論文の公開元へAntifibrotic effect of lung-resident progenitor cells with high aldehyde dehydrogenase activity
概要
(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, and
cell 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 to
evaluate 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 expression
of ALDH isoforms was significantly reduced in the fibrotic lung. When transferred in vivo into bleomycin-pretreated
mice, 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
−
br
are 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 School
of 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 and
have been successfully used for immunomodulation, tissue regeneration, and tissue repair. Several trials using
stem cell therapy have been performed to treat refractory
diseases, with mesenchymal stem cells (MSCs) being the
most frequently used cell type [1]. In particular, MSCs,
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Takahashi et al. Stem Cell Res Ther
(2021) 12:471
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 of
T-cells through secretion of anti-inflammatory cytokines
and 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 an
elastase-induced lung injury mouse model, this population was demonstrated to significantly improve the survival rate and reverse lung damage [6]. Lung Hoechst
33342dim side population (SP) cells are adult stem cells,
which have also been identified to exhibit mesenchymal and epithelial potential [7]. Among the SP cells, the
CD45−/CD31− fraction has been reported to have the
characteristics 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 lung
and 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 of
various normal tissues and is involved in tissue repair
[12]. Moreover, ALDHbr isolated from the human bone
marrow, 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 human
umbilical cord blood was demonstrated to enhance
angiogenesis in the ischemic heart [15]. Given these
findings, 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 of
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lung-resident ALDHbr and to evaluate its possible use as
a 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 of
lung-resident ALDHbr and exploring its usage in cell
therapy, 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 mice
and 52 week old aged female mice) which were purchased from Charles River Laboratories Japan (Yokohama, Japan). The mice were maintained in a specific
pathogen-free environment and randomly assigned to
BLM or control groups. In experiments performed to
confirm 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 the
mCherry 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, and
mRNA expression and subjected to flow cytometry and
histological 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), and
incubated 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 resuspended
in 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.
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