American Journal of Respiratory Cell and Molecular Biology. Vol. 27, pp. 645-651, 2002
© 2002 American Thoracic Society
DOI: 10.1165/rcmb.2002-0056RC
Marrow-Derived Cells as Vehicles for Delivery of Gene Therapy to Pulmonary Epithelium
Joanna E. Grove,
Carolyn Lutzko,
Josef Priller,
Octavian Henegariu,
Neil D. Theise,
Donald B. Kohn and
Diane S. Krause
Departments of Laboratory Medicine and Genetics, Yale University, New Haven, Connecticut; Department of Immunology Research/Bone Marrow Transplantation, Los Angeles Childrens Hospital, Los Angeles, California; Department of Neurology, Charité, Humboldt-University Berlin, Berlin, Germany; and Department of Pathology, New York University School of Medicine, New York, New York
Address correspondence to: Dr. Diane S. Krause, Yale University School of Medicine, 333 Cedar St., New Haven, CT 06520-8035. E-mail: diane.krause{at}yale.edu
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Abstract
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Gene therapy application to pulmonary airways and alveolar spaces holds tremendous promise for the treatment of lung diseases. However, safe and effective long-term gene expression using viral and nonviral vectors has not yet been achieved. Adenoviral vectors, with a natural affinity for airway epithelia, have been partially effective, but are inflammatory and induce only transient gene expression. We investigate the novel approach of using retrovirally transduced multipotent bone marrow–derived stem cells (BMSC) to deliver gene therapy to lung epithelium. We have shown previously that up to 20% of lung epithelial cells can be derived from marrow following BMSC transplantation. Here, irradiated female mice were transplanted with male marrow that had been transduced with retrovirus encoding eGFP. Transgene expressing lung epithelial cells were present in all recipients analyzed at 2, 5, or 11 mo after transplant (n = 10), demonstrating that highly plastic BMSC can be stably transduced in vitro and retain their ability to differentiate into lung epithelium while maintaining long-term transgene expression.
Abbreviations: bone marrow transplant, BMT • bone marrow–derived stem cells, BSMC • fluorescence in situ hybridization, FISH • fluorescein isothiocyanate, FITC • hematopoietic stem cells, HSC • interleukin, IL • long terminal repeat, LTR • phosphate-buffered saline, PBS
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Introduction
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The ideal gene transfer vector for gene therapy would be capable of efficiently delivering a gene to its target, and would not induce an immune response from the recipient. Over the past several years, retroviral vectors have been developed that transduce hematopoietic stem cells (HSC). The transduced cells survive long term in vivo, and have shown tremendous promise for the treatment of genetic diseases of the blood (1). HSC are ideal targets for gene therapy because of their ability both to self-renew and to differentiate into all lineages of blood. Retroviral vectors based on the murine stem cell virus (2) and the murine Maloney leukemia virus, such as the MND series of vectors (3), are designed to achieve consistent, high levels of transgene expression in hematopoietic stem cells and their progeny (4). These murine oncoretrovirus-based vectors stably integrate into target cell DNA and have been effectively used for efficient transduction of murine, canine, nonhuman primate, and human hematopoietic stem and progenitor cells (5–11).
We have shown that a single bone marrow–derived stem cell (BMSC) can reconstitute not only the hematopoietic system, but also can engraft as mature epithelial cells in the liver, lung, GI tract, and skin (12). These data suggest that, if these BMSCs can be stably transduced and maintain their plasticity, then they could be an ideal cell population for the delivery of therapeutic genes to nonhematopoietic tissues. In the experiments reported herein, we tested this hypothesis by performing bone marrow transplantation (BMT) into lethally irradiated female hosts, using cells that had been stably infected with murine retroviruses encoding the eGFP protein. The lethal whole body radiation given to the BMT recipient mice not only causes myeloablation in the marrow, it also causes diffuse alveolar breakdown and can produce tissue necrosis in the lung, which we believe may be the reason why such a large percentage of lung epithelial cells are marrow-derived after transplantation. We have used this BMT model to evaluate the potential for retrovirally transduced bone marrow progenitors to maintain transgene expression while both engrafting the marrow and contributing to lung epithelial tissue.
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Materials and Methods
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Retroviral Vectors and Producer Cell Lines
Experiments in which the recipients were killed at 2 and 5 mo after transplantation used a retroviral vector, MND-eGFP-SN, described previously (13). MND-eGFP-SN (originally called Mp-ncr-dl-neo) is a retroviral vector that has been optimized for long-term expression in embryonic carcinoma and hematopoietic cells by the addition of the enhancer from the myeloproliferative sarcoma virus, inclusion of the primer binding site from the murine retrovirus dl587rev, and the deletion of a negative control region from the long terminal repeat (LTR) (3). It expresses eGFP from the modified viral LTR and neor from the SV40 promoter. Experiments in which the BMT recipients were killed 11 mo after transplantation used the retroviral construct MGirL22Y, described previously (14). In this vector, the LTR drives expression of a bicistronic transgene, which has eGFP cDNA followed by the internal ribosomal entry site from the encephalomyocarditis virus linked to a mutant dihydrofolate reductase gene (L22Y). The ecotropic packaging cell line, GP+E86, was used for the generation of replication incompetent MND-eGFP-SN and MGirL22Y retroviruses (15).
Retroviral Transduction of Murine Bone Marrow
Marrow harvest and retroviral transduction using MND-eGFP-SN were performed as described previously (13). In brief, marrow from the femurs and tibias of 10- to 14-wk-old C57/Bl6 male mice 2 d after intraperitoneal treatment with 5 µg of 5-fluorouracil (American Pharmaceutical Partners, Los Angeles, CA), was resuspended at 1–2 x 106 nucleated cells/ml in basal bone marrow medium (20% fetal bovine serum, 0.1% bovine serum albumin, 2 mM L-glutamine and penicillin-streptomycin in IMDM) supplemented with 50 ng/ml of human interleukin (IL)-6, 10 ng/ml murine IL-3, 2.5 ng/ml murine SCF, and 4 µg/ml polybrene. The marrow was co-cultured with irradiated viral producer cells for 72 h. On the day of transplantation, both the nonadherent and adherent fractions were collected, washed, and counted. The cells were resuspended in Hanks' balanced salt solution (Gibco BRL, Frederick, MD) with 2 U/ml heparin for injection. For retroviral transduction of murine bone marrow using the MGirL22Y construct, bone marrow from 8- to 10-wk-old C57/Bl6 mice (BgVV, Berlin, Germany) was harvested 2 d after treatment with 5-fluoruracil (Sigma, Deisenhofen, Germany) (16). Before injection into recipients, donor bone marrow cells were cultured first for 48 h in DMEM/15% fetal calf serum supplemented with 20 ng/ml recombinant murine IL-3, 50 ng/ml human IL-6 (PromoCell, Heidelberg, Germany), and 50 ng/ml rat SCF (provided by Amgen, Thousand Oaks, CA), and subsequently the cells were co-cultured for an additional 48 h with irradiated viral producer cells.
BMT
Ten- to fourteen-week-old C57/Bl6 female mice (Jackson Laboratories, Bar Harbor, ME) served as bone marrow transplant recipients for MND-eGFP-SN transduced whole bone marrow from syngeneic age-matched male mice. Recipients were irradiated with 600 cGy on two consecutive days and then injected with retrovirally transduced or sham transduced male bone marrow cells by tail vein injection. Five females were transplanted with 3.5–4 x 106 transduced bone marrow cells that were not selected for retroviral expression before injection. Three animals were transplanted with 2 x 105 eGFP positive cells that had been sorted using a FACSVantage flow cytometer (Becton Dickinson, San Jose, CA). Recipient mice were killed 2 or 5 mo after transplantation. Bone marrow transplant procedures were approved by the Animal Care Committee at Childrens Hospital Los Angeles.
For reconstitution of hematopoiesis using MGir22Y transduced bone marrow, 8- to 10-wk-old, syngeneic C57/Bl6 males served as BMT recipients. Recipient mice were irradiated with 1,100 cGy cumulative dose, injected via tail vein with 5 x 106 whole bone marrow cells, and killed 11 mo after transplantation.
Immunohistochemistry and Immunofluorescence
Frozen 3-µm sections of paraformaldehyde fixed tissue were equilibrated to room temperature and air-dried before washing in phosphate-buffered saline (PBS). The tissue was then partially digested with 0.25% trypsin for 5 min at 37°C after which the enzyme was neutralized by placing the slides in PBS/2% fetal bovine serum for 30 s. Sections were then rinsed in PBS, incubated 30 min in 4x SSC/0.1% Tween 20/3% BSA to block nonspecific binding, and incubated with primary antibody for 30 min at 37°C. Rabbit polyclonal anti-GFP, IgG fraction (Molecular Probes, Eugene, OR) was used at a 1:1,000 dilution, and rabbit polyclonal anti-keratin, wide spectrum screening (DAKO, Carpinteria, CA) was used at a 1:200 dilution. Slides were washed three times with 4x SSC/0.1% Tween 20 and then incubated with the secondary antibody (alexa fluor 568 goat anti-rabbit IgG [H+L] conjugate; Molecular Probes, Eugene, OR) at 37°C. The slides were washed twice more with 4x SSC/0.1% Tween 20 at 42°C and counterstained with DAPI. Analysis protocols were approved by the Yale University Institutional Animal Care and Use Committee.
Fluorescence In Situ Hybridization
Fluorescence in situ hybridization (FISH) for murine Y chromosome and murine surfactant B mRNA was performed as previously described (12, 18). Primer pairs were synthesized at positions 3748–3781/4064–4041, 8020–8043/8500–8478 in the SP-B sequence (accession number S78114) and labeled by PCR incorporation of digoxigenin-dUTP. Lung tissues from normal male and female mice were uses as controls for the Y chromosome FISH procedure. Y chromosome staining in male control tissue is never 100 percent because tissue sectioning often cuts through only a portion of each cell's nucleus (18, 19). Therefore not all of the eGFP positive, donor-derived cells stain positively for the Y chromosome.
Fluorescence Microscopy
Images were taken using a Nikon Eclipse TE200 microscope (Kanagawa, Japan) and the DeltaVision microscope system (Applied Precision, Inc., Issaquah, WA) equipped with a CCD camera (Photometrics, Ltd., München, Germany). Images were pseudocolored using image processing software (Adobe Photoshop, San Jose, CA).
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Results
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Engraftment of Transduced Donor Bone Marrow in Hematopoietic Tissue following BMT
Bone marrow cells were transduced with a retroviral vector (MND-eGFP) designed for high transduction efficiency and stable reporter gene expression in bone marrow stem cells (13). The reporter gene, eGFP, is a fluorophore that requires no substrates for its fluorescence, and transgene expression can therefore be detected by direct fluorescence microscopy. Although high levels of this protein may be toxic to cells, at lower levels eGFP is biologically inert and does not influence cellular activities. In mice receiving transduced bone marrow, the engraftment of eGFP-positive circulating blood cells was examined 3–4 wk after transplantation. Mice injected with unselected transduced donor cells (n = 5) had 34–43% eGFP-positive cell engraftment in the peripheral blood, whereas mice that received marrow cells that had been selected for transgene expression (n = 3), had 56–65% eGFP-positive peripheral blood cells.
Recipient mice were killed 2 (n = 6) or 5 (n = 2) mo after transplantation, and engraftment of transduced blood and bone marrow cells was analyzed by flow cytometry. The percentage of eGFP-positive cells in the blood and bone marrow ranged from 19–80% and 11–56%, respectively (Table 1). Three of the mice were transplanted with eGFP-selected bone marrow. However, there was no difference in the level of eGFP-positive cells after 2 mo, suggesting equal levels of engraftment of transduced long-term engrafting bone marrow stem cells despite positive selection for short-term hematopoietic progenitors.
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TABLE 1 Analysis of transduced bone marrow cell engraftment in the peripheral blood, bone marrow, and lungs of lethally irradiated BMT recipients.
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Engraftment of Transduced Donor Bone Marrow–Derived Cells in Nonhematopoietic Tissue following BMT
Figures 1a–1d show co-localization of direct eGFP expression (a, fluorescein isothiocyanate [FITC]) and immunofluorescence for eGFP (c, Cy3.5) when the channels are combined (d). Tissue autofluorescence is shown in the Cy3 channel (b). Note that the 2 eGFP-positive cells indicated by the arrows do not have significant autofluorescence in the Cy3 channel. We found that a greater number of cells could be identified as eGFP-positive using the antibody than using direct fluorescence. This was not surprising, as transduced cells express varying amounts of eGFP, and some cells containing the retroviral construct will fluoresce with low intensity that is undetectable by direct fluorescence microscopy. Although direct eGFP detection can be valid and useful for tracking bone marrow–derived cells, it has the disadvantage of needing to be clearly distinguished from false positive signals that occur due to tissue autofluorescence, which can result from paraformaldehyde fixation, metabolic products within the tissue, experimental reagents (e.g., rubber cement), and dust or tissue debris. Figures 1e–1h show a cell fluorescing in the green channel that could be mistaken for an eGFP-positive cell. Only by overlaying several channels of autofluorescence (FITC, e; Cy3, f; and Cy3.5, g) does it become apparent that this cell is a false positive.
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Figure 1. Verification of fluorescence analysis of eGFP-expressing cells in alveolar tissue. Fluorescence was assessed in the FITC (a, e), Cy3 (b, f), and Cy3.5 (c, g) channels. eGFP fluoresces in the FITC channel, and tissue autofluorescence is captured in the Cy3 channel. Immunofluorescence for the eGFP protein using a rabbit polyclonal antibody and an anti-rabbit alexa fluor 568 antibody is shown in the Cy3.5 channel. DAPI-stained nuclei are shown in blue. The three channels were overlaid in each panel to the produced images in d and h. Original magnification: x60. In the top panel, the two cells marked by arrows are confirmed to be eGFP-positive because they do not autofluoresce in the Cy3 channel (b) and they stain by immunofluorescence for eGFP in the Cy3.5 channel (c). eGFP detected by direct fluorescence microscopy, shown in green, and immunofluorescence for eGFP, shown in red, is co-localized in the combined image (d, arrows). In contrast, lung tissue from a mouse that did not receive eGFP-expressing cells contains background autofluorescence that is similar in all channels (e–g). Original magnification: x60.
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Alveolar tissue from each transplanted mouse was examined using a combination of direct fluorescence to visualize eGFP-expressing cells and immunofluorescence for cytokeratins to identify alveolar epithelial cells, including bronchiolar lining cells and type I and type II pneumocytes. Cells were considered to be eGFP-positive only if the fluorescent signal in the FITC channel was absent from the Cy3 (autofluorescence) channel. Cells with the morphologic appearance of both type I and type II pneumocytes were eGFP-positive, indicating that they were derived from retrovirally transduced donor marrow. Figures 2a–2d show co-expression of eGFP (green) and cytokeratins (red). Figures 2e–2h show the same image without the green channel to allow for better visualization of the underlying cytokeratin staining. The percentage of lung epithelial cells that are marrow-derived transgene expressing cells was determined by focusing on random fields at x60 magnification in the Cy3.5 (cytokeratin) channel and counting all of the clearly cytokeratin-positive cells. Then, for the same field, the FITC and Cy 3 channels were used to count eGFP+ (FITC+, Cy3-) cells. All of the cytokeratin-positive cells in a 160-µm2 region were analyzed for each slide. In mice killed 2 and 5 mo after transplantation with transduced bone marrow cells, from 1–7% of cytokeratin-positive cells in the lung were eGFP+ (Table 1). There is no statistically significant correlation between the percentage of bone marrow cells and the percentage of cytokeratin-positive lung cells that express eGFP.
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Figure 2. Immunofluorescence for cytokeratins and direct fluorescence analysis for eGFP identifies donor-derived transgene-expressing epithelial cells in the lung. (a–d) Lung tissue 2 mo after transplantation of lethally-irradiated recipients with transduced donor bone marrow cells shows eGFP+ cells. eGFP fluoresces in the FITC (green) channel but not in the Cy3 channel (not shown), nuclei are stained with DAPI (blue), and cytokeratins are immunostained with alexa fluor 568 (red). (e–h) The same images are shown in a–d without the green channel. When fluorescence in the green channel is removed, it is clear that the cytokeratin staining is expressed in several eGFP-positive cells (arrows). eGFP can diffuse to the nucleus and is therefore found throughout cell. (a, e) Pneumocytes form rings of alveolar tissue. Two cells are eGFP-positive. The bottom center cell is cytokeratin-positive and the top left cell appears to be, although is not definitely, cytokeratin-positive (arrows). (b, f) Two eGFP-positive, cytokeratin-positive, bone marrow–derived lung cells are evident. (c, g) Enlarged image of an eGFP-positive, cytokeratin-positive cell. (d, h) Two adjacent eGFP-positive cells are also positive for cytokeratin (arrow), whereas others are clearly cytokeratin-negative (arrowheads). Original magnification: x60.
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Long-Term Maintenance of eGFP Expression in Bone Marrow–Derived Lung Cells
To assess long-term transgene expression in the lungs, lung tissue from recipients 11 mo after transplant was analyzed. The retrovirus used in the mice killed at 11 mo is MGirL22Y, which contains the eGFP reporter gene and an antifolate-resistant dihydrofolate reductase gene (17). Brain tissue of the same recipient mice has been shown to have eGFP-positive, functional cerebellar neurons derived from the transduced bone marrow cells (16). In the lung tissue, transgene-positive epithelial cells were detected by direct fluorescence for eGFP and immunofluorescence for cytokeratins (Figure 3).
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Figure 3. Lung images from BMT recipients killed 11 mo after transplantation with transduced bone marrow donor cells demonstrate long-term transgene expression in donor-derived lung cells. (a, b) Lung cells, identified by alexa fluor 586 (red) for cytokeratins express eGFP and fluoresce in the FITC (green) channel (marked with arrows). (c, d) The cytokeratin staining, shown in all panels, is more obvious after fluorescence in the green channel is removed. (a, c) and (b, d) each show a single eGFP-positive, cytokeratin-positive cell (arrows). In (b, d) an eGFP-positive, cytokeratin-negative cell, presumably a blood cell, is marked with an arrowhead. Original magnification: x60.
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Confirmation that eGFP-Positive Epithelial Cells Are Donor-Derived
Bone marrow transplant donors and recipients were sex-mismatched so that FISH for the Y chromosome could be used to confirm that transduced cells were donor-derived. After images taken of BMT recipient lung tissue were analyzed for eGFP and cytokeratin expression (Figures 4a and 4b), Y-chromosome FISH was performed on the same sections (Figure 4c). By detecting Y chromosomes in the eGFP-positive epithelial cells, we can rule out the unlikely possibility that free replication incompetent retrovirus was injected, which then directly infected the female lung cells. eGFP-expressing alveolar cells that stained positively for the Y chromosome were found in all sex-mismatched transplant recipients (n = 8). The mice analyzed 11 mo after transplant had female donors. Alveolar tissue from female BMT recipients injected with nontransduced female bone marrow did not contain any eGFP-positive or Y chromosome positive cells 5 mo after transplantation.
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Figure 4. Sequential immunostaining for cytokeratins and FISH for the Y chromosome confirms that eGFP-expressing epithelial cells in the lung are bone marrow–derived. (a) Lung tissue from a lethally-irradiated female mouse, killed 5 mo after receiving a BMT using transduced male bone marrow, stained with alexa fluor 586 (red in a and b) for cytokeratins. Several eGFP-positive (green) cells are evident. (b) When the image is shown without the FITC (green) channel, it is clear that one eGFP-positive cell is also expressing cytokeratin (arrow). (c) FISH for the Y chromosome (shown in pink within the blue DAPI-stained nuclei) verified that the eGFP-positive, cytokeratin-positive cell is donor-derived (arrow). Original magnification: x60.
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Secondary BMT Recipients Show eGFP Expression in Lung Tissue
Bone marrow harvested from primary BMT recipients was injected into four lethally irradiated secondary recipients. In addition, two lethally irradiated recipients were transplanted with sham-transduced bone marrow cells. The hematopoietic and nonhematopoietic organs of these mice were analyzed 2 mo after transplantation as described above. Transduced eGFP-positive cells were detected in the blood and lung. Cytokeratin-positive, eGFP-expressing cells were present in the lungs of all of the animals transplanted with transduced cells, and not in the control animals that had received sham-transduced cells (data not shown). This confirms previous results showing that long-term repopulating HSC have the ability to differentiate into lung pneumocytes (12).
eGFP-Positive Cells Produce Surfactant B mRNA
To test whether eGFP-positive donor-derived pneumocytes are functional, sections of tissue containing eGFP-positive type II pneumocytes were subjected to FISH for surfactant protein B (SP-B) mRNA. Surfactant, secreted by type II pneumocytes, facilitates gas exchange in the alveoli by acting as a lubricant, allowing for airway expansion during inhalation. Because SP-B mRNA is produced at very high levels, two large transcription centers can be identified in the nuclei of type II pneumocytes (12). Donor-derived, eGFP-positive type II cells expressing SP-B mRNA were found in all BMT recipients analyzed (Figure 5). Thus, functional, transgene-expressing type II pneumocytes were produced in the alveoli of transplanted mice.
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Figure 5. Sequential eGFP fluorescence analysis and FISH for surfactant protein B mRNA confirms that donor-derived, transgene-expressing cells express lung-specific mRNA. Lung tissue from a lethally irradiated female mouse 2 mo after transplantation with transduced male bone marrow cells. (A) The first image is an overlay of the fluorescent images obtained with DAPI (blue nuclei), FITC (green eGFP expression), and Cy3 (autofluorescence) filters. (B) The eGFP-positive cell subsequently analyzed by FISH for surfactant protein B mRNA shows Cy3.5-labeled transcription centers (red) within the blue nuclei. (C) Overlay of images in A and B showing expression of surfactant B mRNA in the eGFP+ cell. Note that the eGFP image was taken before FISH analysis, and there is some swelling and loss of nuclei during the subsequent FISH procedure for surfactant protein B mRNA. Original magnification: x60.
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Discussion
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In this study, we investigated the possibility of using multipotent bone marrow–derived stem cells as delivery vehicles for the administration of gene therapy to lung epithelium. Irradiated female mice were transplanted with male bone marrow cells transduced in vitro with retrovirus encoding eGFP. Approximately one tenth of all eGFP-positive cells in the lung were identified as cytokeratin-positive pneumocytes; these cells comprised an average of 3% of all alveolar epithelial cells in lung tissue analyzed in BMT recipient mice (n = 8). The remaining eGFP-positive cells were likely marrow-derived blood cells including macrophages and lymphocytes. Secondary recipients of marrow harvested from mice that had been transplanted with retrovirally transduced bone marrow cells again showed engraftment of eGFP-positive cells in the bone marrow and eGFP-positive cytokeratin-positive cells in the lung, suggesting that the highly plastic stem cell population survived long-term in the marrow of the primary recipient and retained the ability to differentiate into epithelial cells. These data demonstrate that retrovirally transduced bone marrow stem cells can engraft and differentiate into epithelial cells of the lung with sustained expression of the retroviral transgene.
We conclude that retrovirally transduced bone marrow cells have the potential to be used to target gene therapy to epithelial cells of the lung. Potential clinical applications include gene therapy for surfactant deficiencies or for acute lung damage lung. Whether this approach could be effective for cystic fibrosis remains to be determined. Retroviral transgene expression has not yet been analyzed in pulmonary epithelia from mice with a mutated CFTR gene.
Recently, in a nonmyeloablative transplant protocol, adherent bone marrow–derived cells were demonstrated to engraft as mature type I pneumocytes in the lung (17). In contrast to our findings, no type II pneumocyte or bone marrow engraftment was detected. The differences between the findings reported here and those of Kotton and coworkers are likely due to differences in the experimental protocols. The cell populations used and animal conditioning were different. They used plastic adherent bone marrow–derived cells that had been cultured for 7 d, and they transplanted the cells 5 d following bleomycin injury. We, in contrast, used fresh whole bone marrow infusion after whole body irradiation. Given the plasticity of bone marrow–derived stem cells, it is not surprising that they engraft as different cell types in response to different types and degree of injury.
It has been suggested that circulating bone marrow–derived stem cells support tissue-specific stem cells during periods of severe acute injury in multiple tissues (18–20), but it is not yet known whether lung damage is necessary for bone marrow cells to engraft as lung cells. Nonmyeloablative bone marrow transplantation protocols in which the recipient mice are not irradiated will be necessary to characterize the role of bone marrow stem cells in lung regeneration in the absence of injury and in response to injuries other than irradiation.
Using bone marrow stem cells as an autologous source for gene delivery has several advantages, including their accessibility, their ability to be manipulated in vitro, and their host compatibility. It remains to be determined whether retrovirally-mediated transgene expression in nonhematopoietic tissues is eventually silenced; however, our data showing maintenance of expression as far out as 11 mo after transplant suggest that this expression is relatively long-lived. Future studies using clonal analysis of the retrovirally marked cells can be used to further track the potential of single adult hematopoietic stem and progenitor cells as they engraft different nonhematopoietic tissues.
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Acknowledgments
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The authors are grateful to Paul Lizardi for instruction and use of the fluorescent microscope, to Robert Homer for guidance with lung markers and pathology, and to Helen Seow for assistance with figure preparation. This research was funded in part by NIH grants DK 53037 and HL 63357.
Received in original form May 2, 2002
Received in final form July 31, 2002
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Abstract
Introduction
