to minimize risk of early graft-versus-host disease (28). Adult female Cftr KO (recipient) mice underwent total body irradiation (800 rads), using a cesium-137 cell irradiator, followed by tail vein administration of CD3-depleted total bone marrow cells (20 x 106 cells/mouse).
Naphthalene-induced Lung Injury
Naphthalene (275 mg/kg body weight in sterile corn oil; Mazola; ACH Food Companies, Memphis, TN) was administered by intraperitoneal injection to (1) naive adult female Cftr KO mice 3 d before administration of cultured stromal cells and (2) chimeric adult female Cftr KO mice 1 mo after transplantation with total marrow cells (29).
Assessment of Donor-derived Cells in Recipient Lungs
Recipient female Cftr KO mice were killed by lethal overdose of pentobarbital at the indicated times, the lungs were gravity fixed with 4% paraformaldehyde, and 5-μm paraffin sections were mounted on glass slides. Hematoxylin- and eosin-stained sections were assessed for inflammation and injury.
Donor-derived cells were assessed by fluorescence in situ hybridization for Y chromosome–positive cells followed by immunohistochemical characterization of epithelium and leukocytes, using antibodies directed against Clara cell secretory protein (CCSP), pro–surfactant protein C (pro-SPC), CFTR, cytokeratin, and CD45 (30). Sections were systematically visualized with an LSM 510 META confocal microscope (Carl Zeiss, Oberkochen, Germany) and a BX50 confocal microscope (Olympus America, Melville, NY).
Analysis of Cftr Expression by RT-PCR
Total RNA was isolated from donor marrow cells, mature donor neutrophils, alveolar macrophages, and splenic lymphocytes, and from homogenates of recipient mouse left lung as previously described (31–33). One microgram of total RNA was subjected to reverse transcription and Cftr, glyceraldehyde-3-phosphate dehydrogenase, and -actin cDNAs were amplified (40 cycles) by PCR with Taq polymerase. Cftr was amplified with forward exon 9 primer (mCF11, 5'-CTT GTG GGA AAT CCT GTG CTG AA) and reverse exon 11 primer (mCF12, 5'-CCT TCT CCA AGA ACT GTG TTG TC) (34).
Flow Cytometry
Peripheral leukocytes obtained from chimeric mice 1 mo after transplant were assessed for percent GFP-positive cells (engraftment) with a Coulter Epics XL analyzer (Beckman Coulter, Fullerton, CA). Recipient lung homogenates were assessed for percentage of GFP- and CD45-positive and negative cells after incubation with anti-CD45 antibody (35).
Statistical Analyses
Data are expressed as means ± SD. Poisson analysis for rare events was applied to evaluate the distribution of rare donor-derived cells under the various experimental conditions (36).
See the online supplement for additional details on all methods.
RESULTS
Localization of Stromal Marrow Cells in Transplanted Cftr KO Mouse Lungs
Two approaches were used to determine whether donor bone marrow–derived cells could repopulate Cftr KO mouse lungs (Figure 1). In the first (Figure 1A), plastic-adherent marrow stromal cells from adult male C57BL/6 mice were administered by tail vein injection to adult female Cftr KO mice with intact bone marrow. Mice were assessed 1 wk, 1 mo, and 3 mo after transplantation for the presence of, and to characterize, marrow-derived cells in the lung by simultaneous fluorescence in situ hybridization (FISH) for detection of the Y chromosome (30) and by immunohistochemical detection of hematopoietic (CD45) and lung-related (CCSP, pro-SPC, CFTR, cytokeratin) markers.
At each time point, rare donor-derived cells were observed in recipient mouse lungs (Figures 2A and 2B). The majority of donor-derived cells were CD45 negative, were located predominantly in alveolar walls, and demonstrated morphologic characteristics of alveolar epithelial and/or endothelial cells (Figure 2A). Rare donor-derived cells expressing the Clara cell secretory protein were also detectable in airways of recipient lung at all time points after administration of stromal bone marrow cells (Figure 2B). Positive and negative controls for the detection of the Y chromosome by FISH are also shown for comparison (Figures 2C and 2D).
To determine whether airway remodeling resulting from naphthalene injury would increase donor-derived stromal cell recruitment to the airways, naphthalene was administered to recipient Cftr KO mice 3 d before administration of stromal marrow cells (Figure 1A). Bioactivation of naphthalene by the cytochrome P-450 2F2 isoenzyme results in dose-dependent Clara cell toxicity leading to epithelial cell necrosis (29). Administration of naphthalene resulted in desquamation of airway epithelium within 3 to 5 d of administration, with subsequent epithelial regeneration noticeable at 10 to 20 d (Figure 3B). One month after naphthalene administration, nearly complete airway regeneration was achieved (Figure 3C). Complete airway regeneration was observed 3 mo after naphthalene administration (Figure 3D).
As in non–naphthalene-exposed control mice receiving stromal marrow cells, only rare donor bone marrow–derived cells were found, mostly in alveolar walls. Most of these were CD45 negative and had the morphologic appearance of alveolar epithelial and/or endothelial cells (Figure 4A). Rare donor-derived, CCSP-expressing airway epithelial cells were detected in the lungs of naphthalene-injured mice 1 wk, 1 mo, and 3 mo after injury (Figures 4B and 4C; Figures 5A–5F). Quantitative assessment of the number of Y chromosome–positive, CD45-negative, CCSP-positive cells for each experimental condition is depicted in Table 1. Airway remodeling after naphthalene injury approximately doubled the number of chimeric airway epithelial cells found at each time point, although the increase was not statistically significant. Rare events analysis by Poisson distribution revealed clustering of Y chromosome–positive, CD45-negative, CCSP-positive cells in the condition corresponding to the 3-mo time point after naphthalene treatment (p < 0.05), with 0.025 ± 0.01% of CCSP-positive cells being of donor origin. Approximately 20,000 CCSP+ cells were scored for each lung.
Cftr Expression in Lungs of Cftr KO Mice after Transplantation of Stromal Marrow Cells
At 1 wk, 1 mo, and 3 mo after transplantation, rare donor-derived airway epithelial cells expressing CFTR protein were detected by FISH immunofluorescence (Table 2; Figures 6A–6F). The Cftr-expressing cells also stained positively for cytokeratin and had morphologies characteristic of columnar epithelial cells. Interestingly, Cftr-positive donor-derived airway epithelial cells often showed a clustered pattern of engraftment, with two cells being localized in a single high-magnification field (Figures 6A–6F). See the online supplement for additional images of CFTR staining including positive and negative control antibody staining.
Wild-type Cftr mRNA was found in lung homogenates by RT-PCR at all time points after administration of stromal marrow cells to naphthalene-injured mice (Figure 7). Importantly, neither mature differentiated leukocytes nor marrow cells from the transgenic GFP donors (either cultured stromal cells or fresh isolates of total marrow cells) expressed wild-type Cftr mRNA. This indicates that the marrow-derived hematopoietic cells used in the transplant were not the source of the Cftr mRNA that was detected in recipient lungs, and suggests that a small number of marrow-derived cells were recruited to lung and acquired an epithelial-specific phenotype not found in the donor cells. Wild-type Cftr mRNA was not consistently found in all naphthalene-injured mice receiving stromal cells and there were noninjured mice that also exhibited wild-type Cftr mRNA after stromal cell administration (Table 2). See the online supplement for a depiction of the no-RT control.
Localization of Marrow-derived Cells in Lung of Chimeric Cftr KO Mice
In the second transplantation approach (Figure 1B), CD3-depleted total bone marrow from adult male transgenic GFP-expressing mice was administered to adult female Cftr KO mice previously myeloablated by total body irradiation. As a result, Cftr KO mice chimeras were generated with marrow cells expressing the GFP transgene and bearing the Y chromosome. One month was allowed for chimeras to achieve marrow reconstitution. One month after transplantation recipient mice were engrafted with an average of 93% bone marrow cells of donor origin. The rate of survival of adult female Cftr KO mice myeloablated and transplanted with total marrow cells was 87%.
Photomicrographs of mouse lungs assessed 5 wk after transplantation demonstrated numerous donor-derived cells staining positively for the Y chromosome (Figures 8A and 8B). Most donor-derived cells were located in lung parenchyma and costained for CD45, indicating their identity as donor-derived leukocytes (Figures 8A and 8B). Y chromosome–positive, CD45-negative cells of donor origin were detectable in the interstitium just below the airway epithelium (Figure 8A). Y chromosome–positive, CD45-negative cells that stained for pro-SPC were also located in the alveolar septa, suggesting an immunophenotype consistent with type 2 alveolar epithelial cells (Figure 8B). Similar findings were observed in mouse lungs assessed 8 wk after transplantation (Figure 8C). There was no obvious difference in number or localization of CD45-negative donor-derived cells between lungs harvested 5 or 8 wk after transplantation. No donor-derived airway epithelial cells expressing CCSP or Cftr were detected by FISH immunofluorescence in airways of chimeric mouse lungs. Similarly, no wild-type Cftr mRNA was detectable by RT-PCR in lung homogenates at any of the time points assessed (data not shown). No histologic indication of pneumonitis was observed, suggesting that any radiation-related lung injury induced by total body irradiation at the time of transplantation had resolved by the times of lung assessment (Figure 8D).
To determine whether airway epithelial remodeling could increase donor marrow cell recruitment to the airways in chimeric mice, naphthalene was administered to recipient Cftr KO mice 1 mo after transplantation. Lungs from chimeric female Cftr KO mice were assessed 1 wk and 1 mo after naphthalene administration (corresponding to 5 and 8 wk after transplantation). At both time points lung engraftment by marrow-derived cells was similar to that observed in otherwise noninjured chimeric controls. Most of the donor-derived cells were CD45+ leukocytes. However, quantification of Y chromosome–positive, CD45-negative, pro-SPC–positive cells in alveolar epithelial walls (20 random x60 fields on five sections assessed for each lung) demonstrated that in both non–naphthalene-treated and naphthalene-treated chimeric mice, only approximately 0.1% of CD45-negative, pro-SPC–positive cells were of apparent donor origin (Figure 9A).
In addition, rare CD45-negative cells were observed in airway epithelial walls. Also similar to what was observed in non–naphthalene-treated chimeric mice, these appeared mostly subepithelial in localization and did not stain positively for either CCSP or CFTR (Figures 9B and 9C). As in uninjured chimeric controls, wild-type Cftr mRNA was not detected by RT-PCR. No obvious difference was noted in the number of Y chromosome– positive, CD45-negative, pro-SPC–positive cells in naphthalene-injured or otherwise uninjured chimeric mice ( 0.1% of total pro-SPC–positive cells).
GFP-positive, CD45-negative cells in homogenates of recipient mouse lungs did not reach detectable levels by flow cytometric analysis for any experimental condition evaluated.
DISCUSSION
We have demonstrated that adult bone marrow–derived cells have the potential to engraft in lungs of Cftr KO mice and to acquire phenotypic characteristics of alveolar and airway epithelial cells, including the expression of Cftr mRNA and protein. However, engraftment of marrow-derived cells in airway epithelium proved to be a rare event, and therefore unlikely to contribute to correction of transepithelial CFTR-mediated chloride current by the present approach.
In addition to reports demonstrating that cells derived from bone marrow of adult mice can localize to the lung and either transdifferentiate into or fuse with airway or alveolar epithelial cells, interstitial cells, and vascular endothelial cells, lung specimens from clinical bone marrow transplant recipients and from lung transplant recipients have demonstrated chimerism of both lung epithelial and endothelial cells (37, 38). These reports are based on in situ detection of a marker for the donor cells, in most cases the Y chromosome after sex-mismatched transplants. Further characterization of the donor-derived cells has been by morphologic appearance and/or by coexpression of cell-specific markers. Various reports have described up to 20% of epithelium and 37.5% of pulmonary vascular endothelium as being of donor marrow cell origin in mice and humans (14, 37). Most recently, mesenchymal stem cells obtained from bone marrow of adult patients with CF have been induced in vitro to acquire an epithelial phenotype including partial resumption of CFTR-mediated chloride current (39). These reports collectively suggest that repopulation of lung epithelium with adult marrow origin cells can occur although the relevance has yet to be established.
Gene transfer studies have suggested that approximately 10 to 15% of defective airway epithelium needs to be corrected to allow resumption of normal CFTR-mediated chloride current (24) If so, available data suggest that this level of epithelial repopulation could be achievable with marrow-derived cells. Moreover, observations that preexisting lung injury increases lung repopulation by adult bone marrow–derived cells suggests additional relevance for cystic fibrosis. The CF lung epithelium is chronically injured and is also subject to repeated acute injuries from recurrent infections. This may fortuitously provide a more conducive or permissive environment for adult bone marrow cells to localize to lung epithelium. Importantly, adult bone marrow cells can be genetically manipulated ex vivo with subsequent expression in lung (40). This suggests that the bone marrow of a patient with CF could be removed, treated ex vivo to correct defective CFTR, and subsequently readministered to correct underlying lung disease.
For these reasons, we attempted to transplant wild-type adult marrow cells expressing Cftr in the airway epithelium of Cftr KO mice. However, despite the use of two different transplantation protocols and two populations of marrow-derived cells, the number of donor-derived cells that appeared to acquire lung epithelial phenotype, including Cftr expression, was small when compared with previous reports. Even with robust airway remodeling induced by naphthalene injury in nonirradiated mice, the number of donor-derived airway epithelial cells expressing wild-type Cftr was small ( 0.01%) up to 3 mo after transplantation, and not significantly increased compared with noninjured mice. There was not always good correlation between Cftr RT-PCR and immunohistochemistry results demonstrating CFTR protein expression. This likely resulted from clustering of small numbers of chimeric airway epithelial cells in discrete sporadic locations in the lung. Approximately 50% of chimeric airway epithelial cells exhibited detectable Cftr expression whereas all chimeric airway epithelial cells stained positively for CCSP and/or cytokeratin. The reason for this is unclear as CCSP-positive Clara cells have been described to express CFTR (41, 42). The cytokeratin antibody used was a anti-pancytokeratin that stains several types of epithelial cells including both basal and differentiated airway epithelial cells. Future studies will incorporate more specific cytokeratin antibodies. Initial attempts at flow cytometry to further quantitate the number of Y chromosome–positive, CD45-negative cells in homogenates of recipient lungs were unsuccessful as there were too few cells to detect. The low number of chimeric cells also made it difficult to assess whether fusion of donor-derived cells with epithelium had occurred.
These results contrast published data in several respects. Other reports have described higher levels of chimerism despite the use of similar protocols and marrow cell populations (12–21). In part this may reflect the analytical technique used, particularly the immunohistochemical methods. We have found that the majority of donor-derived cells in lungs of chimeric mice are CD45-positive leukocytes. Overlap of leukocytes with lung cells in histologic sections may occur and can falsely elevate perceived numbers of chimeric lung cells. Furthermore, as radiation-induced lung injury can contribute to the engraftment of lung with marrow-derived cells (14, 16, 17, 21), we had attempted to minimize radiation effects when generating chimeric mice by using a lower dose than is more commonly used in studies reporting higher levels of engraftment (14, 16, 18, 19). This may also have contributed to the lower engraftment levels observed in our study. However, in parallel studies we conducted to evaluate the role of endotoxin and NO2-induced lung injury in repopulation of mouse lung epithelium with adult bone marrow–derived cells, higher doses of radiation, although increasing overall mortality in the recipient mice, did not result in increased chimerism of airway epithelium (43). There are many other potential confounding factors including potential immunogenicity of both the Y chromosome in the recipient female mice as well as the expression of GFP. Further studies will need to be undertaken to assess the influence of these and other factors that can potentially influence cell plasticity.
Plastic-adherent stromal marrow cells administered to naive nonirradiated mice resulted in engraftment of donor-derived airway epithelial cells, particularly after naphthalene-induced airway remodeling, although at low percentages ( 0.025%). In contrast, no donor-derived airway epithelial cells were detectable in irradiated mice transplanted with CD3-depleted total marrow cells. The stromal cells used were a heterogeneous population of cells and not purified mesenchymal stem cells as has been described by others (15, 27). This suggests that the population of adult marrow cells relevant for repopulating lung epithelium may be found in the plastic-adherent stromal cell compartment. These results coincide with findings of several other groups and suggest further study with mesenchymal stem cells and other cell populations isolated from the stromal compartment.
In conclusion, the current study supports the concept that functional airway epithelium can originate from adult bone marrow stem cells. This phenomenon may provide the basis for a therapeutic approach toward cystic fibrosis through replacement of defective epithelium with gene-corrected adult bone marrow–derived stem cells expressing wild-type CFTR. However, because adult bone marrow–derived cells home to airway epithelium with a low frequency by current techniques, further investigation on the molecular mechanisms that govern marrow cell recruitment and phenotypic conversion is needed in order for this approach to be clinically feasible.
Acknowledgments
The authors thank Diane Krause, M.D., Ph.D., for advice; Robert Prenovitz for expert technical assistance; Dr. Matt Poynter, Dr. Laurie Whittaker, Jenna Bement, and Joe Petty for assistance with neutrophil and lymphocyte preparations; Scott Tighe for assistance with flow cytometry; and Dr. Doug Taatjes and the staff of the UVM Imaging and Animal Care facilities.
FOOTNOTES
Supported by NHLBI HL03864, NCRR P20 RR15557, the Cystic Fibrosis Foundation, the American Lung Association, and a New Research Initiative from the University of Vermont.
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org
Originally Published in Press as DOI: 10.1164/rccm.200502-309OC on September 22, 2005
Conflict of Interest Statement: None of the authors have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.
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