2. CALL FOR PAPERS Cellular Mechanisms of Tissue Fibrosis
ABCG2pos
lung mesenchymal stem cells are a novel pericyte subpopulation
that contributes to fibrotic remodeling
Shennea Marriott,1
Rubin S. Baskir,5
Christa Gaskill,1
Swapna Menon,9
Erica J. Carrier,1
Janice Williams,6
Megha Talati,1
Karen Helm,11
Catherine E. Alford,8
Jonathan A. Kropski,1
James Loyd,1
Lisa Wheeler,1
Joyce Johnson,4
Eric Austin,7
Eva Nozik-Grayck,10
Barbara Meyrick,1
James D. West,1,3
Dwight J. Klemm,10
and Susan M. Majka1,2,3,4,5
1
Department of Medicine, Division of Allergy, Pulmonary and Critical Care Medicine, Vanderbilt University, Nashville,
Tennesse; 2
Vanderbilt Center for Stem Cell Biology, Vanderbilt University, Nashville, Tennessee; 3
Vanderbilt Pulmonary
Circulation Center, Vanderbilt University, Nashville, Tennessee; 4
Department of Pathology, Microbiology, and Immunology,
Vanderbilt University, Nashville, Tennessee; 5
Department of Cell and Developmental Biology, Vanderbilt University,
Nashville, Tennesse; 6
Vanderbilt Ingram Cancer Center, Electron Microscopy-Cell Imaging Shared Resource, Vanderbilt
University, Nashville, Tennessee; 7
Department of Pediatrics, Vanderbilt University, Nashville, Tennessee; 8
Department of
Pathology and Laboratory Medicine, Veterans Affairs Tennessee Valley Healthcare System, Nashville, Tennessee; 9
Pulmonary
Vascular Research Institute Kochi and AnalyzeDat Consulting Services, Kerala, India; 10
Department of Pediatrics or
Medicine, Pulmonary and Critical Care Medicine, Gates Center for Regenerative Medicine and Stem Cell Biology, University
of Colorado, Aurora, Colorado; and 11
Cancer Center Flow Cytometry Shared Resource, University of Colorado, Aurora,
Colorado
Submitted 9 April 2014; accepted in final form 5 August 2014
Marriott S, Baskir RS, Gaskill C, Menon S, Carrier EJ, Williams
J, Talati M, Helm K, Alford CE, Kropski JA, Loyd J, Wheeler L,
Johnson J, Austin E, Nozik-Grayck E, Meyrick B, West JD, Klemm
DJ, Majka SM. ABCG2pos
lung mesenchymal stem cells are a novel
pericyte subpopulation that contributes to fibrotic remodeling. Am J
Physiol Cell Physiol 307: C684–C698, 2014. First published August 13,
2014; doi:10.1152/ajpcell.00114.2014.—Genesis of myofibroblasts is
obligatory for the development of pathology in many adult lung
diseases. Adult lung tissue contains a population of perivascular
ABCG2pos
mesenchymal stem cells (MSC) that are precursors of
myofibroblasts and distinct from NG2 pericytes. We hypothesized that
these MSC participate in deleterious remodeling associated with
pulmonary fibrosis (PF) and associated hypertension (PH). To test this
hypothesis, resident lung MSC were quantified in lung samples from
control subjects and PF patients. ABCG2pos
cell numbers were de-
creased in human PF and interstitial lung disease compared with
control samples. Genetic labeling of lung MSC in mice enabled
determination of terminal lineage and localization of ABCG2 cells
following intratracheal administration of bleomycin to elicit fibrotic
lung injury. Fourteen days following bleomycin injury enhanced
green fluorescent protein (eGFP)-labeled lung MSC-derived cells
were increased in number and localized to interstitial areas of fibrotic
and microvessel remodeling. Finally, gene expression analysis was
evaluated to define the response of MSC to bleomycin injury in vivo
using ABCG2pos
MSC isolated during the inflammatory phase postin-
jury and in vitro bleomycin or transforming growth factor-1 (TGF-
1)-treated cells. MSC responded to bleomycin treatment in vivo with
a profibrotic gene program that was not recapitulated in vitro with
bleomycin treatment. However, TGF-1 treatment induced the ap-
pearance of a profibrotic myofibroblast phenotype in vitro. Addition-
ally, when exposed to the profibrotic stimulus, TGF-1, ABCG2, and
NG2 pericytes demonstrated distinct responses. Our data highlight
ABCG2pos
lung MSC as a novel cell population that contributes to
detrimental myofibroblast-mediated remodeling during PF.
ABCG2; fibrosis; lung MSC; myofibroblast; pericyte
PARENCHYMAL AND VASCULAR REMODELING by mesenchymal-de-
rived cells, such as myofibroblasts, likely share mechanisms
that may explain the prevalence of pulmonary hypertension
(PH) in pulmonary fibrosis (PF) and interstitial lung disease
(ILD) patients (4, 28, 65, 75, 77, 99). Changes in tissue
structure, including fibrosis and microvascular remodeling,
result in loss of gas exchange surface area and decreased
pulmonary function. Therefore, defining myofibroblast origins
to abrogate their accumulation during pulmonary disease re-
mains a viable therapeutic strategy.
During lung development the mesenchyme influences the
development of both the epithelium and distal vasculature (25,
26, 30, 60, 81, 85, 96, 101). The intimacy of this relationship
persists into the adult tissue and is recapitulated during organ
repair and regeneration (9, 96). However, the function of
ABCG2pos
mesenchymal cells (MSC) in the adult lung during
adult pulmonary tissue homeostasis and disease remains to be
determined. MSC reside in an interstitial perivascular niche
throughout the alveolar-capillary network in both mouse and
human lungs (50). They exhibit the ability to differentiate into
cells capable of vascular remodeling including endothelium
and myofibroblasts, as well as cells that stabilize microvascular
endothelial tubes: NG2-positive pericytes or smooth muscle
cells (22, 67). MSC have also been identified in bronchoalveo-
lar lavage fluid from patient allograft tissue or tracheal aspi-
rates (42, 57). While MSC populations differ in origin, they are
multipotent and thus capable of both repair and remodeling of
pulmonary tissue.
Address for reprint requests and other correspondence: S. M. Majka,
Vanderbilt Univ., Division of Allergy, Pulmonary and Critical Care Med-
icine, 1161 21st Ave. S, T1218 MCN, Nashville, TN 37232 (e-mail:
Susan.M.Majka@Vanderbilt.Edu).
Am J Physiol Cell Physiol 307: C684–C698, 2014.
First published August 13, 2014; doi:10.1152/ajpcell.00114.2014.
http://www.ajpcell.orgC684
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3. The origin of myofibroblasts that participate in the devel-
opment of fibroblast foci and microvascular remodeling and
how their phenotype is regulated during disease dysfunction
are areas of intense study to develop targets for therapeutic
intervention. To date the origin of myofibroblasts in the lung
are theorized to be epithelial cells (EC) via epithelial-to-
mesenchymal transition as well as bone marrow sources (69,
77, 78, 84, 87, 98). Pericytes are defined by anatomical
location adjacent to vascular endothelium and in capillary
beds contact EC discontinuities in basement membranes
(14, 15). NG2-expressing pericytes in the lung do not
participate in the formation of fibroblast foci following
bleomycin injury (7, 83). These findings were striking given
that pericytes are a predominant source of myofibroblasts
during kidney fibrosis and remodeling (7, 28, 29, 38, 47, 48,
53, 58). These differences in results are likely due to
variation in techniques used to identify and label the cell
both in vitro and in vivo as pericyte heterogeneity has been
defined for multiple organ systems, although it has not been
defined within the lung (44, 45, 70).
Intratracheal administration of bleomycin elicits PF, inflam-
mation, and associated PH, contemporaneous with loss of
extant resident lung MSC. Moreover, loss of endogenous lung
MSC correlated with disease severity. We have reported that
replacement of lung MSC reduced the severity of bleomycin
pulmonary injury and associated PH (50). ABCG2pos
lung
MSC also contributed to the progression of hypobaric hypoxia-
induced pulmonary arterial hypertension (PAH) by differenti-
ating to smooth muscle actin (SMA)-expressing cells that form
the newly muscularized layer around microvessels (22). Taken
together, these results demonstrated that lung MSC protect
lung integrity following injury and when endogenous MSC are
lost due to abnormal differentiation, their protective function is
compromised. In addition to their reparative properties, several
studies indicate that lung MSC can instead mediate pathogenic
changes within the lung (11, 80). Indeed, the behavior of MSC
is highly sensitive to the microenvironment to which these cells
are exposed (100). These results illustrate the importance of
MSC during lung injury.
These studies link deregulated tissue-specific stem cell func-
tion with adult disease. Here we show that perivascular
ABCG2pos
lung MSC are a novel pericyte population distinct
from NG2 pericytes. We addressed the hypothesis that multi-
potent ABCG2pos
lung MSC participate in pathological myo-
fibroblast-mediated remodeling associated with PF and associ-
ated PH. MSC responded to the disease microenvironment in
vivo with a profibrotic and migratory gene program. In vitro
this response was not due to bleomycin injury alone but
required the fibrogenic cytokine transforming growth factor-1
(TGF-1), which stimulated early NG2 expression, followed
by a myofibroblast transition, in the absence of significant
proliferation. Interestingly, NG2 pericytes respond to TGF-1
stimulation with an increased expression of SMA as well as
proliferation, further illustrating the heterogeneity of mesen-
chymal cell subpopulations within the lung and the importance
of studying the roles of specific populations during disease.
These results suggest that lung MSC reside at an intersection
between tissue homeostasis and remodeling and are a potential
therapeutic target to regulate the genesis of myofibroblasts.
METHODS
Histological Analysis
Human tissue sections were obtained from explanted lungs of
transplant or autopsy patients at Vanderbilt University. Collection and
storage of samples were approved by the Vanderbilt University
Institutional Review Boards (Vanderbilt IRB Protocol 9401). Sections
of patient lung tissue were stained with hematoxylin and eosin.
Isolation and Characterization of Primary Human Lung MSC
Human lung adherent cells were isolated from explant lung tissue
postautopsy or transplant by collagenase digest (Vanderbilt IRB
Protocol 9401) to form a suspension. The cells were stained with
antibodies to detect and sort CD45neg
ABCG2pos
cells (lung MSC)
using a BD FACSAria III (BD Biosciences, San Jose, CA). Fluores-
cent minus one (FMO) and IgG2b isotype (12–8888-82; eBioscience,
San Diego, CA) controls were used to set the ABCG2-PE gates. DAPI
was used to exclude dead cells. The compensation controls were
established as cells only, cells ϩ DAPI, cells ϩ APC-CD45 antibody,
and cells ϩ PE-ABCG2 antibody; alternatively, comp beads were
used. The gating strategy routinely included FSC/SSC, single cells
gated by SSC-W/SSC-H, FSC-W/FSC-H, and DAPI ϩ Ter119 to gate
out dead and red blood cells followed by gating on the CD45-negative
population. The sort sample consisted of cells ϩ DAPI ϩ APC-CD45
antibody ϩ PE-ABCG2 antibody. Each sample was mixed well and
incubated for 20 min at room temperature. DAPI was used to exclude
dead cells. Following expansion cells were analyzed by flow cytom-
etry to confirm the presence of CD105, CD106, CD73, ScaI, and
CD44 and the absence of c-kit, CD14, and CD45 using a BD Fortessa
or LSRII (BD Biosciences).
To compare relative growth characteristics of MSC and fibroblast
colony-forming unit (CFU-F), cells were counted using the Countess
(Life Technologies, Grand Island, NY) and diluted to a concentration
of 6 ϫ 103
/ml. One milliliter of the cell suspension was added to
individual gelatin-coated plates containing 10 ml ␣-MEM with 20%
fetal bovine serum. The plates were gently rocked to distribute the
cells evenly. Cells were cultured for 5 days, and media were changed
every 48 h. After colonies were formed, spent medium was removed
and cells washed once with DPBS. Four percent paraformaldehyde
was used to fix the cells for 20 min. Following a PBS wash, Giemsa
stain (cat no. GS500; Sigma Aldrich, St. Louis, MO) was added to
cover cells overnight. Giemsa stain was then removed, and the plates
were gently washed with water. Plates were allowed to air dry, and
colonies of 50 cells or larger were enumerated. Cell enumeration
assays were performed by seeding MSC at 50,000 cells per well in
duplicate for collection time points at 24, 48, and 72 h. At each time
point, the spent medium was removed, and cells washed with DPBS.
Cells were collected, washed with PBS, and resuspended in 0.5 ml
␣-MEM. Ten microliters of the cell suspension were counted using
the Countess (Life Technologies) per manufacturer’s instructions. The
assay was performed in triplicate thrice independently.
Isolation and Characterization of Primary Murine Lung MSC and
NG2 pericytes.
Isolation. Cell sorting was used to isolate murine lung MSC and
NG2 pericyte cells from ABCG2 Cre-ERT2 ϫ mT/mG mice and
NG2-dsRed mice, respectively. Cells were sorted using a Moflo XDP
cell sorter with Summit 5.3 software (Beckman Coulter, Miami, FL).
Sort mode was set to Purify 1. Cells were expanded and analyzed at
passage 7.
Phenotypic analyses. Lung MSC were analyzed on a CyAn ADP
flow cytometer (Beckman Coulter) and analysis repeated twice inde-
pendently. Gating strategies included FSC/SSC, dead cell exclusion
with DAPI, and red blood cell exclusion with Ter119 and doublet
discrimination.
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4. Gene expression. For array analysis MSC were sorted using
Hoechst 33342 staining and isolation of the side population (SP). The
cells were sorted, cultured, and phenotyped as described previously
(22, 32, 50, 59, 62). Gates were set using FMO controls. Cells were
sorted using a Moflo XDP cell sorter with Summit 5.3 software
(Beckman Coulter). Sort mode was set to Purify 1. Bleomycin (5
g/ml) was added to cell culture media for 0–72 h. Colony-forming
assays (CFU-F) were performed by seeding MSC at 6,000 cells per
plate density and plated in duplicate. Cells were fixed and stained after
10 days as previously described. NG2 and ABCG2 growth curves, in
the presence or absence of TGF- (10 ng/ml), were performed as
described above. To determine the cellular response to TGF-, NG2
dsRed pericytes, or ABCG2 MSC were plated at a concentration of
50,000 cells per well in medium containing 20% serum. The cells
were allowed to remain in 20% serum for 24 h. After 24 h, the
medium was changed to starvation medium containing 5% serum. The
cells were allowed to remain in starvation medium for 24 h. After 24
h in starvation medium, the untreated cell lysates were collected for
RNA isolation (0 h posttreatment). Treatment conditions included
untreated or 10 ng/ml TGF--1, and lysate was harvested at 6, 24, and
48 h posttreatment. Cell lysates were collected using lysis buffer
(Qiagen, Valencia, CA) for total RNA isolation and analysis of gene
expression. Quantitative PCR analysis was normalized to HPRT.
Cell-Cell Communication via Calcein Dye Transfer
Plating of cell monolayers on glass slides. Primary murine lung
microvascular endothelial cells (22) or alveolar epithelial type I cells
(AEC; Cell Biologics, Chicago, IL) were plated to form a monolayer
on four-well chamber slides 24 h before the addition of calcein-AM
dye loaded ABCG2 MSC. To label the ABCG2 MSC, calcein-AM
dye (Life Technologies, Grand Island, NY) was prepared as a 1:200
dilution in staining buffer containing PBS with 2% fetal bovine serum.
The spent medium was aspirated from cells. The enhanced green
fluorescent protein (eGFP)-ABCG2 MSC were stained with cal-
cein-AM dye (which fluoresces green) at 37°C and 5% CO2 for 30
min. After 30 min, the medium was removed from the cells and
replaced with staining buffer without calcein. The dye transfer to
monolayers of either primary lung microvascular endothelial cells
(22) or type I AEC was documented using epifluorescence.
Lung MSC Lineage Tracing and Injury
All procedures and protocols were approved by the Institutional
Animal Care and Use Committee at Vanderbilt University. Mice were
on a C57Bl6/B129 background. ABCG2-CreERT2 mice, obtained in
collaboration with Dr. B. P. Sorrentino (31), were crossed to a
fluorescent eGFP reporter [(Cg)-Gt(ROSA)26Sortm4(ACTB-tdTo-
mato,-EGFP)] labeled as Rosa26 mtomato/mGFPlox-stop
(reporter mice;
JAX stock no. 007676; designated mT/mG) strain to facilitate lineage
tracing analysis. Mice were injected intraperitoneally at 8–10 wk of
age with 1 mg tamoxifen (T-5648; Sigma) in sesame oil or in sesame
oil alone (vehicle control). To identify a low dose of tamoxifen that
labeled relatively few cells, we performed a dose titration analysis in
which mice were injected with 0.1–1.0 mg in a single dose (n ϭ 3 for
each dose). For lineage tracing analysis mice were injected with 0.5
mg in a single dose. In all experiments, a single intratracheal admin-
istration of bleomycin (0.15 U) or PBS was performed 2 wk after
injection as described (50). The mice were randomized and distributed
as three to five mice per cage for study. Mice were euthanized
between 14 and 21 days following bleomycin treatment (n ϭ 5–7 per
group). Associated PH was documented by measurement of right
ventricular systolic pressure (RVSP) as previously described (16, 94).
Five independent experiments were pooled for the hemodynamic
measurements. The number of test subjects per RVSP group were five
and six.
Transcriptome Analysis
Lung MSC were isolated by cell sorting as described from vehicle-
or bleomycin-instilled lung tissue (2–4 days postinjury) directly into
RNA lysis buffer (n ϭ 20ϩ bleo mice; n ϭ 15 vehicle). RNA was
isolated from cultured MSC and NG2 cells. Total RNA was prepared
with Qiagen RNA isolation kit reagents (Qiagen, Valencia, CA) and
amplified using a Nugen kit. Complimentary DNA generated from
amplified RNA was hybridized to duplicate Affymetrix (Santa Clara,
CA) Mouse gene 1.0 chips. Array analysis was performed as de-
scribed previously (22, 50). Quantitative RT-PCR assays were per-
formed in triplicate, and levels of analyzed genes were normalized to
glyceraldehyde-3-phosphate dehydrogenase abundance.
Imaging
Epifluorescent and bright-field images were captured with Nikon
Eclipse 90i upright epifluorescence or Nikon Eclipse TS100 micro-
scopes. Confocal imaging was performed using a Nikon Eclipse Ti.
Fluorochromes used included DAB, DAPI (to label nuclei), Alexa 488
or eGFP, Alexa 594 or mTomato, and Cy5 (to detect alpha-SMA).
The camera used to capture the images was a Nikon DS-Fi1 using the
Nikon NIS elements AR 4.11.00 acquisition software.
Transmission Electron Microscopy
Specimens were processed for transmission electron microscopy
(TEM) and imaged in the Vanderbilt Cell Imaging Shared Resource-
Research Electron Microscope facility.
Embedding. Mouse lung tissue samples were fixed in 4% parafor-
maldehyde in 0.1 M cacodylate buffer at room temperature for 1 h and
then washed in ice-cold 0.1 M cacodylate buffer containing 1%
dimethyl sulfoxide (DMSO). The samples were then washed three
times with 0.1 M cacodylate buffer containing 0.1 M glycine, fol-
lowed by wash with 0.1 M cacodylate buffer only. Subsequently, the
samples were incubated for 1 h in 1% tannic acid in 0.1 M sodium
maleate (pH 6.0) followed with two washes with 0.1 M sodium
maleate buffer (pH 6.0). The samples were then dehydrated for 15 min
each through a graded ethanol series containing 1% p-phenylenedi-
amine (PPD). The samples were then infiltrated for 30 min in the cold
with a mixture of 95% ethanol and 1% PPD to Unicryl resin [2:1] and
then 30 min with a [1:1] ratio followed by a [1:2] ratio before
incubating in 100% Unicryl resin for 90 min. Samples were embedded
in gelatin capsules and filled with cold Unicryl resin and polymerized
at 50–55°C for 24 h.
Sectioning, labeling, and imaging. Seventy-nanometer-thin sec-
tions were collected on nickel grids. Sections were blocked with 50
mM glycine in phosphate-buffered saline (PBS; 15 min) to neutralize
any free aldehyde groups. After blocking, the grids with sections were
washed (15 min) in PBS with 0.1% acetylated bovine serum albumin
(BSAc) to further reduce nonspecific antibody binding. The grids then
incubated overnight with anti-GFP [1:250] in PBS with 0.1% BSAc.
Next, the grids were washed in PBS with 0.1% BSAc (30 min) and
then incubated with 40 nm gold anti-rabbit [1:500] for 2 h at room
temperature. Samples were then washed with PBS with 0.1% BSAc
(30 min) and then 1ϫ PBS (30 min). Sections were then postfixed
with 2% gluteraldehyde in PBS (5 min), then washed with PBS (5
min), and finally washed in water (10 min). Sections were then
contrast enhanced with 2% uranyl acetate (5 min), then washed with
water (10 min), and then air-dried. Sections were then imaged using
Philips/FEI Tecnai T12 electron microscope at various magnifica-
tions.
Statistical Analysis
Data analyzed by one-way ANOVA followed by Tukey’s honestly
significant difference post hoc analysis using JMP version 5.0.12.
Significance was defined as *P Ͻ 0.05, **P Ͻ 0.01, or ***P Ͻ 0.001.
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5. RESULTS
PF is characterized by excessive matrix deposition as well as
epithelial and mesenchymal cell abnormalities, including the
accumulation of myofibroblasts and inflammatory cells, signif-
icant vascular and airway remodeling, and loss of alveolar
spaces (Fig. 1) (99). Previous studies in our laboratory have
defined ABCG2 as a marker for primitive MSC associated with
the alveolar-capillary network, capable of differentiating into
contractile myofibroblasts as well as NG2-expressing pericytes
(22). Here we translated these findings to human patient
samples by isolating, expanding, and quantifying CD45neg
ABCG2pos
lung MSC from control, interstitial PF (IPF), and
ILD (excluding PF) explanted human lungs (Fig. 1C). The
number of CD45neg
ABCG2pos
MSC was decreased in cultured
disease samples, similar to what we previously reported using
a murine model of bleomycin fibrosis (50). In vitro MSC
retained a mesenchymal morphology (Fig. 1, D and E). Inter-
estingly, the MSC from control and disease lungs exhibited
similar CFU-F potential (Fig. 1F), demonstrating they are
distinct from lung fibroblasts and likely retain some stem cell
or reparative capacity (5). The MSC expressed characteristic
MSC markers including CD44, CD73, CD105, and CD106.
They also expressed high levels of CD140b/PDGFR. The
cells lacked significant expression of endothelial and hemato-
poietic markers including CD45, CD31, CD34, CD144, CD14,
as well as CD140a/PDGFR␣ (Table 1). A striking difference
between control and IPF-derived MSC was their growth in-
dexes over time. IPF MSC proliferated more rapidly than
control cells by 48 and 72 h (Fig. 1G). ABCG2pos
MSC are
therefore altered during adult lung disease, but whether this is
due to intrinsic differences between the cells themselves or a
result of exposure to the microenvironment is unknown.
Perivascular cells are thought to be a reservoir for MSC in
many adult tissues (23). Perivascular cell populations include
C
ABCG2-PE
CD45-APC
G 0
24 hours
48 hours
72 hours
0
Mean Cell # x105
**
**
Control
5
F
AW
PA
PA
V
A
Control IPF ILD
402 (67.6) 350 (39.0) 459 (33.8)
B AW
AW
AW
PA
V
AW
D
E
Control
IPF
IPF
0
105
105
Control 0.325% (0.09)
0 105
IPF 0.190% (0.06) ILD 0.131% (0.07)
0 105
Fig. 1. Human ABCG2pos
lung mesenchymal stem cells (MSC) are decreased in interstitial pulmonary fibrosis (IPF) and other interstitial lung disease (ILD) lung
tissue. A and B: representative hematoxylin and eosin-stained sections from control and IPF human lung tissue illustrate the accumulation of fibroblasts, matrix,
and inflammatory cells and loss of alveolar structure. PA, pulmonary artery; AW, airway; V, muscularized vessel. Scale bar ϭ 100 M. C: lung fibroblasts were
isolated from explanted human lung tissue via collagenase digest to form a cell suspension. Adherent cells were plated and expanded for 2 passages. At this time
flow cytometric analysis was performed on single cell suspensions of human lung tissue to detect CD45-negative (horizontal axis) and ABCG2pos
(vertical axis)
cells. Patient n ϭ 5 control, 8 IPF, and 6 ILD. The means (SE) are depicted. Variances were unequal at P ϭ 0.0070 by Bartlett’s test statistic for homogeneity
of variances and P ϭ 0.0284 by Levene’s test for equality of variances. D and E: representative bright-field photos demonstrate MSC phenotypes. Scale bar ϭ
10 M. F: representative Giemsa-stained fibroblast colony-forming unit (CFU-F) are presented and did not differ significantly between groups (P Ͻ 0.2). Data
are presented in text as the means (SE). G: changes in lung MSC control vs. IPF cell numbers over a period of 0–72 h were quantitated via automated cell
counting with trypan blue exclusion. Results are presented as total numbers of cells per time point. **P Ͻ 0.01 relative to control at the same time point.
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6. the NG2 pericytes, previously reported to not participate in
deleterious remodeling associated with bleomycin injury (83).
To distinguish ABCG2pos
MSC as a novel pericyte cell popu-
lation distinct from NG2 cells, we compared these two cell
types. Morphologically the cells are distinct (Fig. 2, A and B).
When the global gene expression patterns of ABCG2pos
MSC
were compared with NG2 pericytes relative to lung fibroblasts,
we found that ABCG2pos
MSC were much more similar to
NG2 pericytes than lung fibroblasts (Fig. 2C). The similar gene
expression patterns overlapped on a line (blue). However, there
were 542 genes that differed between the two populations by
two-fold or more (green and red respectively; Supplemental
Table S1; Supplemental Material is available online at the
Journal website). These differences included contractile pro-
teins, extracellular matrix expression, and cell cycle. Both cell
populations expressed similar levels of CD44 and ScaI and
lacked CD45, F480, CD133, CD31/PECAM, and CD144/VE-
cadherin (Table 2). When comparing the growth characteristics
of these cells over time, the ABCG2pos
MSC demonstrated
greater proliferative potential with increased cell numbers at
24–72 h relative to the NG2 pericytes (Fig. 2D). We also
compared CFU-F between these two populations and found
that although NG2 pericytes formed CFU, the ABCG2pos
MSC
had greater colony-forming potential (Fig. 2E). Taken together
these results suggest that ABCG2pos
MSC are a novel pericyte
subpopulation of cells, distinct from previously studied NG2pos
populations. These are the first data to illustrate the existence
of pericyte heterogeneity in the distal lung.
Table 1. Characterization of surface antigens in human lung MSC populations
CD140b, % CD140a, % CD44, % CD73, % CD105, % CD106, % CD31, % CD34, % CD144, % CD14, %
Control 99.0 99.4 98.6 6.5 0.021 0 5.32 0
Control 90.4 0.41 99.9 99.8 99.2 9.91 0.11 0.057 0.024 0.063
Control 99.9 0.37 99.8 100 99.9 0.44 0 0.07 0.07 0
Control 99.5 0.22 99.9 99.9 99.9 0.46 0 0.04 0.05 0.01
Control 98.7 97.3 98.9 0.69 0 0.14 0.04 0
IPF 98.6 0.84 99.3 100 99.6 0.66 0.54 0.07 0.35 0
IPF 91.9 1.3 100 99.9 99.9 2.73 0.36 0.19 0.1 0.0076
IPF 95.2 0.97 100 100 79.3 20.7 0.11 0.18 0.11 0.035
IPF 97.3 1.62 100 100 100 5.80 0.25 0.21 0.30 0.19
IPF 93.6 9.29 100 99.9 99.8 31.7 0.030 0.36 0.41 0.48
IPF 94.6 4.69 100 100 99.9 18.2 0.49 0.54 0.20 0.27
ILD 97.5 11.4 89.1 99.9 100 26.2 0 0.017 0.14 0
ILD 91.9 1.42 93.1 100 100 6.25 0 0 0.095 0
NSIP 97.9 7.04 94.9 100 100 2.02 0 0.014 0.3 0
NSIP 98 8.67 99.3 100 99.9 5.56 0.023 0.2 1.13 0.023
MSC, mesenchymal stem cells; IPF, interstitial pulmonary fibrosis; ILD, interstitial lung disease; NSIP, nonspecific interstitial pneumonia.
CABCG2
NG2
A
B
D E
ABCG2
396 (16)
NG2
178 (2.0)
**
MeanCellNumber
(x105
)
0
7.5
15.0
0 24 48 72
ABCG2
NG2
hours
*
**
**
Fig. 2. Perivascular ABCG2pos
lung MSC are
a distinct population from NG2 pericytes.
ABCG2pos
lung MSC [enhanced green fluo-
rescent protein (eGFP)] and NG2pos
pericytes
(dsRed) were isolated from mouse lungs by
flow cytometry and expanded in culture. A
and B: representative bright-field photos dem-
onstrate distinct phenotypes. C: RNA was
extracted from cultured ABCG2pos
lung
MSC, lung fibroblasts, and NG2pos
pericytes.
cDNA from each sample was hybridized to
Affymetrix mouse whole genome microar-
rays. Analysis of array data showing cluster-
ing of ABCG2pos
lung MSC and NG2pos
peri-
cytes relative to lung fibroblasts. R2
ϭ 0.983.
542 genes, shown in green and red, are Ͼ2-
fold different between MSC and pericytes.
The clustering of the blue genes demonstrated
the MSC and pericytes are more similar to
each other than fibroblasts. Two indepen-
dently isolated pooled cultures each cell line
were used for these analyses. D: changes in
lung MSC (green) or NG2 pericyte (red) cell
number over a period of 0–72 h were quan-
titated via trypan blue exclusion and auto-
mated cell counting. Results are presented as
total numbers of cells per time point. *P Ͻ
0.05, **P Ͻ 0.01 relative to control at the
same time point. E: CFU-F assays were per-
formed twice independently and representa-
tive analyses presented. Data are presented in
text as means (SE). **P Ͻ 0.01.
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7. To further define the localization and potential function of
ABCG2 MSC in vivo, we performed confocal microscopy and
ultrastructural analyses. We employed a low-dose tamoxifen
induction strategy to specifically label few ABCG2pos
MSC
using ABCG2 Cre-ERT2 driver mice crossed to an mT/mGFP
reporter strain. All lung cells were mTomatopos
while the
ABCG2pos
cells and their progeny were labeled with mem-
brane-localized eGFP. Validation of tamoxifen titration was
performed 3 wk postinduction by flow cytometry (Fig. 3, A and
B) as well as confocal microscopy (Fig. 3, B–E). Increasing
doses of tamoxifen labeled an increasing percentage of lung
MSC. Up to 80% of the eGFP-positive MSC also costained
with ABCG2 antibody (Fig. 3B), further illustrating specificity
of the system. The labeled MSC localized to the corners of the
alveolus and displayed large cell bodies and visible processes
(Fig. 3, C–F), which extended into various planes or directions.
Interactions and localization of ABCG2 lung MSC relative
to adjacent cell types were documented by TEM. Distal lung
tissue was sectioned and MSC were identified by immunogold
labeling to detect eGFP driven by ABCG2 (Fig. 4, A–D).
Ultrastructural analysis by TEM confirmed that MSC had large
cell bodies and multiple extending processes (Fig. 4, A and B).
Interestingly, the processes of MSC contacted both the micro-
vascular endothelium as well as type I AEC (Fig. 4A). These
contacts formed functional gap-junctional communication as
defined by calcein dye transfer to both populations of pri-
mary microvascular EC as well as type I AEC (Fig. 4, E and
F). Pericyte communication with endothelium is required
for the generation and maintenance of normal, stable vas-
culature (33, 45). Loss of pericytes results in hemorrhage
and embryonic lethality (40). Taken together, these analyses
define MSC as a population with the potential to regulate the
alveolus by bridging communication between endothelium
and epithelium (Fig. 4G).
To elucidate the role of ABCG2pos
MSC during deleterious
remodeling associated with bleomycin-induced PF and associ-
ated PH we performed lineage tracing analyses. Mice were
induced with tamoxifen and 2 wk later vehicle bleomycin was
introduced via intratracheal inoculation. During peak fibrosis at
14 days postinjury, lungs were harvested for lineage tracing or
flow cytometry. Histological evaluation of frozen sections
using confocal microscopy revealed the presence of ABCG2-
derived eGFPpos
cells in areas of deleterious tissue remodeling
(Fig. 5, A–D). In response to bleomycin injury, we demon-
strated the transition of ABCG2pos
MSCs from a stem cell to a
pro-PF contractile phenotype, expressing alpha-SMA (Fig. 5,
C and D). This transition to a pro-PF phenotype also resulted
in their direct contribution to pathologic microvessel remodel-
ing (Fig. 5E), absent in vehicle-only control lung tissue (Fig.
5F). This pro-PF transition and remodeling were accompanied
Table 2. Characterization of surface antigens in mouse ABCG2pos
lung MSC and NG2 pericyte populations
Purity, % CD44, % ScaI, % CD31, % CD133, % CD144, % CD45, % F480, %
ABCG2 100 99.7 96.2 0.17 0.1 0.2 0 0.04
NG2 100 95.8 76.2 0.10 0 0.1 0 0
C
0.0mg
TAM (mg)A
mTomato
ABCG2-eGFP
xy yz
xz
E
xy yz
xz
D
F
+0.5mg TAM ABCG2
ABCG2
+0.5mg TAM ABCG2
ABCG2
0.5mg
1.0mg
1.42%
0
105
APC-A::Isotype
77.5%
0 104
ABCG2 - eGFP
0
105
APC-A::ABCG2
B
Fig. 3. ABCG2pos
MSC targeted recombination in vivo. ABCG2 mouse lung 3 wk postinduction titrating a low dosage of tamoxifen from 0 to 1.0 mg. A:
recombination resulted in the appearance of membrane eGFP expression detectable by flow cytometry. B: eGFP-positive MSC colabeled with ABCG2-APC
antibody. Isotype-negative control is presented. C–F: representative confocal sections of eGFP-labeled MSC in 3 axis depict a large cell body with elongated
processes. Tamoxifen titration corresponds in a dose-dependent manner to %labeling of MSC in vivo. Scale bar ϭ 100 uM.
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8. by exacerbation of PH in vivo (Fig. 5G) (22). Flow cytometric
quantitation of eGFP-positive cells derived from ABCG2pos
lung MSC indicated there was a significant increase in eGFP-
positive cells following bleomycin treatment (Fig. 5H), sug-
gesting that the ABCG2pos
MSC likely respond to the disease
microenviroment by losing their normal homeostatic functions
and participating in the adverse remodeling associated with
fibrosis. Our data establish that lung MSC play an important
role in the maintenance of pulmonary alveolar and microvas-
cular tissue function as well as structure and when dysfunc-
tional during injury, they participate in the development of
disease. Understanding these processes are crucial to defining
the role lung MSC play during normal microvascular tissue
function and pathological remodeling to define therapeutic
targets for intervention.
To further address this theory, ABCG2pos
MSC were
isolated from murine lungs during the inflammatory phase
early following bleomycin injury and global gene analysis
was performed. We previously demonstrated that MSC
numbers were decreased in murine lung following bleomy-
cin injury (50); therefore, MSC were isolated by flow
cytometry to detect the SP of cells to select MSC that were
expressing ABCG2 (Fig. 6, A and B). The SP phenotype has
been demonstrated, by our laboratory and others, to be
dependent on the presence and activity of the ATP-depen-
dent multidrug resistance transporter ABCG2 (31, 50, 91,
103). ABCG2 has also been characterized as a potential
tissue-specific stem cell marker (31, 91, 103). In the lung
CD45neg
SP-expressing ABCG2 can be utilized to isolate
cells with MSC characteristics and potential (50, 89, 90).
Loss of active ABCG2 expression in vivo likely correlates
to an altered phenotype of these cells and differentiation of
the MSC. The MSC-expressing functional ABCG2 isolated
from bleomycin-exposed murine lungs demonstrated signif-
icant changes in their gene signatures (Fig. 6B). A summary
of genes significantly changed involved in inflammation,
fibrosis, apoptosis, cell cycle, migration, and WNT/TGF-
signaling is given in Table 3 (and Supplemental Table S2).
The stimuli that affect ABCG2pos
MSC function and con-
tribution to deleterious remodeling during fibrosis were next
evaluated in vitro. MSC were treated in vitro with bleomycin
or TGF-1 and analyzed changes in gene expression indicative
of myofibroblast transition. TGF-1 was chosen because it is a
known regulator of fibrosis in many adult tissues as well as in
response to bleomycin injury (11, 64, 71, 73). TGF-1 target
genes were also upregulated in the MSC during the inflamma-
tory phase of bleomycin injury (Fig. 6). We found that bleo-
mycin treatment of the MSC resulted in cell spreading (Fig. 7,
A and B) as well as increased gene expression indicative of
adaptive pericyte differentiation (pdgfr, rgs5, col3aI) as well
as injury (sod3) (Fig. 7C) (20, 63). However, there was no
change in SMA levels traditionally associated with myofibro-
blast transition (acta2), and periostin, associated with fibrosis,
did not increase above baseline (Fig. 7C). These results were in
contrast to ABCG2pos
MSC exposed to TGF- in vitro for 48
h. The MSC underwent a myofibroblast transition (Fig. 8,
A–C). Immunostaining was performed to detect alpha-SMA
and confirmed an increase in protein and stress fiber formation
(Fig. 8, A and B). The increased gene expression of known
myofibroblast markers, alpha-SMA (acta2), col1a1, col3a1,
and snail, indicated a TGF--induced myofibroblast transition
(Fig. 8C). TGF- exposure also induced the increased expres-
sion of NG2, illustrating the transition to a more contractile
pericyte phenotype before a myofibroblast transition (Fig. 8D)
in the absence of significant proliferation (Fig. 8E). Similarly,
NG2 pericytes responded to TGF- with increased alpha-SMA
expression (Fig. 8, F–H). However, in contrast to ABCG2pos
MSC, NG2 cells responded to TGF- with significant in-
MV
LUMEN
Lung
MSC
MV
LUMEN
A B
C
D
E FABCG2 + mvEC ABCG2 + AEC G
Fig. 4. Lung MSC contact and communi-
cate with microvascular (MV) epithelial
cells (EC) and distal lung type I epithe-
lium. Immunogold labeling was performed
to detect eGFP expression to specifically
label ABCG2pos
MSC in normal uninjured
lung tissue (A–D). Immunogold particles
were visualized by transmission electron mi-
croscopy (TEM), which localized MSC as
perivascular and in contact with a microvas-
cular EC (B, enlarged in C and D) as well as
alveolar type I cells in the alveolus. Red
arrows indicate immunogold labeling. Scale
bar ϭ 500 nM. E and F: communication
between cells was evaluated by loading MSC
with calcein-AM dye and culturing them in
contact with a monolayer of either primary
microvascular EC or alveolar epithelial type
I cells (AEC). Calcein-AM (green) dye was
transferred to both primary microvascular
EC and AEC indicating gap junction com-
munication between cells. G: schematic rep-
resentation of ABCG2pos
MSC within the
pulmonary microvasculature.
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9. creased rates of proliferation (Fig. 8I). Therefore, while bleo-
mycin alone is not sufficient to induce a myofibroblast transi-
tion in lung MSC, downstream modulators of injury and
fibrosis, including inflammation and TGF-, clearly play a role
in the maladaptive differentiation of ABCG2pos
MSC. These
data also illustrate the distinct responses of two different lung
pericyte cell populations to injury. Thus the complex microen-
viroment following bleomycin injury during fibrotic remodel-
ing involving TGF- signaling likely influences ABCG2pos
MSC phenotype and function and their contribution to adverse
remodeling at the expense of functional tissue repair via
myofibroblast differentiation.
B
A HC
D
0
15
30
MeanRVSP(mmHg)
Vehicle Bleo Tx
***
E
F
ABCG2
ABCG2 ABCG2 + SMA
ABCG2 + SMA
ABCG2+Bleo
ABCG2+Veh
G
AW
mv
mv
mv
mTOMATO
Vehicle
mTOMATO
m eGFP
+ Bleomycin
Fig. 5. ABCG2pos
lung MSC-derived cells directly contribute to deleterious interstitial remodeling following bleomycin injury in vivo. ABCG2 mice were
exposed to either vehicle or bleomycin, 2 wk postinduction. A–F: representative confocal micrographs of frozen sections from tamoxifen-induced ABCG2 mice
are presented. ABCG2-eGFP lung MSC were lineage traced via their membrane green fluorescence on an mTomato background. Nuclei were visualized with
DAPI. A–D: ABCG2 eGFP-positive-derived cells were present in the areas of deleterious fibroblast remodeling in the bleomycin-treated tissue (A: ϫ200; B–D:
ϫ400 confocal). D: enlargement from boxed area in C; n ϭ 6 mice per group. C and D: confocal imaging localized smooth muscle actin (Cy5) with eGFP-positive
MSC-derived cells. E and F: confocal imaging localized eGFP-positive MSC-derived cells to the lung microvasculature following bleomycin injury. G: right
ventricular systolic pressure (RVSP) was increased following bleomycin injury indicative of associated PH. H: single cell suspensions of murine lung tissue were
analyzed 14 days following vehicle or bleomycin treatment to enumerate the numbers of cells derived from ABCG2 eGFP-labeled MSC. Three mice were pooled
per group. Scale bar ϭ 20 M. ***P Ͻ 0.001.
B Control Bleomycin+Bleomycin Day 2
0
256
0 256
Hoechst Red
HoechstBlue
Vehicle
SP SP
0
256
0 256
Hoechst Red
HoechstBlue
A
-1.5-1.0-0.50.00.51.01.5
Fig. 6. Lung MSC respond directly to bleomycin with increased expression of a injury or profibrotic gene program in vivo. A: RNA was extracted from lung
MSC isolated by flow cytometry to detect Hoechst dye efflux and the side population of cells between days 2 and 4 following bleomycin injury or vehicle
treatment. cDNA from each sample was hybridized to Affymetrix 1.0 ST mouse whole genome microarrays. B: heatmap representation of sample log fold change
of 1,632 genes relative to control. These genes were identified as significantly differentially expressed using the AltAnalyze software, under the criterion of fold
change Ͼ2 in either direction. P Ͻ 0.05; n ϭ 15–20 mice per group.
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10. DISCUSSION
Mesenchymal cell participation and differentiation to peri-
cytes and myofibroblasts during dermal wound healing and
scar formation were first described in 1970 (24). Our recent
studies provide evidence that such a cell resides in the distal
lung and expresses ABCG2. Because dermal wound healing
shares mechanisms common to fibrosis, we hypothesized that
ABCG2pos
MSC participate in deleterious interstitial remodel-
ing associated with PF. Our data suggest that ABCG2pos
MSC
likely respond to their microenvironment, lose their normal
homeostatic function, and subsequently contribute to the ex-
pansion of the myofibroblast pool during tissue remodeling.
We found that in patients with IPF or ILD ABCG2pos
cell
numbers were decreased relative to control. Our current results
extend these observations by showing an increase in eGFP-
expressing cells derived from ABCG2pos
MSC in mouse lungs
14 days following bleomycin injury, during fibrosis. These
eGFP-positive MSC-derived cells localized to interstitial fi-
brotic areas of remodeling as well as microvessels. ABCG2pos
lung MSC responded to bleomycin treatment in vivo with a
profibrotic gene program. In vitro, exposure to TGF- was
necessary for the transition to a myofibroblast, as bleomycin
treatment alone did not increase SMA (acta2) gene expression.
The retention of MSC in disease tissue may provide a resource
to promote repair and rescue tissue function.
Before our ability to lineage trace ABCG2pos
lung MSC
following differentiation and presumptive changes in ABCG2
expression, we demonstrated a significant decrease in the
number of ABCG2-expressing MSC in murine lungs following
bleomycin treatment (50). These lung MSC were identified by
Table 3. Fold change in gene expression: bleomycin-treated vs. vehicle control
Accession Gene Symbol Gene Description
Inflammation
18.66970207 NM_133871 Ifi44 Interferon-induced protein 44
7.619522026 NM_023386 Rtp4 Receptor transporter protein 4
5.20055646 NM_016850 Irf7 Interferon regulatory factor 7
3.320300921 NM_001146275 Iigp1 Interferon-inducible GTPase 1
5.902980189 NM_009855 Cd80 CD80 antigen
3.1412995 NM_029803 Ifi27l2a Interferon, alpha-inducible protein 27 like 2A
3.855736603 NM_175649 Tnfrsf26 Tumor necrosis factor receptor superfamily, member 26
2.388983683 NM_010510 Ifnb1 Interferon beta 1, fibroblast
2.348340323 NM_013654 Ccl7 Chemokine (C-C motif) ligand 7
2.173591758 NM_024290 Tnfrsf23 Tumor necrosis factor receptor superfamily, member 23
1.811826739 NM_021274 Cxcl10 Chemokine (C-X-C motif) ligand 10
3.834492107 NM_009969 Csf2 Colony stimulating factor 2 (granulocyte-macrophage)
3.431933266 NM_009141 Cxcl5 Chemokine (C-X-C motif) ligand 5
1.816850863 NM_011333 Ccl2 Chemokine (C-C motif) ligand 2
1.672674817 NM_021500 Maea Macrophage erythroblast attacher
1.626700891 NM_001001495 Tnip3 TNFAIP3 interacting protein 3
Fibrosis
5.151055198 NM_198095 Bst2 STRO-2 MSC antigen
3.970851751 NM_011150 *Lgals3bp Lectin, galactoside-binding, soluble, 3 binding protein
3.888971063 NM_008827 Plgf Placental growth factor–survival factor
Ϫ4.985910549 NM_009114 S100a9 S100 calcium binding protein A9 (calgranulin B)
2.601723235 NM_001081401 Adamts3 A disintegrin-like and metallopeptidase
Ϫ8.631710626 NM_054038 Scgb3a2 Secretoglobin, family 3A
2.417483428 NM_177290 *Itgb8 Integrin beta 8
2.094371688 NM_001044384 Timp1 Tissue inhibitor of metalloproteinase 1
2.473679344 NM_007950 Ereg Epiregulin
Apoptosis
3.138372285 NM_172689 Ddx58 DEAD (Asp-Glu-Ala-Asp) box polypeptide 58
3.027849505 NM_010786 Mdm2 Transformed mouse 3T3 cell double minute 2
2.2761064 NM_009810 Casp3 Caspase 3
2.074557292 NM_007527 Bax BCL2-associated X protein
1.656629518 NM_013929 Siva1 SIVA1, apoptosis-inducing factor
2.022921729 NM_022032 Perp PERP, TP53 apoptosis effector
1.696639435 NM_023190 *Acin1 Apoptotic chromatin condensation inducer 1
1.879935286 NM_011052 Pdcd6ip Programmed cell death 6 interacting protein
Cell Cycle
1.845472833 NM_183417 Cdk2 Cyclin-dependent kinase 2
1.671665855 NM_009830 Ccne2 Cyclin E2
1.508740827 NM_007669 Cdkn1a Cyclin-dependent kinase inhibitor 1A (P21)
Mobilization/migration
3.193133785 Z31359 Npn2 Neoplastic progression 2
2.346544793 NM_018761 Ctnnal1 Catenin (cadherin associated protein), alpha-like 1
1.666233397 NM_001033335 Serpina3f Serine (or cysteine) peptidase inhibitor, clade A, member 3F
TGF/WNT pathways
1.761019641 NM_010754 Smad2 MAD homolog 2 (Drosophila)
Ϫ1.572305978 NM_010091 Dvl1 Dishevelled, dsh homolog 1 (Drosophila)
2.21303689 NM_001025067 Lrig Leucine-rich repeats and immunoglobulin-like domains 2
Italics indicate gene may be regulated by transforming growth factor- (TGF-). *Protein participates in TGF- signaling.
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11. the appearance of the SP profile by flow cytometry during
active expression of the multidrug resistance protein ABCG2.
The existence of ABCG2 indicates the existence of the “prim-
itive” MSC state before any differentiation during which
ABCG2 expression is decreased. We hypothesized that the loss
of these MSC during bleomycin fibrosis was the result of
differentiation or apoptosis. The current study demonstrated
that indeed loss of the majority ABCG2-expressing SP cells is
due to differentiation to a myofibroblast phenotype. These
ABCG2pos
MSC-derived eGFPpos
cells were present in in-
creased numbers in areas of fibrotic remodeling demonstrated
by confocal microscopy as well as flow cytometry. The cell
surface of expression of ABCG2 was also absent on the
MSC-derived eGFP-labeled cells. Interestingly, the patient
control, IPF, and ILD ABCG2pos
lung MSC retained during
disease remained able to form CFU-F in a colony-forming
assay, indicating some retention of their primitive characteris-
tics. These results also elude to the importance of the local
microenvironment in regulating cell function. Perhaps these
MSC capable of forming colonies are yet capable of repair and
maintenance of a stem cell reservoir during disease.
ABCG2pos
lung MSC colocalized with the alveolar capillary
network in the distal lung of both mouse and humans, an
anatomical feature observed in adult angioblasts, pericytes, and
endothelial precursors (7, 23, 25, 37). We previously demon-
strated their multipotent potential and capacity for microvas-
cular remodeling via myofibroblast/contractile transition using
a murine model of hypoxia-induced PAH (22). These results
were highly significant because while MSC from various
tissues have been shown to express varying levels of pericyte
markers, MSC have not been linked functionally to lung
disease pathogenesis (7, 23, 66). These studies go on to show
that while ABCG2pos
lung MSC are similar to NG2pos
peri-
cytes, they are not the same population of cells and likely
represent pericyte heterogeneity within the lung (7, 45, 70, 86).
We propose that a hierarchy exists, similar to the branches of
the vascular tree, from contractile SMApos
smooth muscle in
larger diameter vessels exposed to high pressure, pericytes
with varying levels of SMApos
and NG2pos
supporting more
moderate pressure and flow to the distal noncontractile mi-
crovessels, supported by ABCG2pos
NG2neg
/SMAneg
perivas-
cular cells.
ABCG2pos
lung MSC associated with the smallest microves-
sels in the lung and represent a small noncontractile subset of
these vascular supporting cells. However, following stimula-
tion, such as TGF-, the ABCG2pos
cells increase expression
of both SMA and NG2, resembling an “activated” pericyte,
followed by a myofibroblast transition, indicated by increased
expression of snail and collagens (22). In contrast, NG2
pericytes significantly increased SMA expression and prolifer-
ated. Heterogeniety between these two populations of perivas-
cular cell types would likely explain why some NG2pos
peri-
cytes expand their population in response to bleomycin injury,
do not become myofibroblasts, and may not participate in
deleterious remodeling associated with bleomycin fibrosis
(83), whereas ABCG2pos
lung MSC clearly localize to areas of
fibrosis and microvasculature. A recent study by Ricard et al.
(82) also demonstrated expansion of an NG2pos
pericyte pop-
ulation in response to chronic hypoxia. In this study we
demonstrate distinct responses of ABCG2pos
lung MSC and
NG2pos
pericytes to the profibrotic stimulus TGF-. Lung
MSC differentiate to myofibroblasts in the absence of prolif-
eration while NG2 pericytes both significantly increase their
expression of SMA and proliferate. While the proliferative
response of pericytes to lung injury is becoming recognized,
the role they play in disease is still relatively unknown. How-
ever, here we demonstrate that not only is their phenotypic and
genomic heterogeneity on the two populations but also in
functional responses during tissue injury.
The transition from a noncontractile ABCG2pos
MSC to an
activated NG2-expressing pericyte may be an adaptive re-
sponse to injury, which may be followed by proliferation,
decreased apoptosis, and transition to a synthetic myofibro-
blast, all leading to pathologic remodeling, adversely impact-
ing the microvasculature. Abnormal pericyte function has been
implicated as a cause for PH in Adams-Oliver syndrome as
well as capillary rarefaction in scleroderma (36, 74). Addi-
tional studies in multiple adult tissues have defined myofibro-
blasts that derived from pericytes as expressing NG2 (58, 92).
Understanding this sequence of events as well as differences
among MSC, NG2 pericytes, and additional lung mesenchymal
cell populations will allow us to inhibit this transition in a
cell-specific manner and promote functional tissue repair.
To date, defining ABCG2 as an appropriate marker to study
lung MSC coupled to an in vivo model system with which to
study these cells has facilitated progress in this area (22).
Previous studies have demonstrated labeling of perivascular
tissue-specific stem cells in the heart and bone marrow using
0
0.8
1.6 col3a1
0
1.5
3 rgs5
*
* *
* *A
B
Control
Bleomycin
0
1
2 sod3
0
0.6
1
1.2
periostin acta2
* *
*
*
*
0
1.5
2.5
pdgfrb
*
*C
0
0.8
1.4
T=0 24hr 48hr 72hr T=0 24hr 48hr 72hr
+ bleomycin+ bleomycin
FoldChangeRelativetoControl
Fig. 7. Bleomycin treatment of lung MSC in
vitro does not result in myofibroblast differ-
entiation. Lung MSC were treated for up to
72 h in vitro with bleomycin (5 ug/ml). A and
B: representative phase micrograph of the
untreated and bleomycin-exposed cells at 72
h. C: RNA was extracted from all groups and
cDNA used to perform quantitative (q)PCR
to analyze expression patterns in genes asso-
ciated with injury and myofibroblast transi-
tion. *P Ͻ 0.05, relative to control.
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12. ABCG2-driven reporter expression (31). MSC, pericytes
(NG2), and lung fibroblasts have very similar cell surface
markers and differentiation potential (22, 50) (Fig. 9). Due to
these similarities, pericytes have been hypothesized to be MSC
in adult tissues (7, 23). Further global gene profiling analyses
in our laboratory demonstrated that ABCG2pos
lung MSC were
distinct from lung NG2 pericytes as well as lung fibroblasts.
Fibroblasts differ from MSC and pericytes in that they do not
form colonies in a colony-forming assay, with clonal growth
being a defining characteristic of MSC (27). A summary of
these distinct lung mesenchymal populations is presented in
Fig. 9. In these studies we begin to delineate these events both
in vitro and in vivo. Based on these analyses we theorize that
ABCG2pos
MSC are a unique subclass of noncontractile peri-
cytes, in that they represent a fraction of cells in the lung that
function to stabilize a large surface area of microvessels and
they lack expression of NG2 and SMA.
Bleomycin treatment alone was not a potent inducer of
myofibroblast transition of lung MSC in vitro. This was evi-
dent by the lack of increase in SMA actin (acta2) expression.
However, there were increased levels of gene expression of
PDGFR, rgs5, col3a1, periostin, and sod3. Enhanced expres-
sion of PDGFR by mesenchymal cells and fibroblasts is
associated with fibrosis to increase the biological responses to
PDGF including cell proliferation and survival (11, 13). Rgs5,
a regulator of G-protein signaling, functions as its name im-
plies and potentiates PDGFR signaling (20, 63). It is consid-
ered a marker of rare pericytes (10), and its expression is
typically not detected in adult lung (72). Its expression has
been associated with vascular remodeling, rarefaction, or loss
of microvessels and regulation of vascular tone (21, 35, 36,
74). Periostin is a profibrotic and proinflammatory protein
hypothesized to be a potential biomarker in IPF (68, 93).
Extracellular superoxide dismutase (sod3) is a potent antioxi-
6 24 48 hr
*** **
**
NG2 (cspg4)
0
2
4
6
FoldChange
OverControl
0
2
4
SMA Col1a1 Col3a1 Snail
***
***
**
**
B
A
FoldChange
OverControl
C
F
G
MSC +TGFβ / SMA
MSC Control / SMA
NG2 Control
NG2 +TGFβ
E
NG2 pericytes
D
0 24 48 72 0 24 48 72
0TX TGF β-1
hrs
MeanCellNumberx105
***
***
ABCG2 MSC
6
0
6 24 48 hr
**
***
SMA (acta2)
0
2
4
6
FoldChange
OverControl
H
I
0
1.5
3
0 24 48 72 0 24 48 72
0TX TGF β-1
hrs
MeanCellNumberx105
Fig. 8. Transforming growth factor- (TGF-) treatment in vitro promotes the transition of MSC to a myofibroblast phenotype. ABCG2pos
lung MSC (A–E) or
NG2 pericytes (F–I) were treated with TGF- for 48 h. A and B: ABCG2pos
lung MSC were fixed and immunostained to detect alpha-smooth muscle actin
(SMA-red) and visualized by epifluorescent microscopy. Scale bar ϭ 50 M. C–E: RNA was collected and amplified by qPCR. Data are presented as means Ϯ
SE. Three individual cell samples were run in triplicate during qPCR analyses. Fold change relative to control was calculated using delta delta-CT. C:
characteristic changes in gene expression indicative of a myofibroblast transition in ABCG2pos
lung MSC. D: ABCG2pos
lung MSC increased expression of NG2.
E: analysis of total ABCG2pos
lung MSC numbers over time demonstrated a decreased rate of proliferation in the presence of TGF-. F and G: representative
phase images of NG2 cells 48 h following treatment with TGF-. H: NG2 pericytes increased expression of SMA (acta2). I: total NG2 cell numbers were
enumerated over time in the presence or absence of TGF-. NG2 pericytes proliferated in response to TGF. **P Ͻ 0.01, or ***P Ͻ 0.001.
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13. dant enzyme, which has previously been shown to attenuate
bleomycin injury in vivo (32, 94). Early in the course of
disease, sod3 has reported to increase as an adaptive response
to oxidative stress. Taken together, these data suggest that
bleomycin induces an injury and profibrotic response in vitro
but lacks the cytoskeletal changes indicative of a migratory
myofibroblast-like transition. Such a transition likely requires
additional components of the in vivo disease microenviron-
ment, such as TGF-.
TGF- is a known regulator of mesenchymal cell differen-
tiation and migration (11, 71), lung fibrosis, proliferation,
apoptosis, matrix turnover, and differentiation in humans (73)
and the murine bleomycin model (64). Our gene profiling data
provide evidence that TGF- signaling is active in lung MSC
during bleomycin injury in vivo. These lung MSC were iso-
lated by active expression of ABCG2 protein and the appear-
ance of the SP profile by flow cytometry, that is to say before
any differentiation during which ABCG2 expression is likely
altered. Smad2 and Itgb8 message upregulation, as well as
decreased scgb3a2 levels, suggests that that TGF- signaling is
active in the lung MSC. Secretoglobin3A2 (scgb3a2) inhibits
TGF--induced myofibroblast differentiation in part via regu-
lation of SMAD2 (56). SMAD 2 mediates TGF- signaling
while integrin subunit beta8 (Itgb8) expression is increased
with inflammation and results in TGF- activation (with ␣v
subunit) by fibroblasts (41, 61). This activation of TGF-
results in myofibroblast transition of fibroblasts as well as EC,
increased collagen deposition (col1a1, col3a1), increased ma-
trix stiffening, and adverse remodeling (41, 46). S100A9,
galectin 3 (Lgals3bp), and placental growth factor (pgf) have
all been identified as biomarkers that indicate the severity of
fibrosis in various tissues including IPF (18, 39, 76, 95).
Interestingly, galectin 3 interacts with NG2 and promotes
migration and invasion of cells (97). ␣-Catulin (ctnnal1) ex-
pression typically parallels that of mesenchymal transition
genes (snail/slug, twist); interacts with the NfB, Rho, and
-catenin; and regulates cytoskeletal reorganization, migra-
tion, inflammation, and apoptosis (55).
TGF- exposure of ABCG2pos
MSC in vitro resulted in a
mesenchymal-myofibroblast transition (2) exhibiting a similar
gene profile to epithelial-mesenchymal transition in response to
TGF- including upregulation of SMA, collagen, and the
mesenchymal transcription factor snail (17, 43, 51, 52, 54, 79).
Snail regulates epithelial mesenchymal transition and cell in-
vasion downstream of TGF- as well as Wnt signaling (88,
102). Taken together our genetic profiling data suggest that
TGF- directly regulates the transdifferentiation of MSC to
invasive myofibroblasts and therefore their contribution to
deleterious remodeling. Common pathways associated with
vascular remodeling in PAH and IPF were recently identified
in patient samples by microarray analyses (75). These path-
ways may regulate ABCG2pos
lung MSC contribution to vas-
cular as well as interstitial remodeling in response to different
stimuli. Whether this response is adaptive or adverse is cur-
rently unknown.
Dysfunction in both the mesenchymal and epithelial com-
partments of the distal lung underlies both IPF and chronic
obstructive pulmonary disease, resulting in inflammation and
deleterious remodeling (19). However, the interactions be-
tween the epithelium and the mesenchyme, as well as endo-
thelium that drive the transition of cells to their participation in
pathological processes are not well understood. There is likely
opportunity for cell-cell regulation of phenotype and function
via direct gap junction communication as well as paracrine
mechanisms. The candidate signaling pathways involved in
tissue renewal vs. remodeling include established molecules
known to regulate lung development TGF- Wnt, PDGF, and
Notch (1, 3, 6, 13, 17, 19). It is of the utmost importance to
understand the “switch” that drives deleterious pathological
remodeling vs. adaptive remodeling and repair as well as the
similarities between various lung diseases.
In conclusion, our studies show that ABCG2pos
lung MSC
represent a perivascular stem cell pool of cells distinct from
previously described lung pericytes and have the potential to
participate in the remodeling associated with bleomycin-in-
duced fibrosis. These cells have been shown to function in the
CD45neg
Lung Side Population (SP)
CD45neg
ABCG2pos
MSC NG2pos
Pericyte
• NG2
pos
/SMA
pos
• Contractile
• Proliferate during TGF-β
stimulation
• Increased collagen,
adhesion and
cytoskeletal
genes
• NG2neg
/SMA
neg
• Non contractile
• Self-renewal
• Upregulate NG2/SMA &
myofibroblast phenotype
with TGF-β stimulation;
Decreased Proliferation
• Increased
immunoregulatory and
mesenchymal genes
CFU-F
Differentiate:
Osteocyte, Adipocyte &
Chondrocyte
Express:
CD44,CD90,
CD105,CD106, CD73,
ScaI, CD140b
Negative:
CD45,CD117,
CD31,CD144,
CD14,CD34
Lung
Fibroblast
Upregulate SMA with
TGF-β stimulation
MSC
Lung Mesenchymal
Cell Populations
Fig. 9. Summary of lung mesenchymal cell
subpopulations. Classically defined MSC
(black box) have traditionally been isolated
based on their ability to adhere to plastic and
a panel of cell surface antigens. This limited
their study in vivo because fibroblasts and/or
endothelial cells express many of the same
markers. However, more recently lung mes-
enchymal subpopulations have been further
delineated by genomic analyses, CFU-F, and
multilineage differentiation potential (22, 34,
49, 50, 57, 62, 89). These distinct subpopu-
lations include the CD45neg
side population
(SP), lung fibroblasts, ABCG2pos
MSC, and
NG2pos
pericytes.
C695LUNG MSC PARTICIPATE IN FIBROSIS
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14. maintenance and repair of the adult murine distal lung micro-
vasculature, pathological remodeling of the lung, and regula-
tion of pulmonary inflammation (50). In understanding
ABCG2pos
MSC function we will be poised to target therapeu-
tics to preserve MSC function and restore microvascular in-
tegrity and surfaces for gas exchange; replace MSC following
injury; and lastly promote the generation of tissue grafts.
Identification of therapeutic strategies directed at preserving
ABCG2pos
MSC function may facilitate the retention of
healthy distal lung microvascular tissue structure and function.
ACKNOWLEDGMENTS
We extend appreciation to Christine Childs, Lester Acosta, and Du Jun for
expert technical assistance and the Research Flow Cytometry Laboratory at the
Nashville Veterans Affairs Medical Center. We thank Dr. Pat D’Amore for
helpful discussion and Dr. Vibha Lama for generously providing control lung
sample.
GRANTS
This work was funded by American Heart Association Grant SDG-
0335052N and National Institutes of Health (NIH) Grants R01-HL-091105 and
R01-HL-11659701 (to S. M. Majka). Experiments were performed using the
University of Colorado Cancer Center (UCCC) Flow Cytometry Shared
Resource [NIH Grant 5-P30-CA-46934; Skin Research Disease Center (SDR)
Grant P30-AR-057212], UCCC Microarray Core (NIH Grant P30-CA-46934–
14), UCCC SDRC Morphology and Phenotyping Core (NIH Grant P30-AR-
057212), Vanderbilt University Medical Center (VUMC) Cell Imaging Shared
Resource (supported by NIH Grants CA68485, DK20593, DK-58404, DK-
59637, and EY-08126), and VUMC Flow Cytometry Shared Resource sup-
ported by the Vanderbilt Ingram Cancer Center Grant P30-CA-68485.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
Author contributions: S. Marriott, R.S.B., C.G., J.L., E.A., E.N.-G.,
B.O.M., J.D.W., D.J.K., and S.M.M. conception and design of research; S.
Marriott, R.S.B., C.G., E.J.C., J.A.W., M.T., K.H., C.E.A., J.A.K., L.W., E.A.,
and S.M.M. performed experiments; S. Marriott, R.S.B., S. Menon, J.A.W.,
K.H., C.E.A., B.O.M., J.D.W., and S.M.M. analyzed data; S. Marriott, R.S.B.,
S. Menon, K.H., C.E.A., J.L., J.J., D.J.K., and S.M.M. interpreted results of
experiments; S. Marriott, R.S.B., S. Menon, J.A.W., J.D.W., and S.M.M.
prepared figures; S. Marriott, R.S.B., C.G., S. Menon, E.J.C., K.H., J.A.K.,
J.L., L.W., E.N.-G., J.D.W., D.J.K., and S.M.M. edited and revised manu-
script; S. Marriott, R.S.B., C.G., S. Menon, E.J.C., J.A.W., M.T., K.H., J.A.K.,
J.L., L.W., J.J., E.A., E.N.-G., B.O.M., J.D.W., D.J.K., and S.M.M. approved
final version of manuscript; S.M.M. drafted manuscript.
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