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Published ahead of print on September 25, 2003, doi:10.1165/rcmb.2003-0140OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 30, pp. 491-499, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2003-0140OC

Lung Cells Transplanted to Irradiated Recipients Generate Lymphohematopoietic Progeny

Shinji Abe, Gina Lauby, Craig Boyer, Lidia Manouilova, Stephen I. Rennard and J. Graham Sharp

Department of Pulmonary and Critical Care Medicine Section, Department of Genetics, Cell Biology and Anatomy, University of Nebraska Medical Center, Omaha, Nebraska

Address correspondence to: J. G. Sharp, Ph.D., University of Nebraska Medical Center, Department of Genetics, Cell Biology and Anatomy, 986395 Nebraska Medical Center, Omaha, NE 68198-6395. E-mail: jsharp{at}unmc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Bone marrow (stem) cells can differentiate into cells in multiple tissues, including lung. Conversely, there are reports that cells of nonhematopoietic tissues (brain, muscle) can give rise to lymphohematopoietic cells. Here we show that the lung contains cells capable of giving rise to lymphohematopoietic cells when transplanted to irradiated recipients. Whole lung cell suspensions, lung side population (SP) cells, and CD45+/- lung cells obtained from male transgenic enhanced green fluorescent protein–expressing mice were transplanted intravenously to total body irradiated female mice. Green fluorescent cells were recovered from the circulation and phenotyped for their expression of lymphohematopoietic markers (CD3, CD4, CD8, B220, Gr-1, and Mac-1). Lung SP cells were composed of heterogeneous populations and had less ability to give rise to lymphohematopoietic cells than did bone marrow SP cells. Furthermore, the ability of cells from the lung of aged mice to generate lymphohematopoietic progeny was equivalent to that of cells from young mice. Cells from lung with radioprotective and lymphohematopoietic reconstituting abilities were CD45+. CD45+ cells in the lung cells have lymphohematopoietic stem/progenitor cell characteristics, and this has implications for cell or gene therapy applications.

Abbreviations: Dulbecco's modified Eagle's medium, DMEM • enhanced green fluorescent protein, EGFP • fluorescence-activated cell sorter, FACS • fetal calf serum, FCS • green fluorescent protein, GFP • Gray, Gy • Iscove's modified Dulbecco's medium, IMDM • phosphate-buffered saline, PBS • side population, SP


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
There is accumulating evidence that the lung contains stem/progenitor cells for its epithelial compartment and that these cells occupy distinct "niches" along the bronchial tree (1–5). The properties of these stem cells are of interest because they are likely targets of carcinogens in the development of lung cancer (6, 7). Additionally, the lung contains a significant connective tissue compartment, including alveolar macrophages that are believed to be derived from stem cells in the bone marrow (8) as well as intraepithelial lymphocytes (9, 10). The progeny of marrow stem cells migrate in the form of monocytes to the lung, where they differentiate terminally to alveolar macrophages (11). Because of its blood pool, the lung in vivo also contains a small number of circulating hematopoietic stem cells at very low frequencies (12).

Recently, progeny of stem cells from adult bone marrow have been shown to contribute, albeit in very low numbers, to other tissues such as muscle, liver, lung, and intestine (13, 14). Conversely, cells from muscle and brain have been shown to generate hematopoietic cells on transplantation to irradiated recipients (15–17).

The extent of this process has been questioned (18) and concerns have been expressed over cell fusion as a basis of stem cell plasticity (19–21). Because hematopoietic stem cells can give rise to progeny in both the epithelial and connective tissue compartments of the lung, this study was devised to test the hypothesis that cells of the lung had the potential to give rise to hematopoietic cells. Lungs were obtained from bovine and enhanced green fluorescent protein (EGFP)-murine donors that had been perfused with several blood volumes of saline to minimize blood stem cell contamination. Lung side population (SP) cells, isolated based on their expression of an ABCG2 efflux pump for the vital Hoechst 33342 dye (22–25), from the bovine were characterized morphologically in vitro. Whole lung and lung SP cells from the murine donors were injected intravenously into irradiated syngeneic recipients. Green fluorescent cells were recovered and evaluated for their expression of hematopoietic and immune cell markers (CD3, CD4, CD8 B220, Gr-1, and Mac-1). Additionally, lung cells from male mice aged either 2 or 25 mo were injected intravenously into seven Gray (Gy)-irradiated syngeneic young adult female mice. The ability of the cells to repopulate circulating leukocytes was tracked and at 7 mo post-transplantation the proportion of Y chromosome positive leukocytes was analyzed. The expression status of CD45+/- on lung cells with lymphohematopoietic repopulating abilities was assessed to ascertain if these cells were lung cells exhibiting plasticity or hematopoietic stem cells in the lung.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Female C57Bl/6 (6–8 wk old) and EGFP C57Bl/6-TgN (ACTbEGFP, 6–8 wk old) mice were obtained from Jackson Laboratories (Bar Harbor, ME). Male C57Bl/6 mice 2 and 25 mo old were obtained from the National Institute of Aging (Bethesda, MD). Lungs from bovines were obtained from a local abattoir.

Lung and Bone Marrow Cell Suspensions
Male C57Bl/6 mice, 2 or 25 mo old, were killed and perfused via the right ventricle with 6 ml sterile phosphate-buffered saline (PBS). Lungs were then removed, minced, and digested in Dulbecco's modified Eagle's medium (DMEM; Sigma, St. Louis, MO) containing 10 U/g porcine pancreatic elastase (Sigma) and 125 U/g collagenase (Sigma blend collagenase; Sigma), as previously described with minor modifications (26). The cell suspension and undigested fragments were dispersed by repeated passage through a 5 ml pipette with DMEM 30% Fetal Calf Serum (FCS; Hyclone, Logan, UT). Then the cells were passed through a 70-µm strainer (Becton Dickinson, Franklin Lakes, NJ), washed twice with sterile PBS, and then resuspended in Iscove's modified Dulbecco's medium (IMDM; Gibco BRL, Grand Island, NY) containing 2% FCS, 1% penicillin-streptomycin (Gibco BRL), and 1 mM HEPES (Gibco BRL) (Hoechst IMDM solution). Bovine lung cells were obtained as described previously (27, 28). Bone marrow cells were extracted from the tibias and femurs of mice using a 25-G needle. Marrow cells were washed once with sterile PBS and resuspended in Hoechst IMDM solution.

Hoechst Staining
Lung and bone marrow cells were stained with the DNA-binding Hoechst 33342 dye (Sigma) as described previously (29, 30), with modifications (25). After overnight incubation at 4°C in Hoechst IMDM, the cell concentration was adjusted to 1 x 106 cells/ml in prewarmed Hoechst IMDM solution with 4 µg/ml Hoechst 33342 dye (Sigma). Cells were incubated at 37°C for 60 min for bovine or murine lung cells, or 90 min for murine bone marrow cells, respectively, with or without 100 µm and 1 mM verapamil (Sigma). Lung and bone marrow cells were also stained with c-kit (PE anti-mouse c-kit, CD117, 2B8; PharMingen, San Diego, CA), Sca-1 (PE anti-mouse Sca-1, Ly-6 A/E, D7; PharMingen), following Hoechst dye staining. The cells were maintained at 4°C until analyzed or sorted.

Isolation of SP Populations
Hoechst-stained cells were sorted using either an FACStarplus or FACSVantage cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA). The Hoechst 33342 dye was excited with an ultraviolet laser operating at 350 nm. Dual wave fluorescence detection was performed using a 450-nm band pass filter (blue) and a 675-nm EFLP optical filter (red). Separation of the two emission wavelengths was performed using a 610-nm DMSP dichronic mirror. The Hoechst blue and red emissions were presented on a linear scale. Forward and side scatter gates were used to eliminate erythrocytes and debris. After 5 x 104 to 1 x 105 events were collected, the side population (SP) was clearly identified as reported previously (25). Two sort gates were established. One sort gate was placed on the SP cell population and the other sort gate was placed on the major stained population of the profile (non-SP cell population). The two cell populations were sorted into tubes containing DMEM/10% FCS. The sorted cells were processed for injection in vivo or for cell culture.

Culture of Bovine Lung SP Cells
Sorted bovine lung SP cells were cultured in a 1:1 mixture of LHC9 (Rockville, MD) and RPMI 1640 media (Gibco BRL) at 1 x 106 cells/ml as described previously (27, 28). These cells were plated on chamber slides (Nunc, Naperville, IL) coated with Vitrogen (Cohesion, Palo Alto, CA) and maintained in an incubator at 37°C in an atmosphere of 5% CO2. The medium was changed every 2–3 d. The adherent cultured cells at 14 d were used for immunohistochemical analysis.

Transplantation Procedure
Female C57Bl/6 recipients were whole body irradiated initially with 7 Gy because the extent of reconstitution was uncertain. Whole lung, lung SP, bone marrow, or bone marrow SP cells were injected intravenously via a tail vein. Engraftment was determined by population of GFP-positive cells in the peripheral blood of the recipient by fluorescence-activated cell sorter (FACS) gating on bright green fluorescent cells. To investigate the effect of CD45-positive lung cell transplantation, lung cells from the EGFP mice were stained with CD45-phycoerythrin (30-F11; PharMingen) and sorted by FACS. Before sorting, forward and side scatter gates were used to eliminate erythrocytes and debris. Sorted CD45+ lung cells (2.5 x 106) or CD45- lung cells (2.5 x 106) were injected intravenously into 9.5 Gy irradiated recipients.

Immunohistochemistry
Bovine lung SP cells were cultured in chamber slides (Nunc) coated with Vitrogen (Cohesion) for 14 d. They were fixed by 4% paraformaldehyde and stained with an avidin–biotin complex method. Primary anti-cytokeratin 14 or anti-vimentin antibodies were purchased from Sigma. A Vectastain kit (Vector Laboratories, Burlingame, CA) was used for visualization. Positive cells exhibited brown staining, whereas the negative cells had no color reaction.

Electron Microscopic Analysis
Bovine lung SP cells were sorted into 4% paraformaldehyde at 4°C. The cells were washed twice in PBS and fixed in 1% OsO4 for 1 h. The pellet was washed again, dehydrated through ascending alcohol, treated twice in 100% ethanol, and transferred to propylene oxide. The pellet was embedded in Epon and araldite mixture and placed overnight at 56°C in a Beam capsule. Semithin sections and then ultrathin sections were cut and viewed using a Philips 410LS-transmission electron microscope, and images were recorded digitally, as reported previously (31).

Phenotyping of Peripheral Blood
Three months after whole lung cell (1 x 106 cells) transplantation, the mice were bled retro-orbitally to assess the phenotype of cells in the blood. After lysis of erythrocytes using WBL 1000 VitaLyse (Bio Ergonomics, St. Paul, MN), white blood cell suspensions were stained with combinations of antibodies (all from PharMingen), including CD3 (PE anti-mouse CD3{epsilon}, CD3{epsilon} chain), CD4 (PE anti-mouse CD4, L3T4 RM4–5), CD8 (PE anti-mouse CD8{alpha}, Ly-2, 53–6.7), Mac-1 (APC anti-mouse CD11b, Integrin {alpha} chain, Mac-1), Gr-1 (APC anti-mouse Ly-6G, Gr-1), and B220 (Cy-chrome anti-mouse CD45R/B220) to phenotype the cells. Samples were analyzed using an FACSCalibur (Becton Dickinson Immunocytometry Systems), and histograms were gated and analyzed for donor and antibody positive and negative cell populations.

FISH Analysis of Cells in the Blood of Recipients
At 7 mo after lung cell transplantation, FISH analysis was performed for the presence of male cells in the blood of recipient female mice using the Y chromosome probe (Cambio, Y mouse Probe 1189-YMF, Cambridge, UK). Blood cells were spread on a glass slide, fixed in methanol-acetic acid. The cells were air-dried at room temperature and fixed in 95% ethanol. The mouse Y chromosome probe was applied to the sections at 37°C. The sections were coverslipped and sealed with rubber cement, then denatured at 72°C for 1 min and incubated overnight at 37°C in a hydrated slide box. The next day, the coverslips were carefully removed in 4x SSC/3% NP-40 buffer for 3 min at 72°C. The sections were washed twice in 2x SSC/1% NP-40 buffer at room temperature and visualized with fluorescent mounting media (Vector Laboratories). Blood from normal C57Bl/6 male and female mice were used as positive and negative controls, respectively. More than 300 cells were counted for each sample.

Phenotyping of CD+45 Lung and Bone Marrow Cells
Whole lung and bone marrow cell suspensions were stained with antibodies (all from PharMingen) of combination CD45 (FITC) with c-kit (PE), Sca-1 (PE), and CD34 (PE) to phenotype CD45+ lung and bone marrow cells. Samples were analyzed by FACS.

Statistical Analysis
All data were reported as means ± SEM. The results were analyzed by Student's t test for comparison between the two groups. Values of P < 0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Isolation and Characterization of SP Cells from Lung and Bone Marrow
To evaluate the presence of SP cells in murine or bovine lungs, cells were stained with the DNA-binding Hoechst 33342 dye and SP cells were isolated. We first examined Hoechst 33342 staining with different concentrations and incubation times to obtain clear SP fractions (data not shown). As a result, optimal conditions were obtained when 4 µg/ml Hoechst 33342 dye for 90 min incubation was used for murine bone marrow (25), and 4 µg/ml Hoechst 33342 dye for 60 min incubation was used for murine or bovine lung cells. As shown in Figures 1A and 1D, similar numbers of SP populations (~ 0.3%) were detected in murine and bovine lung. SP cells of both murine lung and bone marrow cells were sensitive to verapamil (Figures 1B, 1C, and 1F), consistent with the efflux of Hoechst dye in these cells being mediated by the activity of p-glycoprotein multidrug/ATP-binding cassette transporter proteins such as Bcrp1 (ABCG2).




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  		Figure 1. Isolation and characterization of side population (SP) cells from lung and bone marrow. The presence of SP cells in murine lung, bovine lung, and murine bone marrow cells was evaluated by staining with the DNA-binding dye Hoechst 33342. The SP cells in murine lung (A), bovine lung (D), and murine bone marrow (E) are indicated in the boxed areas. The indicated region of bovine lung cells contained ~ 0.3% of SP cells similar to murine cells (D). Both lung and bone marrow SP cells were sensitive to verapamil (B, 100 µM; C, 1 mM for lung; F, 1 mM for bone marrow). Murine lung and bone marrow SP cells were stained with Sca-1 or c-kit. (G) Unstained lung SP cells. (H) Lung SP cells stained with Sca-1. (I) Lung SP cells stained with c-kit. (J) Unstained bone marrow SP cells. (K) Bone marrow SP cells stained with Sca-1. (L) Bone marrow SP cells stained with c-kit. The data are representative of at least three experiments.

 
To characterize lung and bone marrow SP cells, SP cells were stained with c-kit, Sca-1, and CD34. Both lung and bone marrow SP cells had ~ 30–40% of Sca-1–positive cells (Figures 1H and 1L). Although > 80% of bone marrow SP cells were stained with c-kit, only 5–7% of lung SP cells were stained with c-kit (Figures 1I and 1M). Lung SP cells were not stained with CD34 (< 1%). Bone marrow SP cells also had a low expression of CD34 (2–3%). Lung SP cells had fewer c-kit–positive cells than did bone marrow cells.

Detection of GFP-Positive Cells in the Blood after Transplantation
The recovery of circulating leukocytes was tracked in irradiated recipients of murine whole lung (1 x 106 cells) or whole bone marrow (1 x 106 cells) or lung SP (2,000 cells) or bone marrow SP (2,000 cells), and the proportion of donor cells was assayed flow cytometrically, gating on bright green fluorescent cells. The population of GFP-positive cells in the blood of mice receiving whole bone marrow or bone marrow SP cells transplantation was ~ 80% at 5 wk, similar to values in control EGFP mice. Donor whole lung cells contributed to ~ 30% of the leukocyte pool. In contrast, 2,000 lung SP cells only produced a minimal transient lymphohematopoietic repopulation in a small proportion of recipients (Figure 2), potentially because of the low number of transplanted cells.



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  		Figure 2. Detection of GFP-positive cells in the blood after transplantation to irradiated recipients. From 1–9 wk after transplantation of whole bone marrow cells (1 x 106 cells; circles), bone marrow SP cells (2,000 cells; squares), whole lung cells (1 x 106 cells; triangles), or lung SP cells (2,000 cells; diamonds), the mice were bled retro-orbitally to assess the percent of donor cell engraftment in the blood. Data are expressed as a percentage of the GFP-positve cells in the recipient's blood. Each value represents the mean ± SE of at least three mice.

 
Characterization of Lung SP Cells
In order to have a sufficient number of cells for cell culture and electron microscopic analysis, we used bovine lung cells. Morphologically, whole lung cell preparations from bovine and murine, unlike whole bone marrow populations, contained significant proportions of cytokeratin 14–positive cells in addition to vimentin-positive cells (data not shown). Bovine and murine lung SP cells appeared at the light microscopic level as small to medium lymphocyte-like mononuclear cells, very similar in appearance to bone marrow SP cells (data not shown). Also, like bone marrow SP cells, small/medium lymphocyte-like lung SP cells from bovine were immunocytochemically negative for cytokeratin 14 staining (Figure 3A, left panel, arrows). When bovine lung SP cells were grown in culture for 14 d under conditions designed to support the growth and differentiation of lung epithelial cells, unlike bone marrow SP populations, the cells spread and enlarged and generated colonies of cytokeratin 14 positive cells (Figure 3A, right panel). Despite their relatively homogenous appearance at the light microscopic level, when analyzed electron microscopically, sorted bovine lung SP cells were heterogeneous populations containing small epithelial cells (Figure 3B, top left panel) and macrophages (Figure 3B, top right panel), plasmablasts (Figure 3B, bottom left panel), and undifferentiated lymphocyte-like cells (Figure 3B, bottom right panel).



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  		Figure 3. Characterization of lung SP cells. (A) Sorted small/medium lymphocyte-like lung SP cells of bovines immunostained for cytokeratin 14 did not exhibit staining (left panel, arrows; original magnification: x200). Sorted bovine lung SP cells were cultured on chamber slides with a 1:1 mixture of LHC9 and RPMI 1640 media. The cells spread and enlarged and were immunocytochemically positive for cytokeratin 14 at Day 14 (right panel; original magnification: x200). (B) Analysis by electron microscopy showed bovine lung SP cells were a heterogeneous population, containing small epithelial cells (top left panel; original magnification: x10,000) and macrophages (top right panel; original magnification: x10,000), plasmablasts (bottom left panel; original magnification: x10,000), and lymphocyte-like cells (bottom right panel; original magnification: x10,000).

 
The Phenotype of the Circulating White Blood Cells in Recipients of EGFP Donor Lung Cells
The phenotype of cells of the circulating leukocyte pool was analyzed gating on the bright green fluorescence of the donor cell population using markers of T cells (CD3, CD4, CD8), B cells (B220), granulocytes (Gr-1), and macrophages (Mac-1). A representative result is presented in Figure 4A. Transplanted whole lung cells (1 x 106 cells) had the potential to generate all of these lineages in irradiated recipients, yielding population similar to those in the blood of the control (non-green) C57Bl/6 mouse (Figure 4B).




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  		Figure 4. The phenotype of the circulating leukocyte cells in irradiated recipients of lung cells from EGFP donor mice. To evaluate the phenotype of circulating white blood cells in recipients from EGFP donor lung cells, blood was analyzed by antibody staining and flow cytometry for the presence of T cells (CD3, CD4, CD8), B cells (B220), macrophages (Mac-1), and granulocytes (Gr-1). (A–F) Analysis of white blood cells from recipients of lung cells from EGFP donors. (G–L) Analysis of white blood cells from normal female C57/6 mice.

 
The Effect of Aging Donor Whole Lung Cells on Repopulation of Leukocytes in Irradiated Recipients
To investigate if there was an effect of aging on the lung cell lymphohematopoietic repopulating ability, cells were harvested from the lungs of male C57Bl/6 mice aged 2 or 25 mo. One, five, or ten million of these cells were injected intravenously into 7 Gy–irradiated syngeneic female mice. The ability to repopulate circulating leukocytes was analyzed. At 7 mo after lung cell transplantation, FISH analysis was performed for the presence of Y chromosome–positive leukocytes in the blood of female mice to confirm the engraftment of male donor lung cells. Although there was an indication of a slight inverse cell dose response in early repopulation at 3 wk after lung cell transplantation, there were no significant differences evident in the long-term recovery of leukocytes in recipients of aged versus young donor lung cells (Figure 5A). There was a clear dose response in proportions of Y chromosome–positive leukocytes at 7 mo after transplantation (young 10 x 106 cells, 7.62 ± 1.24% at *P < 0.05 compared with young 5 x 106 cells, at {dagger}P < 0.01 compared with young 1 x 106 cells, respectively; aged 10 x 106 cells, 8.28 ± 0.70% at {ddagger}P < 0.01 compared with aged 5 x 106 cells and 1 x 106 cells), but no significant differences between the proportion of Y chromosome–positive leukocytes from the aged and young lung cell donors (Figures 5B and 5C).



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  		Figure 5. Effect of aging on lung cell transplantation. (A) Whole lung cells from young (2-mo-old) and aged (25-mo-old) male mice at different doses (1 x 106, 5 x 106, 10 x 106 cells) were transplanted into irradiated (7 Gy) C57Bl/6 female mice (2 mo old). The recovery of circulating white blood cells was followed to 9 wk after lung cell transplantation. Each value represents the mean ± SE of 8–10 mice. At 7 mo after lung cell transplantation, FISH analysis was performed to detect the presence of donor male cells in the blood of female recipients. (B) Left panel: representative fluorescence microscopic image (original magnification: x100) of FISH for Y-chromosome–positive cells in the blood of female recipients. Right panel: negative control cells in the blood of normal female C57/Bl mice (original magnification: x100). (C) The proportion of Y-chromosome–positive cells both in young and old lung cell transplant recipients. *P < 0.05 compared with young 1 x 106 group, {dagger}P < 0.01 compared with young 1 x 106 group, {ddagger}P < 0.01 compared with old 1 x 106 group.

 
The Effect of CD45+ Lung Cell Transplantation
To determine the ability of CD 45+ lung cells to support irradiated mice, C57Bl/6 mice were irradiated with a lethal dose of 9.5 Gy and injected with CD 45+ lung cells (2.5 x 106), CD 45- lung cells (2.5 x 106) from EGFP mice. At day 19 post-transplantation, retro-orbital blood was evaluated for the presence of green cells to assess the percentage of donor cell engraftment in the blood. As shown in Figure 6A, the proportion of GFP-positive cells in the blood of CD 45+ lung cell transplanted recipients was significantly higher than that of CD45- lung cell transplanted recipients (CD45+, 28.9 ± 7.4% at *P < 0.05 compared with CD45-, 4.9 ± 0.8%). In addition, a significant difference was observed in the mortality of irradiated CD 45- lung cell transplanted mice compared with irradiated CD45+ lung cell transplanted recipients (Figure 6B). The Mantel-Cox log rank test showed that mortality was higher in irradiated CD45- lung cell transplanted-recipients compared with irradiated CD45+ lung cell transplanted-recipients (P = 0.033, n = 10 mice each group). Consequently, the lymphohematopoietic and radioprotective abilities of lung cells are primarily contained in the CD45+ cell fraction.



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  		Figure 6. Lymphohematopoietic reconstitution by CD45+/- lung cells in irradiated recipients. CD45+lung cells (2.5 x 106) or CD45-lung cells (2.5 x 106) from EGFP mice were transplanted into irradiated (9.5 Gy) mice. (A) At Day 19 after transplantation, the mice were bled retro-orbitally to assess engraftment in the blood. Data are expressed as a percentage of the GFP-positive cells in the recipient's blood. Each value represents the mean ± SE of at least three mice. (B) Survival curves for CD45+/- lung cell transplanted mice (n = 10, per group). Closed circles, CD45+ lung cell transplanted mice; open circles, CD45- lung cell transplanted mice.

 
To investigate the phenotypic characterization of CD45+ lung cells, dual staining of the combination CD45 with c-kit, sca-1, or CD34 was performed. Bone marrow cells were also stained for comparison. In whole murine lung cells, ~ 30–40% of whole lung cells were CD45+. About 30% of whole lung cells were stained with both CD45 and Sca-1 (Figure 7B). About 3% of whole lung cells were stained with both CD45 and c-kit (Figure 7C). In contrast, ~ 75% of bone marrow cells were CD45+. About 10% of bone marrow cells were stained with both CD45 and c-kit or CD45 and Sca-1 (Figures 7F and 7G). Both lung and bone marrow cells had a low proportion of cells stained with the combination CD45 and CD34 (Figures 7D and 7H). CD45+ lung cells had more Sca-1+ cells than CD45+ bone marrow cells.



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  		Figure 7. Phenotypes of CD45+ lung and bone marrow cells. Murine lung and bone marrow cells were stained with the combination of CD45 with Sca-1, c-kit, or CD34. (A) Unstained lung cells. (B) Lung cells stained with the combination of CD45 with Sca-1. (C) Lung cells stained with the combination of CD45 with c-kit. (D) Lung cells stained with the combination of CD45 with CD34. (E) Unstained bone marrow cells. (F) Bone marrow cells stained with the combination of CD45 with Sca-1. (G) Bone marrow cells stained with the combination of CD45 with c-kit. (H) Bone marrow cells stained with the combination of CD45 with CD34. The data are representative of at least three experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we demonstrate that cell suspensions of extensively perfused (several blood volumes) whole lung, but not lung SP cells, produced lymphohematopoietic progeny in irradiated recipients. Lung cells with radioprotective and lymphohematopoietic reconstituting abilities were CD45+. Furthermore, the ability of cells from the lungs of aged mice to generate lymphohematopoietic progeny was equivalent to that of lung cells from young mice. Lung SP cells had less ability to give rise to lymphohematopoietic cells, but had the ability to generate cytokeratin-positive epithelial cells in vitro.

"Side population (SP)" cells, originally described in bone marrow, have the majority of bone marrow stem cell activities (29, 30). Cells from nonhematopoietic tissues, including lung, also have been demonstrated to contain SP populations (32, 33). In this experiment, murine or bovine lungs contained SP cells, which shared some morphologic and functional properties with bone marrow SP stem cells. There were also differences in their properties. The expression of Sca-1 in bone marrow or bone marrow SP cells has been reported to be variable between mice (34, 35). In the current study, both lung and bone marrow SP cells had ~ 30–40% of Sca-1+ cells. Bone marrow SP cells had > 80% of c-kit+ cells, which was several-fold that observed in SP cells of lung (5–7%). Lung SP cells were small- to medium-sized mononuclear cells that were cytokeratin 14–negative. The lung SP population was morphologically heterogeneous and included cells, for example, plasmablasts and macrophages, that were likely irreversibly committed differentiated cells. This suggests that epithelial and lymphocyte-like cells were more likely to have stem/progenitor activities. The lung and lung SP cells did not form hematopoietic colonies in short-term in vitro assays but had the ability to generate, under appropriate culture conditions, colonies of cytokeratin 14–positive epithelial cells, indicating epithelial progenitor cell activities. In this study, we used bovine SP cells for culture and analysis by electron microscopy. Although SP cells have been reported in multiple species (30), it is possible that SP cells in different species have different properties, including the heterogeneity observed in the current study.

When transplanted intravenously into mid-lethally irradiated recipients, whole lung, like bone marrow and bone marrow SP cells, contributed to the circulating leukocyte pool, generating cells with the phenotype of myeloid cells, macrophages, B cells, and T cells. The transplanted cells also contributed progeny to various tissues, including bone marrow, spleen, and lung. However, unlike transplanted bone marrow or bone marrow SP cells, cells from the lung did not give rise to significant numbers of cells in the recipients' livers (data not shown). The frequency of the donor progeny was generally lower for equivalent numbers of lung cells transplanted, compared with bone marrow cells, potentially reflecting a lower frequency of lymphohematopoietic stem/progenitor cells in the donor lung. Lung SP cells, unlike bone marrow SP cells, exhibited only transient production of lymphohematopoietic progeny, but were able to generate cytokeratin positive epithelial progeny in vitro. Potentially, the low number of SP cells transplanted and their heterogeneous composition compromised the ability to detect lymphohematopoietic progeny in recipients.

The hematopoietic stem cell compartment in aging mice has been postulated to have a diminished primitive cell compartment and a concomitantly expanded committed stem/progenitor cell compartment (36, 37). In this experiment, the lungs of young and aged C57Bl/6 mice had equivalent abilities to generate lymphohematopoietic progeny in irradiated recipients. There was no evidence for a loss of potency of in vivo lymphohematopoietic repopulating ability of lung cells with aging. The lung appears to contain a reservoir of lymphohematopoietic repopulating stem/progenitor cells throughout life.

It is unlikely that the cells in whole lung with lymphohematopoietic repopulating abilities reflect blood cell contamination of the lungs, because the recipients were extensively perfused with PBS before the harvesting of the lung cells. Based on the frequency of hematopoietic stem cells in the circulation in the absence of cytokine mobilization (38) and the blood volume of mouse lungs even without perfusion, the number of blood stem cells that could be present is too infrequent to account for the results. Rather, the results are consistent with the presence of (stem, or progenitor), cells with lymphohematopoietic reconstituting ability in the lung. Recently, Wagers and coworkers showed a significant number of infused CD45+ hematopoietic cells in the lung parenchyma in a study of parabiosis (11). A CD45+ fraction in lung SP cells has also been reported (32, 33). In the current study, CD45+ lung cells had different proportions of Sca-1+ and c-kit+ cells from those of CD45+ bone marrow cells. The in situ functions of CD45+ lung cells are unclear. However, based on parallels with the intestine (39), lymphohematopoietic progenitor cell populations might differentiate in situ into intraepithelial lymphocyte populations in the lung (8) or like CD45+ cells in muscle, participate in local tissue repair (40).

This study does not generally distinguish the existence of a pluripotential stem cell in the lung that can give rise to hematopoietic or other mesenchymal cells as well as cells of the epithelial compartment. The lack of colony formation in short-term cytokine stimulated assays and delay in generating circulating leukocytes after transplantation suggests that the lung cell responsible for lymphohematopoietic repopulation does not have all of the characteristics of more committed hematopoietic stem or progenitor cells. It may represent a primitive, undifferentiated cell that takes time to acquire a more differentiated phenotype. To produce progeny, it may need time to reprogram when transferred from one environment (the lung) to another (intravenous injection into irradiated recipients). At the same time, the CD45+ phenotype of the lymphohematopoietic reconstituting cell together with the epithelial cell–generating ability of lung SP cells strongly suggests that these are separate but coexisting stem cell populations in the lung.

In summary, these studies demonstrate for the first time that lung contains CD45+ stem cells capable of giving rise to lymphohematopoietic cells in vivo and SP cells capable of generating epithelial compartment in vitro. Furthermore, the lungs of young and aged mice have equivalent abilities to generate lymphohematopoietic progeny. If human lung cells have similar characteristics, such cells could be useful in the cellular or gene therapy of hematopoietic disorders and CD45+ hematopoietic cells, useful in the therapy of lung disorders.


   Acknowledgments
 
The authors thank Dr. C. Kuszynski and L. Wilke of the Cell Analysis Core Facility for performing flow cytometry, Dr. Vasiliy Polosukhin for assistance with electron microscopy, and Ms. Lillian Richards for secretarial support. This research was supported by the Nebraska Research Initiative and this support is gratefully acknowledged.

Received in original form April 21, 2003

Received in final form September 24, 2003


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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