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First published online January 10, 2008
Stem Cells Vol. 26 No. 3 March 2008, pp. 675 -681
doi:10.1634/stemcells.2007-0803; www.StemCells.com
© 2008 AlphaMed Press

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TISSUE-SPECIFIC STEM CELLS

Bone Marrow-Derived Cells Exhibiting Lung Epithelial Cell Characteristics Are Enriched In Vivo Using Methylguanine DNA Methyltransferase-Mediated Drug Resistance

Jane S. Reesea, Justin C. Rothb, Stanton L. Gersona

aCenter for Stem Cell and Regenerative Medicine, Case Comprehensive Cancer Center, Ireland Cancer Center at University Hospitals Case Medical Center, Case Western Reserve University, Cleveland, Ohio, USA
bDivision of Human Gene Therapy, University of Alabama Birmingham, Birmingham, Alabama, USA

Key Words. Methylguanine DNA methyltransferase • Bone marrow-derived stem cell • Enrichment • Gene transfer

Correspondence: Correspondence: Stanton L. Gerson, M.D., Case Comprehensive Cancer Center, Case Western Reserve University, 11100 Euclid Avenue, Cleveland, Ohio 44106, USA. Telephone: 216-844-8565; Fax: 216-844-4975; e-mail: Stanton.gerson{at}case.edu

Received on September 21, 2007; accepted for publication on December 14, 2007.

First published online in STEM CELLS EXPRESS  January 10, 2008.

   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Previous studies have suggested that donor bone marrow-derived cells can differentiate into lung epithelial cells at low frequency. We investigated whether we could enrich the number of donor-derived hematopoietic cells that have type II pneumocyte characteristics by overexpression of the drug resistance gene methylguanine DNA methyltransferase (MGMT). MGMT encodes O6-alkylguanine DNA alkyltransferase (AGT), a drug resistance protein for DNA damage induced by N,N'-bis(2-chloroethyl)-N-nitrosourea (BCNU), and the mutant P140K MGMT confers resistance to BCNU and the AGT inactivator O6-benzylguanine (BG). For this study, we used two MGMT selection models: one in which donor cells had a strong selection advantage because the recipient lung lacked MGMT expression, and another in which drug resistance was conferred by gene transfer of P140K MGMT. In both models, we saw an increase in the total number of donor-derived cells in the lung after BCNU treatment. Analysis of single-cell suspensions from 28 mice showed donor-derived cells with characteristics of type II pneumocytes, determined by surfactant protein C (SP-C) expression. Furthermore, an increase in the percentage of donor-derived SP-C cells was noted after BCNU or BG and BCNU treatment. This study demonstrates that bone marrow cells expressing MGMT can engraft in the lung and convert into cells expressing the type II pneumocyte protein SP-C. Furthermore, these cells can be enriched in response to alkylating agent-mediated lung injury. These results suggest that expression of MGMT could enhance the capacity of bone marrow-derived cells to repopulate lung epithelium, and when used in combination with a gene of interest, MGMT could have therapeutic applications.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Defining the ability of bone marrow-derived stem cells to differentiate into lung parenchyma can potentially affect development of cellular therapies for lung repair. In the last several years, studies have shown that mouse and human bone marrow-derived cells can establish themselves in the lungs and exhibit characteristics of lung epithelial cells [1–7], specifically type II pneumocytes. These data involving the lung and similar studies reporting bone marrow stem cell differentiation across lineage boundaries into tissues such as liver, heart, and brain originally generated a high degree of enthusiasm [8–10]. However, subsequent data have suggested that the mechanism by which bone marrow contributes to other tissues includes fusion with pre-existing tissue-specific cells [11, 12]. Although fusion has not been identified in the lung [13], recent reports indicate that bone marrow-derived type II pneumocytes occur at a much lower frequency than originally reported, and some have suggested that they do not occur at all [14–17]. The controversy surrounding these data can be attributed to the wide range of experimental conditions used and the technical difficulty of identifying tissue-specific donor-derived cells by standard immunohistochemical staining techniques.

Despite the variability in methods and results, the current consensus is that the frequency of bone marrow-derived stem cells that are able to differentiate into lung epithelium is lower than required for clinical significance. One strategy to enrich for a cell of interest is by providing a selective advantage to that cell. Currently, one of the most promising methods to augment the number of genetically altered cells in vivo is the use of the drug resistance gene methylguanine DNA methyltransferase (MGMT), followed by alkylating agent treatment to mediate selection in vivo. Overexpression of MGMT, the gene that encodes the DNA repair protein O6-alkylguanine DNA alkyltransferase (AGT), enhances cellular protection from N,N'-bis(2-chloroethyl)-N-nitrosourea (BCNU) and other alkylating agent toxicity. The point mutant MGMT P140K renders AGT resistant to the inhibitor O6-benzylguanine (BG), allowing selective protection of cells expressing the gene after treatment with the combination BG and BCNU. This strategy has been used successfully to select for transduced cells in mouse and large animal bone marrow [18–23] and has been shown to provide selection for a second therapeutic gene [24, 25]. In addition, BCNU is a known lung toxin, inducing both acute lung injury and chronic fibrosis.

Here, we show that selective expression of the drug resistance gene MGMT protects hematopoietic stem cells in the bone marrow from BCNU-induced DNA damage and cell death and results in enrichment of bone marrow-derived cells in the lung after radiation- and chemotherapy-induced damage. In rare instances, donor marrow cells that engraft in the lung express surfactant protein C (SP-C), a surface marker characteristic of type II pneumocytes. Furthermore, human P140K (hP140K) MGMT conferred by gene transfer into bone marrow-derived cells retains its expression in cells that express SP-C and enhances the capacity of bone marrow-derived cells to repopulate lung alveolar epithelium.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Mice
C57BL/6J mice and transgenic mice expressing green fluorescent protein (GFP) under the control of the chicken β-actin promoter (C57BL/6j-Tg(ACTB-EGFP)1Osb/J) were obtained from Jackson Laboratory (Bar Harbor, ME, http://www.jax.org). C57BL/6J/MGMT–/– mice were generated by breeding MGMT+/– heterozygotes (kindly provided by Dr. Metsuo Sekiguchi, Fukoka, Japan). The genotypes of the offspring were determined by polymerase chain reaction (PCR) using MGMT and neo-specific primers as described previously [26]. GFP mice were identified on the basis of phenotype. All animal studies were approved by the Institutional Animal Care and Use Committee of Case Western Reserve University.

Lung Digests and Cytopsins
Single-cell lung suspensions were prepared according to the method of Corti et al. [27]. Briefly, lungs were perfused free of blood using phosphate-buffered saline (PBS) via the right ventricle. Lungs were then inflated via the trachea with 3 ml of warm Dispase II (Roche Diagnostics, Indianapolis, IN, http://www.roche-applied-science.com). Immediately following this, agarose was infused and allowed to solidify. The lungs were then removed from the thoracic cavity and incubated in Dispase II for 45 minutes at room temperature. The tissue was minced in Dulbecco's modified Eagle's medium/25 mM HEPES with 0.01% DNase (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com). The cell suspension was then filtered through 100- and 30-µm cell strainers (BD Falcon, San Jose, CA, http://www.bdbiosciences.com) and washed, and the remaining red blood cells were lysed. Cells were fixed in 2% ultrapure formaldehyde and cytospun (Shandon Cytospin 3, Thermo Scientific, Waltham, MA, http://www.thermo.com) to a density of 20,000 per slide or stained for surfactant protein C and then cytospun.

In Situ Hybridization
In situ hybridization was performed as described previously [28]. Briefly, tissue sections were deparaffinized in xylene for 10 minutes, rehydrated in graded alcohol solutions, and then washed and treated with Proteinase K. Following additional washes, sections were dehydrated through graded ethanol solutions, denatured probe was placed on each section, and the section was incubated in a humidified chamber at 52°C overnight. The next day, tissues were washed, treated with RNase, and dehydrated. Autoradiography was performed by exposing slides to Kodak NTB-2 photographic emulsions (Kodak, Rochester, NY, http://www.kodak.com) for 1 week at 4°C.

Antibody Staining
Immunohistochemical detection of GFP, SP-A, and human AGT was performed on 3-µm paraffin sections. Sections were dewaxed in xylene and then rehydrated in graded alcohols. Antigen retrieval was performed using Antigen Unmasking Solution (Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com) according to the manufacturer's instructions. Endogenous peroxidase was blocked using 1.6% H2O2 in Tris-buffered saline for 30 minutes and then incubated in primary antibody overnight and secondary antibody for 2 hours. Slides were developed with DAB. Immunofluorescence staining on tissue sections was performed as above, excluding the dewaxing and peroxidase blocking. For immunofluorescence on cytospins, cells were fixed with 2% ultrapure formaldehyde and stained with anti-SP-C 1:2,000 (Chemicon, Temecula, CA, http://www.chemicon.com) or anti-human AGT at 1:40 (Kamiya Biomedical, Seattle, http://www.kamiyabiomedical.com) followed by secondary anti-rabbit or anti-mouse, respectively, conjugated to Alexa-Fluor 488 or Alexa 568 (Molecular Probes, Eugene, OR, http://probes.invitrogen.com). Nuclear counterstaining was performed using 4',6-diamidino-2-phenylindole dihydrochloride (DAPI; Vector Laboratories). Negative controls or isotypes were used for each staining run.

Vectors and Virus Production
The {gamma}-retrovirus used for transduction of mouse bone marrow was MFG-hP140K MGMT. This vector was used in the transplantation studies analyzed using tissue sections. Cells were transduced by coculture with Am-12 producers as described previously [29]. The self-inactivating lentiviral vector containing an internal MND promoter and the central polypurine tract/central termination sequence were kindly provided by D. Kohn and modified to include MGMT-P140K. Virus was generated as described by Zielske et al. [30]. Lentiviral P140K MGMT was used in the transplantation studies analyzed using single-cell suspension cytospins.

Transplants and Transduction
For MGMT–/– recipient studies, whole donor marrow was obtained from 6–8-week-old GFP transgenic mice and transplanted into MGMT–/– mice that were irradiated with 850 cGy using a 137Cs source. For the wild-type recipient/gene transfer studies, marrow was harvested from C57Bl/6J mice (Jackson Laboratory) 2 days after treatment with 150 mg/kg 5-FU. Sca+Kit+lin– (SKL) cells were isolated as described previously [31]. Six-week-old recipient mice were lethally irradiated with 850 cGy as described above and transplanted with 2 x 106 5-FU whole bone marrow cells per mouse or 1,000 SKL cells supported with 2–4 x 106 cells per mouse. For mice receiving transduced cells, whole bone marrow and sorted stem cell populations were transduced for 12 hours (lentivirus) or 48 hours ({gamma}-retrovirus) in {alpha}-minimum essential medium (A-MEM, Cellgro, Manassas, VA, http://www.cellgro.com) containing 20% heat-inactivated fetal bovine serum (HyClone, Logan, UT, http://www.hyclone.com), 2 mM GlutaMAX (Gibco, Grand Island, NY, http://www.invitrogen.com), and 6 µg/ml polybrene in the presence of 20 ng/ml mouse interleukin (IL)-3, 50 ng/ml mouse IL-6, and SCF (R&D Systems Inc., Minneapolis, http://www.rndsystems.com). The transduced cells were transplanted immediately after transduction.

Drug Selection
BCNU was obtained from the Drug Synthesis and Chemistry Branch of the National Cancer Institute (Bethesda, MD), and BG was synthesized by Dr. Robert Moschel at the Frederick Cancer Research Institute (Frederick, MD). For in vivo selection, BCNU was dissolved in ethanol and diluted to 1 mg/ml in PBS, and BG was dissolved to 3 mg/ml in 40% polyethylene glycol (Union Carbide Corp., Danbury, CT, http://www.unioncarbide.com) and 60% PBS (pH 8.0) Those mice receiving BG were injected intraperitoneally with 30 mg/kg BG, followed by 7.5–10 mg/kg BCNU 1 hour later. MGMT–/– mice did not require BG and were treated with 7.5 mg/kg BCNU only, as described above. One to three rounds of selection were carried out at 3-week intervals, beginning at 3 weeks post-transplant for animals transplanted with whole bone marrow and 5 weeks post-transplant for animals transplanted with SKL cells.

DNA Content Analysis
Lungs were harvested and digested with dispase as described above. Single-cell suspensions were stained with PerCP-conjugated anti-CD45 (BD Biosciences, San Diego, http://www.bdbiosciences.com) and 2.5 µg of Hoechst 33432 for 30 minutes at 37°C. GFP-positive cells were analyzed on a BD Aria (BD Biosciences) for DNA content by Hoechst 33432 staining.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Selection of Drug-Resistant Pulmonary Parenchymal Cells In Vivo
To evaluate the potential for BCNU-resistant bone marrow-derived donor cell enrichment in a recipient lung, two models were used. In the first model, we optimized the environment for selection advantage to donor cells by transplanting whole bone marrow from a GFP transgenic mouse expressing wild-type MGMT into MGMT knockout mice (MGMT–/–), which lack AGT expression and are highly sensitive to BCNU. In a second model, we used normal mouse marrow donor cells transduced with a mutant human AGT (hP140K MGMT) that confers resistance to BG and BCNU. In both models, donor cells have a distinct selective survival advantage to BCNU alone in the MGMT–/– recipient setting and to BG and BCNU in the wild-type recipient setting. The combination of BG and BCNU is known to be toxic to untransduced wild-type cells, particularly those expressing low levels of AGT, such as bone marrow, whereas hP140K-MGMT-transduced cells are protected. BCNU is also toxic to the mouse lung [32, 33]. In both models, the recipients were lethally {gamma}-irradiated with a dosage known to eliminate the host cells in the marrow and that has also been shown to facilitate engraftment of marrow-derived epithelial cells [34]. After engraftment with donor cells, mice were treated two or three times with BCNU alone for the MGMT–/– recipients of normal marrow or with BG and BCNU for the wild-type recipients of P140K MGMT-transduced marrow. Mice were sacrificed at between 2 and 11 months and evaluated for percentage of donor marrow-derived cells in the lung and in bone marrow to define the degree of enrichment and selection of donor cells.

Marrow Donor-Derived Cells Are Selected in the Bone Marrow After Alkylating Agent Treatment
In all mice tested, recipient bone marrow was reconstituted with donor whole bone marrow or SKL cells after selection with BCNU or BG and BCNU. MGMT–/– recipient mice were repopulated with 99% GFP+ cells (n = 4) after two cycles of BCNU, detected by PCR for GFP. PCR of the GFP gene is more accurate than relying on engraftment based on GFP expression, because in the GFP transgenic donor strain used, GFP is expressed in only approximately 50% of bone marrow cells at any point in time when determined by flow cytometry (data not shown). In the second model, bone marrow cells transduced with {gamma}-retroviral hP140K MGMT were selected in normal, lethally irradiated recipients and represented 99.9% ± 2.4% (n = 4) of total marrow cells after two cycles of BG and BCNU, based on AGT expression by flow cytometry (Fig. 1). Donor marrow cells transduced with lentiviral hP140K MGMT were enriched to 74.4% ± 2.1% (n = 4) of the marrow population after two cycles of BG and BCNU. These data confirm strong selection for bone marrow cells expressing MGMT.


Figure 1
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  		Figure 1. Selection for retroviral human P140K-methylguanine DNA methyltransferase-expressing cells in recipient mouse bone marrow. The percentage of P140K-expressing donor cells in a BG- and BCNU-treated recipient mouse was assessed by flow cytometry using the anti-AGT antibody mT3.1. The increase in the percentage of transgene-expressing cells is shown. Abbreviations: AGT, O6-alkylguanine DNA alkyltransferase; BCNU, N,N'-bis(2-chloroethyl)-N-nitrosourea; BG, O6-benzylguanine; hMGMT, human methylguanine DNA methyltransferase.

 
Marrow Donor-Derived Cells Are Selected in the Lung After Alkylating Agent Treatment
We next evaluated whether donor marrow-derived cells present in the lung could show an enrichment similar that observed in bone marrow after treatment with BCNU. Immunohistochemistry was used to quantify the number of donor-derived cells before and after drug treatment and to visualize their phenotype and morphology. Paraffin sections of distal lung were stained with primary antibodies to donor-specific GFP or human AGT, depending on the model being analyzed, followed by horseradish peroxidase (HRP)-conjugated secondary antibodies. All mice examined at 2–11 months post-transplant showed an increase in the total number of donor-derived cells after drug treatment. Cells constituting the donor-derived population were hematopoietic, such as neutrophils and alveolar macrophages, and lung parenchymal in origin. Interestingly, we observed some donor-derived cells with a morphology consistent with type II pneumocytes (Fig. 2A). Type II cells are typically located in the alveolar walls, distinct from alveolar macrophages. In the MGMT–/– recipient mice, the total number of donor-derived cells in lung tissue increased 1.7-fold from 19% ± 1.9% (n = 849 cells from four mice) in untreated mice to 32% ± 5.9% (636 cells from four mice; p = .0001) after two rounds of BCNU. Similarly, in wild-type recipients, the proportion of all donor-derived (hP140K MGMT-transduced) cells increased 2.2-fold from 21% ± 9.7% after two rounds of BG and BCNU (n = 4,580 cells from four mice; p = .0001) (Fig. 3). These data demonstrate that BCNU selects for donor-derived cells expressing human or mouse MGMT in the lung. The higher degree of bone marrow cell-derived selection in the gene transfer model compared with the MGMT–/– model likely results from the under-representation of genotypic GFP+ cells using the immunohistochemistry assessment of GFP expression, a practical limitation of this model.


Figure 2
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  		Figure 2. Morphology of donor-derived cells in paraffin sections from methylguanine DNA methyltransferase–/– distal lung. (A): Mice transplanted with green fluorescent protein (GFP)+ donor marrow were stained with anti-GFP and horseradish peroxidase conjugated secondary antibody. Cells located in the alveolar wall indicated by black arrows possessed the phenotype of a type II pneumocyte. Cells indicated by red arrows had the phenotype of an alveolar macrophage. (B): Confocal microscopy demonstrated dual staining for surfactant protein A (red) and GFP (green).

 


Figure 3
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  		Figure 3. Selection of donor-derived cells in the lung after drug treatment. Paraffin sections of distal lung from mice transplanted with GFP-expressing or O6-alkylguanine DNA alkyltransferase-expressing donor marrow were stained with the respective primary antibodies followed by a horseradish peroxidase-conjugated secondary antibody. The numbers of cells used in the analyses are shown, and the p value represents the difference from untreated mice. Abbreviations: BCNU, N,N'-bis(2-chloroethyl)-N-nitrosourea; BG, O6-benzylguanine; GFP, green fluorescent protein.

 
Marrow Donor-Derived Cells with Characteristics of Type II Pneumocytes Can Be Detected in the Lung After Radiation and BCNU Injury
Our ability to select for donor cells expressing MGMT after treatment with BCNU or BG and BCNU prompted us to investigate the parenchymal phenotype of the donor cells in the lung. To detect mRNA expression of the type II pneumocyte-specific SP-B, we performed in situ hybridization and immunohistochemistry on tissue sections from MGMT–/– mice that were reconstituted with GFP donor marrow. Surfactant is secreted by type II pneumocytes and acts as a lubricant to facilitate gas exchange, thus allowing airway expansion during inhalation. For in situ hybridization, adjacent 3-µm paraffin sections of treated MGMT–/– lung were subjected to either immunohistochemistry using anti-GFP followed by HRP-conjugated secondary antibody and DAB staining or radioactive mRNA in situ hybridization. Adjacent sections were then aligned and identified as donor-derived type II pneumocytes if a radioactive signal and positive staining for GFP were present in the same cell. Using this method, we detected cells that appeared to express both SP-B and GFP (Fig. 4).


Figure 4
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  		Figure 4. Donor-derived surfactant protein B (SP-B) cells are detected in the lung after N,N'-bis(2-chloroethyl)-N-nitrosourea injury. Three-micrometer distal lung paraffin sections were hybridized with radioactive SP-B mRNA (left), and an adjacent section was stained with an antibody to GFP (right). (A–C): Magnified areas containing cells positive for both SP-B mRNA and GFP (indicated by arrows). Abbreviations: GFP, green fluorescent protein; IHC, immunohistochemistry; SP-C, surfactant protein C.

 
As a second method to determine the phenotype of donor cells, we performed immunofluorescence on paraffin sections using dual staining with an antibody to other surfactant proteins (SP-A or SP-C) combined with either anti-AGT or anti-GFP, depending on the donor-recipient pair being evaluated. In normal mice that Received wild-type donor cells transduced with hP140K MGMT, donor-derived cells with characteristics of type II pneumocytes were identified as cells that displayed positive staining in the nucleus corresponding to human AGT and positive staining in the cytoplasm corresponding to SP-A or SP-C. In the second model, in which MGMT–/– mice received wild-type donor marrow cells expressing GFP, donor-derived cells with type II characteristics were identified using confocal microscopy to detect dual expression of both GFP (green) and SP-A (red) in the cytoplasm (Fig. 2B). Using this method, we observed few donor-derived SP-A-expressing cells randomly distributed in lung sections from treated MGMT–/– mice transplanted with GFP+ marrow and from wild-type mice transplanted with human MGMT P140K-expressing marrow. Although both in situ hybridization and immunohistochemical methods suggested the presence of donor-derived type II pneumocytes, neither technique was optimal because of the limitations associated with tissue sections. Nonspecific antibody staining was excluded by the use of appropriate isotypes (i.e., only sections with no background were included in analysis); however, cell overlay of a hematopoietic cell on a nonhematopoietic cell in the convoluted physical structure present in the lung precluded accurate cell type designation. Although confocal microscopy was capable of identifying dual-stained cells, it was not feasible to use for screening up to 1,500 cells per mouse, which is necessary to quantify low-frequency dual-positive cells.

Detection of Donor-Derived SP-C+ Cells in the Lung by Single-Cell Analysis and Enrichment After BCNU Injury
To overcome the technical limitations of phenotyping and quantification in tissue sections, we prepared single-cell suspensions from fresh lungs to analyze enrichment of SP-C+ cells. Freshly isolated lungs were inflated with warm dispase, dissociated and filtered into a single-cell suspension, and cytospun onto slides. Slides were then sequentially stained using antibodies to SP-C, followed by anti-GFP in the case of MGMT–/– mice transplanted with GFP+ wild-type donor cells or antibodies to SP-C followed by AGT for recipient mice transplanted with hP140K MGMT-transduced cells. Table 1 shows microscopy analyses of single cells from the lungs of representative mice transplanted and selected under both conditions. Twenty-eight mice were analyzed using this method. Quantification of cell number, lung epithelial cell type, and evidence of donor phenotype were based on DAPI cell counts from 5–10 microscopic fields consisting of 500–1,500 cells per mouse and characterization of the phenotypic staining. In the absence of BCNU selection, 16% ± 0.14% of cells present in the lung of MGMT–/– mice receiving GFP transgenic whole bone marrow were of donor origin (n = 5 mice) at 3 months after transplantation. Donor cells included hematopoietic and parenchymal cells. Donor-derived SP-C+ cells were detected in two of those mice at frequencies of 0.10% and 0.09%, respectively. The three remaining mice showed no evidence of donor-derived SP-C+ cells. After 2 BCNU treatments, the total number of donor-derived cells increased to 20.8% ± 1.6% (n = 6 mice). Two of the six mice demonstrated SP-C+ cells of donor origin, and the frequency in these two mice after selection increased to 0.35% of total cells (8.3% of all SP-C+ cells) and 0.39% of total cells (10.7% of all SP-C+ cells), respectively. Four other mice showed no evidence of SP-C+ cells. Analysis of single-cell suspensions of lung cells from wild-type mice reconstituted with hP140K MGMT-transduced SKL cells showed similar results. In untreated mice, 20.3% ± 0.45% of cells harvested from the lung were donor-derived (n = 6 mice) at 3 months after transplantation, and two of the six mice had donor-derived SP-C+ cells at frequencies of 0.06% and 0.005%. After BG and BCNU treatment, 25% ± 0.7% of cells from the lung were of donor origin; 2 of the 11 mice receiving this treatment exhibited donor-derived SP-C+ cells. Nine other mice showed no evidence of SP-C+ cells of donor origin. In mice with evidence of donor-derived SP-C+ cells, the frequency increased after selection to 0.38% of total cells or 5.4% of all SP-C+ cells in one mouse and to 0.12% of total cells, or 1.96% of all SP-C+ cells, in the second mouse. Of note, the relative cell size and granular SP-C staining pattern of the dual-positive cells were consistent with those of a type II pneumocyte (Fig. 5). These results suggest that occasional bone marrow-derived cells may differentiate into cells with lung epithelial characteristics, and their numbers can be increased by treatment with BCNU. However, the lack of detection of these cells in all mice suggests that the capable progenitor is present at low frequency, perhaps rare under conditions of transplantation.


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  		Table 1. Donor-derived SP-C cells are rare and can be enriched with BCNU treatment

 


Figure 5
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  		Figure 5. Detection of donor-derived type II pneumocytes by dual immunofluorescence on single-cell suspensions isolated from recipient lung. (A): Cells expressing SP-C (red) and GFP (green) in the cytoplasm. (B): Cells with nuclear AGT expression (red) and characteristic cytoplasmic SP-C staining (green). Cells exhibiting dual staining are indicated by arrows. Abbreviations: AGT, O6-alkylguanine DNA alkyltransferase; DAPI, 4',6-diamidino-2-phenylindole dihydrochloride; GFP, green fluorescent protein; SP-C, surfactant protein C.

 
Donor-Derived Cells Residing in the Lung Have No Evidence of Cell-Cell Fusion
Recent studies have shown that bone marrow can repopulate various organs by fusing with pre-existing cells. Although rigorous studies have not detected this phenomenon in the lung, it has not been assessed after drug selection. To address this issue in the lung parenchyma of our mice, the DNA content of engrafted GFP+ donor-derived cells in four BCNU transplant recipients was analyzed. Single-cell suspensions of lung cells, dissociated using the dispase method, were evaluated by Hoechst staining followed by flow cytometry (Fig. 6). In all mice tested, more than 99% of GFP+ cells, including hematopoietic and lung parenchymal cells, exhibited 2N DNA. CD45 staining was performed to identify and exclude hematopoietic cells from the analysis, but since the percentage of donor cells of nonhematopoietic origin was very low, we assessed DNA content in all donor cells. Since essentially all cells had no more than 2N DNA, we conclude that donor-derived cells with epithelial cell characteristics are not a result of cell-cell fusion events.


Figure 6
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  		Figure 6. Analysis of DNA content of donor-derived cells in the lung of a mouse reconstituted with green fluorescent protein (GFP)+ bone marrow. Lung tissue was dissociated with dispase into a single-cell suspension and stained with Hoechst dye 33432. The histogram represents DNA content in GFP+ donor cells and indicates that >99% of these cells contain 2N DNA.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 Acknowledgments
 References
 
This study demonstrates that MGMT-mediated drug selection results in enrichment of bone marrow-derived cells in the lung that express a marker characteristic of type II pneumocytes. The precedent for using the MGMT gene followed by drug selection to enrich for a cell of interest is extensive in bone marrow, where the endogenous levels of AGT are low [18–23, 35]. We and others have shown in a nonmyeloablative mouse model that even limited numbers of BG and BCNU-resistant MGMT-transduced cells can be enriched in the bone marrow up to 120-fold with one cycle of drug treatment [18]. We further demonstrated that the total population of MGMT-expressing donor marrow-derived cells can also be enriched in the lung up to twofold and make up more than 40% of all cells after two treatments of BCNU. More interestingly, we observed an increase in selectively resistant donor marrow-derived SP-C+ cells, suggesting conversion to type II pneumocytes.

The data were consistent across two different models of transplantation and drug selection. One model used donor cells that had a strong selection advantage because the recipient lung lacked MGMT expression, whereas the other used selection derived from expression of the transduced hP140K MGMT in the proper progenitor at the appropriate time. Were there to be a clinical application, it is likely that the gene transfer approach would be used. In this context, a phase I safety study of transduction of drug-resistant MGMT by {gamma}-retroviral gene transfer into CD34+ cells has been completed and was well tolerated (unpublished results).

Defining epithelial cell engraftment has been a complex task for a number of cell transfer studies, in large part because the convenience of immunohistochemical assessment is compromised by the dense, intricate nature of cell contact in tissues. We strove to overcome this through complementary and confirmatory single-cell analysis. Our initial studies to detect donor-derived epithelial cells were performed using tissue sections so that their morphology, location, and distribution in situ could be evaluated. Although this was not optimal for cell type identification or quantification, it was informative, and it verified that the morphology and tissue location of a portion of the donor-derived cells were consistent with type II pneumocytes. Furthermore, these experiments provided information regarding the tissue distribution of donor-derived cells. We found that the donor-derived cells with type II morphology and location were distributed randomly throughout the lung rather than in clusters that might suggest clonal type expansion. This suggests that cells were moderately differentiated when they populated the lung, or that only rare cells proceeded down the type II cell differentiation pathway and had lost any clonogenic potential by the time they had done so.

The drawback to an in situ approach, which has been described by others, is the potential for artifacts related to the tissue or the microscopy [13–15, 36]. Specifically, complex interdigitation and invagination of a hematopoietic cell on a type II cell may lead to the erroneous conclusion of a single cell with colocalized makers. To rule out tissue-specific issues, we performed additional analysis of donor-derived type II cells by single-cell evaluation on cytospins obtained from dispase-digested lung tissue. In these analyses, 2 of 11 wild-type mice transplanted with marrow transduced with hP140K MGMT and treated twice with BG and BCNU exhibited detectable levels of donor-derived SP-C+ cells. In the drug-treated mice, the percentages of hP140K MGMT donor-derived SP-C+ cells were 5.4% and 1.96% of total SP-C+ cells, an increase compared with untreated mice, in which two of five mice exhibited 0.6% and 1% SP-C+ cells. The selection was even more pronounced in MGMT knockout mice, possibly due to the increased BCNU sensitivity of the lung parenchyma due to their lack of AGT expression. In these mice, two of six treated mice had 10.7% and 8.3% donor-derived SP-C+ cells, compared with 2.1% and 1.7% from untreated mice. This provided evidence that marrow-derived cells can give rise to lung epithelium and that this process can be enriched after drug selection. The possibility cannot be excluded, however, that the costaining of donor (AGT+) cells and SP-C+ cells is a result of donor-derived cell phagocytosis of surfactant or a surfactant producing cell. The most promising approach to eliminate this variable is the use of donor cells harboring expression of a donor-specific gene under the control of an SP-C promoter [14, 15].

The spectrum of published results regarding the detection of marrow-derived lung epithelial cells suggests that additional experimental factors affect the interpretation of results. Although we did not observe significant differences in the frequency of donor-derived cells related to the time of harvest relative to the transplant or the cell population infused (whole marrow vs. SKL), it is possible that variables such as mouse strain, nature of lung injury, timing of the harvest, age at the time of transplant, cell type infused, and method of analysis can influence the degree of donor cell differentiation plasticity or the ability to detect it [14, 15, 34].

The specific mechanism and microenvironmental factors that facilitate bone marrow differentiation into alveolar type II cells are not yet clear. In normal lung tissue, parenchymal cell turnover is low; however, lung injury is well known to facilitate growth of new lung tissue. Type II pneumocytes are classically known as the tissue-specific stem cell for the alveolus and play a role in the re-epithelialization of the distal lung by proliferation and differentiation into type I cells in response to injury. The low number of donor-derived type II-like cells present in our analysis suggests that most of the repair in the lung after irradiation and BCNU injury originated from the host. After drug treatment, the number of donor-derived SP-C+ cells did increase; however, the lack of clonal pattern observed on the in situ analysis indicates that the origin of these cells was not from the local donor-derived type II cells. Rather, it suggests that the bone marrow harboring donor-derived cells, in response to cues from the damaged tissue, migrates to the lung. In addition, it appears that the donor-derived bone marrow cells contribute for a limited time after the BCNU or BG and BCNU treatment, since we did not detect an increase in donor cells over time.

In summary, we demonstrate that cells in the bone marrow expressing the drug resistance gene MGMT can engraft in the lung and convert into cells expressing the type II pneumocyte protein SP-C. Furthermore, the number of these cells can be increased in response to alkylating agent-mediated lung injury. These findings raise the possibility of using bone marrow-derived cells expressing a gene of interest in tandem with a drug resistance gene for the treatment of lung diseases.


   DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
Acknowledgments
 References
 
The authors indicate no potential conflicts of interest.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
References
 
We thank Dr. Claire Doerschuk and Dr. Alan Fine, for helpful discussions. This work was supported by Public Health Service Grant CA73062 and used the Flow Cytometry, and Histology Core Facilities of the Case Comprehensive Cancer Center (P30CA43703). BCNU was obtained from the Drug Synthesis and Chemistry Branch of the National Cancer Institute (Bethesda, MD). BG was obtained from the Frederick Cancer Research Institute (Frederick, MD).


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 

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