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International Immunology, Vol. 29, No. 1, pp. 11–19
doi:10.1093/intimm/dxx002
Advance Access publication 10 February 2017
© The Japanese Society for Immunology. 2017. All rights reserved.
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Mesenchymal stem cell-derived extracellular
vesicles: a glimmer of hope in treating Alzheimer’s
disease
Lee Chuen Liew1,2, Takeshi Katsuda1, Luc Gailhouste1, Hitoshi Nakagama2,3 and Takahiro Ochiya1
Division of Molecular and Cellular Medicine, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku,
Tokyo 104-0045, Japan
2
Department of Pathology, Immunology and Microbiology, Graduate School of Medicine, The University of Tokyo,
7-3-1 Hongo, Bunkyou-ku, Tokyo 113-0033, Japan
3
National Cancer Center, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan
Correspondence to: T. Ochiya; E-mail: [email protected]
Received 2 October 2016, editorial decision 5 January 2017, accepted 13 January 2017
Abstract
One of the pathological hallmarks of Alzheimer’s disease (AD) is the presence of extracellular plaques
resulting from the accumulation of beta-amyloid peptide (Aβ). To date, a definitive cure for this disease is
still lacking as the currently approved drugs used are mainly symptomatic treatments. The revolutionary
discovery of extracellular vesicles (EVs) has shed new light on the development of disease-modifying
treatments for AD, owing to their potential in delivering the therapeutic agents to the brain. The feasibility
of harnessing EVs for clinical applications is highly dependent on the donor cell, which determines the
intrinsic properties of EVs. The merit of mesenchymal stem cells (MSCs) as therapeutic delivery vehicles,
and the proven therapeutic effects of the EVs derived from these cells, make researchers esteem MSCs
as ideal producers of EVs. Therefore, MSC-derived EVs (MSC-EVs) emerge to be an appealing therapeutic
delivery approach for the treatment of AD. Here, we discuss perspectives on the therapeutic strategies
using MSC-EVs to treat AD and the associated challenges in clinical application.
Keywords: Alzheimer’s disease, exosomes, extracellular vesicles, mesenchymal stem cells, neurodegenerative disease
Introduction
The remarkable progress in understanding the molecular
mechanisms of human diseases has generated new excitement in biomedical research, which in turn had led to the
discovery of potential therapeutic approaches. In the past
several decades, the development of stem cell therapy and
its continuous advancement has gained immense attention.
Among all stem cell types, mesenchymal stem cells (MSCs),
which have been intensively studied, hold great promise in
disease treatments, particularly in the fields of regenerative
medicine and therapeutic gene delivery. The therapeutic efficiency of MSCs was initially attributed to their migration and
engraftment to the target site. However, studies on the bio-distribution of MSCs after systemic infusion have indicated that
the localization of MSCs at target tissues is rare (1–3), likely
reflecting the fact that the therapeutic effects of these cells
are not predominantly imputed to their presence at the target
site and could be related to the factors secreted by the cells.
Recently, accumulating evidence has shown that extracellular vesicles (EVs) that are secreted from cells play a
vital role in cell-to-cell communication (4). MSC-derived EVs
(MSC-EVs) were reported to exert therapeutic effects similar
to those of their whole-MSC counterparts, thereby indicating
a role of EVs as key mediators of the therapeutic efficacy of
MSCs (5, 6). These nano-sized (50–150 nm in diameter) vesicles, with the potential to transport a wide range of biomolecules over a long distance between cells (7), coupled with
their ability to cross biological barriers (8), have ignited the
light of hope in treating intractable neurodegenerative diseases such as Alzheimer’s disease (AD). In this review, we
outline the current knowledge in the role of EVs in AD treatment. We also discuss the therapeutic perspective of utilizing
MSC-EVs in treating AD as well as the possible challenges of
using EVs as a novel cell-free therapy.
MSCs and their characteristics
Slightly more than half a century ago, scientists discovered
multipotent bone marrow cells. Since then, these cells have
been given many different names (9–11) but the term ‘mesenchymal stem cells’, which was first coined by Arnold Caplan
REVIEW
1
12 MSC-derived EVs in Alzheimer’s disease
in 1991 (12), has remained popular in current everyday use.
Such MSCs can be isolated from various tissues and organs,
such as peripheral blood (13), umbilical cord blood (14),
adipose tissue (15), amniotic fluid (16), placenta (17) and so
forth (18–21); yet, bone marrow remains the most abundant
source of MSCs (22).
The multipotency of MSCs confers them great capacity to
undergo both self-renewal and multilineage differentiation.
Studies have shown that MSCs are able to differentiate into
adipocytes, osteoblasts, chondrocytes (23), muscle cells
(24–26), endothelial cells (27), neurons (28), keratocytes (29),
hepatocytes (30, 31) and pancreatic β-cells (32). In addition to their multilineage differentiation potential, MSCs also
exhibit immunoprivileged properties (33). MSCs are thought
to be non-immunogenic or hypo-immunogenic because they
do not express MHC class II and co-stimulatory molecules
(CD80, CD86 or CD40) (34). In addition, the ability of MSCs to
suppress T lymphocyte activation and proliferation (35, 36),
to impair dendritic cell differentiation and maturation (37, 38)
as well as to inhibit B cell proliferation (39) also contributes to
their immunosuppressive effect.
Homing and migration are other distinctive features of
MSCs. Numerous studies have demonstrated the homing
potential of MSCs to the site of injury while also exerting their
therapeutic effects on the damaged tissues at the same time
(40–42). This homing ability is restricted not only to the site of
injury, but also toward tumor environments (43). Although the
underlying homing mechanisms remain unclear, it has been
postulated to be similar to the concept of leukocyte migration
during inflammation (44).
The therapeutic potential of MSCs
Over the past few decades, tissue regeneration and therapeutic gene delivery have received much research and clinical interest. These new generations of cellular therapy hold
the promise of treating a wide range of diseases including
both genetic and acquired diseases, leading to an improved
quality of life.
MSCs, with their distinctive properties of differentiation
potential as well as the homing and migration abilities, are
ideal candidates for use in regenerative medicine (45, 46).
The emerging evidence on the immunomodulatory properties
of MSCs has made them even more attractive for use in tissue and organ repair as the rejection of transplanted cells in
recipients has always been an issue in transplantation (47).
For instance, there is growing enthusiasm among researchers to evaluate MSCs as an alternative approach for treating myocardial infarction (MI) and osteogenesis imperfecta
(OI) owing to their myogenic and osteogenic differentiation
potential, respectively (34, 48, 49). The therapeutic potential
of MSCs in the repair of other tissues has also been demonstrated in multiple independent studies. Currently, extensive
research and clinical trials are underway to exploit MSCs as
a tool to treat diseases including type-1 diabetes (50), neurological diseases (51) and kidney disease (52).
On top of that, MSCs are also highly favored in gene therapy, a powerful therapeutic approach that may offer a cure
for severe diseases refractory to conventional treatment (53).
Cancer is the most extensively evaluated disorder for gene
therapy applications. The standard treatments for cancer
such as surgery, radiotherapy and chemotherapy, are often
highly invasive and less effective as a result of cancer recurrence and metastasis. MSCs can be readily transduced with
most of vectors and can efficiently express and secrete the
therapeutic agents, thus contributing to their versatility as the
most promising cellular vehicles in gene delivery (54–56).
Moreover, the tumor-homing ability of MSCs, which allows
them to specifically deliver anti-cancer agents to the target
sites, is likely to make the cells more effective in treating
both isolated tumors and metastatic diseases (57). Several
research groups have reported that genetically engineered
MSCs show significant anti-cancer effects in various animal
tumor models (58–60).
Despite the promising results in animal models that have
opened up the path of novel therapeutic strategies for cancers with the use of genetically engineered MSCs, concerns have been raised against these cells for their recently
reported tumorigenic roles. There are conflicting reports in
terms of the effect of MSCs in tumor progression. Several
studies showed that MSCs exert inhibitory effects on tumor
progression by increasing inflammatory infiltration, inhibiting
angiogenesis and suppressing cellular signaling. However,
there is also plentiful evidence supporting the role of MSCs
in promoting tumor growth. For example, MSCs have been
shown to migrate to the tumor site and integrate into tumorassociated stroma. Cancer cell-derived cytokines have also
been shown to induce MSCs to secrete soluble factors, which
then promote tumor growth in a paracrine manner (61–64).
In addition, differentiation of MSCs into endothelial cells and
pericytes also contributed to tumor angiogenesis (65). This
cross-talk between cancer, MSCs and other neighboring cells
is believed to create a tumor niche that enables tumor progression, invasion and metastasis. To date, the reason for the
contradictory role of MSCs in tumor growth remains unknown.
In spite of the presence of ambiguity of the exact role
of MSCs, a decent amount of research that delves into its
beneficial role is still going on. It was initially thought that
the physically close proximity of transplanted MSCs and
their subsequent differentiation or gene expression contributed to the substantial therapeutic effects mentioned above.
However, it has recently been suggested that the mechanism
underlying the therapeutic efficacy of MSCs could be executed by paracrine effects. This hypothesis stemmed from
the findings of studies that suggest secreted factors in MSCderived conditioned medium, rather than cell incorporation,
could be accountable for the significant therapeutic impact,
in both in vitro and in vivo settings (66–68).
In addition to these studies, Gnecchi et al. (69) have successfully demonstrated that the intramyocardial injection of
conditioned medium from Akt-overexpressing MSCs reduces
the infarct size in an acute MI (AMI)-rodent model and, more
importantly, this effect is found to be similar as that seen in
the animals injected with the parental cells. Furthermore,
the observation of transplanted MSCs being trapped in a
wide variety of organs, instead of the target side, also supports the notion that the therapeutic efficacy of MSCs is not
solely dependent on cell engraftment (70). MSCs were also
reported to secrete large amount of EVs, leading to considerable attention being paid to investigate the possible role of
MSC-derived EVs in Alzheimer’s disease 13
this lipid membrane vesicle as an active therapeutic factor
secreted by MSCs.
Extracellular vesicles
EVs were described in the 1980s by Pan and Johnstone
(71). These vesicles can be categorized into different subtypes, mainly based on their origin of biogenesis. Presently,
exosomes are the most well characterized and widely studied EVs. In general, they are defined as nano-sized vesicles
ranging from 50 to 150 nm in diameter and have a floating
density of 1.10–1.18 g ml−1 on a sucrose density gradient.
Exosomes are generated from the inward budding of late
endocytic compartments, named as multivesicular bodies
(MVBs), and are subsequently released to the extracellular
milieu as membranous vesicles upon fusion with the plasma
membrane. This endows them with a plasma membrane
similar to that of their parental cells and enriches them with
lipid rafts of cholesterol, sphingomyelin and ceramide (72–
74). Despite being classified according to their biogenesis,
the precise terminology for membranous vesicles is often
ambiguous and the terms ‘extracellular vesicles’, ‘exosomes’,
‘microvesicles’, ‘shedding vesicles’, etc. are used interchangeably (75). To avoid confusion, throughout this review
we will use the term ‘extracellular vesicles’ to represent all
secreted vesicles in the extracellular space.
After many years of intense research, EVs are no longer
being viewed as disposal vehicles with the sole function of
eliminating unwanted proteins and biomolecules. They are
increasingly recognized as important messengers in facilitating intercellular communication. It has been known that
nearly all cell types are capable of secreting EVs. Owing to
their stable lipid bilayer membrane structure and the ability
to traffick in biological fluids, EVs are capable of transporting and transferring bioactive molecules (proteins and RNAs)
between cells (7). Through this exchange of proteins and
genetic materials, they have been reported not only to function in normal physiological processes (76, 77), but also to
be associated with the pathogenesis of various diseases and
cancers (78–80).
EVs are also found to play an essential role in cell–cell communication in the central nervous system (CNS) (81). Taken
together with their ability to cross the blood–brain barrier
(BBB) (8), these vesicles have come into the limelight as a
potential therapeutic tool for the treatment of neurodegenerative diseases such as AD.
Association of EVs with AD
Pathogenic/detrimental role of EVs in AD
AD is the most common type of dementia, accounting for
up to 75% of all cases. It is a progressive neurodegenerative disease characterized by the overproduction and/or
impaired clearance of beta-amyloid peptide (Aβ) in the brain.
The deposition of Aβ has been suggested to be the causative
factor for the destruction and death of neurons, leading to the
gradual erosion of memory and subtle decline in cognitive
ability (82–84).
EV production in the brain has been suggested to
contribute to the pathogenesis and progression of AD.
The association between EVs and AD was first raised from
the finding that showed the increment and accumulation of
Aβ in MVBs within the nerve terminals of AD transgenic mice
and the human brain (85). It was then further described by
Rajendran et al. (86) that the intracellularly accumulated Aβ
in MVBs is routed to the extracellular environment via EVs as
a result of fusion with the plasma membrane. In addition, they
observed that neutritic plaques in the hippocampal section
of AD patients are enriched with EV marker, Alix. This phenomenon was not observed in the hippocampus of a normal
control brain, indicating the correlation between the secretion
of Aβ and EVs (86).
Since then, research interest on EVs in the pathogenesis of
AD has increased, with the aim of identifying the role of EVs
in the metabolism of Aβ as well as the effect of Aβ-associated
EVs on neuronal functions through their interaction with the
resident cells in the brain. The aggregation of Aβ has been
shown to be dependent on EV secretion. The inhibition of
neutral sphingomyelinase 2 (nSMase2), which regulates EV
biosynthesis, prevents the secretion of ceramide and EVs,
thus leading to the reduction of amyloid plaque formation in
the brains of an AD mouse model (87). In contrast, ceramidetreated mice, compared with the controls, show increased
levels of serum EVs and enhanced amyloid plaque formation
(88), suggesting that EVs are associated with the metabolism
of Aβ in AD.
Non-neuronal cell-derived EVs have also been reported to
exert a pathogenic role in AD. EVs secreted from microglia
were shown to promote the formation of neurotoxic Aβ and
act as a potent driver in damaging neuronal cells (89). This
is, however, contradictory to the previously reported physiological function of microglia in controlling extracellular Aβ
aggregation via phagocytosis and degradation. The authors
suggested that the secretion of neurotoxic Aβ-carrying EVs
from microglia might occur when the intracellular pathway of
Aβ degradation becomes saturated. Therefore, the release
of EVs to their extracellular environment is the only way for
microglia to eliminate un-degraded Aβ. Although the precise mechanism is yet to be defined, previous studies have
reported the re-secretion of phagocytosed Aβ from microglia
(90).
Aβ is well known for its toxicity to neurons, but little is
known about its effect on non-neuronal cells, such as astrocytes, which play a supportive role for neurons in the CNS.
The dysfunction or death of astrocytes might have implications in neurons and their functions, consequently contributing to neurodegeneration in AD. A study done by Wang et al.
has shown that treatment of Aβ triggers the secretion of EVs
enriched with ceramide and prostate apoptosis response 4
(PAR4) from astrocytes via an nSMase2-dependent pathway,
which exerts a pro-apoptotic effect on astrocytes in the mice
(91).
Tau, another AD-related protein, was also reported to be
associated with EV secretion. In the study, Asai et al. (92)
speculated that microglia phagocytosed Tau aggregates and
facilitated the transfer of EV-bounded Tau to neurons via exocytosis. The authors emphasized the importance of microglia
and nSMase2 activity in the propagation of Tau protein and
neurotoxicity in the brain. Depletion of microglia in two different tauopathy mouse models via intracerebroventricular
14 MSC-derived EVs in Alzheimer’s disease
infusion of clodronate liposome or PLX3397 treatment, as well
as inhibition of EV synthesis through silencing of nSMase2
expression or inhibiting nSMase activity showed remarkable
reduction of Tau contents in EVs as compared with the control
group.
Neuroprotective role of EVs in AD
There are two sides to every coin—although the role of EVs
in the pathogenesis of AD has been proposed, accumulating
evidence shows that EVs might also play a beneficial role in
the clearance of Aβ in the AD brain. Yuyama et al. (93) have
conducted an elegant study on the neuroprotective functions
of EVs, highlighting a novel mechanism of Aβ clearance by
microglia in association with neuronal EVs. They demonstrated that (i) sphingolipid-metabolizing enzyme is essential
for the secretion of EVs from neurons; (ii) these extracellular
neuronal-EVs further promote Aβ amyloidgenesis through
glycosphingolipids (GSLs) on their surface; and (iii) EV–Aβ
fibril complexes are subsequently internalized by microglia
and transported to lysosomes for degradation (93).
The suggested machinery was further validated in their
subsequent in vivo study (94). Indeed, in AD transgenic
mice, intracerebrally injected neuroblastoma-derived EVs
were shown to bind to Aβ and subsequently be incorporated
into microglia. Interestingly, the continuous injection of exogenous EVs shows a marked reduction of Aβ deposition and
Aβ-associated synaptotoxicity in the animals. This finding
suggests the rapid clearance of EV-bounded Aβ by microglia
before the formation of Aβ deposits (94). Nevertheless, the
authors also acknowledged the importance of normal phagocytic activity of microglia in this EV-mediated Aβ clearance.
Without the normal function of microglia, the free EV-bounded
Aβ protein could resume a pathological role in plaque formation or even the onwards dissemination of the protein.
In addition to the EV-mediated Aβ clearance by microglia,
the biomolecules contained in naive EVs might also contribute to their protective effect in AD. In a previous study, we
have observed that EVs secreted from human adipose tissue
derived-MSCs (ADSCs) are enriched with neprilysin (NEP), an
Aβ-degrading enzyme in the brain. Co-culture of ADSCs with
a genetically modified neuroblastoma cell line that overproduced human Aβ (N2a cells) shows a significant decrease
of both extracellular Aβ and intracellular Aβ in N2a cells. We
suggest that NEP-enriched EVs are released from ADSCs and
incorporated into N2a cells, thus leading to the decreased
level of intracellular Aβ observed in the recipient cells (95).
The neuroprotective effect of NEP in the stem cell
secretome is further supported by a study done by Ahmed
et al., in which functional NEP was detected in the dental
pulp stem cell (DPSC) secretome and was shown to degrade
Aβ1–42 and reduced Aβ-induced toxicity on neuroblastoma
cells (SH-SY5Y) in vitro. The expression of NEP in DPSCs
was reported to be higher than in both BMSCs and ADSCs
(96). Although concrete evidence for either the pathogenic
or seemingly favourable aspects of EVs in AD remains elusive, recent findings have nonetheless helped to improve the
current understanding of the mechanisms of AD onset and
facilitate the subsequent development of potential therapeutic approaches.
Therapeutic perspectives of MSC-EVs in the treatment
of AD
Most of the drugs currently available for the treatment of AD
in the market merely alleviate the manifestation of the disease and slow down its progression. These drugs are by no
means able to reverse or cure the disease. Therefore, there
is a pressing need to develop a disease-modifying treatment
that could directly tackle the pathogenic process of the disease, with the hope not only to completely stop the disease
progression, but also to restore the function of the damaged
brain. In this context, EVs could be exploited as useful natural
vehicles for therapeutic delivery of a wide variety of potential
disease-modifying molecules, owing to their possible ability to cross the BBB for horizontal transferring of biologically
active molecules between cells (97).
From the clinical and manufacturing point of view, we propose the use of MSCs as the cellular source in producing EVs
for therapeutic application in AD treatment. The availability of
MSCs in a wide range of tissues and the ease of their isolation, as well as their large capacity in in vitro propagation (98)
have made these cells highly desirable as potential producers of EVs (Fig. 1). Such EVs, on top of sharing a conserved
set of proteins, are also known to acquire donor-derived properties (99, 100). Proteomic analysis of MSC-EVs has shown
consistent immune tolerance properties similar to those of
MSCs (101, 102), further adding their value as allogenic and
autologous natural delivery vehicles. In addition, MSCs are
the preferred producers of EVs, owing to the amenability of
these cells to gene modification, thereby conferring them the
ability to produce EVs that are enriched with desired therapeutic factors (103, 104).
Most importantly, MSC-EVs have shown encouraging therapeutic effects in a wide spectrum of tissue injuries, such as
those in the liver, lungs, kidneys, heart and brain [Reviewed in
refs. 105, 106]. The first report describing the potential therapeutic use of MSC-EVs was in an acute kidney injury (AKI)
mouse model. The EVs derived from MSCs have been demonstrated to induce the proliferation of tubular cells, thereby
promoting renal functional and morphologic recovery in the
mice (6). Moreover, in brain injuries, systemic administration of MSC-EVs has been reported to significantly improve
the neurologic outcome in a rat model of stroke (107). The
MSC-EVs were shown to induce neurite outgrowth, an effect
believed to be mediated through the transfer of miR-133b
from MSCs to neurons via secreted EVs in both in vitro and
in vivo settings (104, 108). Altogether, the evidence suggests
an optimistic outlook for utilizing MSC-EVs as a cell-free therapy in treating AD.
To further pursue the development of MSC-EVs as a therapeutic delivery vehicle to treat AD, MSCs can be genetically
modified to enhance the therapeutic effect of EVs, by employing the reduction of the Aβ level as the core strategy (Fig. 2).
There are two ways to decrease Aβ levels in the brain: (i)
termination of Aβ peptide production; and (ii) promotion of
the clearance of accumulated Aβ peptides, for example by
increasing degradation of Aβ peptides. Neurotoxic Aβ peptides are derived from the cleavage of Aβ precursor protein
(APP) by β- and γ-secretases (109–111). These enzymes
are therefore attractive targets that can effectively block the
MSC-derived EVs in Alzheimer’s disease 15
Fig. 1. Therapeutic effects of MSCs in tissue repair. MSCs exert their therapeutic effects in tissue repair through various mechanisms, including (i) transfer of therapeutic biomolecules via EVs; (ii) differentiation of MSCs into multiple cell types followed by engraftment at the side of the
damaged tissue; and (iii) secretion of bioactive trophic factors that promote tissue repair.
Fig. 2. Schematic representation of the proposed therapeutic strategy of MSC-derived EVs in AD. MSCs can be genetically modified to produce EVs that are enriched with therapeutic factors, for example through transfection of siRNAs that target secretase enzymes or through
over-expression of Aβ-degrading enzymes. It is optional to enhance specific delivery to the brain by modification of the EV membrane. Purified
MSC-derived EVs can be administered into patients via the intranasal route, thereby exerting their therapeutic effect either by inhibition of the
production of neurotoxic Aβ-peptides or by degradation of soluble Aβ-peptides.
production of Aβ through inhibition of their proteolytic activities. In this context, MSCs can be genetically modified to
secrete EVs enriched with therapeutic factors, such as small
interfering RNA (siRNAs) that target β- and γ-secretase
enzymes.
This principle of EV-mediated siRNA delivery to the brain
has recently been confirmed by Alvarez-Erviti et al. (97)
The authors showed that the delivery of β-site APP-cleaving
enzyme 1 (BACE-1) siRNA, which targets β-secretase,
resulted in 60% of BACE-1 gene knockdown, thus leading
to a 55% reduction of Aβ level in the animals (97). Based on
this proof of concept study, siRNA targeting of γ-secretase
could also be encapsulated into MSCs and delivered to the
brain region via MSC-EVs. Indeed, γ-secretase inhibitors
have been put forward for consideration as a treatment for
AD since their effects in reducing Aβ production have been
demonstrated in APP-overexpressing transgenic mice (112).
However, γ-secretase is also reported to be involved in
other signaling pathways such as Notch signaling, which is
essential for cell development and differentiation, thereby raising concerns about potential side-effects that may result from
the affected Notch signaling (113). Thus, before γ-secretase
can be successfully utilized as a therapeutic target, the differential mechanism of γ-secretase function in APP cleavage
and the Notch signaling pathway must be clearly elucidated
in order to develop γ-secretase inhibitors that specifically
16 MSC-derived EVs in Alzheimer’s disease
affect APP and that simultaneously spare the Notch signaling
(114). On the other hand, transfer of Aβ-degrading enzymes
via EVs to the brain may also possibly improve the clearance
of Aβ and therefore impede the progress of AD. For instance,
the Aβ-degrading enzymes, insulin-degrading enzyme (IDE)
and NEP, which have been shown to reduce Aβ levels (115),
could be selectively enriched in EVs through overexpression
in the parental MSCs.
Apart from that, to maximize the therapeutic outcome of
MSC-EVs, enhancing delivery specificity and minimizing
undesirable off-target effects are also essential points to
be pondered upon. These goals may be achieved through
the modification of the EV membrane, thereby augmenting
the docking of EVs to neuron-specific receptors or ligands.
Successful delivery to the brain has been demonstrated by
targeting the acetylcholine receptor through fusion of EV
membrane protein, Lamp2b, with the neuron-specific RVG
peptide (97).
Instead of docking to neurons, specific delivery of EVs can
also be achieved through targeting non-neuronal resident
cells in the brain. For example, the fusion of EV membrane
proteins to peptides that recognize the gap junction protein,
connexin 43, which is highly expressed in astrocytes (116),
might enhance the tropism of EVs toward the brain (117,
118). In essence, it is worthy to delve into the search for other
new cell-specific ligand targets that can precisely guide EV
migration toward the desired destination. Nonetheless, determining the optimal administration method of EVs, one of the
success-limiting steps, is crucial to maximize the efficacy of
EV-mediated treatment. For example, intra-nasal administration has been shown to rapidly deliver EVs to the brain,
and the repeated administration of these vesicles continued
to exerts a therapeutic effect without causing any toxicity or
behavioral abnormalities in the mice (8).
Challenges in the clinical application of MSC-EVs
Large-scale production of clinically compliant MSCs for the
generation of EVs is by all means necessary in translating
MSC-derived EVs into therapeutic reality. The feasibility of
large-scale production has been demonstrated by Chen et al.
(119) on the basis of their work that successfully immortalised
human embryonic stem cell-derived MSCs (hESC-MSCs) by
overexpressing the MYC gene through lentivirus transduction. Although this appears to be an infinite source for EV
production, the slight phenotypic changes in the transformed
cells observed in the study, as well as other safety issues that
are likely to arise from any unidentified effects of MYC overexpression, should not be overlooked. Therefore, other potential
methods to enhance the production of EVs from MSCs, such
as the overexpression of nSMase2 and Rab family members
(Rab27a, Rab27b and Rab35) may be further explored.
Moreover, the precise content of MSC-EVs remains largely
unknown because these vesicles contain many molecules
that are yet to be identified. This may lead to the possible
concurrent transport of these unidentified endogenous biomolecules together with the exogenous therapeutic agent to
the recipient cells via MSC-EVs, thus causing unwanted side
effects. Therefore, a more in-depth and rigorous characterization of the contents of these vesicles is needed to ensure
their safety. Last, but not least, prior to clinical application, it is
also crucial to examine the optimal dosage of MSC-EVs and
the effect of their repeated administration to achieve the best
therapeutic efficiency while minimizing undesired toxicity and
other adverse effects.
Conclusions
Thus, the development of genetically modified MSC-EVs
might open a new horizon for therapeutic strategies in AD and
other neurodegenerative diseases. The MSC-EVs are superior
to cell-based therapy in terms of safety as there is no involvement of cells that could potentially elicit unknown side-effects.
However, its seemingly promising therapeutic potential
should not overshadow the unaddressed issues, particularly
because research in this field is still in its infancy. Hence, additional studies are needed to meet these challenges in order to
improve the therapeutic intervention for AD treatment.
Funding
This work was financially supported in part by Developing
Key Evaluation Technology: Manufacturing Technology for
Industrialization in the Field of Regenerative Medicine by
Japan Agency for Medical Research and Development, a
Grant-in-Aid for the Japan Science and Technology Agency
(JST) through the Center of Open Innovation Network for
Smart Health (COINS) initiated by the Council for Science,
by a Grant-in-Aid for Basic Science and Platform Technology
Program for Innovative Biological Medicine and by a Project
for Cancer Research and Therapeutic Evolution (P-CREATE).
Conflict of interest statement: The authors declare that they have no
conflict of interest.
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