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Published ahead of print on March 1, 2007, doi:10.1164/rccm.200607-941OC
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American Journal of Respiratory and Critical Care Medicine Vol 175. pp. 1158-1164, (2007)
© 2007 American Thoracic Society
doi: 10.1164/rccm.200607-941OC

Original Article

Lung Cells from Neonates Show a Mesenchymal Stem Cell Phenotype

Kenneth T. Hennrick1, Angela G. Keeton1, Suparna Nanua1, Theresa G. Kijek1, Adam M. Goldsmith1, Umadevi S. Sajjan1, J. Kelley Bentley1, Vibha N. Lama2, Bethany B. Moore2, Robert E. Schumacher1, Victor J. Thannickal2 and Marc B. Hershenson1,3

Departments of 1 Pediatrics and Communicable Diseases, 2 Internal Medicine, and 3 Molecular and Integrative Physiology, University of Michigan, Ann Arbor, Michigan

Correspondence and requests for reprints should be addressed to Marc B. Hershenson, M.D., Medical Science Research Building II, Room 3570B, 1150 West Medical Center Drive, Box 0688, Ann Arbor, MI 48109-0688. E-mail: mhershen{at}umich.edu


   ABSTRACT
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 ABSTRACT
AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Rationale: Mesenchymal stem cells have been isolated from adult bone marrow, peripheral blood, adipose tissue, trabecular bone, articular synovium, and bronchial submucosa.

Objectives: We hypothesized that the lungs of premature infants undergoing mechanical ventilation contain fibroblast-like cells with features of mesenchymal stem cells.

Methods: Tracheal aspirate fluid from mechanically ventilated, premature (< 30 wk gestation) infants 7 days old or younger was obtained from routine suctioning and plated on plastic culture dishes.

Measurements and Main Results: A total of 11 of 20 patients studied demonstrated fibroblast-like cells, which were identified as early as 6 hours after plating. Cells were found to express the mesenchymal stem cell markers STRO-1, CD73, CD90, CD105, and CD166, as well as CCR2b, CD13, prolyl 4-hydroxylase, and {alpha}-smooth muscle actin. Cells were negative for the hematopoietic and endothelial cell markers CD11b, CD31, CD34, or CD45. Tracheal aspirate monocyte chemoattractant protein-1/CCL2 levels were ninefold higher in aspirates in which fibroblast-like cells were found, and cells demonstrated chemotaxis in response to monocyte chemoattractant protein. Placement of cells into appropriate media resulted in adipogenic, osteogenic, and myofibroblastic differentiation. Patients from whom mesenchymal stem cells were isolated tended to require more days of mechanical ventilation and supplemental oxygen.

Conclusions: Together, these data demonstrate that tracheal aspirate fluid from premature, mechanically ventilated infants contains fibroblasts with cell markers and differentiation potential typically found in mesenchymal stem cells.

Key Words: bronchopulmonary dysplasia • fibroblast • monocyte chemoattractant protein • prematurity



   AT A GLANCE COMMENTARY
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Scientific Knowledge on the Subject
Mesenchymal stem cells have been shown in animal studies to home to sites of lung injury and reduce lung inflammation. Mesenchymal stem cells have been isolated from the human bronchial submucosa.

What This Study Adds to the Field
Cells with a mesenchymal stem cell phenotype can be isolated from the lungs of infants with respiratory distress syndrome.

 
Adult bone marrow contains a minority population of mesenchymal stem cells that are believed to contribute to the regeneration of connective tissues, such as bone, cartilage, muscle, ligaments, tendons, fat, and stroma. Cells with characteristics of mesenchymal stem cells have been isolated from adult bone marrow (1, 2), peripheral blood (3), adipose tissue (4), articular synovium (5), and trabecular bone (6). Most recently, such cells have been isolated from the bronchial submucosa (7). Mesenchymal stem cells tend to rapidly proliferate in culture; are devoid of hematopoietic and endothelial markers (e.g., CD34, CD45); express variable levels of CD90 (Thy-1), CD105 (SH2 or endoglin), and STRO-1; and undergo differentiation to fat, bone, and cartilage.

Approximately 30% of infants with birth weights less than 1,000 g develop bronchopulmonary dysplasia (BPD), a severe fibrotic lung disease requiring ventilation and/or supplemental oxygen for months or years (8). The pathogenesis of BPD is not completely understood, although animal models have shown that the preterm lung can be injured by either oxygen or mechanical ventilation, resulting in interference with lung alveolar and vascular development. We hypothesized that mesenchymal stem cells are present in the tracheal aspirates of premature infants undergoing mechanical ventilation for the treatment of respiratory distress syndrome (hyaline membrane disease).

Some of the results of these studies have been previously reported in the form of an abstract (9).


   METHODS
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 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients
We examined tracheal aspirates from 20 infants admitted to the University of Michigan C.S. Mott Children's Hospital Newborn Intensive Care Unit between July 2005 and September 2006. Entry criteria included gestational age at birth less than 30 weeks old, mechanical ventilation for respiratory distress, and age of 7 days or less. Infants with acute sepsis were excluded. This study was approved by the institutional investigational review board.

Tracheal Aspirate Collection and Cell Culture
Aspirates were collected during routine suctioning, as described (10). Details on this method are provided in the online supplement. Specimens were centrifuged (1,200 x g for 5 min at 15°C) and supernatants stored at –80°C. The cell pellet was resuspended in 2 ml Dulbecco's modified essential medium supplemented with 10% fetal bovine serum, 1% penicillin–streptomycin, 1% L-glutamine, and 0.5% amphotericin B. Adherent cells were incubated at 37°C and in 5% CO2 and grown to confluence. Cells of passage number 3 were used for subsequent analysis.

Flow Cytometry
After trypsinization, the cell pellet was suspended in phosphate-buffered saline (PBS) at 1 x 106 cells/ml. For direct staining, 10 µl of primary monoclonal antibody against CD11b, CD13, CD31, CD34, CD45, CD73, or CD166 (BD Pharmingen, San Diego, CA) were placed in 100-µl cell suspensions. For indirect staining, 10 µl of primary antibody for STRO-1 (R&D Systems, Minneapolis, MN), CD90, prolyl hydroxylase, CCR2b (all from Calbiochem, San Diego, CA), or CD105 (Abcam, Cambridge, MA) were added for 30 minutes at 4°C. Cells were washed and the appropriate Alexa fluor 488-tagged secondary antibody (Molecular Probes, Portland, OR) was added at 1:1,000 dilution for 30 minutes at 4°C in the dark. Cells were washed, suspended in 800 µl PBS, and immediately analyzed in a flow cytometer (FACSCalibur; BD, Franklin Lakes, NJ). Cell viability was assessed by trypan blue staining and was greater than 95%.

Immunocytochemistry
Cells grown on D-lysine–coated glass coverslips were fixed in 4% paraformaldehyde, permeabilized in 1% Triton-X-100 in PBS, and stained with anti–{alpha}-smooth muscle actin (Calbiochem).

Immunoblotting
Cell lysates were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to nitrocellulose. Membranes were probed with an antibody against {alpha}-smooth muscle actin (Calbiochem).

Monocyte Chemoattractant Protein-1/CCL2 Levels
Aspirate monocyte chemoattractant protein (MCP)-1/CCL2 was measured by ELISA (R&D Systems).

Chemoattractant Assay
Migration of cells to MCP-1 and two individual tracheal aspirates was assessed after overnight incubation in a 12-well Boyden chamber (Neuroprobe, Gaithersburg, MD), as described (11).

Differentiation
For adipogenic differentiation, cells were cultured in standard medium supplemented with dexamethasone (10 µM), isobutylmethylxanthine (100 µg/ml), indomethacin (50 µM), and insulin (10 µg/ml) (1). For osteogenic differentiation, cells were cultured in medium supplemented with dexamethasone (0.1 µM), beta-glycerophosphate (10 mM), and L-ascorbic acid (50 µg/ml) (1). Cells were stained with oil red O and alizarin red S for identification of fat vacuoles and calcium deposits, respectively. For myofibroblast differentiation, cells were treated with transforming growth factor (TGF)-beta, 10 ng/ml, for 72 hours.


   RESULTS
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DISCUSSION
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Cell Isolation
Aspirates were collected from 20 premature infants born at less than 30 weeks' postconceptual age. All aspirates contained round cells, with the total cell count ranging from 3 x 104 to 1 x 107/ml. Trypan blue staining showed an average viability of 91%. However, nearly all of these cells failed to adhere firmly to plastic and were eventually lost. On the other hand, 11 aspirates eventually showed the presence of adherent, thin, fibroblast-like cells (Figure 1A). These cells were noted as early as 6 hours after plating, but sometimes they did not become apparent until days later. Cells formed one to two individual colonies per well (Figure 1B), which were allowed to coalesce and grow to confluence (Figure 1C). Upon confluency, cells were passaged by trypsinization. Thus far, cells have been passaged as many as 23 times without demonstrating signs of senescence.


Figure 1
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  		Figure 1. (A) Morphology of adherent cells from tracheal aspirate of a mechanically ventilated, premature infant after 6 hours of culture. Cells reveal a typical spindle shape. (B) Typical colony of fibroblast-like cells observed 14 days after initial plating of tracheal aspirate; original magnification, x25. (C) Fibroblast-like cells at subconfluency.

 
Characterization of Fibroblast-like Cells
Confluent cells from passage 3 were harvested for analysis of surface markers by flow cytometry. Typical results for the isolates are shown in Figure 2. Cells were positive for the following: STRO-1, a marker for stromal cell precursors (8/10 isolates tested); CD73 (SH3, 10/10 isolates); CD90 (Thy-1), a marker of hematopoietic progenitors and fibroblasts (3/3 isolates); CD105 (SH2 or endoglin), expressed on cultured mesenchymal progenitor cells (10/10 isolates); CD166 (leukocyte cell adhesion molecule, 10/10 isolates); CD13 (aminopeptidase N, 6/10 isolates); prolyl 4-hydroxylase (PH), a key enzyme involved in collagen synthesis (3/3 isolates); and C-C chemokine receptor (CCR)-2b, the MCP-1 receptor (3/3 isolates). Cells were negative for the following: CD34, a hematopoietic stem cell antigen (0/9 isolates); CD45, a pan-leukocyte marker (0/10 isolates); CD11b, a macrophage marker (also called Mac-1, 0/2 isolates); and CD31, an endothelial cell marker (also called platelet endothelial cell adhesion molecule, 0/2 isolates). Unstimulated cells also expressed low levels of {alpha}-smooth muscle actin (Figure 3). Together, this pattern of markers is consistent with a mesenchymal stem cell phenotype.


Figure 2
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  		Figure 2. Immunophenotypic analysis of fibroblast-like cells by flow cytometry. (A) Two-color analysis shows almost all cells are Stro-1+, CD45–. (B) Cells were stained for the following: Stro-1, CD73, CD73, CD105, and CD166, each of which have been identified on mesenchymal stem cells but not hematopoietic stem cells; CD90 (Thy-1), a fibroblast marker; CD13, a myeloid marker; prolyl hydroxylase (PH), a key enzyme involved in collagen synthesis; CCR-2b, the receptor for monocyte chemoattractant protein (MCP)-1 (CCL2); CD34, CD45, and CD11b, hematopoietic cell markers typically found on "fibrocytes"; and CD31, an endothelial cell marker. Specific signals are shown as black lines. Control reactions were performed with irrelevant isotype IgG controls (gray lines). Typical results from individual subjects are shown.

 

Figure 3
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  		Figure 3. Mesenchymal cell expression of {alpha}-smooth muscle actin. (A) Immunocytochemical stain of unstimulated cells. (B) Cells treated with transforming growth factor (TGF)-beta showed increased {alpha}-smooth muscle expression, typical of myofibroblasts. (C) {alpha}-Smooth muscle actin immunoblot confirming increased expression after TGF-beta treatment. Ten micrograms of protein were loaded per lane. These results were characteristic of three isolates tested.

 
Tracheal Aspirates Hold Chemoattractant Activity for Fibroblast-like Cells
We examined tracheal aspirates for the CCR2b ligand MCP-1. MCP-1 levels were ninefold higher in aspirates from which fibroblast-like cells were isolated (1.91 ± 1.61 vs. 17.35 ± 18.98 ng/ml, mean ± SD). Using a Boyden chamber, we found substantial migration of fibroblast-like cells in response to 1 ng/ml MCP-1 (Figure 4A). Cell migration was also found in response to a tracheal aspirate from which fibroblast-like cells were isolated (Figures 4A–4C).


Figure 4
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  		Figure 4. Chemotaxis of fibroblast-like cells in response to monocyte chemoattractant protein (MCP)-1. Migration of fibroblast-like cells in response to various stimuli was assessed after overnight incubation in a 12-well Boyden chamber. Chemotaxis membranes were fixed for 30 minutes and stained with hematoxylin and eosin. (A) Group mean data for medium control, MCP-1 (1 ng/ml) and fibronectin (FN, 50 µg/ml). Results from a typical experiment are shown. This experiment was repeated twice. (B) Migration of cells in response to a tracheal aspirate from which fibroblast-like cells were not isolated (patient 2; original magnification, x64). (C) Chemotaxis in response to a tracheal aspirate from which fibroblast-like cells were isolated (patient 11; original magnification, x48). Note clumps of cells traversing the chamber membrane (insert; original magnification, x320).

 
Fibroblast-like Cells Demonstrate Adipogenic, Osteogenic, and Myofibroblast Differentiation
To examine the ability of fibroblast-like cells to differentiate, cells were placed in adipogenic or osteogenic media. Adipogenic differentiation, as evidenced by accumulation of lipid droplets within the cells, was revealed by staining with oil red O (Figures 5A and 5B). Osteogenic differentiation, as evidenced by calcium deposits, was demonstrated by alizarin red S staining (Figures 5C and 5D). After treatment with TGF-beta, cells expressed significant levels of {alpha}-smooth muscle actin (Figure 3).


Figure 5
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  		Figure 5. The differentiation capability of fibroblast-like cells from patients 1 and 3 was determined. (A) Adipogenic differentiation–accumulation of lipid droplets within the cells was revealed by staining with oil red O (original magnification, x100). (B) Adipogenic differentiation (original magnification, x300). (C) Osteogenic differentiation–calcium deposits were revealed by staining with alizarin red S (original magnification, x100). (D) Osteogenic differentiation (original magnification, x300).

 
Clinical Data
As noted above, fibroblast-like cells were isolated from 11 of 20 patients studied. There were no differences in gestational age at birth or birth weight between the two groups (Table 1). However, patients from whom mesenchymal cells were isolated tended to require more days of mechanical ventilation and supplemental oxygen, and to have more BPD.


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  		TABLE 1. SUMMARY OF CLINICAL OUTCOMES FOR PARTICIPATING INFANTS

 

   DISCUSSION
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Mesenchymal stem cells undergo differentiation to various connective tissue cell types, including fat, bone, and cartilage. They are plastic-adherent, heterogeneous, fibroblast-like cells that form colonies and rapidly proliferate in culture, although they exhibit a finite lifetime and do not display properties of immortalized cells. Although no single marker is definitive, mesenchymal stem cells are devoid of hematopoietic and endothelial markers (e.g., CD34, CD45, CD11, CD31) and express variable levels of CD73 (SH3/SH4), CD90 (Thy-1), CD105 (SH2 or endoglin), CD166 (SB10 or leukocyte cell adhesion molecule), and STRO-1 (12). STRO-1, CD73, CD105, and CD166 each react with antibodies raised against human mesenchymal stem cells (13–16); these antibodies do not react with hematopoietic cells. Cells with characteristics of mesenchymal stem cells have been isolated from adult bone marrow (1, 2), peripheral blood (3), adipose tissue (4), articular synovium (5), trabecular bone (6), and, most recently, bronchial submucosa (7). Thus, it is conceivable that adult tissues contain populations of cells that have the capacity for renewal after disease, trauma, or aging. CD105+, CD90+ cells have also been isolated from 15- to 22-week-old fetal tissues, including bone marrow, liver, lung, and spleen (17), suggesting that mesenchymal stem cells are present during embryologic development.

In the present study, we hypothesized that the lungs of premature infants undergoing mechanical ventilation contain fibroblast-like cells with features of mesenchymal stem cells. We found that fibroblast-like cells can be isolated from the tracheal aspirates of premature infants undergoing mechanical ventilation for respiratory distress syndrome. These cells became apparent as early as 6 hours of plating on plastic. Cells formed one to two individual colonies, which were allowed to coalesce and grow to confluence. After growth to confluence, they demonstrated the mesenchymal stem cell markers STRO-1, CD73, CD105, and CD166, and the fibroblast marker CD90, but they did not express the hematopoietic progenitor marker CD34, the leukocytic markers CD45 or CD11b, or the endothelial marker CD31. Cells also expressed PH, a key enzyme involved in collagen synthesis. These cells appear to have a significant proliferation potential, and also undergo adipogenic, osteogenic, and myofibroblast differentiation. Together, these data suggest that mesenchymal stem cells participate in the response to neonatal lung injury and/or repair.

Fibroblastic cells have previously been implicated in the pathogenesis of asthma, pulmonary fibrosis, adult respiratory distress syndrome, and acute lung injury. Bronchial biopsy of surgical lung specimens yields fibroblasts that are devoid of the hematopoietic and endothelial markers CD34 and CD45 but positive for the fibroblast marker CD90 (Thy-1) and the mesenchymal progenitor cell marker CD105 (SH2 or endoglin). These cells also weakly express STRO-1, a marker for bone marrow stromal cell precursors, and undergo adipogenic, osteogenic, and chondrogenic differentiation, consistent with a mesenchymal stem cell phenotype (7). Bronchial biopsies from patients with allergic asthma demonstrate fibroblast-like cells that are positive for CD34, collagen I, and {alpha}-smooth muscle actin, and are believed to originate from "fibrocytes," circulating monocytes which develop fibroblast morphology when recruited into murine lung tissue after allergen exposure (18). Circulating fibrocytes isolated from normal volunteers traffic to the lungs in response to bleomycin challenge, suggesting a role in the pathogenesis of pulmonary fibrosis (19). PH and {alpha}-smooth muscle actin–positive cells have been found in the lungs of patients with adult respiratory distress syndrome (20). Circulating endothelial progenitor cells have been associated with survival in acute lung injury in humans (21). Together, these data suggest that fibroblastic cells may be recruited to areas of lung damage and participate in lung repair and, in some cases, fibrosis.

The precise origin of the fibroblast-like cells we isolated from premature infants undergoing mechanical ventilation for respiratory distress syndrome is unclear. These multipotent lung mesenchymal cells may represent bone marrow–derived fibrocytes, bone marrow–derived mesenchymal stem cells, or, alternatively, multipotent cells arising from the lung itself. For the following reasons, we favor the last possibility. First, the absence of hematopoietic markers suggests that these cells are not typical fibrocytes. Second, in contrast to our results, bone marrow–derived CD45–, collagen I+ mesenchymal cells isolated from the lungs of rats undergoing bleomycin-induced lung injury fail to undergo myofibroblast differentiation (22). Finally, we have recently demonstrated the presence of mesenchymal stem cells of donor-sex identity in lung allografts (23), consistent with an origin in the lung, perhaps from peribronchial or perivascular adventitia.

MCP-1/CCL2, a monocyte and fibrocyte chemoattractant that also stimulates collagen synthesis in fibroblasts (11), has been previously demonstrated to be increased in children with respiratory distress syndrome (24) and interstitial lung disease (25). MCP-1 has been implicated in the recruitment of fibrocytes to the alveolar space after fibrotic injury (11). We therefore examined the concentrations of MCP-1 in the tracheal aspirates of premature infants undergoing mechanical ventilation for respiratory distress syndrome. MCP-1 levels were increased in aspirates in which fibroblast-like cells were found, and MCP-1 holds chemoattractant activity for mesenchymal cells, consistent with the notion that this chemokine plays a role in the translocation of mesenchymal cells to the airspaces. Other chemokines may also play an important role, including stroma-derived factor-1 (CXCL12), which has been shown to traffic circulating fibrocytes to the lung (19).

The precise role of the observed mesenchymal cells in the pathogenesis of neonatal lung disease is unclear. There appears to be a trend toward increased days of mechanical ventilation, days of O2 supplementation, and incidence of BPD in patients from whom mesenchymal stem cells were isolated, consistent with the notion that these cells play a physiologic role in lung repair. However, additional subjects would be needed to establish such a relationship. We performed a sample size calculation estimating the number of patients needed to find a significant difference in days of O2 supplementation between the two groups, assuming a power of 0.9, standard deviations of 65 and 24 days for the two groups, and an effect size of 30 days. Although this is a large effect size, it would be important to seek a difference in O2 supplementation between the two groups that is clinically, as well as statistically, significant. Our power calculation suggests that nearly 100 patients would be required for such an analysis. Fewer patients would be needed to power a study using days of mechanical ventilation as a primary outcome variable.

If indeed there was an association between the isolation of mesenchymal stem cells and adverse clinical outcome, this would not necessarily suggest that the cells are harmful to the lung. Lung injury may be necessary for homing of mesenchymal stem cells to the airspaces (26), where they are accessible to lavage. In addition, isolation of these cells could simply be a biomarker of oxidant injury. Alternatively, mesenchymal stem cells could actually promote lung repair. Infusion of mesenchymal stem cells has been shown to protect the mouse lung from bleomycin-induced lung injury (26, 27). Preliminary data from oxygen-induced newborn rats suggest that intratracheal administration of mesenchymal stem cells prevents stunted alveolar growth (28). Rat pulmonary interstitial lipofibroblasts have been demonstrated to produce growth factors that induce differentiation of epithelial type II cells and surfactant phospholipid synthesis, thereby promoting lung development (29, 30). Finally, our preliminary studies suggest that mesenchymal stem cells isolated from the tracheal aspirates of premature infants undergoing mechanical ventilation for respiratory distress synthesize keratinocyte growth factor, a respiratory epithelial mitogen that accelerates wound closure and decreases hyperoxia-induced mortality in rats (31–34), and vascular endothelial cell growth factor, an angiogenic factor deficient in infants with BPD (35).

We conclude that tracheal aspirate fluid from premature, mechanically ventilated infants contains fibroblasts with cell markers and differentiation potential typically found in mesenchymal stem cells. Further study is required to determine their role in the development of chronic lung disease in these infants.


   FOOTNOTES
 
Supported by the Frederick G. L. Huetwell Professorship for the Cure and Prevention of Cystic Fibrosis.

This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org

Originally Published in Press as DOI: 10.1164/rccm.200607-941OC on March 1, 2007

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

Received in original form July 12, 2006; accepted in final form February 23, 2007


   REFERENCES
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 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
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