Tanabe et al. BMC Molecular and Cell Biology
https://doi.org/10.1186/s12860-024-00507-7(2024) 25:9

BMC Molecular and Cell
BiologyOpen Access

RESEARCH

Development of an in vitro human alveolar
epithelial air-liquid interface model usinga small molecule inhibitor cocktail
Ikuya Tanabe1, Kanae Ishimori1 and Shinkichi Ishikawa1*
Abstract
Background The alveolar epithelium is exposed to numerous stimuli, such as chemicals, viruses, and bacteriathat cause a variety of pulmonary diseases through inhalation. Alveolar epithelial cells (AECs) cultured in vitro are avaluable tool for studying the impacts of these stimuli and developing therapies for associated diseases. However,maintaining the proliferative capacity of AECs in vitro is challenging. In this study, we used a cocktail of three small
molecule inhibitors to cultivate AECs: Y-27632, A-83-01, and CHIR99021 (YAC). These inhibitors reportedly maintain the
proliferative capacity of several types of stem/progenitor cells.
Results Primary human AECs cultured in medium containing YAC proliferated for more than 50 days (over ninepassages) under submerged conditions. YAC-treated AECs were subsequently cultured at the air-liquid interface(ALI) to promote differentiation. YAC-treated AECs on ALI day 7 formed a monolayer of epithelial tissue with strong
expression of the surfactant protein-encoding genes SFTPA1, SFTPB, SFTPC, and SFTPD, which are markers for type II
AECs (AECIIs). Immunohistochemical analysis revealed that paraffin sections of YAC-treated AECs on ALI day 7 weremainly composed of cells expressing surfactant protein B and prosurfactant protein C.
Conclusions Our results indicate that YAC-containing medium could be useful for expansion of AECIIs, which arerecognized as local stem/progenitor cells, in the alveoli.
Keywords Alveolar epithelial cells, Air-liquid interface, Y-27632, A-83-01, CHIR99021, Surfactant proteins

Background
Cellular respiration is a vital process for energy production in all living organisms. Human cells mainly carry out
aerobic respiration, which involves the use of oxygen to
create energy through oxidation of glucose into carbon
dioxide and water. Breathing, therefore, plays a vital rolein human survival by facilitating the exchange of oxygen*Correspondence:
Shinkichi Ishikawa
shinkichi.ishikawa@jt.com
1
Scientific Product Assessment Center, R&D Group, Japan Tobacco Inc.,
6-2 Umegaoka, Aoba-ku, Yokohama, Kanagawa 227-8512, Japanand carbon dioxide gases in the pulmonary alveoli, which
are located at the distal ends of the respiratory tract.
However, inhalation of stimuli in the ambient air (e.g.,chemicals, viruses, and bacteria) can also lead to respiratory diseases. Assessment of the impacts of these stimuli
on the alveoli is important to explore and develop new
therapeutic methods to treat the associated diseases.
Studies on the alveoli are generally conducted using
animals or human cell models [1]. In recent years, in vitro
studies in human cells have become increasingly common, not only because they provide advantages suchas simplicity, speed, and inexpensiveness, but because
they reflect changing viewpoints on the ethics of animal© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use,sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) andthe source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in thisarticle are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not includedin the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you willneed to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The
Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available
in this article, unless otherwise stated in a credit line to the data.

Tanabe et al. BMC Molecular and Cell Biology(2024) 25:9

research [2]. Several in vitro models of human alveoli
have been developed that primarily focus on the alveolar epithelium, which mainly comprises alveolar epithelial cells (AECs) of types I and II (AECIs and AECIIs,
respectively) in vivo [3]. AECIs are thin, flat cells that are
responsible for gas exchange, while AECIIs are cuboidal
cells that secrete lung surfactant, which is essential fornormal lung function [4–6]. The A549 cell line, derived
from human lung adenocarcinoma, is commonly used
as an in vitro model of alveolar epithelium [7]. Like
most immortalized and tumor-derived cell lines, however, A549 cells exhibit limited characteristics of in vivo
alveolar epithelium [8, 9]. Thus, there is a need for thedevelopment of human AEC lines that are more physiologically relevant [10].
The use of primary human AECs is another option
for the development of in vitro alveolar epithelial models because they are expected to exhibit properties thatclosely resemble those of normal tissue. However, AECIs
are terminally differentiated and lack proliferative capacity. Therefore, expansion of AECIIs, the progenitors of
AECIs, is necessary for the development of an in vitro
alveolar epithelial model using primary human cells.
Several methods for culturing human AECIIs have beenreported in the literature. For example, a combinationof feeder cells and the Rho-associated protein kinase(ROCK) inhibitor Y-27632 has been reported to maintainthe proliferative capacity of human AECIIs [11]. Proliferation of human AECIIs can also be maintained without
feeder cells using spheroid cultures supplemented withvarious factors [12]. Furthermore, there are methods
for inducing AECs from human pluripotent stem cells[13–17]. However, the reported methods for maintaining
primary AEC proliferation are complex, while those forinducing AECs from pluripotent stem cells are also complex, in addition to being time-consuming and expensive.
Therefore, a simple and universal culture method for proliferating primary human AECs should be included in thedevelopment of in vitro alveolar epithelial models.
The objective of this study was to develop a human invitro alveolar epithelial model through the establishment of a simple culture method to maintain the proliferative capacity of human AECs. To achieve this, we
utilized a cocktail of three inhibitors, Y-27632, A-8301, and CHIR99021 (YAC), which have previously beenfound to be effective in maintaining the proliferation of
rat hepatocytes or their progenitors [18, 19]. We hypothesized that YAC would also be useful in maintaining theproliferation of primary human AECs, especially AECIIs,
which are recognized as local stem/progenitor cells in thealveoli [20]. Y-27632 is a ROCK inhibitor [21], A-83-01
is an inhibitor of transforming growth factor-β (TGF-β)signaling [22], and CHIR99021 is an activator of Wnt signaling via inhibition of glycogen synthase kinase-3 [23].

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YAC has been utilized in various combinations to culture human stem cells [24], and previous studies used
at least one of these inhibitors to maintain proliferationof primary human AECIIs [11, 12, 25]. We obtained primary human AECs and initially cultured them in flasks
containing medium supplemented with YAC under submerged conditions, followed by culturing at the air-liquidinterface (ALI). ALI culture mimics the in vivo environment of the alveolar epithelium and is known to promote
the differentiation of AECs [26]. The YAC-treated, ALIcultured AECs were then evaluated for the expression ofalveolar epithelium-specific phenotypes. For comparison,
we also evaluated ALI-cultured A549 cells that were prepared in accordance with previously described methods.
A549 cells are conventionally used as an in vitro model of
AECs [27, 28].

Methods
Cell culture

Human pulmonary AECs (HPAEpiCs) were purchased as
AECs from ScienCell Research Laboratories (Carlsbad,
CA, USA). AEC medium (AEpiCM, ScienCell ResearchLaboratories), which is composed of basal medium, fetal
bovine serum (FBS), penicillin/streptomycin solution,
and Epithelial Cell Growth Supplement (EpiCGS), wasused for cultivation. The final concentration of penicillin and streptomycin were 100 units/mL and 100 µg/
mL, respectively. AEpiCM was supplemented with 10µM Y-27632, 0.5 µM A-83-01, and 3 µM CHIR99021 (allfrom FUJIFILM Wako Pure Chemical, Osaka, Japan). The
HPAEpiCs used in this study were from three lots, each
derived from a different fetus 20 weeks gestation: #27000,#28696, and #33625. A549 cells (American Type Culture
Collection, Manassas, VA, USA) were cultured in Dulbecco’s modified Eagle medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% FBS(MP Biomedicals, Santa Ana, CA, USA), as described inprevious studies [27, 28]. Cells were seeded in collagentype I-coated T75 flasks (Corning, Corning, NY, USA)at 0.5–1.0 × 106 cells/flask, incubated at 37 °C in 5% CO2,
and passaged when they reached 80–90% confluency.
The population doubling level (PDL) of YAC-treated
HPAEpiCs at each passage was calculated as follows:
PDL = PDL0 + 3.322(logCf– logCi), where PDL0 is initial
PDL, Ci is initial cell number seeded into flasks, and Cf isfinal cell yield, or the number of cells at each passage.
ALI culture

YAC-treated HPAEpiCs and conventional A549 cells
at passage 3 were used for ALI culture. The cells wereseeded at 1 × 105 cells/cm2 on the apical surface of Transwell inserts (6.5-mm diameter, 0.4-µm pores; Corning)in 24-well plates. Cells were cultivated in AEpiCM containing YAC under submerged conditions for 4–9 days

Tanabe et al. BMC Molecular and Cell Biology(2024) 25:9

until they reached confluency. The culture medium wasthen removed from the apical surface to establish an ALI
culture, and the cells were further cultured for 7 daysin AEpiCM containing YAC. The medium was changed
every 2–3 days throughout the culture period.
Measurement of tissue integrity

Transepithelial electrical resistance (TEER) values wereassessed using a Millicell ERS-2 Voltohmmeter (Merck
Millipore, Burlington, MA, USA) to assess tissue integrity during ALI culture. TEER (Ω × cm2) was calculated asfollows: TEER = (resistance of tissue insert– resistance ofblank insert) × surface area of the insert.
Histological analysis

Cells on ALI day 7 were fixed with 4% paraformaldehyde(FUJIFILM Wako Pure Chemical), embedded in paraffin, sectioned using a rotary microtome RX-860 (Yamato
Kohki, Saitama, Japan), deparaffinized, and subjected tohematoxylin and eosin or immunohistochemical staining.
Deparaffinized sections were processed for immunohistochemical staining by incubation in the following seriesof solutions: (1) 10 mM citric acid buffer (LSI Medience,
Tokyo, Japan) for 15–30 min at 95 °C; (2) blocking buffer(phosphate-buffered saline [PBS, Thermo Fisher Scientific] containing 1% bovine serum albumin [Merck Millipore], and 0.1% Triton X-100 [FUJIFILM Wako Pure
Chemical]) for 30 min at room temperature (RT); (3)primary antibodies against cytokeratin 19 (CK19, diluted
1:500, ab52625; Abcam, Cambridge, UK), SOX2 (1:400,
ab97959; Abcam), SOX9 (1:1000, ab185966; Abcam),aquaporin-5 (AQP-5, 1:200, ab92320; Abcam), surfactant protein B (SP-B, 1:200, ab40876; Abcam), or prosurfactant protein-C (proSP-C, 1:200, ab90716; Abcam)
for 90 min at RT; (4) Alexa 488-conjugated secondary
antibody (1:1000, ab150077; Abcam) or Alexa 555-conjugated secondary antibody (1:500, A-31572; Thermo
Fisher Scientific) for 60 min at RT for single-labelingstudies; anti-HT1-56 antibody (1:100, TB-29AHT1-56;
Terrace Biotech, San Francisco, CA, USA) for 90 min
at RT and Alexa 488-conjugated secondary antibody(1:500, ab150105; Abcam) for 60 min at RT for double
staining studies; and (5) DAPI solution (1:1000, Dojindo,
Kumamoto, Japan) for 15 min at RT. The sections wererinsed twice with PBS between each step. Images wereacquired with an AXIO Imager or Observer (Carl Zeiss,
Oberkochen, Germany).
Immunocytochemical analysis

Cells on ALI day 7 were fixed with 4% paraformaldehyde,then incubated in the following series of solutions: blocking buffer for 60 min at RT; antibodies against E-cadherin (1:500, ab40772, Abcam), AQP-5 (1:100, ab92320,
Abcam), or proSP-C, (1:200, ab90716, Abcam) for 90 min

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at RT; Alexa 488-conjugated secondary antibody (1:500,
ab150077, Abcam) for 60 min at RT; and DAPI solution(1:1000, Dojindo) for 15 min at RT. The cells were rinsed
twice with PBS between each step. Images were acquiredby a confocal laser scanning microscope (LSM880, Carl
Zeiss).
Quantitative PCR

Total RNA was extracted from YAC-treated HPAEpiCs
and A549 cells (passage 3) cultured under submergedand ALI conditions using an RNeasy Mini Kit (Qiagen,Hilden, Germany). The cDNA was synthesized using
SuperScript IV VILO Mastermix (Thermo Fisher Scientific). Quantitative real-time PCR was performed using
a 7900HT Fast Real-Time PCR system (Thermo Fisher
Scientific) with the following TaqMan gene expression
assay kits (Thermo Fisher Scientific): Hs00387048_m1(AQP5), Hs00831305_s1 (SFTPA1), Hs00167036_m1(SFTPB), Hs00161628_m1 (SFTPC), and Hs01108490_
m1 (SFTPD). The following thermal cycling profile
was applied to all samples: 2 min at 50 °C, 20 s at 95 °C
followed by 40 cycles of 1 s at 95 °C and 20 s at 60 °C.
Relative expression levels were calculated by the ΔΔCt
method using 18 S rRNA (4319413E, Thermo Fisher Scientific) as an internal control. When cycle threshold (Ct)values were undetermined, calculations were performed
with a Ct value of 40. If the Ct values were undetermined
for an entire set of samples, expression of the gene wasconsidered to be undetectable under those conditions.
HPAEpiCs (lot #27000) cultured under submerged conditions (passage 1) were used as a calibrator (relative
expression = 1).
Statistical analysis

Data are expressed as mean ± standard error of three
independent experiments, with six inserts (ALI culture)or a single flask (submerged culture) in each experiment. Statistical analysis was performed using GraphPad
Prism 9 (GraphPad Software Inc., La Jolla, CA, USA)
employing one-way analysis of variance (ANOVA) or
two-way ANOVA with Dunnett’s multiple comparison
test. A p value < 0.05 was considered to indicate statisticalsignificance.

Results
Maintenance of AEC proliferation

HPAEpiCs were purchased from ScienCell Research Laboratories as primary human AECs and cultured undersubmerged conditions in AEpiCM supplemented with
YAC (Fig. 1A). According to the manufacturer, HPAEpiCs comprise AECIs and AECIIs and are unsuitable for
expansion or long-term culture because the cells differentiate to become AECIs immediately after plating. Normally, AECIs do not proliferate in culture. We measured

Tanabe et al. BMC Molecular and Cell Biology(2024) 25:9

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Fig. 1 Maintenance of proliferative capacity of HPAEpiCs in AEpiCM containing YAC under submerged culture conditions. (A) Schematic diagram of theexperimental procedure. Each plot represents the date of passaging, with over nine passages during the culture period. (B) Time course of PDLs of YACtreated HPAEpiC cultures. (C) Phase contrast images of YAC-treated HPAEpiCs (passage 3). Scale bar: 100 μm

the PDL for more than 50 days (over nine passages) todetermine whether the proliferative capacity of HPAEpiCs was maintained under treatment with YAC (Fig. 1A).
Indeed, HPAEpiCs continued to proliferate throughout
the entire period, regardless of the lot number (Fig. 1B).
Furthermore, there were no distinct lot-to-lot differencesin cell morphology at passage 3 (Fig. 1C).

number (Fig. 2B). These tissues comprised cells expressing the epithelial cell marker CK19 (Fig. 2B). By contrast, A549 cells, which are frequently used as an in vitro
alveolar epithelial model, formed multilayered structureswith sparse expression of CK19 when cultured at the ALI(Fig. 2B).

Tissue structure during ALI culture

Next, we assessed the integrity of epithelial tissues
composed of YAC-treated HPAEpiCs formed at the
ALI. TEER values, which reflect tight junction formation, exceeded 100 Ω × cm2 throughout the ALI culture
period, regardless of the lot number (Fig. 3A). By contrast, ALI-cultured A549 cells showed very low TEER
values. The epithelial tissues derived from two HPAEpiC
lots (#27000 and #33625) exhibited significantly higher
TEER values than those derived from ALI-cultured A549

First, we confirmed that HPAEpiCs were able to be passaged in AEpiCM containing YAC. Next, to confirm
that these passaged cells had the ability to differentiate
into alveolar epithelial tissue, we cultured passage 3 cells
under ALI conditions for 7 days in AEpiCM containing
YAC (Fig. 2A). We confirmed that YAC-treated HPAEpiCs formed monolayered tissues on the porous membrane of commercial Transwell inserts, regardless of lot

Tissue integrity during ALI cultureTanabe et al. BMC Molecular and Cell Biology(2024) 25:9

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Fig. 2 Comparative histological analysis of YAC-treated HPAEpiCs and conventional A549 cells under ALI culture conditions. (A) Passage 3 cells wereused for 7-day ALI culture. (B) Representative images of hematoxylin and eosin staining (left panels) and immunohistochemical staining with anti-CK19
antibody (right panels) on day 7 of culture are shown. Blue color indicates nuclei (DAPI), and green color indicates CK19 (Alexa 488). Scale bar: 50 μm

Fig. 3 Comparative tissue integrity of YAC-treated HPAEpiCs and conventional A549 cells under ALI culture conditions. Passage 3 cells were used for ALI
culture. (A) TEER values during ALI culture. Data expressed as mean ± standard error (n = 3, three independent experiments). Data obtained from each lotof HPAEpiCs were compared with those from A549 cells using two-way repeated measure ANOVA with Dunnett’s multiple comparison test; **p < 0.01.
(B) E-cadherin expression in ALI-cultured, YAC-treated HPAEpiCs and conventional A549 cells. Blue color indicates nuclei (DAPI), and green color indicates
E-cadherin (Alexa 488). Scale bar: 50 μm

Tanabe et al. BMC Molecular and Cell Biology(2024) 25:9

cells (Fig. 3A). The results of immunocytochemistry
revealed that the epithelial tissues derived from all three
YAC-treated HPAEpiC lots expressed E-cadherin, whichreflects the formation of adherens junctions, at day 7 of
ALI (Fig. 3B). E-cadherin expression was sparse in ALIcultured A549 cells (Fig. 3B).
Investigation of alveolar epithelial marker expression

Quantitative PCR was performed to compare the expression levels of alveolar epithelial marker genes in YACtreated HPAEpiCs and conventional A549 cells cultured
under submerged or ALI conditions. AQP5 was selected
as a marker for AECIs, while SFTPA1, SFTPB, SFTPC,
and SFTPD were selected as markers for AECIIs. Under
submerged conditions, AQP5 and SFTPB expression was
detected in YAC-treated HPAEpiCs, but not in A549 cells(Fig. 4A). SFTPA1, SFTPC, and SFTPD were expressedin both YAC-treated HPAEpiCs and conventional A549
cells under submerged conditions, but none of the genes
showed significantly higher expression in YAC-treated
HPAEpiCs than in A549 cells, regardless of lot number(Fig. 4A). The expression levels of all five genes were drastically increased in all three lots of YAC-treated HPAEpiCs under ALI conditions, and were significantly higher
in YAC-treated HPAEpiCs than in A549 cells, except for
SFTPD in HPAEpiC lot #27000 (Fig. 4A). Immunocytochemical analysis of YAC-treated HPAEpiCs cultured
under ALI conditions revealed that AQP-5 was expressedin some cells, while proSP-C was expressed in most cells(Fig. 4B). Expression of both AQP-5 and proSP-C was
weak in ALI-cultured A549 cells (Fig. 4B).
Investigation of differentiation status of ALI-cultured
HPAEpiCs

Further characterization of the alveolar epithelial tissues composed of YAC-treated HPAEpiCs formed at the
ALI was performed. The tissues were fixed on ALI day
7 and paraffin sections were prepared for immunohistochemical analysis. There were no cells with detectable
expression levels of SOX2, a marker of undifferentiatedproximal lung progenitor cells (Fig. 5), whereas SOX9, amarker of undifferentiated distal lung progenitor cells,
was expressed in a small fraction of cells, regardless of
the lot number (Fig. 5). While the expression of theseundifferentiated cell markers was limited, the expressionof SP-B and proSP-C, which are differentiation markers
for AECIIs, was detected strongly throughout the tissue,
regardless of the lot number (Fig. 5). Limited expression of HT1-56, a differentiation marker for AECIs,
was observed in a very small fraction of cells in the tissue derived from only one HPAEpiC lot (#33625; Fig. 5).
Furthermore, expression of these HT1-56 molecules did
not co-localize with that of AQP-5, another AECI marker(Fig. 5).

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Discussion
In this study, we aimed to establish an in vitro humanalveolar epithelial model for research on the pulmonary
toxicity of inhaled substances and associated diseases.
Using commercially available HPAEpiCs, we demonstrated that the use of YAC-containing medium maintained the proliferative capacity of the AECs for morethan 50 days (over nine passages) under submerged culture, allowing their expansion. Furthermore, under ALI
culture, YAC-treated HPAEpiCs formed well-organized,
monolayered epithelial tissue that exhibited higher TEER
values and higher expression of surfactant-encoding
genes compared with A549 cells. These findings suggestthat our in vitro model of ALI-cultured, YAC-treated
HPAEpiCs mimics the human alveolar epithelium moreclosely than the traditional A549 model.
The idea that the YAC cocktail could be useful for
maintaining proliferative capacity in human stem/progenitor cells was suggested by the authors of previous
studies demonstrating YAC-promoted proliferation of
rat hepatocytes or their progenitors [18, 29]. Therefore,
we anticipated that HPAEpiC cultures grown in media
supplemented with YAC might maintain the proliferative capacity of the AECIIs, which serve as local stem/
progenitor cells in the alveoli. Although our YAC-treated
HPAEpiCs proliferated for more than 50 days (overnine passages) under submerged culture, the details of
how these small molecule inhibitors individually affect
HPAEpiCs remain unclear.
Y-27632 is well known as a ROCK inhibitor [21], andis thought to reduce stress fiber formation through relaxation of the actin cytoskeleton in several cell types [30].
The possibility that this effect was involved in maintaining proliferative capacity of our AECIIs is supported byrecent studies indicating that actin remodeling and cytoskeletal strain are important for terminal differentiationof AECIIs into AECIs [31, 32]. In addition to inhibiting
ROCK-mediated terminal differentiation of AECIIs,
Y-27632 may promote the activation of signaling pathways involved in cell proliferation and survival (e.g., Akt,
ERK, and JNK) [33–35]. Y-27632 reportedly also works in
combination with fibroblasts as feeder cells to maintain
AECII proliferation [11], confirming its critical importance in our novel in vitro human alveolar epithelial
model.
The fact that Y-27632 interacts with fibroblasts to maintain AECII proliferation suggests that A-83-01 (inhibitor of TGF-β signaling) and CHIR99021 (activator of
Wnt signaling) in YAC may have substituted for the roleof fibroblasts in our experiments. Lung fibroblasts havebeen shown to promote AECII proliferation by secreting
essential soluble factors such as keratinocyte growth factor (KGF), which reportedly suppresses AECII terminal
differentiation [36, 37]. KGF has also been reported toTanabe et al. BMC Molecular and Cell Biology(2024) 25:9

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Fig. 4 Expression of AEC markers in YAC-treated HPAEpiCs and conventional A549 cells under both submerged and ALI culture conditions. Passage 3 cells
were used for analysis. (A) Comparative gene expression analysis of A549 cells and each lot of HPAEpiCs using AQP5 as a marker for AECIs, and SFTPA1,SFTPB, SFTPC, and SFTPD as markers for AECIIs. Data expressed as mean ± standard error (n = 3, three independent experiments). Data obtained from each
lot of HPAEpiCs were compared with those from A549 cells using one-way ANOVA with Dunnett’s multiple comparison test; *p < 0.05; ND, not detected.
(B) Immunocytochemical analysis of ALI-cultured, YAC-treated HPAEpiCs and conventional A549 cells. Blue color indicates nuclei (DAPI), and green color
indicates AQP-5/proSP-C (Alexa 488). Scale bar: 50 μm

stimulate AECII proliferation [38], which can be antagonized by TGF-β [39]. Based on these reports, it is possible that A-83-01 promoted AECII proliferation in thisstudy by blocking the antagonistic TGF-β signal. In addition to KGF, lung fibroblasts also reportedly secrete Wnt

protein [40], and Wnt released from fibroblasts has beenshown to act on neighboring AECIIs to control their proliferation and differentiation [41]. Therefore, CHIR99021
may have substituted for fibroblasts by supplying Wnt to
the AECII niche.

Tanabe et al. BMC Molecular and Cell Biology(2024) 25:9

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Fig. 5 Fluorescent immunohistochemical analysis of paraffin sections obtained from YAC-treated HPAEpiCs on ALI day 7 using antibodies against markers of undifferentiated and differentiated lung epithelial cells. SOX2 and SOX9 are markers of undifferentiated progenitor cells of the proximal and distallung bud, respectively. SOX9-positive cells are indicated by arrows. SP-B and proSP-C are markers of differentiated AECIIs. HT1-56 and AQP-5 are expressedin differentiated AECIs. HT1-56-positive cells are indicated by arrows, while AQP-5-positive cells are indicated by arrowheads. Blue color indicates nuclei(DAPI). Scale bar: 50 μm

Another important finding of this study was that the
YAC-treated HPAEpiCs formed well-differentiated epithelial tissue at the ALI. Under submerged culture conditions, YAC-treated HPAEpiCs, but not A549 cells,
expressed AQP5 and SFTPB. The expression levels ofother genes (SFTPA, SFTPC, and SFTPD) were notclearly different between YAC-treated HPAEpiCs andconventional A549 cells under submerged conditions. By
contrast, at the ALI, YAC-treated HPAEpiCs expressedhigher levels of all five marker genes compared with
A549 cells.
In addition to gene expression, we also compared
protein expression between YAC-treated HPAEpiCs
and A549 cells at the ALI using immunocytochemical
analysis. YAC-treated HPAEpiCs expressed AQP-5 and
proSP-C more strongly than A549 cells. These markers
have been previously well-characterized: AQP-5 in fully
differentiated AECIs and proSP-C in fully differentiated
AECIIs [42, 43].
Additional immunofluorescence analysis, using antibodies against both undifferentiated and differentiatedlung cells, was performed to further characterize thedifferentiation status of YAC-treated HPAEpiCs at the
ALI. SOX2 and SOX9 are typical markers expressed in
the proximal and distal part of undifferentiated lungbud progenitor cells, respectively [44]. We found that
SOX2 was not expressed in YAC-treated HPAEpiCs at
the ALI, consistent with the fact that alveolar epithelial tissue is formed from distal lung buds. While SOX9

was expressed in a small fraction of cells, typical AECII
markers such as SP-B and proSP-C were also strongly
expressed in most of the cells. This is consistent with aprevious study showing that ALI culture promotes differentiation of AECs derived from induced pluripotent
stem cells, decreases expression of SOX9, and increases
expression of SFTP genes [45]. Expression of the AECI
marker HT1-56 was observed in a very small fractionof cells, indicating that epithelial tissue derived from
HPAEpiCs mainly comprised SP-B/proSP-C-positive
AECIIs. However, this HT1-56 expression was not colocalized with that of AQP-5, another marker for AECIs.
One possible explanation for this is the existence of several different types of AECI-like cells that express various
combinations of markers, which would include intermediate cells between AECIs and AECIIs [46]. We analyzed
the expression of these markers using paraffin sections
obtained from YAC-treated HPAEpiCs on ALI day 7.
However, it is crucial to note that the expression patternsof these markers may undergo dynamic changes during
the ALI culture. To better understand the types of cells
that comprise YAC-treated HPAEpiCs and their dynamicchanges during the ALI culture, we plan to perform a single-cell analysis in the future.
The observation that YAC-treated HPAEpiCs mainlycomprise AECII-like cells is consistent with our hypothesis that YAC maintains the proliferation capacity of
AECs, especially AECIIs. Considering that ∼ 96% of thesurface area of human alveoli is covered by AECIs [47],Tanabe et al. BMC Molecular and Cell Biology(2024) 25:9

our in vitro model may not fully replicate the physiological contributions of AECIs and AECIIs in vivo. Therefore, it will be worth investigating whether YAC-treated
HPAEpiCs, which mainly comprise AECII-like cells,
can differentiate into AECI-like cells. One approach forpromoting the differentiation of YAC-treated HPAEpiCs into AECI-like cells is to add the Wnt signal inhibitor XAV939 to the ALI culture medium. This approachreportedly promoted the differentiation of AECIIs into
AECIs [13, 25]. We anticipate that optimizing the ALI
culture medium of YAC-treated HPAEpiCs will lead toratios of AECI and AECII that more closely resemble
those in vivo.
Although we successfully developed a well-differentiated alveolar epithelial model derived from human primary AECs, there are several limitations to this study.
First, while we demonstrated proliferation of HPAEpiCs for more than 50 days (over nine passages), we only
confirmed expression of AEC markers in YAC-treated
HPAEpiCs at passage 3. Therefore, it is necessary to
determine the number of passages for which the differentiation potential of YAC-treated HPAEpiCs can be maintained. Additionally, the duration of ALI culture should
also be investigated further. YAC-treated HPAEpiCs were
subjected to 7 days of ALI culture in this study. It would
be interesting to examine the effect of longer durations of
ALI culture on the differentiation status of the cells.
Second, further research is needed to determine thecontribution and detailed mechanism of each YAC component in maintaining the proliferation of AECIIs in
HPAEpiC. For this purpose, it will be necessary to test
the effects of all combinations of the three YAC inhibitors
to facilitate understanding of the detailed mechanisms ofaction and relative impacts of each. Third, we tested theeffect of YAC on AECs using HPAEpiCs purchased from
ScienCell Research Laboratories, which derived fromthe fetal lung. It is important to test if the effect of YAC
observed in this study is also reproducible with AECs
derived from the adult lung because some fetal and adultcells are known to have different characteristics.

Conclusions
This study demonstrated that the addition of a cocktailof three small molecule inhibitors, YAC, could be used
to maintain proliferative capacity of HPAEpiCs. Thisapproach is simpler than previously reported methods
using feeder cells and spheroid culture. The expanded
HPAEpiCs were subjected to ALI culture to promote
differentiation. YAC-treated HPAEpiCs formed a monolayered epithelial tissue, predominantly comprising thecells expressing markers for AECIIs. AECIIs are known
to secrete lung surfactant to prevent alveolar collapse,
and abnormalities in these cells are associated with various respiratory diseases. Therefore, this model will be

Page 9 of 10

useful for testing the inhalation toxicity of various substances and developing therapeutic methods for associated diseases.
Abbreviations
AECs	alveolar epithelial cells
AECIs	alveolar epithelial cells of types I
AECIIs	alveolar epithelial cells of types II
AEpiCM	alveolar epithelial cell medium
ALI	air-liquid interface
ANOVA	analysis of variance
AQP-5	aquaporin-5
Ct	cycle threshold
CK19	cytokeratin 19
EpiCGS	Epithelial Cell Growth Supplement
FBS	fetal bovine serum
HPAEpiCs	human pulmonary alveolar epithelial cells
PDL	population doubling level
proSP-C	prosurfactant protein-C
ROCK	Rho-associated protein kinaseRT	room temperature
TEER	transepithelial electrical resistance
TGF-β	transforming growth factor-β
YAC	Y-27632,A-83-01,and CHIR99021
Acknowledgements
The authors are grateful to Michelle Kahmeyer-Gabbe, PhD from Edanz(https://en-author-services.edanzgroup.com/) for editing a draft of thismanuscript.
Author contributions
S.I. conceived and designed the study. I.T. and K.I. acquired the data. I.T. and
S.I. analyzed and interpreted the data. I.T. drafted the manuscript. All authors
reviewed and revised the draft manuscript. All authors approved the final
version of the manuscript for publication.
Funding
This research was funded by Japan Tobacco Inc. and all authors are employees
of Japan Tobacco Inc.
Data availability and materials
The datasets generated and/or analyzed during the current study are available
from the corresponding author on reasonable request.

Declarations
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Received: 20 October 2023 / Accepted: 11 March 2024

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