(2021) 12:471
Takahashi et al. Stem Cell Res Ther
https://doi.org/10.1186/s13287-021-02549-6

Open Access

RESEARCH

Antifibrotic effect of lung‑resident
progenitor cells with high aldehyde
dehydrogenase activity
Hiroshi Takahashi1, Taku Nakashima1* , Takeshi Masuda1, Masashi Namba1, Shinjiro Sakamoto1,
Kakuhiro Yamaguchi1, Yasushi Horimasu1, Shintaro Miyamoto1, Hiroshi Iwamoto1, Kazunori Fujitaka1,
Hironobu Hamada2 and Noboru Hattori1

Abstract
Background: Aldehyde dehydrogenase (ALDH) is highly expressed in stem/progenitor cells in various tissues, andcell populations with high ALDH activity ­(ALDHbr) are associated with tissue repair. However, little is known about
lung-resident ­ALDHbr. This study was performed to clarify the characteristics of lung-resident A­ LDHbr cells and toevaluate their possible use as a tool for cell therapy using a mouse model of bleomycin-induced pulmonary fibrosis.
Methods: The characteristics of lung-resident/nonhematopoietic ­(CD45−) ­ALDHbr cells were assessed in control
C57BL/6 mice. The kinetics and the potential usage of ­CD45−/ALDHbr for cell therapy were investigated in bleomycininduced pulmonary fibrosis. Localization of transferred ­CD45−/ALDHbr cells was determined using mCherry-expressing mice as donors. The effects of aging on ALDH expression were also assessed using aged mice.
Results: Lung ­CD45−/ALDHbr showed higher proliferative and colony-forming potential than cell populations with
low ALDH activity. The ­CD45−/ALDHbr cell population, and especially its ­CD45−/ALDHbr/PDGFRα+ subpopulation,
was significantly reduced in the lung during bleomycin-induced pulmonary fibrosis. Furthermore, mRNA expressionof ALDH isoforms was significantly reduced in the fibrotic lung. When transferred in vivo into bleomycin-pretreatedmice, ­CD45−/ALDHbr cells reached the site of injury, ameliorated pulmonary fibrosis, recovered the reduced expression of ALDH mRNA, and prolonged survival, which was associated with the upregulation of the retinol-metabolizing
pathway and the suppression of profibrotic cytokines. The reduction in ­CD45−/ALDHbr/PDGFRα+ population was
more remarkable in aged mice than in young mice.
Conclusions: Our results strongly suggest that the lung expression of ALDH and lung-resident C­ D45−/ALDHbr cells−brare involved in pulmonary fibrosis. The current study signified the possibility that C­ D45 /ALDH cells could find application as novel and useful cell therapy tools in pulmonary fibrosis treatment.
Keywords: Aldehyde dehydrogenase, Bleomycin, Cell therapy, Profibrotic cytokines, Pulmonary fibrosis, Stem cells*Correspondence: tnaka@hiroshima-u.ac.jp
1
Department of Molecular and Internal Medicine, Graduate Schoolof Biomedical and Health Sciences, Hiroshima University, 1‑2‑3 Kasumi,
Minami‑ku, Hiroshima 734‑8551, Japan
Full list of author information is available at the end of the article

Background
Tissue-resident stem cells are valuable in cell therapy andhave been successfully used for immunomodulation, tissue regeneration, and tissue repair. Several trials using
stem cell therapy have been performed to treat refractorydiseases, with mesenchymal stem cells (MSCs) being themost frequently used cell type [1]. In particular, MSCs,© The Author(s) 2021. 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 theoriginal author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images orother third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of thislicence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​
mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Takahashi et al. Stem Cell Res Ther

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shown to exhibit pluripotency toward the nonhematopoietic cell lineage, can be isolated from various organs,including the bone marrow, adipose tissue, skeletal muscle, and the umbilical cord [1]. Bone marrow-derived
MSCs, isolated from the most orthodox cell source of
MSCs [2, 3], have been shown to have immunomodulatory effects such as the inhibition of the proliferation ofT-cells through secretion of anti-inflammatory cytokinesand growth factors [4]. In a mouse model of bleomycin(BLM)-induced lung injury, administration of bone marrow-derived MSCs was reported to improve lung injury
by exerting an anti-inflammatory effect [5]. With respect
to lung resident stem cells, the ­Sca1+/CD45−/CD31− cell
population has been identified as lung tissue stem cells
capable of differentiating into endothelial and lung epithelial cells in vitro. Moreover, when transferred into anelastase-induced lung injury mouse model, this population was demonstrated to significantly improve the survival rate and reverse lung damage [6]. Lung Hoechst­33342dim side population (SP) cells are adult stem cells,
which have also been identified to exhibit mesenchymal and epithelial potential [7]. Among the SP cells, the­CD45−/CD31− fraction has been reported to have thecharacteristics of lung resident MSCs, due to their ability
to differentiate into smooth muscle, bone, fat, and cartilage [8, 9]. Furthermore, the number of lung resident SP
cells was shown to be significantly reduced in mice with
BLM-induced lung injury, and this reduction was correlated with the pathology of the lung injury. When administered intravenously into the lung, lung SP cell therapy
was shown to reduce BLM-induced pulmonary fibrosis
and pulmonary arterial hypertension [10]. These results
suggest the existence of tissue-specific MSCs in the lungand their involvement in lung injury.
Aldehyde dehydrogenases (ALDH) are a group of
enzymes that catalyze the oxidation of aldehydes to carboxylic acids, with 19 different isoforms in humans [11].
A cell population with high ALDH activity, called ALDH
bright cells (­ALDHbr), is associated with the stemness ofvarious normal tissues and is involved in tissue repair[12]. Moreover, ­ALDHbr isolated from the human bonemarrow, reported to have a higher colony-forming capacity when compared to a cell population with low ALDH
activity ­(ALDHdim) [13], was shown to be a progenitor
population for epithelial, endothelial, and mesenchymal
lineages [14]. When administered in a mouse model of
myocardial infarction, ­ALDHbr collected from the humanumbilical cord blood was demonstrated to enhance
angiogenesis in the ischemic heart [15]. Given thesefindings, the existence of lung resident A­ LDHbr and its
contribution to tissue repair were speculated; however,
little is known about lung resident ­ALDHbr. The objectives of this study were to clarify the characteristics ofPage 2 of 14

lung-resident ­ALDHbr and to evaluate its possible use asa tool for cell therapy in a mouse model of BLM-induced
pulmonary fibrosis.

Methods
Animals and BLM‑induced pulmonary fibrosis

This study, aimed at elucidating the characteristics oflung-resident ­ALDHbr and exploring its usage in celltherapy, was performed in accordance with the protocols approved by the Animal Ethics Committee of Hiroshima University (A19-122 and 28-29-2). In this study,
pulmonary fibrosis was induced as previously described[16] in C57BL/6J mice (6–8-week-old young female miceand 52 week old aged female mice) which were purchased from Charles River Laboratories Japan (Yokohama, Japan). The mice were maintained in a specificpathogen-free environment and randomly assigned to
BLM or control groups. In experiments performed toconfirm the localization of transferred cells, C57BL/6Gt (ROSA)26Sor < tm1.1 (H2B-mcherry) Osb > heterozygotic mice (mCherry mouse, BRC No. RBRC06036,
RIKEN, Tokyo, Japan) [17] systemically expressing themCherry protein in their nuclei were used as a donor
population. On day 0, after intraperitoneal injection of
mixed anesthesia with medetomidine hydrochloride(0.3 mg/kg body weight; Kyoritsu Seiyaku, Tokyo, Japan),midazolam (4 mg/kg body weight, Sandoz K.K., Tokyo,
Japan), and butorphanol tartrate (5 mg/kg body weight,
Meiji Seika Pharma, Tokyo, Japan), pulmonary fibrosis
was induced by endotracheal injection of BLM (2 mg/kg
of body weight, Nippon Kayaku, Tokyo, Japan). Control
mice received the same amount (2 mL/kg body weight) of
phosphate-buffered saline (PBS, Nacalai Tesque, Kyoto,
Japan) alone. For survival analysis, a higher dose of BLM(5 mg/kg) was used. At 7 and 14 days after BLM administration, both lungs were removed from each animal
and the lung tissue was assessed for hydroxyproline, andmRNA expression and subjected to flow cytometry andhistological analysis.
Cell isolation

The lungs were removed and minced in 1-mL Roswell
Park Memorial Institute 1640 medium (Thermo Fisher
Scientific, Waltham, MA, USA) supplemented with collagenase A (1 mg/mL, Roche, Basel, Switzerland), andincubated at 37 °C for 30 min. Following lysis of red
blood cells with ACK Lysing Buffer (Life Technologies,
Grand Island, NY, USA), the cells were resuspendedin 2 mL of PBS containing 0.5% bovine serum albumin(Sigma-Aldrich, St. Louis, MO, USA) and 2 mM ethylenediaminetetraacetic acid (Sigma-Aldrich), and cell counting was performed.

Takahashi et al. Stem Cell Res Ther

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Antibodies and ALDH staining

Antibodies (all purchased from BioLegend, San Diego,
CA, USA) used for flow cytometry and cell sorting areshown in Additional file 1. After staining for cell surface
proteins using the aforementioned antibodies, ALDH
activity was expressed as fluorescent intensity using the
ALDEFLUOR™ Kit (STEMCELL Technologies Inc.,
Vancouver, Canada) according to the manufacturer’s
protocol, as previously reported [18]. A separate tube
containing 5 µL of diethylaminobenzaldehyde (DEAB,
provided in the ALDEFLUOR™ Kit), a specific inhibitorof ALDH, was prepared to determine ­ALDHbr gating.

Page 3 of 14(STEMCELL Technologies Inc.). Consecutively, 2 to
3 weeks after the start of culture, the number of proliferated colonies was counted.
Cell viability assay

Cells were seeded into 96-well plates at a density of
5.0 × ­103 cells/well and the medium was changed every
3–4 days. After 3–4 weeks from the start of the culture,cell proliferation was evaluated using Cell Counting Kit-8(Dojindo, Kumamoto, Japan).
Cell transfer to recipient mouse

Hoechst 33,342 staining of lung cells was performed
as previously reported [19]. After suspended in 1 mL
DMEM (Thermo Fisher Scientific) with 5% FBS (SigmaAldrich), 1.0 × ­106 cells were stained with 4 µL Hoechst 33,342 (Invitrogen, Carlsbad, CA, USA) alone or in
combination with 30 µL verapamil (Sigma-Aldrich) for
90 min at 37 °C with mixing every 20 min during staining. Antibody and ALDH staining were performed as
described above after Hoechst 33,342 staining.

Sorted 1.0 × ­105 ­CD45−/ALDHbr and ­CD45−/ALDHdim
cells were dissolved in 100 µL PBS and administeredintravenously via the tail vein to recipient BLM-pretreated mice on day 2 (2 days after treatment with BLM).
To confirm the localization of transferred cells, 5.0 × ­104
­mCherry+ ­CD45−/ALDHbr and ­CD45−/ALDHdim cells
were administered intravenously into recipient BLM-pretreated C57BL/6 mice on day 2. On the following day and5 days after the injection (on days 3 and 7), the recipient
mice were sacrificed, and lung samples were subjected toflow cytometry and histology analyses.

Flow cytometry and cell sorting

Hydroxyproline assay

Hoechst staining

Flow cytometric analysis of lung cells was performed
using the following method, referring to a previous report[20]. Flow cytometry and cell sorting were performed
using the FACS Aria II system (BD Biosciences, San Jose,
CA, USA) and LSRFortessa X-20 (BD Biosciences). Data
were analyzed using the FACS Diva (BD Biosciences) andthe FlowJo (version 10.7.1, BD Biosciences) software. For
the isolation of ­ALDHbr, unnecessary cell populations
were pre-depleted using magnetic cell sorting (MACS)
cell separation using a Stem Cell Pre-Enrichment kit(Miltenyi Biotec, Bergisch Gladbach, Germany) prior to
FACS according to the manufacturer’s protocol. Cell sorting from mCherry-expressing donor mice and analysis of
injected donor ­mCherry+ cells was performed using the
SORP Aria (BD Biosciences) and LSRFortessa X-20 (BDBiosciences) systems, respectively.
Cell culture and colony‑forming assay

Sorted cells were seeded into 96-well plates at a densityof 5–10 × ­103 cells/well and cultured in Dulbecco’s modified Eagle medium (DMEM, Thermo Fisher Scientific)
and 10% fetal bovine serum (FBS, Sigma-Aldrich) supplemented with or without 20 ng/mL epidermal growth
factor (EGF, BioLegend) or 20 ng/mL fibroblast growth
factor-2 (FGF2, BioLegend) or both. The medium waschanged every 3–4 days. For colony formation, 5.0 × ­103
cells were seeded into 6-well plates using MethoCult

The left lungs were removed and the sample was homogenized in 1 mL of PBS and hydrolyzed with 1 mL of HCl
for 16 h at 120 °C. The supernatant was centrifuged at
10,000g for 5 min (Model 3740, KUBOTA, Tokyo, Japan),and 5 µL of the supernatant was aliquoted into a 96-wellplate. After dispensing 5 µL hydroxyproline standard(Sigma-Aldrich) into each well of the 96-well plate, 5µL citrate/acetate buffer (deionized distilled water supplemented with 238 mM Citric acid, Sigma-Aldrich,
1.2% glacial acetic acid, Sigma-Aldrich, 532 mM sodium
acetate, Sigma-Aldrich, and 850 mM sodium hydroxide, Nacalai Tesque) and 100 µL chloramine T solution(1.0 mL deionized distilled water supplemented with
0.141 g chloramine T, Sigma-Aldrich, 1.0 mL 1-propanol,
Sigma-Aldrich, and 8.0 mL citrate/acetate buffer) wereadded. After 30 min of incubation at 25 °C, 100 µL of
Ehrlich’s reagent (2.5 g 4-dimethylaminobenzaldehyde,
Sigma-Aldrich, 9.3 mL 1-propanol, and 3.9 mL 70% perchloric acid, Sigma-Aldrich) was added and the mixture
was incubated at 65 °C for 30 min. After 5 min at 25 °C,
the absorbance was measured at 550 nm using a plate
reader (iMARK, Bio-Rad, Hercules, CA, USA), as previously described [21].
PCR and agarose gel electrophoresis

The sorted cells and the excised lungs were homogenizedusing 1 mL TRIzol reagent (Life Technologies) and total

Takahashi et al. Stem Cell Res Ther

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RNA was extracted using the RNeasy Mini Kit (QIAGEN,
Venlo, Netherlands). The extracted RNA was reverse transcribed into cDNA using the High Capacity RNA-to-cDNA
Kit (Applied Biosystems, Foster City, CA, USA). Real-timequantitative PCR was performed using the Applied Biosystems 7500 Fast Real-Time PCR System (Applied Biosystems) and the TaqMan Gene Expression Assays (Applied
Biosystems) as previously described [16]. The expression of
Actb (β-actin, Mm02619580_g1; Applied Biosystems) wasused as an endogenous control. The TaqMan Gene Expression Assays were used as shown in Additional file 2. To
distinguish the mCherry-heterozygotic mice the from wildtype mice, mouse-tail DNA was extracted using the DNeasy
Blood and Tissue Kit (QIAGEN). The extracted DNA was
subjected to PCR using the primers shown in Additional
file 2. PCR conditions were as follows: 120 s at 94 °C, 10 s
at 98 °C, 30 s at 60 °C, 120 s at 68 °C, repeated for 30 cycles.
Amplified products were stained with SAFELOOK™ (Fujifilm Wako Junyaku, Osaka, Japan), and bands were confirmed using electrophoresis on a 1% agarose gel.
Histological analysis

Lung tissue sections were fixed in 2% formalin solution(Nacalai Tesque, Kyoto, Japan) and embedded in paraffin, followed by hematoxylin–eosin (HE) and Masson’s
trichrome staining. Immunostaining for ALDH1A1 andmCherry was performed using an anti-ALDH1A1 rabbit
polyclonal antibody (dilution factor 1:500; GTX123973,
GeneTex, Irvine, CA, USA) and anti-mCherry rabbit
polyclonal antibody (dilution factor 1:400; ab167453,
Abcam, Cambridge, UK), respectively, as the primary
antibodies and a peroxidase-conjugated anti-rabbit goat
IgG polyclonal antibody (ready to use; #424144, Nichirei,
Tokyo, Japan) as the secondary antibody.
Statistical analyses

All experiments were performed 2 or 3 times and therepresentative data are shown as median ± interquartile range except for mRNA data, which is shown asmean ± SEM to ensure the visibility of the graph. The
Kruskal–Wallis test for median values was used to assess
the statistical significance between groups. Correlation
coefficients for parameters were calculated using theSpearman’s rank correlation coefficient analysis. Kaplan–
Meier analysis and log-rank test were used for survival
analysis. A P value < 0.05 was considered significant. All
statistical analyses were performed using JMP Pro 14(SAS Institute Inc., Cary, NC, USA).

Results

Detection of ALDHbr in mouse lung

Following the determination of the appropriate ­ALDHbr
gating using ALDEFLUOR staining with the DEAB

Page 4 of 14

ALDH inhibitor, we observed a rare ­ALDHbr populationin the whole lung of mice (Fig. 1A). When we divided
the whole lung cells into ­CD45+ hematopoietic cells−and ­CD45 nonhematopoietic cells (Fig. 1B), we notedthat both fractions contained ­ALDHbr (Fig. 1C, D). To
assess lung resident ­ALDHbr, we focused on the nonhematopoietic ­CD45−/ALDHbr fraction. Analysis of thesenonhematopoietic cells, that is, the lung resident ­CD45−/
ALDHbr fraction, revealed that this fraction was further divided into mesenchymal (platelet-derived growth
factor receptor α positive, PDGFRα+) and epithelial(epithelial cell adhesion molecule positive, ­EpCAM+)
phenotypes (Fig. 1E).
Characteristics of lung CD45−/ALDHbrTo determine the characteristics of lung C­ D45−/ALDbr−br
H , we collected C­ D45 /ALDH (n = 3) and C­ D45−/dim
ALDH(n = 3) cells using FACS. As shown in Additional file 3, pre-depletion of unnecessary cell populations prior to FACS resulted in the enrichment of the­CD45−/ALDHbr fraction. To confirm if sorted ­CD45−/
ALDHbr cells truly expressed high levels of ALDH mRNAand to determine the isoforms of ALDH that were mainly
expressed in C
­ D45−/ALDHbr cells, we performed realtime quantitative PCR. Our results showed that the levels of mRNA expression of ALDH1a, ALDH2, ALDH3a1,
ALDH4a1, ALDH7a1, and ALDH18a were significantly
higher in the ­CD45−/ALDHbr than in the ­CD45−/ALDHdim cells (Fig. 2A, P = 0.049 for ALDH1a1, P = 0.049
for ALDH1a2, P = 0.049 for ALDH1a3, P = 0.037 for
ALDH1a7, P = 0.049 for ALDH2, P = 0.046 for ALDH3a1,
P = 0.049 for ALDH4a1, P = 0.037 for ALDH7a1, and
P = 0.049 for ALDH18a). We further observed that whenboth cell populations were cultured, the ­CD45−/ALDHbr population showed higher proliferative ability thanthe ­CD45−/ALDHdim population (Fig. 2B, C, P = 0.009
between ­CD45− and C­D45−/ALDHbr and P = 0.009−
between ­CD45 /ALDHdim and ­CD45−/ALDHbr). To−
brexamine whether ­CD45 /ALDH cells maintained ahigh ALDH activity in culture, sorted ­CD45−/ALDHbr
cells were further cultured, harvested, and reexamined
for ALDH activity. As shown in Fig. 3A, most proliferated
cells were A­ LDHdim, with A­ LDHbr cells accounting for
approximately 5% of the total proliferative cells.
Next, we examined the colony-forming ability of­CD45−/ALDHbr cells using a colony-forming assay. We
found that ­CD45−/ALDHbr cells formed larger (Fig. 2D)
and higher number (Fig. 2E) of colonies than the ­CD45−/
ALDHdim cells. Although a similar pattern of colony formation was observed for C­ D45−, the size and the number of colonies were relatively small, suggesting thatthe colony-forming ability of the C­ D45− population
depended to a large extent on the ­CD45−/ALDHbr cells.

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Page 5 of 14

Whole lung cells
ALDH

ALDH + DEAB

ALDH

SSC

(C)

ALDH

SSC

(B)

Lung CD45+ cells
ALDH + DEAB

ALDH

SSC

CD45

(E)

PDGFRαALDH(D)Lung CD45- / ALDHbr

EpCAM

ALDH + DEAB

SSC

(A)

Lung CD45- cells

ALDH

Fig. 1 Detection of cell population with high ALDH activity in the mouse lung. Lung cells obtained from 6-to-8-week-old wild-type C57BL/6 mice
were analyzed using flow cytometry. Data were analyzed using the FACS Diva (BD Biosciences) and FlowJo (version 10.7.1, BD Biosciences) software.
A ­ALDHbr gating determined by comparing samples stained with ALDEFLUOR alone and with ALDEFLUOR and diethylaminobenzaldehyde (DEAB),
a specific inhibitor of ALDH. B–D ­ALDHbr cells observed in the whole lung, in the nonhematopoietic/lung-resident C­ D45− fraction, and in thehematopoietic ­CD45+ fraction. E ­CD45−/ALDHbr cells divided further into ­EpCAM+ and PDGFRα+ fractions

As the ­CD45−/ALDHbr population seemed to be a heterogeneous cell population and ­ALDHbr is associated withstemness in various tissues, we evaluated the expression of surface antigens associated with the mesenchymal cells, fibroblasts, and the stem cells in the ­CD45−/
ALDHbr population. As shown in Additional file 4, notall ­CD45−/ALDHbr cells expressed the representative
markers of bone marrow-derived MSCs (CD44, CD73,
CD90, and CD105). It was notable that the stage-specificembryonic antigen-4 (SSEA4) stem cell marker was solely
expressed in C
­ D45−/ALDHbr cells in the mouse lung.
When we divided the ­CD45−/ALDHbr population−brinto ­CD45 /ALDH /PDGFRα+ and C­D45−/ALDHbr/+
EpCAM population, and investigated their characteristics, we observed that the ­CD45−/ALDHbr/PDGFRα+population exhibited a fibroblast-like spindle shape,whereas the ­CD45−/ALDHbr/EpCAM+ population
exhibited a flat and round shape (Fig. 2F). We also foundthat growth factors led to an increase in the number of­CD45−/ALDHbr/PDGFRα+ cells but not that of ­CD45−/
ALDHbr/EpCAM+ cells, suggesting that C­ D45−/ALDHbr/+PDGFRα fraction contributed to the high proliferative
potential of the C­ D45−/ALDHbr population (Fig. 2F).
Next, we examined the expression of ALDH in primary cultured lung fibroblasts (Fig. 3A). These primarycultured lung fibroblasts obtained from BLM-untreatedwild-type C57BL/6 mice were shown to frequently
express PDGFRα, but not CD45 or EpCAM, suggestingthat these cells were truly fibroblasts (Fig. 3B). As shownin Fig. 3C, both fluorescent microscopy and flow cytometry revealed that the percentage of A
­ LDHbr cells in primary cultured lung fibroblasts was approximately 5%.
Similarly, we noted that the percentage of ­ALDHbr cells
in fibroblast cell lines was also less than 5% (Fig. 3D).
Kinetics of CD45−/ALDHbr in BLM‑induced pulmonary
fibrosis

To investigate the kinetics of ­CD45−/ALDHbr in fibrotic
lungs, we used endotracheal administration of BLM(2 mg/kg body weight) to generate a mouse model of pulmonary fibrosis. We found that the levels of hydroxyproline were significantly elevated in the BLM group (n = 9)
14 days after BLM administration (Fig. 4A, P = 0.005)
compared with the PBS group (n = 4). On days 7 and 14,
the percentage of total ­ALDHbr cells in the lung was significantly elevated compared with that on day 0 (Fig. 4B),
whereas the percentage of C
­ D45−/ALDHbr cells and−br+­CD45 /ALDH /PDGFRα cells, but not ­CD45−/ALDHbr/EpCAM+ cells, were significantly decreased (Fig. 4B).
Among the ­ALDHbr populations in the lung obtained on

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Page 6 of 14

✱

✱

✱

✱
✱

✱
N.D.

15

Number of colonies

0.4

C
D
45 - CD/ A 45 CL
D
45 - DH d
im
/A
LD
H br

H

0.6

0.0

D

(E)

0.8

0.2

N.D. N.D.

1a
1
1
AL a2
D
H
AL 1a3
DH
AL 1a7
D
H
1
AL b1
D
H
1l
AL 1
D
AL H2
D
H
AL 3a1DH
AL 4a1DH
AL 7a1
D
H
AL 8a
1
D
H
18
a1

N.D.

D

✱✱

1.0

✱

Absorbance

✱

ns

1.2

AL

AL

(D)

CD45- / ALDHdim
CD45- / ALDHbr

✱

0.21
0.15
0.09
0.03
0.021
0.015
0.009
0.003
0.003
0.002
0.001
0.000

✱✱

(C)

(B)

H

Relative mRNA expressions(/β-actin)(A)

Medium alone
EGF
FGF
Both

10

5

LD
H br
C
D
45 /A

C

D

45 /A

C

LD

D

H di

m

45 -

0

(F)

Fig. 2 A Real-time quantitative PCR analysis of the mRNA expression levels of ALDH isoforms in sorted C­ D45−/ALDHbr (n = 3) and ­CD45−/ALDHdim
cells (cell population with low ALDH activity, n = 3). Values are expressed relative to the expression of the endogenous control β-actin mRNA. Data
are shown as mean ± SEM. *P < 0.05, N.D., not detectable. B Representative image of cultured cells (left, low-power field, scale bar, 400 µm, right,
high-power field, scale bar, 100 µm). C The proliferative capacity of cells using a CCK-8 kit (n = 5/group), as shown in (B). **P < 0.01. D Representative
image of colonies derived from the colony-forming assay using MethoCult supplemented with or without growth factors. Scale bar, 400 µm. E
The number of colonies derived from the colony-forming assay in (D). F Representative image of sorted cell populations cultured with or without
growth factors. The bright field and cells stained with crystal violet are shown. Scale bar, 70 µm. G Sorted C­ D45−/ALDHbr/PDGFRα+ cells induced for
differentiation into adipocytes and stained with Oil red O. Scale bar, 70 µm

day 14, the percentage of C
­ D45−/ALDHbr/PDGFRα+−cells, but not that of ­CD45 /ALDHbr cells or C
­ D45−/
br+
ALDH /EpCAM cells, inversely correlated with thelevels of hydroxyproline (Fig. 4C).
Real-time quantitative PCR analysis revealed that themRNA expression of ALDH1a1, ALDH1a7, ALDH1l1,
ALDH2, ALDH3a1, ALDH4a1, and ALDH7a1 wassignificantly lower in the fibrotic lung obtained on
day 14 (Fig. 4D, P = 0.006 for ALDH1a1, P = 0.006
for ALDH1a7, P = 0.014 for ALDH1l1, P = 0.006
for ALDH2, P = 0.006 for ALDH3a1, P = 0.006 for
ALDH4a1, and P = 0.009 for ALDH7a1). Consistent
with the reduced number of ­C D45−/ALDHbr cells andthe reduced expression of ALDH1a1 mRNA in the
fibrotic lung, the expression of ALDH1a1 was reduced
throughout the alveolar epithelia, especially in theareas of fibrosis, as demonstrated using immunostaining (Additional file 5).
The decrease in number of cells observed in ­C D45−/
ALDHbr cells during BLM treatment was a featureobserved in lung SP cells as well [10]. Therefore, weinvestigated the possibility of an overlap between­ALDHbr and lung SP cells. After Hoechst staining, ALDH staining was performed, followed by flow
cytometry, which revealed that ­C D45−/ALDHbr population is completely different from C­ D45− lung SP cells(Additional file 6).

Takahashi et al. Stem Cell Res Ther

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ALDH + DEAB

ALDH

(C)
Bright Field

ALDH + DEAB

ALDH

ALDH + DEAB

ALDH

SSC

(A)

Page 7 of 14

CD45

EpCAM

PDGFRαPrimary lungfibroblasts

SSC

(B)

SSC

ALDH

ALDH

(D)

ALDH + DEAB

ALDH

markers

MRC

SSC

3T3

ALDH

Fig. 3 ALDH activity in cultured cells. A ALDH activity in cultured lung ­CD45−/ALDHbr. Sorted ­CD45−/ALDHbr cells were cultured and reexamined
for ALDH activity. B Surface markers on primary cultured lung fibroblasts. C ALDH activity in primary cultured lung fibroblasts. (Upper)
Representative image of primary cultured lung fibroblasts stained with either ALDEFLUOR alone or ALDEFLUOR and DEAB. (Lower) Flow cytometry
to determine ALDH activity in primary cultured lung fibroblasts. C ALDH activity in 3T3 and MRC fibroblast cell lines

Effect of CD45−/ALDHbr cell therapy on BLM‑induced
pulmonary fibrosis

In the preceding experiments, we presumed that C­ D45−/
br
ALDH cells were depleted during pulmonary fibrosis; therefore, we assessed the possible usage of C
­ D45−/
br
ALDH cells in cell therapy for BLM-induced pulmonary fibrosis. Our results showed that both the levels ofhydroxyproline (Fig. 5A, P = 0.023) and the degree oftissue fibrosis (Fig. 5B for HE staining, Additional file 7
for Masson’s trichrome staining) in the lung obtainedon day 14 were significantly lower in the C­ D45−/ALDbr−
H i.v. group (n = 4) than in the ­CD45 /ALDHdim i.v.
group (n = 7). In the C­ D45−/ALDHbr i.v. group (n = 7–8),
the mRNA expression of interleukin 6 (IL6) and transforming growth factor β1 (TGFb1) genes in lung tissues
obtained on day 7 was significantly suppressed comparedwith the C­ D45−/ALDHdim i.v. group (n = 9–10) (Fig. 5C,
P = 0.042 for IL6, and P = 0.013 for TGFb1). Interestingly, we noted that the percentage of C
­ D45−/ALDHbr/+PDGFRα cells, which was lowered, was recovered in the­CD45−/ALDHbr i.v. group (n = 4) in the lung obtainedon day 14 (Fig. 5D). Furthermore, the expression levelsof ALDH1a1 and ALDH4a1 mRNAs, which were significantly reduced after treatment with BLM (Fig. 4D), werealso recovered in the C­ D45−/ALDHbr i.v. group (n = 4)
on day 14 (Fig. 5E, P = 0.008 for ALDH1a1, and P = 0.038
for ALDH4a1).
Among the ALDH family of enzymes, the ALDH1a
family (ALDH1a1, ALDH1a2, and ALDH1a3), also
known as retinal dehydrogenases or retinaldehyde
dehydrogenases (RALDH), convert retinal/retinaldehyde to all-trans retinoic acid (ATRA) (Fig. 5F) [22].

Takahashi et al. Stem Cell Res Ther

(2021) 12:471

(B)

(A)

Page 8 of 14

ALDHbr in whole lung

CD45-/ALDHbr

CD45-/ALDHbr/EpCAM+

**

*

N.S.

1.5

**

✱✱

% from total lung cells

Hydroxyproline on day 14 (µg)

150

100

50

0

N.S.

0.20

N.S.

**

0.20

N.S.

0.15

0.15

0.10

0.10

0.05

0.05

**

N.S.

0.5

0.5

BL
M

*

1.0

1.0

0.0

PB
S

1.5

N.S.

CD45-/ALDHbr/PDGFRα +

0

7

14

0.0

0

7

14

0.00

Days

Days

0

7

0.00

14

0

7

14

Days

Days

Hydroxyproline on day 14 (µg)

(C)
160

160

160

120

120

120

80

80

80

40

40
0
0.0

rs = 0.420
P = 0.260
0.2

0.4

0.6

0
0.00

0.8

0.19

0.05

0.10

0
0.00

0.15

CD45- / ALDHbr / EpCAM+ (% of total lung)

✱✱

0.02

0.04

0.06

0.08

CD45- / ALDHbr / PDGFRα + (% of total lung)

✱✱

PBS
BLM

0.13
✱✱

0.07

✱✱

0.01
0.006

✱✱

0.004
0.002

rs = -0.891
P = 0.001

✱
✱✱

✱✱

0.000

AL
D
H
AL 1a1DH
AL 1a2
DH
AL 1a3
DH
AL 1a7
D
H
1
AL b1
D
H
1l
AL 1
D
AL H2
D
H
AL 3a1DH
AL 4a1DH
AL 7a1
D
H
AL 8a
1
D
H
18
a1

(D)

Relative mRNA expressions on day 14(/β-actin)

CD45- / ALDHbr (% of total lung)40
rs = -0.151
P = 0.698

Fig. 4 Decreased ­CD45−/ALDHbr during pulmonary fibrosis. A Lung hydroxyproline content on day 14 in the PBS (n = 4) and the BLM (n = 9)groups. **P < 0.01. B Percentages of A
­ LDHbr, ­CD45−/ALDHbr, ­CD45−/ALDHbr/EpCAM+, and ­CD45−/ALDHbr/PDGFRα+ cells in total lung cells during
BLM-induced pulmonary fibrosis (n = 6/group). *P < 0.05, **P < 0.01. ns, not significant. C Correlations between lung hydroxyproline content on
day 14 and percentages of ­ALDHbr, ­CD45−/ALDHbr, ­CD45−/ALDHbr/EpCAM+, and ­CD45−/ALDHbr/PDGFRα+ cells in total lung cells. D Real-timequantitative PCR analysis of the mRNA expression levels of ALDH isoforms in the lungs obtained from the PBS (n = 4) and BLM (n = 9) groups on day
14. Values are expressed relative to the expression of the endogenous control β-actin mRNA. Data are shown as mean ± SEM. *P < 0.05, **P < 0.01

Page 9 of 14

(B)

1.5

✱

1.0

0.5

0.5

40

0.0

D
45 /A
LD
H dim
C
D
45 /A
LD
H br

C

C

LD
H br

D
45 /A

Type of injected cells

Type of injected cells

C

D
45 /A

D
45 /A
LD
H dim
C
D
45 /A
LD
H br

0.0

0

✱

1.5

1.0

80

LD
H dim

Hydroxyproline on day 14 (µg)

120

C

(C)

✱

Relative IL6 mRNA expression on day 7(A)(2021) 12:471

Relative TGFb1 mRNA expression on day 7

Takahashi et al. Stem Cell Res Ther

Type of injected cells

0.13
0.05

Retinal/Retinaldehyde
RALDH(ALDH1a1, 2, and 3)

0.01
0.005
0.004
0.003
0.002
0.001
0.000

ATRA

✱

D
AL

RAR

AL

H
D

AL

1a
2

H
D

AL

1a
3

H
D
AL

1a
1

CRABP

CD45- / ALDHbr i.v.

80

Survival (%)

0.006
0.004
✱

✱

P = 0.014

60
CD45- / ALDHdim i.v.

40
20

R
ar
g

R
ar
b

0
R
ar
a

Retinol/Vitamin A
STRA6

0.09

100

0.008

0.002
0.00100
0.000750.000500.000250.00000(F)
CD45- / ALDHdim iv
CD45- / ALDHbr iv

✱✱

H
1a
7
D
H
1b
AL
1
D
H
1l
AL 1
D
AL H2
D
H
AL 3a1DH
AL 4a1DH
AL 7a1DH
AL 8 a
1
D
H
18
a1

(H)

CD45- / ALDHdim iv
CD45- / ALDHbr iv

St
ra
6
C
ra
bp
1
C
ra
bp
2

Relative mRNA expressions on day 14(/β-actin)

(G)

(E)

Relative mRNA expressions on day 14(/β-actin)(D)

P = 0.615
PBS i.v.

0

5

10

15

Time (days)

Fig. 5 CD45−/ALDHbr cell therapy ameliorates BLM-induced pulmonary fibrosis ­CD45−/ALDHbr cell therapy ameliorates BLM-induced pulmonary
fibrosis. A Lung hydroxyproline content on day 14 in BLM-treated mice transferred with C­ D45−/ALDHdim (n = 7) and ­CD45−/ALDHbr (n = 4) cells
on day 2. *P < 0.05. B HE-staining of lung tissue sections obtained from PBS-treated and BLM-treated mice transferred with C­ D45−/ALDHdim and­CD45−/ALDHbr cells. C Real-time quantitative PCR analysis of the mRNA expression levels of TGF-β1 and IL-6 in lung tissues on day 7 obtained from
BLM-treated mice transferred with ­CD45−/ALDHdim (n = 9–10) and ­CD45−/ALDHbr (n = 7–8) cells. Values are expressed relative to the expressionof the endogenous control β-actin mRNA and normalized to the mean value of the C­ D45−/ALDHdim i.v. group set as 1. *P < 0.05. D Representative
images of flow cytometry of lung ­CD45−/ALDHbr cells in BLM-treated mice transferred with PBS alone, ­CD45−/ALDHdim cells, and C­ D45−/ALDHbr
cells on day 14. E Real-time quantitative PCR analysis of the mRNA expression levels of ALDH isoforms in the lungs obtained from BLM-treated micetransferred with ­CD45−/ALDHdim (n = 7) and C­ D45−/ALDHbr (n = 4) cells on day 14. Values are expressed relative to the expression of endogenous
control β-actin mRNA. Data are shown as mean ± SEM. *P < 0.05. F Schematic summary of the retinol-metabolizing pathway. Circulating retinol/
vitamin A is taken up into a cell via the cell membrane receptor (stimulated by retinoic acid 6, STRA6). It is converted into retinal/retinaldehyde
within the cytoplasm, which is further converted into all-trans-retinoic acid (ATRA) by retinal dehydrogenase/retinaldehyde dehydrogenase (RALDH,
ALDH1a family). Subsequently, it is transported into the nucleus by the cellular retinoic acid-binding protein (CRABP), where ATRA binds to theintranuclear retinoic acid receptor (RAR) and promotes gene transcription. G Real-time quantitative PCR analysis of the mRNA expression levelsof retinol-metabolizing pathway-related genes in the lung tissues obtained from BLM-treated mice transferred with C­ D45−/ALDHdim (n = 7) and­CD45−/ALDHbr (n = 4) cells on day 14. Values are expressed relative to the expression of the endogenous control β-actin mRNA. Data are shown asmean ± SEM. *P < 0.05. (H) Survival rate in high-dose BLM-treated mice transferred with PBS alone (n = 8), ­CD45−/ALDHdim (n = 8) cells, and ­CD45−/
ALDHbr (n = 5) cells

To investigate the effect of the transferred C­ D45−/
br
ALDH cells on the retinol-metabolizing pathway in
BLM-induced pulmonary fibrosis, we examined the retinol-metabolizing pathway-related mRNA expression in
BLM-treated lung tissue. As shown in Fig. 5G, the mRNA
expression levels of cellular retinoic acid-binding protein

1 (Crabp1) and retinoic acid receptor beta (Rarb) weresignificantly increased in the C­ D45−/ALDHbr i.v. group(n = 4) compared with the ­CD45−/ALDHdim i.v. group(n = 7) (P = 0.019 for Crabp1, and P = 0.038 for Rarb).
In addition, we assessed the effect of C
­ D45−/ALDHbr
cell therapy on survival using BLM-induced pulmonary

Takahashi et al. Stem Cell Res Ther

(2021) 12:471

Page 10 of 14

fibrosis with a higher dose of BLM (5 mg/kg bodyweight). We observed that the higher dose of BLM led
to approximately 80% mortality on day 14 in both the­CD45−/ALDHdim (n = 8) and the PBS i.v. groups (n = 8),
whereas, surprisingly, no death was observed in mice thatreceived ­CD45−/ALDHbr cell therapy (n = 5) (Fig. 5H).

conditions were determined using appropriate
positive and negative controls (Fig. 6D), and we notedthat mCherry-positive ­CD45−/ALDHbr and ­CD45−/
br+
ADLH /PDGFRα cells were also found histologically inthe recipient lung-transferred ­CD45−/ALDHbr (Fig. 6E).

Detection of transferred donor CD45−/ALDHbr in therecipient lung

Effects of aging on ALDH activity−

To distinguish and trace the injected donor C­ D45 /
ALDHbr cells in the lungs of recipient mice, mCherryknock-in mice were used as donors. After mCherry heterozygosity was confirmed using tail PCR (Additional
file 8), donor ­CD45−/ALDHbr or ­CD45−/ALDHdim cells
were sorted from these mCherry-expressing mice using
FACS (Fig. 6A) and transferred into wild-type C57BL/6
recipients pretreated with BLM. We observed that flow
cytometry could detect donor mCherry-positive ­CD45−/
ALDHbr (Fig. 6B) and ­CD45−/ALDHbr/PDGFRα+(Fig. 6C) cells in the recipient lungs more frequently inthe ­CD45−/ALDHbr i.v. group than in the C­ D45−/ALDdim
H
i.v. group. Appropriate mCherry immunostaining

Finally, we examined the role of aging on the ­CD45−/
ALDHbr population. As shown in Fig. 7A, the percentages of whole ­CD45−/ALDHbr cell population and that ofits ­CD45−/ALDHbr/PDGFRα+ subgroup in the lung were
not significantly different between aged and young micethat were not treated with BLM (on day 0). On the contrary, the percentage of ­CD45−/ALDHbr/PDGFRα+ cells,
but not C­ D45−/ALDHbr cells, in the lung obtained 7 days
after treatment with BLM was significantly decreased inaged mice (n = 4–6) compared with that in young mice(n = 4–6). In a similar fashion, the percentage of A
­ LDHbrcells in cultured PDGFRα-predominant (as shown in
Fig. 3B) primary lung fibroblasts obtained from thelung 7 days after treatment with BLM was significantly

Fig. 6 Detection of transferred donor ­CD45−/ALDHbr cells in the recipient lung. A Representative images of flow cytometry of lung C­ D45− cells indonor mCherry-expressing mice. B Flow cytometry of lung ­CD45− cells in recipient BLM-treated C57BL/6 mice transferred with PBS alone, donor­mCherry+/CD45−/ALDHdim cells, and donor m­ Cherry+/CD45−/ALDHbr cells. Recipient lungs were harvested on the day following the cell transfer(day 3, 3 days after BLM-treatment). C Flow cytometry of lung ­CD45−/ALDHbr cells in recipient BLM-treated C57BL/6 mice transferred with PBS
alone, donor ­mCherry+/CD45−/ALDHdim cells, and donor m­ Cherry+/CD45−/ALDHbr cells. Recipient lungs were harvested the next day and 5 days
after cell transfer (days 3 and 7). D Histological analysis of mCherry immunostaining in lungs obtained from mCherry-expressing mice (positive
control) and wild-type C57BL/6 mice (negative control). E Representative images of mCherry immunostaining in lungs obtained from recipient
BLM-treated wild-type C57BL/6 mice transferred with ­mCherry+/CD45−/ALDHbr cells

Takahashi et al. Stem Cell Res Ther

(A)-

(2021) 12:471

CD45-/ALDHbr/PDGFRα +br

CD45 /ALDH
1.5

Page 11 of 14

0.20

✱✱

0.5

5

✱

0.15

Young
Aged

0.10

0.05

ns

✱✱

4

Frequency (%)

ns% from total lung cells% from total lung cells

ns

ns

ns

✱✱

✱✱

✱

1.0

(B)

✱✱

ALDHbr in cultured fibroblasts

Young
Aged

3
2
1

0.0

7
D
ay

D

0

7
D
ay

0

ay

0
ay

ay
D

D

ay
D

0

7

0.00

Fig. 7 Effect of aging on ALDH activity. A Percentages of ­CD45−/ALDHbr and ­CD45−/ALDHbr/PDGFRα+ cells in total lung cells during BLM-induced
pulmonary fibrosis in young and aged mice (n = 4–6). *P < 0.05, **P < 0.01. ns, not significant. B ALDH activity in primary cultured lung fibroblasts
obtained from young and aged mice (n = 5–10) before and 7 days after treatment with BLM. **P < 0.01. ns, not significant

decreased in aged mice (n = 5–10) compared with young
mice (n = 5–10) (Fig. 7B).

Discussion
The present study identified and characterized the nonhematopoietic/lung resident ­ALDHbr cell populations in
the mouse lung. The lung ­CD45−/ALDHbr population
and, the ­CD45−/ALDHbr/PDGFRα+ subpopulation arecell populations with high proliferative capacity. These
population significantly reduced in pulmonary fibrosis. The high levels of expression of ALDH observedin ­CD45−/ALDHbr cells was mainly attributed to the
ALDH1a subfamily, also known as RALDH, which wassignificantly reduced in BLM-treated lungs. When used
as a tool for cell therapy, transferred C­ D45−/ALDHbr
cells reached the site of lung injury and ameliorated
BLM-induced pulmonary fibrosis. Thus, this study demonstrated ­CD45−/ALDHbr cells as a novel lung-resident
stem cell population and suggested their potential therapeutic use in pulmonary fibrosis.
Although ­ALDHbr cells with stem cell properties havebeen detected in various normal tissues, including thebone marrow [14, 23], umbilical cord blood [24, 25],
mammary glands [26, 27], heart [28], and adipose tissue[29], little is known about lung-resident A­ LDHbr cells.
A study showed that isolated murine airway basal andsubmucosal gland duct A­ LDHbr cells exhibited stem cellproperties in normal/healthy lungs [30]. No previous
study has investigated the significance of lung-resident­ALDHbr cells in respiratory diseases, such as pulmonary

fibrosis. In the current study, lung-resident ­CD45−/ALDHbr were rare and heterogeneous population with epithelial and mesenchymal lineages. The percentages of­ALDHbr in both primary cultured lung fibroblasts andfibroblast cell lines were low at approximately 5%, andhence, we assumed that ­ALDHbr cells lost their activity during differentiation and proliferation, consistent
with the findings of a previous report [24]. Similar to the­CD45−/ALDHbr/PDGFRα+ population in the current
study, ­CD45− lung SP cells have been reported to express
mesenchymal markers and exhibit MSC properties [8],and have been shown to be decreased in BLM-induced
pulmonary fibrosis [10]. However, in our study, we foundthat ­CD45−/ALDHbr is a novel population that is completely different from lung SP cells (­ CD45−/CD31−/Hoechstdim). Therefore, it is reasonable that the expressionof the surface markers of MSCs found in lung ­CD45−/
ALDHbr cells differed from that in the SP cells. Instead,
SSEA4, a marker for mesenchymal progenitors [31], wasdemonstrated to be solely expressed on C
­ D45−/ALDHbr
cells in the mouse lung. These results suggest that the­CD45−/ALDHbr population might contain mesenchymal
progenitors and C­ D45−/ALDHbr/PDGFR+ cells maintained the ability to differentiate into the mesenchymal
lineage.
During BLM-induced pulmonary fibrosis, we observeda downregulation in the expression of a broad spectrumof ALDH mRNAs in lung tissues. We also found thattransferred ­CD45−/ALDHbr cells ameliorated BLMinduced pulmonary fibrosis by suppressing IL-6 andTakahashi et al. Stem Cell Res Ther

(2021) 12:471

TGF-β. As an evidence, intravenously administered­CD45−/ALDHbr cells were shown to reach the site oflung injury using mCherry-expressing mice as donors.
Additionally, these lung-protective effects of transferred­CD45−/ALDHbr were accompanied by a recovery in thelevels of ALDH, which had been decreased during fibrosis, suggesting that ALDH was involved in the mechanism of pulmonary fibrosis. Although little is known
about the association of ALDH isoforms with lung diseases, ALDH1a1 and ALDH3a1 have been reported to
be expressed in the human airway epithelium [32]. Jangand coworkers reported that the expression of ALDH3a1
was markedly increased in human airway epithelial cells
exposed to cigarette smoke extract and that ALDH3a1
exerted protective action against smoking-induced airway epithelial damage [33]. In the current study, theexpression of both ALDH1a1 and ALDH4a1 were upregulated in ­CD45−/ALDHbr cells and downregulated in
the fibrotic lung after BLM administration, parallelingthe reduction in the number of ­CD45−/ALDHbr cells.
Likewise, intravenous administration of C
­ D45−/ALDbr
H cells was shown to significantly recover the expression of ALDH1a1 and ALDH4a1 in the fibrotic lung.
Therefore, we speculated that mesenchymal ALDH1a1
and ALDH4a1 might protect against BLM-induced pulmonary fibrosis. Indeed among ALDH family members, RALDHs (ALDH1a1, ALDH1a2, and ALDH1a3)
catalyze the conversion of retinol to ATRA [22], supporting the self-renewal and cell differentiation of stem
cells [34]. Several lines of evidence have suggested that
ATRA exerted protective action against radiation pneumonitis and BLM-induced lung injury in mice throughanti-inflammatory effects by activating protein kinase C
δ (PKC-δ), inhibiting mitogen-activated protein kinase
P38 α (p38MAPK) and nuclear factor kappa-light-chainenhancer of activated B-cells (NF-kB), and suppressing
the production of IL-6 and TGF-β [35–38]. In the current
study, we observed the upregulation of retinol-metabolizing pathway molecules, recovery of the expression of
RALDH, and suppressed expression of IL-6 and TGF-β
in BLM-induced pulmonary fibrosis treated with ­CD45−/
ALDHbr cell therapy. On the other hand, the significanceof ALDH4a1 in lung injury is currently unknown and further investigation is required.
In the fibrotic lung, after BLM administration, we
observed a reduction in the number of cells in the­CD45−/ALDHbr population, especially of its C­ D45−/
br+
ALDH /PDGFRα subpopulation. This reduction was
more remarkably observed in aged mice than in young
mice. These results suggested that aging led to a decreasein the number of A
­ LDHbr cells in the lungs, especially inthe lung PDGFRα+ fibroblasts. As fibrotic lung diseases,
especially idiopathic pulmonary fibrosis (IPF), commonly

Page 12 of 14(A)
Healthy lung

Fibrotic lung

ALDHbr cells↓ALDHbr cells

Retinol-metabolizing pathway
ATRA production↓Retinol-metabolizing pathway↓ ATRA production

Cytokine production
IL-6 & TGF-β↑ Cytokine production
IL-6 & TGF-β

Aging

ALDHbr transfer↑Fibrosis

Fig. 8 Summary of the study. Lung injury (e.g., bleomycin-induced
lung injury) triggers reduction of ­ALDHbr cells in the lung, resulting in
a suppressed retinol-metabolizing pathway, elevated concentrationsof profibrotic cytokines (e.g., IL-6 and TGFβ1), and exacerbation of
pulmonary fibrosis. Aging accelerates the injury-induced reductionin ­ALDHbr cells. ­ALDHbr cell therapy restores the impaired antifibrotic
effects of A
­ LDHbr cells. Solid and dotted arrows indicate promotion
and inhibition, respectively

occur in the elderly [39] and stem cell senescence is oneof the suggested causes of IPF [40], it is speculated thatthe decreased number of A
­ LDHbr cells in the lungs might
accelerate fibrotic lung diseases in the elderly.
The limitation of this study is the difficulty in collecting
sufficient number of cells. Because of the infrequency of
existence of A
­ LDHbr cells, many donor mice lungs werenecessary to acquire a sufficient number of A
­ LDHbr cells,signifying the challenge in applying the methods and theresults of the present study to human lung diseases. If thecollected cells could be proliferated while maintaining
ALDH activity, the burden on donor could be minimized.
To apply the current results to human translational studies in the future, development of less invasive methods
for collecting ­ALDHbr cells is required.

Conclusions
Our results strongly suggest that the lung expression of
ALDH and lung-resident ­CD45−/ALDHbr are involvedin pulmonary fibrosis. (Figure 8 summarizes the findingsof the current study.) When administered intravenously,­CD45−/ALDHbr ameliorated BLM-induced pulmonary
fibrosis, signifying the possibility for ­CD45−/ALDHbr
cells to find application as novel and useful cell therapy
tools in pulmonary fibrosis treatment.
Abbreviations
ALDH: Aldehyde dehydrogenase; ALDHbr: Cell populations with high ALDH
activity; ALDHdim: Cell population with low ALDH activity; ATRA​: All-transretinoic acid; BLM: Bleomycin; CRABP: Cellular retinoic acid-binding protein;
EpCAM: Epithelial cell adhesion molecule; FACS: Fluorescence activated cell

Takahashi et al. Stem Cell Res Ther

(2021) 12:471

sorting; HE: Hematoxylin–eosin; IL: Interleukin; IPF: Idiopathic pulmonary fibrosis; MSCs: Mesenchymal stem cells; NF-kB: Nuclear factor kappa-light-chainenhancer of activated B-cells; PDGFR: Platelet-derived growth factor receptor;
PKC-δ: Protein kinase C δ; p38MAPK: Mitogen-activated protein kinase P38 α;
RALDH: Retinal dehydrogenase/retinaldehyde dehydrogenase; RAR​: Retinoic
acid receptor; SP: Side population; SSEA: Stage-specific embryonic antigen;
TGF: Transforming growth factor.

Supplementary Information
The online version contains supplementary material available at https://​doi.​
org/​10.​1186/​s13287-​021-​02549-6.
Additional file 1. Antibodies used in flow cytometric experiments.
Additional file 2. Primers used in this study.
Additional file 3. Effect of magnetic-activated cell sorting for the enrichment of ­CD45−/ALDHbr cells. Representative image of lung ­CD45−/
ALDHbr cells before and after magnetic-activated cell sorting using the
Tissue Stem Cell Pre-Enrichment Kit. ­ALDHbr gating was determined bycomparing samples stained with ALDEFLUOR alone and with ALDEFLUOR
and diethylaminobenzaldehyde (DEAB), an ALDH inhibitor.
Additional file 4. Expression of cell surface proteins in C
­ D45−/ALDHdim
cells and ­CD45−/ALDHbr cells. Sorted ­CD45−/ALDHdim cells and C­ D45−/
ALDHbr cells were examined for cell surface markers associated withmesenchymal stem cells (MSCs), fibroblasts, and stem cells.
Additional file 5. ALDH1a1 immunostaining in lung tissue. Representative images of ALDH1a1 immunostaining in PBS- or BLM-treated lungtissue sections on day 14.
Additional file 6. Double staining of A
­ LDHbr cells and side population
cells in lung tissue. (A) Representative image of lung C­ D45−/CD31−/ALDHbr live cells. (B) Representative image of lung C­ D45−/CD31−/Hoechstdim(side population, SP) live cells. (C) The gating of SP cells was confirmed
using verapamil, which is an inhibitor for Hoechst staining. (D) SP cells inlung ­CD45−/CD31−/ALDHbr live cells. (E) A­ LDHbr cells in SP cells.
brAdditional file 7. ­CD45 /ALDH cell therapy ameliorates BLM-induced
pulmonary fibrosis. Masson’s trichrome staining of lung tissue sections
obtained on day 14 from BLM-treated mice transferred with ­CD45−/ALDHdim and ­CD45−/ALDHbr cells.
−

Additional file 8. Discrimination of mCherry-heterozygotic mouse. DNA
extracted from the tails of mice was amplified by PCR using primers
shown in Table S2. The expression of the mCherry-heterozygotic band wasevaluated using agarose gel electrophoresis.
Acknowledgements
We would like to thank Dr. Kazuo Yamagata (Kindai University) for providing mCherry mice, Yoko Hayashi and Yukari Iyanaga for excellent technicalassistance, Editage (www.​edita​ge.​com) for English language editing. A part ofthis work was carried out at Research Facilities for Laboratory Animal Science,and Natural Science Center for Basic Research and Development, Hiroshima
University. This work was supported by the Program of the Network-type Joint
Usage/Research Center for Radiation Disaster Medical Science.
Authors’ contributions
HT involved in collection of data, data analysis and interpretation, andmanuscript writing. TN involved in conception and design, collection of data,
financial support, data analysis and interpretation, and manuscript writing.
TM, MN and SS involved in data analysis and interpretation, reviewing themanuscript for important intellectual content and approving for submission.
KY, YH, SM, HI, KF and HH involved in reviewing the manuscript for important
intellectual content and approving for submission. NH involved in conception and design, financial support, reviewing the manuscript for important
intellectual content and approving for submission. All authors have read andapproved the manuscript.

Page 13 of 14

Funding
This work was supported by JSPS KAKENHI (Grand-in-Aid for Scientific
Research) Grant Numbers 17K09654 and 20K08519, and GSK Japan Research
Grant 2015. The funders had no role in study design, data collection and analysis, decision to publish, or presentation of the manuscript.
Availability of data and materials
The data that support the findings of this study are available from the corresponding author upon reasonable request.

Declarations
Ethics approval and consent to participate
All experimental procedures were approved by the Institutional Animal Careand Use Committee of Hiroshima University (permit number, A19-122 and28-29-2).
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Author details
1
Department of Molecular and Internal Medicine, Graduate School of Biomedical and Health Sciences, Hiroshima University, 1‑2‑3 Kasumi, Minami‑ku,
Hiroshima 734‑8551, Japan. 2 Department of Physical Analysis and Therapeutic
Sciences, Graduate School of Biomedical and Health Sciences, Hiroshima
University, 1‑2‑3 Kasumi, Minami‑ku, Hiroshima 734‑8553, Japan.
Received: 9 May 2021 Accepted: 8 August 2021

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