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Published ahead of print on March 31, 2005, doi:10.1165/rcmb.2005-0024OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 33, pp. 32-40, 2005
© 2005 American Thoracic Society
DOI: 10.1165/rcmb.2005-0024OC

Embryonic Lung Side Population Cells Are Hematopoietic and Vascular Precursors

Ross Summer, Darrell N. Kotton, Simon Liang, Kathleen Fitzsimmons, Xi Sun and Alan Fine

The Pulmonary Center, Boston University School of Medicine, Boston, Massachusetts

Correspondence and requests for reprints should be addressed to Ross Summer, The Pulmonary Center, R-304, Boston University School of Medicine, 80 East Concord St., Boston, MA 02118. E-mail: rsummer{at}lung.bumc.bu.edu


   Abstract
Top
 Abstract
Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Side population (SP) cells are a select cell population identified by a capacity to efflux Hoechst dye that are highly enriched for stem/progenitor cell activity. In this study, we found that SP cells comprised of CD45(+) and CD45(–) subtypes are present in the embryonic lung (E-SP) at levels varying with gestational age. Long-term in vivo competitive blood reconstitution studies demonstrated that hematopoeitic stem cell capacity resided within the CD45(+) E-SP cell subset. Immunophenotyping of CD45(–) E-SP cells determined that this population consists of two subtypes: CD31(–) and CD31(+). Limited gene expression profiling indicated that CD45(–)/CD31(–) E-SP cells have features of smooth muscle precursors, and give rise to smooth muscle in culture. On the other hand, CD45(–)/CD31(+) E-SP cells express genes characteristic of endothelium, but by themselves do not grow or differentiate in culture. Co-culture of CD45(–)/CD31(+) and CD45(–)/CD31(–) E-SP cells, however, resulted in the formation of complex tubular networks that express markers of endothelium. Together, these findings illustrate that embryonic lung SP cells are heterogeneous, composed of hematopoeitic and nonhematopoeitic progenitors, and may play a key role in the formation of the lung vasculature.

Key Words: endothelium • mesenchyme • smooth muscle • stem cells


   Introduction
Top
 Abstract
 Introduction
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
The structure of the mammalian lung consists of an array of branching epithelial tubes that is enveloped by a network of cells derived from the embryonic mesoderm. These mesodermal-derived cells form the vascular structures associated with the branching airways, and provide the structural support of the lung. Several studies have suggested the presence of specific endothelial and smooth muscle progenitor cell populations within the developing lung (1–3). To date, the ability to isolate purified populations of these cells has not been possible, due in part, to the absence of specific markers.

In other systems, stem and progenitor cells have been identified based on the expression of unique surface proteins and the ability to exclude vital dyes (4, 5). In particular, the ability to exclude Hoechst dye is a feature shared by various stem and progenitor cell types (6, 7). Cells that efflux Hoechst dye are referred to as side population (SP) cells because of their characteristic appearance on density dot plots generated during flow cytometry (4). SP cells appear "off to the side" of the main population (MP) of cells due to their relative absence of staining. This unique staining property has been used to isolate cell populations enriched for stem or progenitor cell activity.

SP cells were first identified in the adult murine bone marrow (BM), where they comprise less than 1% of all cells (4). These cells share various characteristics with hematopoeitic stem cells (HSC) including the expression of Sca-1, c-kit, and the panhematopoeitic marker CD45, as well as the absence of expression of mature hematopoeitic cell markers (4, 8). Consistent with this phenotype, BM SP cells are highly enriched for HSC activity. In fact, a single BM SP is capable of fully reconstituting the bone marrow after radioablation (9).

SP cells have also been identified in various nonhematopoeitic adult tissues including the heart, skeletal muscle, lung, testes, and breast (7, 10, 11). Tissue SP cells are comprised of CD45(+) and CD45(–) cell populations (10). CD45(+) SP cells can be distinguished from CD45(–) SP cells on the basis of differences in morphology, and gene expression (12). In vitro and in vivo assays of adult tissue SP cells indicate that only the CD45(+) population has hematopoeitic progenitor activity (10, 13). Other studies suggest that adult tissue CD45(–) SP cells are enriched for endothelial and muscle progenitor cell activity. In this regard, CD45(–) skeletal muscle SP cells can engraft as skeletal muscle, and endothelial cell types in vivo, while adult and embryonic heart SP cells have the capacity to differentiate into cardiomyocytes in vitro (7, 14, 15).

In this study, we set out to identify and characterize SP cells in the developing mouse lung. We speculated that by isolating these cells we would identify lung progenitor cell types. As expected, we found that embryonic (E) lung SP cells are comprised of CD45(+) and CD45(–) subsets. Limited gene expression profiling along with functional assays indicates that CD45(+) SP cells possess hematopoeitic stem cell capacity. In contrast, CD45(–) E-SP cells lack hematopoietic activity, but can serve as progenitors of smooth muscle and endothelial cell types.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Animals
Lung suspensions were prepared from timed-pregnant mice of various gestational ages. Mouse strains used were of the CD1 strain obtained from Charles River (Wilmington, MA). For bone marrow transplantation studies C57BL/6J (CD45.2) lung E-SP cells were transplanted into lethally irradiated B6.SJL (CD45.1) congenic mice (6–8 wk old; Jackson Laboratories, Bar Harbor, ME). In co-culture studies, cells expressing green fluorescent protein were isolated from C57BL/6-Tg(ACTBEGFP)1Osb/J mice (Jackson Laboratories). All mice were killed by isoflurane anesthesia followed by cervical dislocation. Animal studies were conducted according to protocols approved by the National Institutes of Health and the Boston University Animal Care and Use Committee.

Cell Preparations and Staining
Cell suspensions were obtained from enzyme-digested lungs as previously described (16). Briefly, lung digestion was performed by finely mincing tissue with a razor blade in the presence of collagenase 0.1%, dispase 2.4 U/ml (Roche Diagnostics, Indianapolis, IN) and CaCl2 (2.5 mM) at 37°C for 1 h. Removal of nonspecific debris was accomplished by sequential filtration through 70- and 40-µm filters. Cells were re-suspended at a concentration of 10 x 106 cells/ml. Hoechst 33342 5 µg/ml (Sigma-Aldrich, St. Louis, MO) staining of cell suspensions were performed in the presence and absence of verapamil (50 mM). The concentration of Hoechst dye was based on our work with adult lung SP cells (16). Staining was performed at 37°C for 90 min in Dulbecco's modified Eagle's medium supplemented with 2% fetal calf serum, 10 mM HEPES, and 1% penicillin/streptomycin. At the completion of staining, cells were immediately placed on ice. Immunostaining studies were performed at 4°C in the dark. Antibodies used for these studies were fluorochome-conjugated monoclonal rat anti-mouse antibodies reactive to CD45, CD31, CD34, CD11b, CD3, CD4, CD8a, CD11c, NK1.1, Gr-1, Ter119, and Sca-1, and mouse anti-mouse antibody to CD45.2 (BD Pharmingen, Lexington, KY). Antibody to VE-Cadherin was unconjugated and required the use of a fluorochrome-conjugated anti-rat secondary antibody for detection (BD Pharmingen). Dead cells were excluded from analysis based on propidium iodide staining (2 µg/ml), and comprised < 10% of total cells. For all studies, isotype control antibodies were employed as negative controls and to establish gating parameters for positive cells.

Bone Marrow Transplantation Studies
Lung cells were isolated from mice expressing CD45.2 and transplanted into CD45.1 mice. Thirty thousand lung CD45(+)/lineage(–), CD45(+)/CD11b(+) and CD45(–) E-SP cells were isolated by flow cytometry from embryonic lung (E17.5) digests and injected by tail vein into lethally irradiated CD45.1 mice. Unfractionated bone marrow cells (2 x 105 CD45.1) were injected along with lung E-SP fractions to serve as competitors. Recipient mice were exposed to 9.5 Gy in a single dose given 4 h before cell infusion. Four months after cell infusion, peripheral blood was isolated from the retro-orbital vein. After red blood cell lysis (Sigma-Aldrich), cells were incubated with FITC-conjugated anti-CD45.2 mAb before analysis of blood chimerism by flow cytometry. Twenty thousand events were analyzed per sample.

Fluorescence-Activated Cell Sorting
Flow cytometry analysis of Hoechst stained cells was performed on a triple laser instrument (MoFlo; Cytomation Inc., Fort Collins, CO). An argon multi-line ultraviolet (333–363 nm) laser was used to excite Hoechst dye. Fluorescence emission was collected with a 405/30 band pass (BP) filter (Hoechst blue) and a 660 long pass filter (Hoechst red). A second 488-nm argon laser was used to excite PE, FITC, and PI. Data analysis was performed using Summit software (Cytomation Inc.). The purity of SP cells isolated from a single sort was ~ 90–95% (data not shown).

Reverse Transcription-Polymerase Chain Reaction
cDNA was generated from RNA extracts using a reverse transcription (RT) kit (Promega, Madison, WI) on 10,000 freshly isolated CD45(–)/CD31(–), CD45(–)/CD31(+) lung E-SP cells, and CD45(–) main population cells (positive control). RNA was also isolated from CD45(–)/CD31(–) E-SP cells 7 d after culture. Polymerase chain reaction (PCR) was done using the following primers: Myocardin 5'-TGTCTTAAGGACTCGATTGGG-3' and 5'-TCAATGGAGGAAAGCACGATA-3', {alpha} SM 5'-AGCTTTGGGCAGGAATGATTTGG-3' and 5'-AAGATCATTGCCCCTCCAGAACG-3', Sm 22 {alpha} 5'-GGTGGGATCTCCACGGTAGTT-3' and 5'-AGGAGCTGGAGGAGCGACTAG-3', Sm-MHC 5'-GAAAGTCATACAGTACTTGGCTGTG-3' and 5'-GAGTTGTCGTTCTTGACGGTT-3', Tie2 5'-CTTCCATCATCATCCGGTATA-3' and 5'-CTATAGTTCAGTGATTCGATTGC-3, Flk-1 5'-TTGACATGCACAGTCTACGC -3' and 5'-AGCAGAGCAAACATAGTCGC-3', flt-1 5'-GGATGCAGGGGACTATACGAT-3' and 5'-AGGAAACGTGAAAGCCATTCG -3', PDGFR-{alpha} 5'-CAAAAA CCTCCTTTCGGACGA-3' and 5'-AAGTGACACCGATGTACGCAT-3' PDGFR-ß 5'-GATCTCATAGATCTCGTCGGA-3' and 5'-CACGGAATACTGCCGATACGG-3', PECAM (CD31) 5'-GTGCACAGGACTCTCGCAATC-3' and 5'-GAAGCCAACAGCCATTACGGT-3' Ang-1 5'-CATTCTTCCAGAACACGACG-3' and 5'-TGTCGTACTGTGAGTAGGCTCG-3', ß-actin primers 5'-GCTCGTTGCCAATAGTGATG-3' and 5'-AAGAGAGGTATCCTGACCCT-3'. For all PCR reactions, 35 cycles were performed. Annealing temperatures were as follows: 52°C for myocardin, flk-1, flt-1, PDGFR{alpha}, and PDGFRß; 55°C for {alpha}-smooth muscle actin ({alpha}-Sma) and Sm22{alpha}; 58°C for SmMHC; 50°C for Tie2; 59°C for CD31; and 53°C for ß-actin and Ang-1. Extension time for all reactions was 1 min.

Immunohistochemistry
Lung E-SP cells were cytospun onto charged slides, fixed with acetic acid/methanol (1:3) for 5 min and washed in phosphate-buffered saline. Slides were quenched with sodium borohydride to quench autofluorescence. Fluorescent staining on E-SP cells was performed using a fluorochrome-conjugated monoclonal antibody to pan-cytokeratin (Sigma-Aldrich) or a fluorochrome-conjugated monoclonal antibody to {alpha}–Sma (Sigma-Aldrich) for 1 h at room temperature. After staining, slides were washed in phosphate-buffered saline, and nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI) before evaluation by fluorescent microscopy. Immunostaining of paraffin-embedded lung sections for {alpha}-Sma, {alpha} smooth muscle tropomyosin, and von Willebrand factor (vWF) was performed per published protocols (16, 17). Labeled cells were then visualized using the Zeiss NH BO 103 microscope (Carl Zeiss, Thornwood, NY) equipped with mercury vapor short-arc-lamp with double band-pass filters set at 390/460 nm, 475/530 nm, and 560/630 nm. Images were captured with Axiocam MR CCD camera (Carl Zeiss) and then processed using Axiovision 3.1 software (Carl Zeiss).

Primary Cell Cultures
Murine lung E-SP and non-SP cells were cultured on fibronectin-coated 8-well chamber slides (10,000 cells/well) at 37°C, 5% CO2. To promote smooth muscle growth, cells were cultured in media (SmGm-2) containing EGF, bFGF, insulin, and fetal bovine serum (Cambrex, Baltimore, MD). To promote endothelial cell growth, E-SP cells were cultured in media (EGM-2) supplemented with EGF, VEGF, IGF-1, bFGF, fetal bovine serum, hydorcortisone, ascorbic acid, heparin (Cambrex, Baltimore, MD), and antimicrobial agents.


   RESULTS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
SP Cells Exist in the Embryonic Lung
In previous work, we identified SP cells in the adult murine lung, and found that these cells comprise only a small fraction (0.03–0.07%) of total cells (16). We speculated that since lung development is a time of increased cell turnover, SP cells would be detected at greater frequency. To test this, we performed Hoechst staining of enzyme-digested embryonic (E) lungs from mice of several gestational ages (13.5, 15.5, 17.5, 18.5 d after conception). In these studies, E-SP cells were detected at all developmental time points (Figure 1). We found that the fraction of E-SP cells varied with age, peaking at embryonic day 17.5, and steadily declining thereafter. In previous work, we found that the percentage of lung SP cells remains fixed from postnatal day 5 to 1 yr of age (16). As with other SP cell populations, the detection of lung E-SP cells could be blocked by verapamil (50 mM).



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  		Figure 1. Embryonic lung SP cells exist at various gestational time points. Representative density dot plot of embryonic (E) lung digests after staining with Hoechst dye. E-SP cells (boxed area) were detected at all time points. The fraction of E-SP cells varied with age, peaking at E17.5. Detection of E-SP cells could be blocked by verapamil (right).

 
Embryonic Lung SP Cells Are Comprised of CD45(+) and CD45(–) Fractions
In adult mouse, tissue SP cells are comprised of CD45(+) and CD45(–) fractions (4, 10). We examined the expression of CD45 on embryonic lung E-SP cells and found that they are also comprised of CD45(+) and CD45(–) subsets (Figure 2). Of interest, the ratio of CD45(+) to CD45(–) E-SP cells varied during development.



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  		Figure 2. Embryonic lung SP cells are heterogeneous in the expression of the panhematopoeitic marker CD45. (A) Left: Representative density dot plot of Hoechst stained embryonic (E) lung digest. E-SP cells are displayed in boxed area. Right: CD45 expression analysis demonstrates that lung E-SP cells consist of CD45(+) and CD45(–) subsets. (B) Histogram showing the fraction of CD45(+) and CD45(–) SP cells in the adult and developing lung (n = 3 at all developmental time points). Error bar reflects standard deviation.

 
CD45(+) Lung E-SP Cells Are Heterogeneous
To further characterize E-SP cells, we examined their expression of mature hematopoeitic markers. In these studies, we focused on cells derived from the E17.5 lung. To do this, we evaluated Hoechst stained lung digests (E17.5) with antibodies to differentiated blood cell lineage (lin) markers (B220, CD3, NK1.1, CD4, CD8a, CD11b, CD11c, Gr-1, and Ter119). We found that 100% CD45(–) SP cells were lin-negative and 15% (SD ± 5.9, n = 3) of CD45(+) cells were also lin-negative. The majority of CD45(+) E-SP cells (86% SD ± 5.9, n = 3) were, however, positive for the macrophage marker CD11b (Figure 3). These results demonstrate that CD45(+) E-SP cells are not a uniform population, but rather consist of at least two distinct types (Table 1).



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  		Figure 3. CD45(+) E-SP cells are a diverse cell type. Left panel: Density dot plot demonstrating the presence of E-SP cells (boxed area) in the E17.5 lung. Right panel: Hoechst-stained lung digests were stained with fluorochrome-conjugated antibodies to CD45 and CD11b. Representative dot plot showing that CD45 (+) E-SP cells (boxed areas) are comprised of CD11b(+) and CD11b(–) subsets. E-SP cells stained negative for all other lineage markers (data not shown). Middle panel: Isotype staining. Results were used for establishing gating for positive cells.

 

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  		TABLE 1. Summary of protein expression profiling in e-sp cells

 
CD45(+) Lineage(–) E-SP Cells Have the Capacity to Reconstitute the Bone Marrow after Lethal Irradiation
To examine the hematopoeitic capacity of lung E-SP cells, we transplanted 30,000 CD45(+)/CD11b(+), CD45(+)/lin(–), or CD45(–) E-SP cells (obtained from E17.5 CD45.2 mice) into lethally irradiated CD45.1 mice. Two hundred thousand CD45.1 whole bone marrow cells were injected along with each lung E-SP cell subset. Four months after transplantation, blood chimerism was tested in each group of mice (Figure 4, n = 3 each group). In mice transplanted with either CD45(+)/CD11b(+) or CD45(–) E-SP cells, no donor-derived cells were detected in the peripheral blood. In contrast, in mice transplanted with CD45(+)/lin(–) E-SP cells, > 90% of peripheral blood cells were donor-derived at 4 mo; these cells demonstrated multilineage hematopoeitic reconstitution capacity (Figure 4).



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  		Figure 4. CD45(+) lineage(–) E-SP cells have hematopoeitic stem cell activity. Thirty thousand CD45(+) lineage(–), CD45(+)CD11b (+), or CD45(–) Ly 5.2 E-SP cells were transplanted in lethally irradiated Ly 5.1 mice and peripheral blood (PB) chimerism was examined 4 mo after transplantation. Donor-derived (CD45.2) blood cells are not detected in the PB of mice transplanted with (A) CD45(–) or (B) CD45(+)/CD11b(+) lung E-SP cells. (C) CD45(+)/lineage(–) E-SP cells were capable of providing long-term blood reconstitution. Multi-lineage engraftment by CD45(–)/lineage(–) E-SP cells was demonstrated by immunostaining for the myeloid marker CD11b, B-cell marker B220, T cell markers CD4 and CD8, and the erythroid marker Ter119 (boxed dot plots on right).

 
CD45(–) Embryonic Lung SP Cells Are Heterogeneous
In the adult lung, 30% of CD45(–) lung SP cells express the endothelial marker CD31(+) (16). We thus examined expression of CD31 on E-SP cells. Using a flourochrome-conjugated antibody, we analyzed immunostained lung suspensions by flow cytometry. In these studies, we found that like the adult lung, CD45(–) E-SP cells are comprised of CD31(+) (16% ± 2.9 SD, n = 3) and CD31(–) (84% ± 2.9 SD, n = 3) subsets (Figure 5). The expression of CD31 led us to speculate that E-SP cells may be enriched for a cell with endothelial features. To test this, we examined the surface expression of known lung endothelial markers (VE-cadherin, Stem cell antigen-1 [Sca-1], CD34) on surface of CD31(+) and CD31(–) E-SP cells (18, 19). In these studies, we found that all three endothelial markers examined were expressed on CD31(+) E-SP cells (Figure 5), but were not detected on the surface of the CD31(–) subtype (data not shown).



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  		Figure 5. CD45(–) embryonic lung SP cells consist of CD31(+) and CD31(–) subsets. Top: Density dot plot of Hoechst stained embryonic (E) lung digest. E-SP cells are depicted in boxed area (left). Density dot plot of E-SP cells after staining with fluorochrome-conjugated antibody to CD31 and CD45. CD45(–) E-SP cells consist of CD31(–) and CD31(+) subsets (right). Bottom: Density dot plot demonstrating CD45(–)/CD31(+) E-SP cells co-express VE-Cadherin, CD34, and Sca-1. Gating was determined based on isotype staining (data not shown). Expression of VE-Cadherin, CD34, and Sca-1 was not detected on CD45(–)/CD31(–) E-SP cells (data not shown).

 
Next, we focused on characterizing the CD45(–)/CD31(–) E-SP cell. As indicated, these cells do not express markers of endothelial or hematopoeitic cell types. CD45(–)/CD31(–) E-SP cells were cytospun onto glass slides and immunostained with fluorochrome-conjugated monoclonal antibodies that recognize acidic cytokeratins, or the mesenchymal marker {alpha}–Sma. In parallel, immunostaining was performed on CD45(–)/CD31(+) E-SP cells. In these studies, we did not detect any apparent cytokeratin-positive cytospun CD45(–)/CD31(–) E-SP cells. We found, however, that the vast majority of cells expressed {alpha}-Sma protein (Figure 6). On the other hand, CD45(–)/CD31(+) E-SP cells stained negative for both markers (Table 1).



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  		Figure 6. CD45(–)/CD31(–) E-SP cells uniformly express {alpha}-Sma. Cytospun CD45(–)/CD31(+) and CD45(–)/CD31(–) E-SP cells were immunostained with fluoroscently-conjugated antibodies to {alpha}-Sma and cytokeratin. Bottom row: CD45(–)/CD31(–) E-SP cells uniformly express {alpha}-Sma (green). Top row: CD45(–)/CD31(+) stained negative for {alpha}-Sma. Both CD45(–)/CD31(+) and CD45(–)/CD31(–) E-SP cells stained negative for cytokeratin (data not shown). DAPI (4',6-diamidino-2-phenylindole) nuclear stain was performed to visualize cell nuclei (blue).

 
CD45(–)/CD31(+) and CD45(–)/CD31(-) E-SP Cells Have Gene Expression Profiles Consistent with Endothelial and Mesenchymal Cell Types, Respectively
To extend our findings, we examined gene expression in CD45(–)/CD31(+) and CD45(–)/CD31(–) E-SP. We focused on the expression of genes associated with endothelial and smooth muscle cell phenotypes. To do this, RT-PCR was performed on RNA derived from an equal number (10,000) of CD45(–)/CD31(+) E-SP, CD45(–)/CD31(–) E-SP cells, and CD45 (–) non-SP cells (Table 2). This latter population served as a positive control for all reactions. Consistent with our previous findings, we found that CD45(–)/CD31(+) E-SP cells expressed endothelial associated genes, including CD31, Tie-2, and flt-1. Expression of these genes was absent from the CD45(–)/CD31(–) E-SP cells. However, CD45(–)/CD31(–) E-SP cells did express the smooth muscle associated gene myocardin and the receptor PDGFR-{alpha}.


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  		TABLE 2. Endothelial and smooth muscle–specific gene expression in CD45(–)/CD31(+) and CD45(–)/CD31(–) embryonic lung sp cells as determined by rt-pcr

 
Growth of CD45(–) E-SP Cells
Based on the expression profile of CD45(–)/CD31(+) E-SP cells, we speculated that these cells could serve as progenitors of endothelial cell types in vitro. To this end, we cultured purified populations of CD45(–)/CD31(+) E-SP cells in endothelial growth conditions. In parallel, primary endothelial cells were cultured as a positive control. After 2 wk, no growth was detected in cultures of CD45(–)/CD31(+) E-SP cells. Rather, these cells maintained their small round phenotype (Figure 7C). In addition, these cells did not grow or differentiate in smooth muscle growth conditions.



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  		Figure 7. Culture of CD45(–)/CD31(–) and CD45(–)/CD31(+) E-SP cells. (A) Purified populations of CD45(–)/CD31(–) E-SP cells were cultured on fibronectin coated plates in smooth muscle growth media. Left: 24 h after culture. Right: after 7 d in culture cells displayed morphologic characteristics of mature smooth muscle. Immunostaining for {alpha}-smooth muscle tropomyosin demonstrates uniform staining for this marker in all cells. (B) RT-PCR of freshly isolated CD45(–)/CD31(–) E-SP cells and after 7 d in culture. Induction of various smooth muscle genes is found during culture of CD45(–)CD31(–) E-SP cells. (C) Culture of CD45(–)/CD31(+) E-SP cells in endothelial and smooth muscle growth conditions. CD45(–)/CD31(+) E-SP cells failed to grow or differentiate in either condition after 2 wk.

 
CD45(–)/CD31(–) E-SP cells have a gene expression profile consistent with a smooth muscle precursor cell. We therefore, explored whether CD45(–)/CD31(–) E-SP cells may be smooth muscle progenitors. To test this, we isolated and cultured CD45(–)/CD31(–) E-SP cells in the presence of smooth muscle growth factors (see MATERIALS AND METHODS). In these conditions, we found that CD45(–)/CD31(–) E-SP cells differentiated into cells with the morphologic characteristics of smooth muscle cells (large, flat, stellate) (Figure 7A). Notably, immunostaining of cultured CD45(–)/CD31(–) E-SP cells for {alpha}-smooth muscle tropomyosin demonstrated uniform staining at 7 d (Figure 7A). Expression of this gene, however, was absent from fresh isolates of CD45(–)/CD31(–) E-SP cells (Figure 7B). To extend these findings, we compared gene expression profiles of freshly isolated and cultured CD45(–)/CD31(–) E-SP cells (Figure 7B). While only {alpha}-Sma and myocardin was detected in fresh isolates of CD45(–)/CD31(–) E-SP cells, we found that all smooth muscle genes examined were present in cultured cells, including those thought to be characteristic of mature smooth muscle (i.e., myosin heavy chain) (20, 21).

Co-Culture of CD45(–)/CD31(–) and CD45(–)/CD31(+) E-SP Cells Results in Formation of Smooth Muscle and Vascular Tube–Like Structures
In vitro and in vivo studies have demonstrated that vascular formation requires the presence of both smooth muscle and endothelial-derived cell types (22–24). Based on these studies, we hypothesized that co-culture of CD45(–)/CD31(+) and CD45(–)/CD31(–) E-SP cells may result in the formation of vascular tube–like structures in vitro. To test this, we co-cultured these cells in smooth muscle and endothelial growth conditions. In smooth muscle growth conditions, co-culture of CD45(–)/CD31(+) and CD45(–)/CD31(–) E-SP cells yielded only cells with the morphologic appearance of smooth muscle (Figure 8). This was confirmed by the uniform expression of {alpha}-smooth muscle tropomyosin at 7 d. In contrast, co-culture of CD45(–)/CD31(+) and CD45(–)/CD31(–) E-SP cells in endothelial growth conditions yielded a distinct growth pattern (Figure 8). After 1 wk in culture, long thin cells became the predominant phenotype. Prolonged culture (> 2 wk) resulted in the interconnection of these long thin cells, and the formation of a complex network of tube-like structures. High-power view of these structures demonstrated multiple nuclei. Consistent with a possible vascular structure, these tube-like structures stained uniformly for the endothelial marker vWF. These findings suggest that an interplay exists between CD45(–)/CD31(+) and CD45(–)/CD31(–) E-SP cells, and that critical soluble and/or membrane bound factors are supplied during co-culture that facilitates the development of these structures. Importantly, the formation of tube-like structures was not detected in co-cultures of CD45(–)/CD31(+) and CD45 (–)/CD31(–) non-SP cells (data not shown).



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  		Figure 8. Co-culture of CD45(–)/CD31(–) and CD45(–)/CD31(+) E-SP cells. Left: Co-culture of cells in smooth muscle growth media results in differentiation of cells to smooth muscle cell types. (A) Twenty-four hours after culture. (B) Seventy-two hours after culture. (C) Seven days in culture (low power) cells uniformly stain for {alpha} smooth muscle tropomyosin. (D) High-power view of cells in C. (E) Isotype staining of CD45(–)/CD31 (–) E-SP cells after 7 d in culture. (F) Staining of paraffin-embedded lung for {alpha} smooth muscle tropomyosin demonstrating specificity of antibody. Right: Co-culture of CD45(–)/CD31(+) and CD45 (–)/CD31 (–) E-SP cells in endothelial growth conditions. (A) Vascular tube–like networks form after 2 wk in culture. (B) High-power view of A; multiple nuclei are visible within these structures. (C) Consistent with a vascular structure, cells stain positive for von Willenbrand factor (vWF). (D) Isotype staining. (E) Staining of paraffin-embedded lung section for vWF demonstrates specificity of antibody. (F) Isotype staining of paraffin-embedded lung section.

 
Vascular Tube–Like Networks Are Formed from CD45(–)/CD31(–) E-SP Cells
Based on the above findings, we set out to determine the physical contribution of CD45(–)/CD31(+) and CD45(–)/CD31(–) E-SP cells to the formation of tubular networks in culture. To do this, we cultured equal numbers of GFP-expressing CD45(–)/CD31(+) E-SP cells with wild-type CD45(–)/CD31(–) E-SP cells, or wild-type CD45(–)/CD31(+) E-SP cells with GFP-expressing CD45(–)/CD31(–) E-SP cells. In these studies, we observed GFP+ networks during co-culture of GFP+/CD45(–)/CD31(–) E-SP cells with wild-type CD45(–)/CD31(+) cells (Figure 9B). Although GFP+/CD45(–)/CD31(+) cells could be identified, they represented a small fraction of total cells after co-culturing, and did not contribute to the formation of cellular networks (Figure 9A). When CD45(–)/CD31(–) E-SP cells were cultured alone in endothelial growth conditions, cells grew rapidly, and did not form any readily apparent organized tubular network.



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  		Figure 9. CD45(–)/CD31(–) E-SP cells are the predominant cell type contributing to the formation of vascular networks in vitro. (A) Co-culture of GFP-expressing CD45(–)/ CD31(+) and wild-type CD45(–)/CD31(–) E-SP cells. GFP-expressing cells represent a small fraction of total cells in culture, and are located in a site outside the vascular network. (B) Phase contrast image of A. (C) Co-culture of GFP-expressing CD45(–)/CD31(–) and wild-type CD45(–)/CD31(+) E-SP cells. Findings demonstrate that vascular tube–like structures are principally derived from CD45(–)/CD31(–) E-SP cells. (D) Phase contrast image of C. (E) Low power magnification of GFP-expressing CD45(–)/CD31(–) E-SP cells cultured in the absence of CD45(–)/CD31(+) E-SP cells. Formation of vascular networks is impaired when CD45(–)/CD31(–) E-SP cells are cultured alone. (F) High-power view of E.

 

   DISCUSSION
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
This is the first study to identify and characterize SP cells in the developing lung. We found that E-SP cells exist in the lung throughout murine development, and that the fraction of these cells varies with gestational age. Our results show that the percentage of E-SP cells peaks at embryonic day 17.5, a time point characterized by rapid expansion of the distal pulmonary vascular network (25). We speculate based on this work that E-SP cells likely play an important role in this critical developmental process.

Similar to the adult, lung E-SP cells are comprised of CD45(+) and CD45(–) subsets. In the adult, CD45(+) SP cells lack expression of mature hematopoeitic markers (4). In contrast, only 15–35% of CD45(+) E-SP cells were found to be lineage-negative. The majority of CD45(+) E-SP cells expressed the myeloid-associated integrin CD11b. In functional assays, these cells lack in vivo hematopoeitic potential, a finding which suggests that CD45(+)/CD11b(+) E-SP cells are lineage committed. Overall, CD11b(+) E-SP cells represent only a small fraction (< 10%) of the total CD11b(+) population in the developing lung (data not shown). At this time, CD11b(+) E-SP cells can only be distinguished from the majority of CD11b(+) cells based on their capacity to efflux Hoechst dye. Whether CD11b(+) E-SP cells are myeloid precursors for macrophages or other resident lung cells of hematopoietic origin has yet to be determined.

In striking contrast, CD45(+)/lin(–) lung E-SP cells were found to have hematopoeitic stem cell activity. In competitive engraftment studies, these cells demonstrate long-term multilineage hematopoeitic reconstituting capacity. One possibility is that these hematopoeitic stem cells traffick through the pulmonary circulation. However, studies demonstrating that extramedullary hematopoeisis can occur within the lung may provide support for the existence of a resident hematopoeitic progenitor (26).

In the adult, CD45(–) tissue SP cells have been shown to be progenitors of nonhematopoeitic cell types (15, 27). To date, the differentiation capacity of these cells appears to be restricted to cells derived from the embryonic mesoderm. For example, CD45(–) skeletal muscle SP cells engraft as skeletal muscle and differentiated endothelium, while testicular SP cells differentiate into testosterone-producing interstitial mesenchymal cells (7, 27). The broad differentiation capacity of CD45(–) SP cells may not be attributable to a single cell type. Indeed, our findings show that CD45(–) E-SP cells are heterogeneous, and that at least two putative progenitor cell types may exist. Interestingly, these populations can be distinguished, in part, by the expression of CD31. Possibly, these observations apply to CD45(–) SP cells derived from other organs. In this regard, gene expression profiling indicates that CD45(–) adult and embryonic heart SP cells express endothelial and cardiomyocyte-specific genes (15).

Further characterization of CD45(–)/CD31(+) E-SP cells determined that these cells have a gene expression profile consistent with an endothelial cell type. Notably, CD45(–)/ CD31(+) E-SP cells comprise only a small fraction (< 1%) of the total CD45(–)/ CD31(+) cells present in the developing lung (data not shown). Interestingly, these cells express the vascular endothelial growth factor receptor flt-1. This molecule is believed to play a critical role in the differentiation of endothelial cells, in part by inhibiting cell division (28, 29).

On the other hand, limited gene expression analysis of CD45(–)/CD31(–) E-SP cells was highly consistent with a smooth muscle precursor (29). These cells lacked expression of late smooth muscle genes, and were found to express the critical regulator myocardin. This transcription factor is restricted to smooth muscle and cardiac muscle cell populations, playing a key role in activating muscle differentiation programs (30, 31). Consistent with this, myocardin positive CD45(–)/CD31(–) E-SP cells were shown to have the capacity to differentiate into cells with the genetic and morphologic features of mature smooth muscle. Furthermore, CD45(–)/CD31(–) E-SP cells express the PDGF-{alpha} receptor, a key signaling molecule that is thought to be selectively expressed in progenitor cells residing within the distal lung mesenchyme (1). Together, these findings lead us to speculate that CD45(–)/CD31(–) E-SP cells are lung vascular smooth muscle progenitors in the developing lung. It is also conceivable that the CD45(–)/CD31(–) E-SP cell is involved in bronchial smooth muscle formation.

The formation of vascular structures requires the interaction of both smooth muscle and endothelial cell types. A variety of key receptor–ligand interactions may explain this cell interdependence. For example, Tie2, an endothelial receptor, is activated by its ligand, Ang1, which is secreted by smooth muscle cells (22, 32). In mice, absence of either receptor or ligand results in severe vascular defects (22). In this work, we found that co-cultured CD45(–)/CD31(+) and CD45(–)/CD31(–) E-SP form complex vascular-like structures. Similar findings were not observed during co-culture of CD45(–)/CD31(+) and CD45(–)/CD31(–) non-SP cells. Our work indicated that CD45(–)/CD31(–) E-SP cells were the predominant cell type contributing to the formation of these structures; when these cells were grown in the absence of CD45(–)/CD31(+) E-SP cells, however, vascular formation was impaired. Together, these findings suggest that interactions between CD45(–)/CD31(+) and CD45(–)/CD31(–) E-SP cells might be important during the development of the pulmonary vasculature.

In conclusion, we identified and characterized SP cells within the developing murine lung. Like SP cells isolated from adult tissues, we determined that lung E-SP cells are comprised of CD45(+) and CD45(–) subtypes. Functional assays indicate that these cells have hematopoeitic and mesenchymal progenitor activity, respectively. Overall, these data suggest a strategy for the isolation and study of viable populations of progenitor cells during organ development.


   Acknowledgments
 
The authors thank Alan Ho for his assistance with cell sorting experiments.


   Footnotes
 
This work was supported by National Heart, Lung, and Blood Institute Grants RO1 HL-69148, R21 HL 72205, PO1 AI50516, and the American Lung Association Research Fellowship Training Award.

Conflict of Interest Statement: None of the authors have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form January 12, 2005

Received in final form March 31, 2005


   References
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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