A Novel Method for Isolating Individual Cellular
Components from the Adult Human Distal Lung
Naoya Fujino1, Hiroshi Kubo1, Chiharu Ota1, Takaya Suzuki2, Satoshi Suzuki5, Mitsuhiro Yamada3,
Toru Takahashi1, Mei He1, Takashi Suzuki4, Takashi Kondo2, and Mutsuo Yamaya1
1Department of Advanced Preventive Medicine for Infectious Disease,2Department of Thoracic Surgery, Institute of Development, Aging, and
Cancer,3Department of Infection Control and Laboratory Diagnostics, and4Department of Pathology and Histotechnology, Tohoku University
Graduate School of Medicine, Aobaku, Sendai, Japan; and5Department of Thoracic Surgery, Japanese Red Cross Ishinomaki Hospital, Hebita,
A variety of lung diseases, such as pulmonary emphysema and idio-
pathic pulmonary fibrosis, develop in the lung alveoli. Multiple cell
and endothelial cells. These resident cells participate in the patho-
genesis of lung disease in various ways. To elaborate clearly on the
mechanisms of these pathologic processes, cell type–specific analy-
ses of lung disease are required. However, no method exists for in-
dividually isolating the different types of cells found in the alveoli.
We report on the development of a FACS-based method for the
direct isolation of individual cell types from the adult human distal
pared single-cell suspension. After depleting CD45-positive cells,
a combination of antibodies against epithelial cell adhesion mole-
in the EpCAMhi/T1a2subset, whereas the EpCAM1/T1a2/lowsubset
and bronchiolar epithelial cells. The EpCAM2/T1a2subset included
both microvascular endothelial and mesenchymal cells, and these
were separated by immunoreactivity to VE-cadherin. Lym-
phatic endothelial cells existed in the EpCAM2/T1ahisubset.
Isolated cells were viable, and further cell culture studies could be
lungs, and is capable of elucidating phenotypes specific to certain
alveolar cell types indicative of lung disease.
Keywords: cell isolation; fluorescence-activated cell sorting; pulmonary
alveoli; cell culture
The lung is a complex organ containing more than 30 different cell
end of this structure, and are thus difficult to approach. A variety
of lung diseases develop in the alveoli, including pulmonary em-
physema (2), idiopathic pulmonary fibrosis (3), pneumonia caused
by microorganisms (4), acute lung injury/acute respiratory distress
syndrome (5), and lung cancer (6). In the pathologic processes of
these lung diseases, alveolar cells may initially be injured by var-
ious insults, including noxious gases, oxidants, and inflammatory
cells. The repair process after an injury relies on the protective
effects of various types of alveolar cells, and the loss of these
protective effects can lead to the development of lung disease.
Thus, to yield insights into the molecular basis of cellular pheno-
types and the pathologic processes in these lung diseases, an in-
vestigation of the roles of specific cell types in the alveoli in health
and disease is necessary (1).
To isolate specific cell types in normal and diseased human
lungs, laser capture microdissection (LCM) and FACS have been
used. Previous studies reported that LCM can be used to isolate
bronchiolar epithelial cells from lung tissue affected by chronic ob-
structive pulmonary disease (7–9). However, LCM is unsuitable
for the isolation of alveolar component cells for two reasons. The
first involves technical limitations in isolating individual alveolar
component cells using LCM, because alveolar cells are firmly
ensconced within the thin alveolar walls (10). Secondly, micro-
array data derived from samples prepared by LCM showed a sig-
nificantly higher level of contamination than occurred with the
use of FACS when neural populations from the murine brain
were isolated (11). In contrast, FACS is thought to be useful
for the isolation of specific cell types from mixed cell popula-
tions, based on the assumption that specific combinations of cell
surface markers are already well characterized. Recently, con-
siderable progress has been made in nanotechnology and geno-
mic and other “-omic” approaches, increasingly facilitating the
characterization of cell surface markers among different cell
types in the alveoli, and enabling the establishment of method-
ologies for cell type–specific isolation (1).
Here, we report on the development of a FACS-based
method for the direct isolation of individual cell types from
the adult human distal lung. The combination of three surface
markers, epithelial cell adhesion molecule (EpCAM), T1a,
and vascular endothelial (VE)-cadherin, allowed for the isolation
of alveolar Type II (ATII) cells, as well as microvascular endothelial
cells, lymphatic endothelial cells, and mesenchymal cells, in a vi-
able condition. We further cultured isolated cells and charac-
terized the phenotypes of each component. Some of the results
of this study were previously presented in abstract form (12).
(Received in original form May 26, 2011 and in final form October 21, 2011)
This work was supported by Japanese Society for the Promotion of Science grant
Author Contributions: N.F. and H.K. designed and performed the experiments and
M. Yamaya performed the experiments. Takaya S., S.S., and T.K. obtained informed
consent from the patients and contributed to the analyses of clinical data. Takashi S.
performed histological analyses and contributed to the electron microscopic evaluation
Correspondence and requests for reprints should be addressed to Hiroshi Kubo,
M.D., Ph.D., Department of Advanced Preventive Medicine for Infectious Disease,
Tohoku University Graduate School of Medicine, 2-1 Seiryoumachi, Aobaku, Sendai
980-8575, Japan. E-mail: firstname.lastname@example.org
This article has an online supplement, which is accessible from this issue’s table of
contents at www.atsjournals.org
Am J Respir Cell Mol Biol
Copyright ª 2012 by the American Thoracic Society
Originally Published in Press as DOI: 10.1165/rcmb.2011-0172OC on October 27, 2011
Internet address: www.atsjournals.org
Vol 46, Iss. 4, pp 422–430, Apr 2012
We developed a novel technique of cell isolation from hu-
manlung tissue, usinga combination of cell surface antigens.
these cells. Because alveoli are the main targets of many
pulmonary diseases, analyzing the component cells in alveoli
is useful for understanding the pathophysiology of disease
development, epigenetic analyses, and drug discovery.
MATERIALS AND METHODS
Patients and Preparation of Tissue Samples
Human lung tissue was obtained from patients who underwent lung
resections at the Department of Thoracic Surgery at Tohoku University
Hospital (Aobaku, Sendai, Japan) or at the Ishinomaki Red Cross Hos-
from the tumors. Through histopathologic study, we confirmed that the
harvested tissue did not contain tumor lesions and did not exhibit em-
physema, fibrosis, or inflammatory changes. In addition, these patients
manifested normal lung function, as determined by spirometry. This
study was approved by the Ethics Committees at Tohoku University
gave informed consent.
Preparation of Single-Cell Suspensions from Human
Humanlung cells were isolatedas previouslydescribed,with some mod-
Flow Cytometry and Sorting of Lung Component Cells
We used phycoerythrin-conjugated anti-human EpCAM antibody (cat-
alogue number 12-9236, clone 1B7; eBioscience, San Diego, CA), Alexa
Figure 1. Epithelialcelladhesion
and T1a-delineated subpopu-
lations of adult human distal
lung cells. (A) A representative
FACS dot plot shows the ex-
pression of EpCAM and T1a in
a live and single-cell–gated
CD45-negative fraction from
normal lung tissue. The dot
plot is a representation of the
results from 40 patients. (B)
cence images of cells isolated
from normal lung tissues. (C)
An electron micrograph of an
EpCAMhi/T1a2cell. ATII, alve-
olar epithelial Type II; ATI, alve-
olar epithelial Type I; pan-CK,
pan-cytokeratin; pro-SP-C, pro-
aquaporin 5; CCSP, Clara cell–
specific protein. Scale bars: B,
20 mm; C, 2 mm.
Fujino, Kubo, Ota, et al.: Isolation of Human Alveolar Cellular Components423
Fluor 647–conjugated anti-human T1a antibody (catalogue number
337008, clone NC-08; Biolegend, San Diego, CA), and FITC-conjugated
anti-human VE-cadherin antibody (catalogue number 560411, clone 55-
7H1; BD Pharmingen, San Diego, CA). To discriminate between live and
dead cells, we used 7-amino actinomycin D (catalogue number 00-6993;
eBioscience). We sorted live and single-cell–gated subpopulations, based
on their staining patterns with EpCAM, T1a, and VE-cadherin, using
a FACS Aria II Cell Sorter and FACS Diva, version 6.1 (BD Biosciences,
San Jose, CA). FACS analyses were performed using the FlowJo software
package (Tree Star, Ashland, OR).
Immunofluorescence Staining and Immunohistochemistry
For immunofluorescence staining, the cytospun cells or cultured cells
were fixed with 4% paraformaldehyde, blocked, and permeabilized.
We stained samples with primary antibodies, as shown in Table E1 in
taken using the Nikon C2 system (Nikon, Tokyo, Japan).
Electron Microscopy Analysis
Electron microscopy analysis was performed as previously described
Isolation of ATII Cells According to a
ATII cells were isolated from lung cell suspensions using a density-
gradient method, as previously reported (14).
Culture of Alveolar Epithelial Type II Cells Isolated
from Human Lungs
Sorted EpCAMhi/T1a2cells were plated on collagen I–coated culture
slides (Thermo Fisher Scientific, Waltham, MA) with a SAGM Bullet-
kit (Lonza, Basel, Switzerland) containing 1% FBS, as previously de-
scribed, with some modifications (15).
Culture of Microvascular Endothelial and Mesenchymal Cells
Isolated from Human Lungs
Sorted VE-cadherin1and VE-cadherin2cells were plated on fibronectin-
coated plates (24 wells; BD Falcon, San Jose, CA) and cultured with two
types of media, EGM-2-MV BulletKit (Lonza) and Dulbecco’s Modified
Eagle’s Medium/10% FBS/penicillin/streptomycin/amphotericin B.
In Vitro Angiogenesis Assay
A tubeformation assay was performed according to previously reported
methods, with some modifications (16).
Statistical analyses were performed with GraphPad Prism, version 5.0b
(GraphPad Software, La Jolla, CA). Data were compared using an un-
paired t test. Statistical significance was defined as P , 0.05.
Identification of Distinct Subpopulations in the Human
To isolate specific cell types from distal lung tissue, we char-
acterized cell surface markers and sought to develop a FACS-
tissue, and prepared single-cell suspensions. We collected whole
lung cells of 1.3 3 1076 7.1 3 106cells/g tissue (mean 6 SD;
n ¼ 11). The viability of the freshly collected cells was determined
using trypan blue. The percentage of trypan blue–negative cells
was 87.7% 6 5.7% (mean 6 SD; n ¼ 11). CD45-expressing
hematopoietic cells containing alveolar macrophages (17) were
depleted from the single-cell suspensions, using anti-human
CD45 antibody–coated microbeads. We confirmed the complete
depletion of CD451cells from the single-cell suspension by flow
cytometry, using another clone of an antibody against CD45 (data
not shown). The percentage of collected CD452lung cells among
the whole lung cells was 22.7% 6 8.1% (mean 6 SD; n ¼ 24).
We fractionated CD452lung cells into four subpopulations,
using antibodies specific for EpCAM and T1a (Figure 1A). The
yield of each subset is shown in Table 1. Freshly sorted and
cytospun cells were stained with each lineage marker by immu-
nofluorescence. We found that ATII cells were enriched in
the EpCAMhi/T1a2subset, and that the EpCAM1/T1a2/low
subset contained alveolar Type I (ATI) cells and bronchiolar
epithelial cells, including Clara cells. Immunofluorescence staining
demonstrated that the EpCAMhi/T1a2subset expressed pro–
surfactant protein–C (pro–SP-C, an ATII cell marker) and pan-
cytokeratin (an epithelial marker), but did not express Clara
cell–specific protein (CCSP, a Clara cell marker) or aquaporin 5
(AQP5, an ATI cell marker) (Figure 1B and Table 2). In addition,
electron microscopy showed that the sorted EpCAMhi/T1a2
cells displayed lamellar bodies that contained pulmonary sur-
factants (18) (Figure 1C). In contrast, the EpCAM1/T1a2/low
subset expressed pan-cytokeratin, CCSP, and AQP5, indicating
that the EpCAM1/T1a2/lowsubset is a mixed cell population
consisting of ATI cells and bronchiolar epithelial cells, including
Clara cells (Figure 1B and Table 2). The EpCAM2/T1a2and the
EpCAM2/T1ahisubset expressed vimentin but not other epithelial
markers, suggesting that these subsets contained mesenchymal
cells and endothelial cells (Figure 1B). A previous study reported
that T1a was expressed by lymphatic endothelial cells but not
vascular endothelial cells (19). Therefore, the EpCAM2/T1ahi
subset shown in Figure 1A predominately contained lymphatic
endothelial cells, and the remaining EpCAM2/T1a2subset
contained vascular endothelial cells and mesenchymal cells such
as fibroblasts, pericytes, and smooth muscle cells.
To isolate ATII cells, previous studies used a method com-
bined with a density-gradienttechnique, the depletion of alveolar
macrophages using anti-CD14 antibody–coated microbeads, and
TABLE 1. THE YIELD OF DIFFERENT CELL TYPES (PER GRAM OF
TISSUE) ISOLATED FROM ADULT HUMAN LUNGS (MEAN 6 SD;
n ¼ 4)
Surface Antigen ExpressionNumber of Cells
4.2 3 1066 3.8 3 106
1.2 3 1066 7.8 3 105
1.4 3 1066 8.4 3 105
2.0 3 1066 1.0 3 106
3.9 3 1056 2.2 3 105
Definition of abbreviations: EpCAM, epithelial cell adhesion molecule; VE-
cadherin, vascular endothelial cadherin.
TABLE 2. ANALYSIS OF IMMUNOFLUORESCENCE STAINING OF
SUBPOPULATIONS DEFINED BY EPCAM AND T1a
98.1 6 0.4
94.0 6 1.6
1.1 6 1.0
0.4 6 0.7
1.8 6 0.7
96.8 6 1.2
3.0 6 3.2
7.5 6 6.2
4.3 6 2.9
1.1 6 0.6
0.0 6 0.0
0.2 6 0.4
0.4 6 0.7
0.0 6 0.0
93.9 6 3.1
0.0 6 0.0
0.3 6 0.5
0.0 6 0.0
0.0 6 0.0
98.6 6 1.0
Values represent mean percentages 6 SD of cells positive for the indicated
markers. Three different samples were evaluated for each marker. pro–SP-C,
pro–surfactant protein–C; CCSP, Clara cell–specific protein.
424AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 462012
the incubation of cells on human IgG–coated dishes (14, 20). We
compared the percentages of pro-SP-C1cells isolated by our
FACS-based method and by the density-gradient method. We
found that cells isolated via the density-gradient method con-
tained not only pro-SP-Chibut also pro-SP-Clowcells, compared
with cells isolated by the FACS-based method (Figure E1). The
percentage of pro-SP-C1cells, including both SP-Chiand SP-
Clowcells, was significantly higher in our FACS-based method
(n ¼ 3) than in the density-gradient method (n ¼ 5) (93.7% 6
1.7% versus 34.9% 6 13.5%, respectively, mean 6 SD, P ¼
0.0003). We further confirmed and characterized the phenotypes
of the EpCAMhi/T1a2subset (an ATII cell population) under
culture conditions. After the sorting procedure, the percentage of
trypan blue–negative cells in EpCAMhi/T1a2cells was 84.3% 6
9.9% (mean 6 SD, n ¼ 4). Cultured cells derived from the
EpCAMhi/T1a2subset attached on culture slides. These cells
displayed a cuboidal shape on Day 2 (Figure 2A). However,
on Day 7, the cultured cells showed a flattened and broad shape,
and grew to confluent monolayers (Figure 2A). To verify the
immunophenotypes of cultured cells, we performed immunoflu-
orescence staining using antibodies against pro-SP-C, AQP5,
and T1a. We confirmed that the cells on Day 2 expressed
pro-SP-C, but not both AQP5 and T1a, indicating that cultured
cells on Day 2 still retained the phenotypes of ATII cells. How-
ever, cultured cells on Day 7 expressed AQP5 and T1a, but lost
their expression of pro- SP-C, suggesting that the cultured cells
differentiated into ATI-like cells on collagen I under culture
conditions, without any specific growth factors (Figure 2B).
These data demonstrated that the sorted EpCAMhi/T1a2cells
had ATII cell phenotypes, with the potential to differentiate
spontaneously into ATI-like cells in vitro. In addition, the
cultured cells forming confluent monolayers on Day 7 expressed
E-cadherin and tight-junction proteins (zonula occludens–1 and
occludin) (Figure 2C). Immunostaining examinations of E-cadherin
and tight-junction proteins verified that the isolated ATII cells
formed epithelial monolayers during the culture period.
Figure 2. Cells isolated as EpCAMhi/T1a2show ATII
cell phenotypes in vitro. (A) Phase contrast images
of cultured EpCAMhi/T1a2cells. Left: Cells on Day
2. Right: Cells on Day 7. (B) Immunofluorescence
staining of pro-SP-C, AQP5, and T1a on Day 2 (left)
and Day 7 (right). (C) Immunofluorescence staining
of E-cadherin, zonula occludens–1 (ZO-1), and
occludin of cultured cells on Day 7. Scale bars: A,
100 mm; B and C, 50 mm.
Fujino, Kubo, Ota, et al.: Isolation of Human Alveolar Cellular Components 425
Localization of EpCAM-Expressing or T1a-Expressing Cells
in the Human Distal Lung
To verify the localization of EpCAM and T1a in normal adult
human lungs, we performed immunohistochemical and immu-
nofluorescence staining, using specific antibodies. As previously
described (21), alveolar and bronchiolar epithelial cells stained
positive for EpCAM (Figures E2A and E2B), whereas endo-
thelial and mesenchymal cells did not express EpCAM. We
confirmed that both ATII and ATI cells expressed EpCAM
through the costaining of EpCAM with either pro-SP-C or
AQP5 (Figures 3A and 3B).
T1a was shown to be expressed by ATI cells in rat lungs (22),
and by lymphatic endothelial cells in adult human lungs (19). We
found that T1a was expressed not only by ATI cells and lym-
phatic endothelial cells, but also by bronchiolar epithelial cells,
in human lungs (Figures E1C–E1E). In addition, co-immunos-
taining demonstrated that T1a1cells expressed AQP5 but not
pro–SP-C (Figures 3C and 3D), indicating that T1a1cells lo-
cated in the alveolar epithelium were ATI cells. To determine
whether T1a1luminal cells in the interstitium observed via im-
munohistochemistry (Figure E1E) were lymphatic endothelial
cells, we performed immunofluorescence staining, using antibod-
ies against T1a and other markers for lymphatic endothelial cells
(Prospero homeobox–1 [Prox-1] and lymphatic endothelial hya-
luronan receptor–1 [LYVE-1]). We found that T1a1luminal
cells in the lung interstitium expressed Prox-1 and LYVE-1 (Fig-
ure 4). These data demonstrate that the T1a1luminal cells in the
interstitium were pulmonary lymphatic endothelial cells. In addi-
tion, immunostaining showed that lymphatic endothelial cells
expressed T1a more strongly than did ATI cells (Figures 4A
and 4B). This observation was consistent with results obtained
from FACS analyses (Figure 1A).
Separation of Microvascular Endothelial Cells from
Mesenchymal Cells in the EpCAM2/T1a2Subset
To separate the vascular endothelial population from the mesen-
chymal population in the EpCAM2/T1a2subset, we performed
triple staining for EpCAM, T1a, and VE-cadherin. Dot plots
showed that the VE-cadherin1and VE-cadherin2subsets were
distinct subpopulations (Figure 5A). The number of cell types
isolated is shown in Table 1. In contrast, we did not observe
a distinct subpopulation in FACS scattergrams using anti-platelet/
endothelial cell adhesion molecule 1 (PECAM1), anti–vascular
endothelial growth factor receptor–2, anti-CD34, or anti-endoglin
antibodies (data not shown). To verify that the EpCAM2/T1a2/
VE-cadherin1subset and the EpCAM2/T1a2/VE-cadherin2
subset predominately contained vascular endothelial cells and
mesenchymal cells, respectively, we cultured each subset and
characterized the cell phenotypes (n ¼ 6). Cells sorted from
the EpCAM2/T1a2/VE-cadherin1subset were cultured in en-
dothelial culture medium containing several growth factors (e.g.,
vascular endothelial growth factor). The cultured VE-cadherin1
cells expressed vascular endothelial markers (PECAM1 and Tie-
2),but not mesenchymal markers (a-smooth muscle actin
[a-SMA] and CD90) (Figures 5B and E3). In contrast, the
EpCAM2/T1a2/VE-cadherin2subpopulation cultured under the
same endothelial culture conditions did not express PECAM1 or
Tie-2 (data not shown). The VE-cadherin2subset cultured in
mesenchymal culture medium containing FBS, but not specific
growth factors, displayed spindle-shaped cells expressing
Figure 3. Immunofluorescence staining of EpCAM and T1a on alveolar walls. EpCAM was expressed by pro-SP-C1cells (ATII cells; A, arrows) and
AQP51cells (ATI cells; B, arrows). Pro-SP-C1cells did not express T1a (C, arrows). AQP51cells expressed T1a (D, arrows). Scale bars, 20 mm. Data are
representative of five patients. DAPI, 4’,6-diamidino-2-phenylindole; DIC, differential interference contrast image.
426AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 462012
a-SMA and CD90, but not endothelial markers (Figures 5B and
E3). The freshly isolated VE-cadherin1subpopulation died af-
ter several days in the mesenchymal medium.
An in vitro angiogenesis assay demonstrated that cells de-
rived from the VE-cadherin1subset formed capillary-like tubes
in Matrigel (Figure 5C). On the other hand, the VE-cadherin2
subset formed corded aggregates, but not capillary-like tubes
(Figure 5C), similar to dermal fibroblasts (23), suggesting that
the VE-cadherin2cells which expanded in the endothelial me-
dium did not have the potential to become endothelial progen-
We developed a FACS-based method for the direct isolation of
individual component cell types from the alveoli of normal or
diseased lungs. Most lung diseases develop in the distal lung.
However, little cell-based knowledge is available, for example,
about intercellular signaling or epigenetic changes in each cell
type, because the disease sites are situated deep in the lungs
and are thus difficult to approach. This difficulty motivated
our development of a method to isolate individual ATII cells, mi-
crovascular endothelial cells, mesenchymal cells, and lymphatic
tissue. Our method uses a combination of antibodies against
EpCAM, T1a, and VE-cadherin (Figure 6). We also demon-
strated a practical application for this method in the primary
culture of each cell type. In addition, the approach described
here can be extended to cell-specific analyses in health and dis-
ease, which will provide insights into pathologic processes and
perhaps identify new therapeutic targets.
ATII cells play a critical role in lung homeostasis and the re-
pair process after injury (24, 25). Methodologies to separate
ATII cells from lung cell suspensions have been devised for
more than two decades (14, 20, 26–28). Notably, Demling and
colleagues described magnetic cell sorting, using anti-EpCAM
antibody–coated microbeads combined with a discontinuous
Percoll density gradient to enable ATII cells to be more con-
centrated (15). We found that EpCAM was expressed by
ATII cells more strongly than by other lung epithelial cells
(Figures 1 and 3). Taken together, the isolation strategy using
EpCAM is an efficient method for the separation of ATII cells.
However, the expression of pro-SP-C was diminished in ATII cells
isolated by the density-gradient method compared with the
FACS method (Figure E1). Mechanical stress during centrifu-
gation within a high-density solution may alter the function of
ATII cells. The isolation method using a FACS is based on the
expression level of EpCAM and T1a, and does not need a high-
density solution. Therefore, we propose that our approach can
Figure 4. Immunofluorescence staining of T1a in
lymphatic endothelial cells. (A) T1a1luminal cells
in lung interstitium expressed Prospero homeobox–
1 (Prox-1) in the nuclei (arrowheads). Prox-1 is a tran-
scription factor known to be expressed by lymphatic
endothelial cells. Notably, lymphatic endothelial cells
expressed T1a more strongly than did ATI cells
(arrows). (B) T1a1luminal cells co-expressed lym-
phatic endothelial hyaluronan receptor–1 (LYVE-1,
a surface marker for lymphatic endothelial cells) in
the perivascular region (asterisks). Alv, alveolus.
Scale bars: A, 20 mm; B, 50 mm. Data are represen-
tative of five patients.
Fujino, Kubo, Ota, et al.: Isolation of Human Alveolar Cellular Components 427
preserve ATII cell function more rigorously than previous methods.
In addition, we showed that ATII cells were isolated in a viable
condition, and had a potential to generate ATI-like cells under
culture condition (Figure 2). These results indicate that our
methods for isolating and culturing ATII cells could apply to
a variety of in vitro studies about ATII biology, such as toxicol-
ogy, drug screening, and microbiology.
Not only ATII cells, but other cell types, were isolated using
our FACS-based method. We separated microvascular endothe-
lial cells and mesenchymal cells, both of which were identified in
the EpCAM2/T1a2subset. We tried using several surface anti-
gens to delineate a microvascular endothelial subpopulation.
These surface markers included established pulmonary vascular
endothelial markers such as VE-cadherin (29), PECAM1 (30),
and CD34 (31). We found that only VE-cadherin delineated
distinct subpopulations (Figure 5A). A subsequent culture and
in vitro angiogenesis assay showed that VE-cadherin distinctly
separated microvascular endothelial cells from mesenchymal cells
(Figures 5B and 5C). However, the microvascular endothelial cells
and mesenchymal cells found in the human distal lung are
Figure 5. The EpCAM2/T1a2
subset was fractionated into
a vascular endothelial popula-
tion and a mesenchymal pop-
ulation, using an antibody
Representative dot plots show
the expression of VE-cadherin
in the EpCAM2/T1a2subset.
(B) Phase-contrast images and
(a-SMA and CD90) in cells de-
rived from VE-cadherin–positive
and VE-cadherin–negative sub-
populations. (C) An in vitro an-
basement membrane extract.
Data are representative of six
patients. Scale bars: B, 100 mm;
C, 500 mm. PECAM1, platelet/
a-SMA, a–smooth muscle actin.
428 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGYVOL 46 2012
composed of heterogeneous subpopulations (24, 32). Further
studies will be required to better characterize the cellular
phenotypes in microvascular endothelial cells and mesenchy-
Lymphatic endothelial cells have received increasing atten-
tion, because they are thought to be associated with severe lung
diseases such as idiopathic pulmonary fibrosis (33, 34), lym-
phangiomyomatosis (35), and metastatic cancer (36). In this
study, we showed that the T1ahipopulation located in the lung
interstitium consisted of lymphatic endothelial cells, based on
markers (Prox-1 and LYVE-1; Figure 4). Prox-1 was recognized
as a master regulator of lymphatic endothelial phenotypes (37–
39). LYVE-1 is a hyaluronan receptor, and is expressed on the
surface of lymphatic endothelial cells (40). Our histological data
support the concept that the EpCAM2/T1ahisubset in FACS
predominately contains lymphatic endothelial cells. However,
a previous report demonstrated that pulmonary lymphatic
endothelial cells consisted of two types of cells that ware func-
tionally different (41). Endothelial cells in the initial lymphatics
were oak leaf–shaped cells with button-like junctions, and were
important in fluid uptake and the migration of leukocytes.
Endothelial cells in collecting lymphatics, on the other hand,
were conventional and continuous cells with zipper-like junc-
tions to allow lymphatic fluid to pass through (41). We speculate
that both types of endothelial cells might be contained in the
EpCAM2/T1ahisubset. Therefore, further characterization will
be needed to separate lymphatic endothelial cells that are basi-
In this study, we could not separate ATI cells from bronchi-
olar epithelial cells. ATI cells are thin, flat cells responsible for
gas exchange (24). T1a is a specific marker for ATI cells in rat
lungs (22), but as shown here, T1a was not a specific marker for
ATI cells in human lungs (Figures 3 and E2). Thus, a better
characterization of cell surface markers for ATI cell–specific
isolation from human lungs is required.
In conclusion, we demonstrated a novel method for the direct
and individual isolation of lung component cells from distal lung
tem biology may provide the molecular bases for pathologic pro-
cesses in the development of lung disease. In addition, isolated
lung cells from patients can be applied to the recently described
triple cell co-culture model (42) by means of reconstruction
with diseased epithelial, mesenchymal, and endothelial cells.
Our new isolation method is a unique and promising tool for the
analysis of individual cells residing in distal lungs, and may shed
light on the cellular biology within this borderland.
Author disclosures are available with the text of this article at www.atsjournals.org.
Acknowledgments: The authors are grateful to Mr. Katsuhiko Ono (Department of
Pathology and Histotechnology, Tohoku University Graduate School of Medicine)
for assistance with transmission electron microscopy, and Professor Ryouichi
Nagatomi (Department of Biomedical Engineering, Tohoku University Graduate
School of Biomedical Engineering) for his advice on this work. The authors also
thank the Biomedical Research Core of Tohoku University Graduate School of
Medicine for technical support.
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