論文の公開元へ高純度間葉系幹細胞は機能的ミトコンドリア投入に優れている
概要
(2023) 14:40
Yang et al. Stem Cell Research & Therapy
https://doi.org/10.1186/s13287-023-03274-y
Stem Cell Research & Therapy
Open Access
RESEARCH
Highly‑purified rapidly expanding clones,
RECs, are superior for functional‑mitochondrial
transfer
Jiahao Yang1† , Lu Liu1,4†, Yasuaki Oda1†, Keisuke Wada1, Mako Ago1, Shinichiro Matsuda2, Miho Hattori1,
Tsukimi Goto1, Yuki Kawashima1, Yumi Matsuzaki3 and Takeshi Taketani1*
Abstract
Background Mitochondrial dysfunction caused by mutations in mitochondrial DNA (mtDNA) or nuclear DNA,
which codes for mitochondrial components, are known to be associated with various genetic and congenital
disorders. These mitochondrial disorders not only impair energy production but also affect mitochondrial functions
and have no effective treatment. Mesenchymal stem cells (MSCs) are known to migrate to damaged sites and
carry out mitochondrial transfer. MSCs grown using conventional culture methods exhibit heterogeneous cellular
characteristics. In contrast, highly purified MSCs, namely the rapidly expanding clones (RECs) isolated by single-cell
sorting, display uniform MSCs functionality. Therefore, we examined the differences between RECs and MSCs to assess
the efficacy of mitochondrial transfer.
Methods We established mitochondria-deficient cell lines (ρ0 A549 and ρ0 HeLa cell lines) using ethidium bromide.
Mitochondrial transfer from RECs/MSCs to ρ0 cells was confirmed by PCR and flow cytometry analysis. We examined
several mitochondrial functions including ATP, reactive oxygen species, mitochondrial membrane potential, and
oxygen consumption rate (OCR). The route of mitochondrial transfer was identified using inhibition assays for
microtubules/tunneling nanotubes, gap junctions, or microvesicles using transwell assay and molecular inhibitors.
Results Co-culture of ρ0 cells with MSCs or RECs led to restoration of the mtDNA content. RECs transferred more
mitochondria to ρ0 cells compared to that by MSCs. The recovery of mitochondrial function, including ATP, OCR,
mitochondrial membrane potential, and mitochondrial swelling in ρ0 cells co-cultured with RECs was superior
than that in cells co-cultured with MSCs. Inhibition assays for each pathway revealed that RECs were sensitive to
endocytosis inhibitor, dynasore.
Conclusions RECs might serve as a potential therapeutic strategy for diseases linked to mitochondrial dysfunction by
donating healthy mitochondria.
Keywords Mesenchymal stem cells (MSCs), Rapidly expanding clones (RECs), Mitochondrial transfer, Mitochondrial
dysfunction
†
Jiahao Yang, Lu Liu, and Yasuaki Oda contributed equally to this work
*Correspondence:
Takeshi Taketani
ttaketani@med.shimane-u.ac.jp
Full list of author information is available at the end of the article
© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
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Yang et al. Stem Cell Research & Therapy
(2023) 14:40
Background
Mitochondria drive cellular metabolism and are
crucial regulators of life and death [1, 2]. They are
essential for processes such as ATP synthesis, oxidative
phosphorylation (OXPHOS), and cellular signal
transduction [3]. Several mitochondria-specific proteins,
rRNAs, and tRNAs are encoded by the mitochondrial
genome (mtDNA) [4]. However, mitochondrial damage
can occur due to the absence of protective histones,
lack of post-injury repair capacity, proximity to the cell’s
main source of reactive oxygen species (ROS), and its
susceptibility to damage. In a self-sustaining damage
cycle, mitochondrial ROS (mtROS) damages mtDNA,
hence producing less effective mitochondria, which in
turn produce more mtROS [5, 6]. Therefore, therapies
that attempt to reduce mitochondrial malfunction and
maintain mtDNA stability are crucial.
Page 2 of 22
There is limited knowledge about the mechanisms
governing mitochondrial transport between cells [7]. The
integration of mitochondrial genes or the mitochondria
themselves into the recipient cell occurs during their
transfer. The host’s biological energy level may be
significantly altered by this phenomenon, along with
changes in cell differentiation, inflammatory reactions,
cell survival, and even treatment resistance. Transcellular
mitochondrial transport has been demonstrated to be
possible in a number of structures, including tunneling
nanotubes (TNTs) [8–10], connexin 43 (Cx43) gap
junctions (GJs) [11, 12], microvesicles (MVs) [9, 13, 14],
cell fusion [15, 16], internalization [17, 18] (Fig. 1A–E).
Researchers have been interested in exogenous
replacement of damaged mitochondria to prevent cell
death [19]. In 2006, it was revealed that mitochondria
from human mesenchymal stem cells (MSCs) might
Fig. 1 Schematic diagram demonstrating routes of mitochondrial transfer from RECs to recipient cells. A TNTs are membranous tubules that extend
from the plasma membrane and measure 50–1500 nm in diameter. It is the most well-known route for mitochondrial transfer between cells. B
Connexin 43 (CX43) is a gap junction protein that regulates a cell connection channel and facilitates organelle exchange or cell migration. CX43
is an essential regulator of mitochondrial transfer. C Another pathway for mitochondrial transfer has been observed as microvesicles generated
by blebbing the cellular plasma membrane. D Cell fusion, possible route for mitochondrial transfer. E Without carriers, free mitochondria can be
extruded or internalized, suggesting a plausible mechanism for intercellular mitochondrial transfer. Make figure by Figdraw
Yang et al. Stem Cell Research & Therapy
(2023) 14:40
be transported into defective cells through TNTs
[20]. Furthermore, MSCs isolated from bone marrow
(BM) [9, 19, 21, 22] and fat [21, 23] is likely to repair
the mitochondrial activity of the recipient cells by
transferring their own mitochondria. However, the
conventionally isolated bone marrow-derived MSCs
(BMSCs) used in clinical research has shown different
ability for cellular proliferation and differentiation and
even contradictory results because these BMSCs always
contain undifferentiated cells leading to a heterogeneous
cell population with inconsistent functions. Our previous
work reported that rapidly expanding clones (RECs)
were isolated as a single clone from CD90high/CD271high
population in bone marrow mononuclear cells [24].
This clonally expanded and ultra-purified BM-MSCs,
RECs displays all the properties of MSCs, such as plastic
adherence, differentiation capacity, and cell surface
antigens, and does not exhibit lot-related variations in
clinical applications.
RECs have a possibility to offer many potential benefits
as transplantable cells for treating several disorders
related to bone, heart, peripheral nerves, brain, and other
organs [24, 25]. However, whether RECs can restore the
bioenergetics of the cell remains unknown. Further, the
health of the mitochondria transferred into mtDNAdeficient cells needs to be assessed. This study aimed
to identify REC-dependent mitochondrial transport
pathways and investigate whether this transfer restores
cellular functions in mtDNA-deficient cells.
Methods
Cell culture, ρ0 cells and MSCs
Human cancer cell lines A549 and HeLa cells were
purchased from RIKEN CELL BANK and cultured in
Roswell Park Memorial Institute (RPMI) 1640 medium
(Wako, Japan) supplemented with 1% penicillin–
streptomycin and 10% fetal bovine serum (FBS; HyClone,
USA) and were maintained at 37 ℃ in an atmosphere of
5% CO2-95% air. Rho 0 A549 (ρ0 A549) and Rho 0 HeLa
(ρ0 HeLa) cells were established as previously described
by culturing in the presence of ethidium bromide[26]
(EtBr; Sigma, USA) (50 ng/mL), 1 × sodium pyruvate
solution (Wako, Japan) (110 μg/mL), and uridine (Sigma,
USA) (50 μg/mL) in RPMI 1640 supplemented with 10%
FBS for more than 20 passages. Control parental A549
and HeLa cells were maintained in standard culture
medium for the same period. We prepared three clones
of BMSCs, MSC 1, 2, and 3 using BMSCs from Lonza
(Basel, Switzerland). Human BMSCs were isolated as
described previously [24] and cultured in DMEM/Ham’s
F-12 medium (Wako, Japan) containing 15% FBS, 1%
penicillin–streptomycin, and 10 ng/mL basic fibroblast
Page 3 of 22
growth factor (bFGF; Abcam; USA) (in 5% C
O2, at 37 °C)
until 80% confluent.
Preparation of highly‑purified MSCs (RECs)
Frozen GMP-compliant RECs provided by PuREC Co.,
Ltd. from Shimane University, were used in this study.
RECs were isolated as a single clone from
CD90high/
high
CD271
population in BM mononuclear cells. For
the comparative analysis of RECs and normal human
BMSCs, we prepared three clones of RECs: REC 1, REC
2, and REC 3. The cells were thawed in a warm bath at
37 °C prior to culturing and were directly used after
thawing. ...
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