論文の公開元へHuman bone marrow-derived mesenchymal stromal cells cultured in serum-free media demonstrate enhanced antifibrotic abilities via prolonged survival and robust regulatory T cell induction in murine bleomycin-induced pulmonary fibrosis
概要
(2021) 12:506
Takao et al. Stem Cell Res Ther
https://doi.org/10.1186/s13287-021-02574-5
Open Access
RESEARCH
Human bone marrow‑derived mesenchymal
stromal cells cultured in serum‑free media
demonstrate enhanced antifibrotic abilities
via prolonged survival and robust regulatory
T cell induction in murine bleomycin‑induced
pulmonary fibrosis
Shun Takao1, Taku Nakashima1* , Takeshi Masuda1, Masashi Namba2, Shinjiro Sakamoto1,
Kakuhiro Yamaguchi1, Yasushi Horimasu1, Shintaro Miyamoto1, Hiroshi Iwamoto1, Kazunori Fujitaka1,
Hironobu Hamada3, Shinya Takahashi4, Ayumu Nakashima5 and Noboru Hattori1
Abstract
Background: Mesenchymal stromal cells (MSCs) are a potential therapeutic tool for pulmonary fibrosis. However,
ex vivo MSC expansion using serum poses risks of harmful immune responses or unknown pathogen infections in the
recipients. Therefore, MSCs cultured in serum-free media (SF-MSCs) are ideal for clinical settings; however, their efficacy in pulmonary fibrosis is unknown. Here, we investigated the effects of SF-MSCs on bleomycin-induced pulmonary inflammation and fibrosis compared to those of MSCs cultured in serum-containing media (S-MSCs).
Methods: SF-MSCs and S-MSCs were characterized in vitro using RNA sequence analysis. The in vivo kinetics and
efficacy of SF-MSC therapy were investigated using a murine model of bleomycin-induced pulmonary fibrosis. For
normally distributed data, Student’s t test and one-way repeated measures analysis of variance followed by post hoc
Tukey’s test were used for comparison between two groups and multiple groups, respectively. For non-normally distributed data, Kruskal–Wallis and Mann–Whitney U tests were used for comparison between groups, using e Bonferroni’s correction for multiple comparisons. All tests were two-sided, and P < 0.05 was considered statistically significant.
Results: Serum-free media promoted human bone marrow-derived MSC expansion and improved lung engraftment
of intravenously administered MSCs in recipient mice. SF-MSCs inhibited the reduction in serum transforming growth
factor-β1 and the increase of interleukin-6 in both the serum and the bronchoalveolar lavage fluid during bleomycininduced pulmonary fibrosis. SF-MSC administration increased the numbers of regulatory T cells (Tregs) in the blood
and lungs more strongly than in S-MSC administration. Furthermore, SF-MSCs demonstrated enhanced antifibrotic
effects on bleomycin-induced pulmonary fibrosis, which were diminished by antibody-mediated Treg depletion.
*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
© 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 the
original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or
other 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 this
licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco
mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
Takao et al. Stem Cell Res Ther
(2021) 12:506
Page 2 of 16
Conclusions: SF-MSCs significantly suppressed BLM-induced pulmonary inflammation and fibrosis through
enhanced induction of Tregs into the lungs and corrected the dysregulated cytokine balance. Therefore, SF-MSCs
could be a useful tool for preventing pulmonary fibrosis progression without the demerits of serum use.
Keywords: Mesenchymal stromal cells, Serum-free, Bleomycin, Pulmonary fibrosis, Regulatory T cells, Interleukin-6,
Transforming growth factor-β
Background
Idiopathic pulmonary fibrosis (IPF) is a severe pulmonary fibrotic disease that presents with short life expectancy and a high mortality rate [1]. Since patients with
IPF who are treated with antifibrotic agents show inhibition of forced vital capacity (FVC) decline and improved
survival, antifibrotic agents such as pirfenidone and nintedanib have been conditionally recommended for IPF
treatment [2]. However, these agents cannot halt disease
progression; moreover, they have adverse effects, including gastrointestinal disorders, skin-related problems,
and liver damage [3]. Thus, considering the lack of more
effective options for treating IPF, further therapeutic
approaches are being explored.
Mesenchymal stromal cells (MSCs) are pluripotent
cells in the bone marrow and are now known to be isolated from various sources, including adipose tissue,
umbilical cord, peripheral blood, and muscle tissue [4].
Systemically administered MSCs home to the site of
injury and exert anti-inflammatory effects by modulating various immune cells. Furthermore, MSCs secrete
cytokines and growth factors with proliferative and
angiogenic effects and support tissue repair through
paracrine effects [5]. With the expectation of applying
these favorable effects to therapy, several preclinical and
clinical studies are ongoing using human MSCs to treat
chronic diseases including autoimmune, inflammatory,
degenerative, and cardiovascular diseases [6]. Regarding
lung diseases, MSC-based therapies have been reported
as effective in preventing experimental models of pulmonary fibrosis [7]. Based on successful studies with animal
models, clinical trials of MSC-based therapies for interstitial lung diseases, mainly human IPF, are underway
worldwide [8]. Some clinical trials have shown that MSCbased cell therapy provides a protective effect against
FVC decline over time in patients with IPF [9, 10]. In
addition, no serious adverse events related to MSC-based
cell therapy have been reported in these trials, suggesting the safety and tolerance of this therapy [8, 10]. These
findings suggest that MSC-based cell therapy could be a
new potential therapeutic option for treating IPF.
In vitro expansion of MSCs is necessary before their
transplantation into recipients of MSC-based therapies.
MSC growth in vitro generally requires culture media
supplemented with fetal bovine serum (FBS) or human
serum to provide the factors essential for cell growth. In
fact, most studies on MSCs have used serum-containing media [11]. However, using serum in culture media
poses various potential disadvantages including pathogen contamination (e.g., unknown viruses, mycoplasma,
and prions), harmful immunizing effects [11, 12], inhibition of cell growth [13], uneven quality between lots,
global shortage of supply, and high costs [14]. Therefore,
defined culture conditions without sera are ideal as a tool
for MSC therapy in humans. Recently, several chemically
defined serum-free media for experimental MSC cultures
have been commercialized [11, 14]. Wu et al. showed
that human MSCs cultured in serum-free medium (SFMSCs) exhibited strong immunomodulatory activity and
secreted higher levels of immunoregulatory factors compared with MSCs cultured in serum-containing medium
(S-MSCs) in vitro, and showing improved therapeutic
activity in a rat model of pulmonary arterial hypertension
in vivo [15]. Moreover, other animal studies have also
reported better therapeutic efficacy of SF-MSCs compared with that of S-MSCs in a mouse model of acute
colitis [16], a rat model of renal fibrosis [17] and peritoneal fibrosis [18]. Thus, using SF-MSCs not only avoids
the disadvantages of using sera, but SF-MSCs may also
be more useful as a therapeutic tool than S-MSCs. Previous preclinical studies of SF-MSCs used for lung disease
reported that xenogeneic SF-MSCs demonstrated therapeutic effects in a rat model of Escherichia coli-induced
[19] and ventilator-induced lung injury [20]. However,
the therapeutic effects of SF-MSCs on pulmonary fibrosis
have not been investigated. Considering the progress of
IPF clinical trials with MSCs, clarifying the efficacy of SFMSCs in experimental pulmonary fibrosis is essential for
developing MSC-based therapies in human IPF. Therefore, in the current study, we investigated the effects of
SF-MSCs on an experimental mouse model of lung fibrosis and compared these effects with those of S-MSCs.
Methods
MSCs
Human bone marrow-derived MSCs were collected
from the sternum of consenting patients during thoracic
surgery with the approval of the Ethics Committee of
Hiroshima University Hospital (E-1089). MSCs were cultured in Dulbecco’s modified Eagle’s medium (DMEM)
Takao et al. Stem Cell Res Ther
(2021) 12:506
(Sigma-Aldrich, St. Louis, MO, USA) supplemented with
10% FBS (Sigma-Aldrich) for S-MSCs, or with serumfree STK2 medium (KBDSTC102; DS Pharma Biomedical) for SF-MSCs. The MSCs were dissociated with
Accutase (Innovative Cell Tech, San Diego, CA, USA)
and passaged 5–6 times before use. For the proliferation
assay, MSCs were seeded into 24-well plates at a density
of 5 × 103 cells/well and cultured as S-MSCs or SF-MSCs.
After staining with Trypan blue (Sigma, St. Louis, MO,
USA), the cell number per well was counted at 24, 48,
72, 96, and 120 h after the start of the cell culture using an
automated cell counter (TC-20, Bio-Rad, Hercules, CA,
USA). Cell morphologies were also recorded at the indicated time points using a Nikon Diaphot 300 microscope
(Nikon, Tokyo, Japan). ...
この論文で使われている画像
