Blue-200902-0179OC – R1 American Journal of Respiratory and Critical Care Medicine
Airway Delivery of Mesenchymal Stem Cells prevents Arrested Alveolar
Growth in Neonatal Lung Injury in Rats
Timothy van Haaften1, Roisin Byrne1, Sebastien Bonnet2, Gael Y Rochefort3, John Akabutu4,
Gloria J Rey-Parra1, Jacques Galipeau5, Alois Haromy6, Farah Eaton1, Ming Chen7,
Doris Abley4, Greg Korbutt8, Stephen L Archer9, Bernard Thébaud1
1. Department of Pediatrics, Women and Children Health Research Institute, University of
Alberta, Edmonton, Alberta, Canada
2. Department of Medicine, Laval University, Québec City, Québec, Canada
3. Université François Rabelais, Tours, France
4. Alberta Cord Blood Bank, Edmonton, Alberta, Canada
5. Lady Davis Institute for Medical Research, Jewish General Hospital, McGill University,
Montreal, Québec, Canada
6. Department of Medicine and Vascular Biology Group, University of Alberta, Edmonton,
7. Surgical Medical Research Institute, Electron Microscopy Unit, University of Alberta,
Edmonton, Alberta, Canada
8. Alberta Diabetes Institute, University of Alberta, Edmonton, Alberta, Canada
9. Section of Cardiology, University of Chicago Medical Centre, Chicago, Illinois
Dr. Bernard Thébaud MD, PhD
407 Heritage Medical Research Centre
University of Alberta
Edmonton, Alberta, Canada, T6G 2S2
Phone: (780) 492-7130
Fax: (780) 492-3603
"This article has an online data supplement, which is accessible from this issue's table of content online at
Footnote: Dr. Thébaud is a Canada Research Chair and supported by the Canada Foundation for
Innovation, the Alberta Heritage Foundation for Medical Research (AHFMR), the Canadian
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AJRCCM Articles in Press. Published on August 27, 2009 as doi:10.1164/rccm.200902-0179OC
Copyright (C) 2009 by the American Thoracic Society.
Institutes for Health Research (CIHR) and the Stollery Children’s Hospital Foundation. TvH was
supported by Tomorrow’s Research Cardiovascular Health Professionals (TORCH) and the
Maternal Fetal Neonatal Health Training Program sponsored by CIHR-IHDCYH. Dr. Archer is
supported by NIH-RO1-HL071115.
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Running Head: Stem cells and lung injury
Descriptor number that best classifies the subject of this manuscript: 14.03 Neonatal Lung
Disease & BPD
Word count for the body of the manuscript, excluding abstract, references and online
supplementary information = 3770
Word count for the Abstract = 243; Methods = 1215
At a Glance Commentary
Scientific Knowledge on the subject:
Currently there is no effective preventative therapy for arrested alveolar and lung vascular
growth in chronic lung disease of prematurity. Mesenchymal stem cells (MSC) show promise for
What this study adds to the field:
MSC preserve alveolar development in an oxygen-induced model of chronic lung disease in
newborn rats. This effect seems mainly due to a paracrine effect. MSCs may have therapeutic
potential for preventing neonatal lung diseases characterized by alveolar damage.
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Rationale. Bronchopulmonary dysplasia (BPD) and emphysema are characterized by
arrested alveolar development or loss of alveoli; both are significant global health problems and
currently lack an effective therapy. Bone marrow-derived mesenchymal stem cells (BMSCs)
prevent adult lung injury, but their therapeutic potential in neonatal lung disease is unknown.
Objectives. We hypothesized that intratracheal delivery of BMSCs prevents alveolar destruction
in experimental BPD. Methods, Measurements and Main Results. In vitro, BMSCs developed
immunophenotypic and ultrastructural characteristics of type II alveolar epithelial cells (AEC2)
(surfactant protein-C expression and lamellar bodies) when co-cultured with lung tissue, but not
with culture media alone or liver. Migration assays revealed preferential attraction of BMSC
towards oxygen-damaged lung vs normal lung. In vivo, in a hyperoxia-induced model of BPD in
newborn rats, air space enlargement and loss of lung capillaries was associated with a decrease in
circulating and resident lung BMSCs. Intratracheal delivery of BMSCs at postnatal day 4
improved survival and exercise tolerance while attenuating alveolar and lung vascular injury and
pulmonary hypertension. Engrafted BMSCs co-expressed the AEC2 specific marker surfactant
protein-C. However, engraftment was disproportionately low for cell replacement to account for
the therapeutic benefit, suggesting a paracrine-mediated mechanism. In vitro, BMSC-derived
conditioned media prevented O2-induced AEC2 apoptosis, accelerated AEC2 wound healing and
enhanced endothelial cord formation. Conclusions. BMSCs prevent arrested alveolar and
vascular growth in part through a paracrine activity. Stem cell-based therapies may offer new
therapeutic avenues for lung diseases that currently lack efficient treatments.
Key Words: Stem cells, Aging, Lung, Oxygen
Word count: 243
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Acute and chronic lung diseases pose a growing socio-economic burden (1). Preterm birth is
on the rise and the leading cause of perinatal mortality and morbidity, accounting for more than
85% of all perinatal complications and death (2). Improvements in perinatal care have increased
the survival of extremely premature newborns (born at less than 28 weeks of gestation) (3).
These infants however, are at high risk for long-term injury to both lung and brain (3). Each year,
5000-10,000 newborns suffer from bronchopulmonary dysplasia (BPD)
(http://www.nhlbi.nih.gov/health/dci/Diseases/Bpd/Bpd_WhoIsAtRisk.html), a chronic lung
disease that follows ventilator and oxygen therapy for acute respiratory failure after premature
birth (4). BPD has long term respiratory and neuro-developmental consequences that extend
beyond childhood and result in increased health care costs (3). Further advances are required to
increase survival free of neonatal morbidity or neurodevelopmental impairment. Recent alarming
reports suggest that BPD results in early onset emphysema (5, 6). Emphysema, defined as
airspace enlargement distal to terminal bronchioles, is a major component of chronic obstructive
pulmonary disease, the fourth leading cause of death in the US (1). BPD and emphysema are
characterized by interrupted development and loss of alveolar structures respectively, and
therapy is palliative.
Increasing attention has been focused on the use of adult derived stem cells to regenerate
damaged organs. Systemically injected mouse bone marrow (BM)-derived mesenchymal stem
cells (MSCs) have been demonstrated to differentiate into parenchymal cells of various non-
hematopoietic tissues including the lung (7). These properties have already been harnessed to treat
various diseases, including among others inborn errors of metabolism, neurodegenerative
disorders, and cardio-vascular diseases (8, 9). In contrast, relatively little information is available
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on the therapeutic potential of MSCs in lung diseases characterized by alveolar damage (reviewed
in (10-12)). In the adult lung, studies have focused on proof of principle that BM-derived cells can
home to the lung and adopt various lung cells phenotype in various injury models. These findings
have been called into question (13, 14). Recent reports now suggest that BM-derived stem cells
can prevent adult lung injury (15-18). The therapeutic benefit of these cells in arrested alveolar
development has not been explored.
Here, we provide evidence that BMSCs improve survival and exercise capacity and prevent
alveolar growth arrest in a well-established, chronic O2-induced model of BPD characterized by
otherwise irreversible alveolar and lung capillary rarefaction. We also provide direct in vitro
evidence that the beneficial effects of MSCs might be mediated through a paracrine activity.
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Expanded methods are available in the online-only Data Supplement. All procedures and
protocols were approved by the Animal Health Care Committee of the University of Alberta.
MSC Harvest and Cell Culture. BM from adult Sprague-Dawley rats was plated into tissue
culture flasks. Adhered cells were allowed to grow to ~75% confluency, then trypsinized and
reseeded at a density of 105 cells/cm2. This procedure was performed for 2 passages. PASMCs,
used as control cells, were obtained from adult Sprague-Dawley as previously described (19).
Fluorescence-Activated Cell Sorting of Cell Surface Markers. Phycoerythrin labeling for
rat monoclonal antibodies against CD31-CD34-CD44-CD45-CD54-CD73 and CD90 (Santa
Cruz, Santa Cruz, California, USA) were utilized according to the manufacturer’s protocol and
were selected in accordance with the position statement for the minimal criteria to define a MSC
from the International Society for Cellular Therapy (20). Cells were analyzed using a FACScan
(Becton Dickinson, Franklin Lakes, NJ, USA) and CellQuest Software as described (21).
Stem Cell Lineage Differentiation Assay. The lineage differentiation assay was performed
in accordance with the position statement for the minimal criteria to define a MSC from the
International Society for Cellular Therapy (20).
Adipogenic, osteogenic and chondrogenic induction was performed on passage 2 BMSCs as
previously described (21).
RT-PCR Analysis for Lineage Conversion. Total RNA was extracted from undifferentiated
(control) and differentiated BMSCs and analyzed by RT-PCR with primers specific for
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adipogenic (Pparg2, Lpl), osteogenic (Bglap, Runx2), and chondrogenic (Col2a1 and Col10a1)
Stem Cell Migration Assay. BMSCs were seeded in the upper chamber of a modified
Boyden chamber (Millipore, Temecula, CA) on a 8 µm mesh. Cells then were allowed to migrate
for 6 hours in a 37oC incubator towards either DMEM, DMEM+normoxic lung tissue, or
DMEM+hyperoxic lung tissue in the lower chamber.
Co-Culture Assay. BMSCs were seeded in the bottom of a modified Boyden chamber
(Corning Inc, Corning, NY) with a 0.4 µm mesh separating the upper and lower chambers and
exposed to various culture conditions. Cells were stained for SP-C and DAPI and imaged with a
confocal microscope. The TaqMan One-Step RT-PCR Master Mix reagent kit (Applied
Biosystems, Foster City, CA, USA) was used to quantify the copy number of cDNA targets as
described in (22).
Animal Model. Experimental BPD was induced as previously described (22). Sprague-
Dawley rats (Charles River, Saint Constant, QC, Canada) were exposed to normoxia (21% O2) or
hyperoxia (95% O2, BPD model) from birth to postnatal day 14 (P14) in sealed plexiglass
chambers with continuous O2 monitoring (BioSpherix, Redfield, NY).
In Vivo Experimental Design. Newborn rat pups were randomized to four groups: 1)
normoxia (21% O2, control group); 2) hyperoxia (95% O2, BPD group); 3) hyperoxia+BMSCs;
and 4) hyperoxia+PASMCs. Cells were administered at P4 (prevention studies) or P14
(regeneration studies) via an intra-tracheal injection (1.0 x 105/cells per animal). Before
administration, BMSCs were labeled with the intra-vital green fluorescent dye 5(6)-
Carboxyfluorescein diacetate N-succinimidyl ester (CFSE) (Sigma Canada, Oakville, ON,
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Canada) according to the manufacturer’s protocol. Animals were harvested at P21 (prevention
studies) or P45 (regeneration studies).
Exercise Capacity. Rats were run according to a predetermined protocol. Exhaustion was
defined as the animal running exclusively on the lower third of the treadmill coupled with hitting
of the shock panel twice within 30 seconds.
Lung Morphometry. Lungs were inflated and fixed via the trachea with a 4% formaldehyde
solution at a constant pressure of 20 cm H2O (22). Lungs were paraffin embedded, cut in 4 µm
thick serial sections and lung sections were stained with hematoxylin and eosin. Alveolar
structures were quantified using the mean linear intercept as described (22).
Barium Angiogram. Barium was instilled into the pulmonary vasculature as previously
described (22). The barium was imaged with a rodent SPECT-CT (FLEX Pre-clinical platform)
using Amira software package (Gamma Medica, Northridge, CA, USA).
Mercox Vascular Casting and Scanning Electron Microscopy. Mercox vascular casting
were prepared and imaged as previously described (22).
Right Ventricular Hypertrophy (RVH). The right ventricle free wall was separated from
the left ventricle and the septal wall. The tissue was dried overnight and weighed the following
Pulmonary Artery Acceleration Time. Pulmonary artery acceleration time (PAAT), a valid
measure of mean pulmonary arterial pressure in rodents, was assessed with Doppler
echocardiography as previously described (19, 23).
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MSC Engraftment. Lungs from P14 and P21 animals (n=3/time point) were inflated with
Tissue-Tek Optimal Cutting Temperature (OCT) Media (Ted Pella, Inc. Redding, CA) and
subsequently frozen in a block of OCT. Lungs were stained for the type 2 alveolar epithelial cell
(AEC2) specific marker surfactant protein-C (SP-C) and pro-SP-C and cellular nuclei (DAPI)
and imaged using a confocal microscope. BMSCs labeled with the cytoplasmic dye CFSE were
manually counted in twenty five random fields throughout the lung for a total cell count of 10
624 cells at P14, and 8224 cells at P21. In addition, we used deconvolution microscopy (Volocity
restoration and classification modules, Improvision®) to assess engraftment.
Generation of BMSC conditioned media (CdM). Passage 2 BMSCs were grown to ~80%
confluency. Media was aspirated and cells were rinsed 3 times with PBS. Cells were cultured in
serum-free media for 12hours. CdM was removed and filtered through a 50 µm mesh to remove
AEC2 Isolation. AEC2 were isolated from time-dated fetal day 19.5 rat lungs as described
using serial differential adhesions to plastic and low-speed centrifugations (24).
O2-induced Injury of fetal rat AEC2. 106 cells/mL AEC2 were seeded onto poly-L lysine-
coated glass coverslips in 24-well cell culture plates. Cells were cultured in DMEM or CdM and
exposed to 21% or 85%O2 for 36 hours. DNA damage was assessed with a dual color APO-
BRDU-immunohistochemistry kit (Millipore, Temecula, CA) that labels DNA breaks and total
cellular DNA. Ten random fields of view were taken for each group. To assess apoptosis we
performed immunoblotting for cleaved caspase 3 (ab13847, Abcam, Cambridge, MA, USA). The
intensity of the bands was normalized to the intensity of a reporter protein (actin) using the
Kodak Gel-doc system (Kodak, Rochester, NY).
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Wound Healing Assay with fetal rat AEC2. 106 cells/mL AEC2 were seeded into a plastic
24-well cell culture plate. At ~80 hours, the cell monolayer was scraped with a p200 pipette tip
and media replaced with CdM or DMEM. The surface area of the wound was recorded over time
using OpenLab (Quorum Technologies Inc, ON, Canada).
Endothelial network formation assay. The formation of cord-like structures by rat lung
microvascular endothelial cells (RLMVEC, VEC Technologies Inc., Rensselaer, NY, USA) was
assessed by seeding RLMVEC (80,000 cells/well) into 24-well plates coated with Matrigel (BD
Biosciences, Mississauga, ON) (22, 23), supplemented with DMEM or BMSC CdM and
incubated at 37 °C for 8–12h in 21% or 85%O2. Cord-like structures were observed using an
inverted phase contrast microscope (Olympus, Melville, NY) and quantified by measuring the
number of intersects and the length of structures in random fields from each well.
Statistics. Values are expressed as the mean±SEM. Intergroup differences were assessed
by Student’s paired t-test. Analysis of variance (ANOVA) with post hoc analysis (Fisher’s
probable least significant difference test (Statview 5.1, Abacus Concepts) was used to assess the
differences between multiple groups. Survival curves were derived by the Kaplan-Meier method
and differences evaluated by log-rank tests. A value of P<0.05 was considered statistically
significant. All evaluations were done by investigators blinded to the experimental groups.
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BMSCs migrate towards injured lung and adopt features of AEC2 in vitro. MSCs
extracted from the BM formed a homogeneous population of cells by the second passage. The
analysis of cell surface phenotype indicated that the MSC population was positive for CD44,
CD54, CD73 and CD90 while negative for CD31 and CD45 (Figure 1A), consistent with the
International Society for Cellular Therapy position statement for the minimal criteria to define a
MSC (20). These MSCs differentiated into three mesenchymal lineages (adipo-, osteo-, and
chondrocytes) when grown in specific media for each lineage as shown by
immunohistochemistry (Figure 1B) and RT-PCR (Figure 1C). These cells were also found in the
circulating blood and the lung.
In migration experiments (Figure 1D), BMSCs were preferentially attracted to O2-damaged
lung placed in the bottom of a modified Boyden chamber as compared to culture media or non-
injured lung, suggesting that the injured lung recruits MSCs to modulate lung injury.
MSCs co-cultured in vitro with normoxic (not shown) or hyperoxic lung tissue, but not with
culture media alone or liver (not shown), adopted immunophenotypic and ultrastructural
characteristics of AEC2. MSCs co-cultured with lung expressed SP-C mRNA (Figure 1E) and
showed the organized and granular distribution of SP-C protein expression (Figure 1E).
Transmission electron microscopy visualized lamellar bodies (a characteristic structure found
only in AEC2) in MSCs co-cultured with lung tissue (Figure 1E). Freshly isolated fetal AEC2
provided a reference for the structure and distribution of lamellar bodies.
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Irreversible O2-induced lung injury is associated with a reduction of lung resident
MSCs. Following two weeks exposure to hyperoxia, the lung had a consistent, irreversible
histological pattern of alveolar simplification (e.g larger but fewer alveoli, Figure 2A). This was
associated with a reduced number of circulating and resident MSCs (expressed as colony
forming unit-fibroblasts, CFU-F show in Figure 2B) in the lung without a reduction in the BM
population of MSCs (Figure 2C).
Intrapulmonary delivery of BM-derived MSCs improves survival and exercise capacity.
These data formed the rationale to test the therapeutic potential of MSC in this model. Kaplan-
Meier analysis demonstrated that intratracheal delivery of MSCs given at postnatal day 4
significantly improved survival as compared with both the hyperoxic group and the
hyperoxic+PASMC group (Figure 3A).
Exercise capacity, using a graduated treadmill exercise protocol by a blinded observer, was
significantly decreased in untreated O2 exposed rat pups (Figure 3B). MSC treatment at postnatal
day 4 (prevention) or day 14 (regeneration, see online supplement Figure 1) significantly
increased the total distance ran. PASMCs had no effect.
Intrapulmonary delivery of BM-derived MSCs preserves alveolar and vascular
development in irreversible O2-induced BPD. Hyperoxia induced a histological pattern
reminiscent of human BPD, characterized by air space enlargement with simplified and fewer
alveolar structures (Figure 4). Intratracheal administration of MSCs at day 4 significantly
improved alveolarization as quantified by the mean linear intercept. Conversely, PASMCs had
no protective effect on lung architecture. Intratracheal MSCs administration at day 14 did not
significantly improve lung architecture (see online supplement Figure 2).
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Capillary rarefaction is another hallmark of BPD and emphysema. Barium angiograms
using CT-scan showed a severe rarefaction of pulmonary capillaries in chronic hyperoxia.
Likewise, scanning electron microscopy revealed a dense vascular network with relatively large,
rounded, smooth, and well-organized vessels in control lungs (Figure 5A). Hyperoxic exposure
caused severe capillary rarefaction, thinning and scaring of the vasculature. The pulmonary
vasculature from MSC-treated lungs demonstrated greater organization, as well as larger and
rounder vessels, resembling more the normal lung, although some scaring was still present.
Quantification of barium-gelatin injected lungs confirmed the severe decrease in
pulmonary vascular density in the hyperoxic group (Figure 5B). MSCs, but not PASMC,
significantly increased pulmonary vascular density.
Intrapulmonary delivery of BM-derived MSCs reduces pulmonary hypertension.
Pulmonary hypertension is one of the most important co-morbidites of severe BPD and
recapitulated in the chronic hyperoxia model as assessed by echocardiographic (PAAT; Figure
6A) and morphological (RVH; Figure 6B) features. MSC therapy normalized the PAAT to near
control levels and reduced RVH; PASMCs had no effect.
Engrafted MSCs adopt a distal lung cell phenotype. CFSE labeled MSCs accounted for
3.7% ± 2.9 of cells counted at P14 and 2.9% ± 2.6 of cells counted at P21 in hyperoxic animals
(Figure 7). Engraftment in room air housed animals was only 0.1%±0.06.
To determine if MSCs adopt the phenotype of the host organ, we co-stained lung sections for
SP-C, a specific marker for AEC2. Confocal microscopy revealed SP-C co-localization with
CFSE labeled MSCs (Figure 7). Furthermore, 75.3%±24.5 of MSCs at P14 converted to the
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AEC2 phenotype (co-expression of SP-C based on immunofluorescence) and 72.2%±28.0 at
Deconvolution microscopy (movie in online supplement) provided additional evidence that
CFSE labeled cell co-express pro-SP-C.
Protective effects of BM-derived MSC CdM in vitro. Because of the discrepancy between
the therapeutic benefit and the low rate of MSC engraftment and accumulating evidence in the
literature suggesting a paracrine activity of MSCs, we explored the potential protective effect of
BMSC CdM in three in vitro assays.
First, AEC2 were cultured with either DMEM or CdM and exposed to either 21% or 85% O2.
CdM prevented O2-induced AEC2 DNA damage (Figure 8A) and apoptosis (Figure 8B)
compared to AEC2 cultured in DMEM.
Second, AEC2 underwent a wound scratch assay (Figure 8C). After 6 hours, AEC2 wound
closure was significantly higher with CdM compared to DMEM (52% vs 37%, p<0.05).
Third, RLMVECs suspended in DMEM or CdM were exposed to room air or 85%O2 in a
serum-free Matrigel and assessed for the formation of vessel-like networks (Figure 8D).
Hyperoxia significantly decreased endothelial cord-like structure formation when cultured in
DMEM whereas BMSC CdM significantly counteracted the effect of O2 and promoted
endothelial network formation.
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