Cells: Mesenchymal Stem For Immunomodulatory Therapeutics and Skin Regeneration

cells
Review
Mesenchymal Stem/Stromal Cell-Derived Exosomes
for Immunomodulatory Therapeutics and
Skin Regeneration
Dae Hyun Ha 1,† , Hyun-keun Kim 1,† , Joon Lee 2 , Hyuck Hoon Kwon 3 , Gyeong-Hun Park 4 ,
Steve Hoseong Yang 5 , Jae Yoon Jung 6 , Hosung Choi 7 , Jun Ho Lee 1 , Sumi Sung 1 ,
Yong Weon Yi 1, * and Byong Seung Cho 1, *
1 ExoCoBio Exosome Institute (EEI), ExoCoBio Inc., Seoul 08594, Korea; dh.ha@exocobio.com (D.H.H.);
hyunkeun.kim@exocobio.com (H.-k.K.); junho.lee@exocobio.com (J.H.L.); sumi.sung@exocobio.com (S.S.)
2 School of Chemical and Biological Engineering, Seoul National University, Seoul 08826, Korea;
purequill@naver.com
3 Oaro Dermatology Clinic, Seoul 13620, Korea; banbury@hanmail.net
4 Department of Dermatology, Dongtan Sacred Heart Hospital, Hallym University College of Medicine,
Hwasweong-si, Gyeonggi-do 18450, Korea; jin66666@hanmail.net
5 Guam Dermatology Institute, Tamuning, GU 96913, USA; guamderm@gmail.com
6 Oaro Dermatology Clinic, Seoul 01695, Korea; jaeyoon007@hanmail.net
7 Piena Clinic, Seoul 06120, Korea; hosungeee@naver.com
* Correspondence: yongweon.yi@exocobio.com (Y.W.Y.); ceo@exocobio.com (B.S.C.);
Tel.: +82-2-2038-3915 (B.S.C.)
† These authors contributed equally to this article.

Received: 20 February 2020; Accepted: 4 May 2020; Published: 7 May 2020
Abstract: Exosomes are nano-sized vesicles that serve as mediators for cell-to-cell communication.
With their unique nucleic acids, proteins, and lipids cargo compositions that reflect the characteristics
of producer cells, exosomes can be utilized as cell-free therapeutics. Among exosomes derived from
various cellular origins, mesenchymal stem cell-derived exosomes (MSC-exosomes) have gained
great attention due to their immunomodulatory and regenerative functions. Indeed, many studies
have shown anti-inflammatory, anti-aging and wound healing effects of MSC-exosomes in various
in vitro and in vivo models. In addition, recent advances in the field of exosome biology have enabled
development of specific guidelines and quality control methods, which will ultimately lead to clinical
application of exosomes. This review highlights recent studies that investigate therapeutic potential
of MSC-exosomes and relevant mode of actions for skin diseases, as well as quality control measures
required for development of exosome-derived therapeutics.
Keywords: anti-aging; anti-inflammation; hair growth; immunomodulation; mesenchymal stem cells

(MSCs); MSC-exosomes; skin barrier; therapeutics; regenerative aesthetics; wound healing
1. Introduction
The discovery of extracellular vesicles (EVs) or exosomes goes back to the 1940s, and these
tiny vesicles were ignored as cellular garbage bins for a long time [1–3]. They only began to draw
significant attention around the mid-2000s after re-discovery of exosomes as messengers for cell-to-cell
communications [1,4–6]. It is no exaggeration to say that we are at the dawn of the exosome era.
There were more than three thousand publications on EVs or exosomes and related subjects in PubMed
annually in 2018 and 2019 [1]. The race toward commercialization of exosome-based therapeutics
has already begun [7–10]. The top four exosome start-up companies, Codiak Biosciences, Exosome
Cells 2020, 9, 1157; doi:10.3390/cells9051157 www.mdpi.com/journal/cells

Cells 2020, 9, 1157 2 of 45
Diagnostics, Evox Therapeutics, and ExoCoBio have received approximately $386.2 million in investor
funding [8]. In addition, several big deals have been made between exosome start-ups and big pharma
companies [10].
Exosomes are nano-sized extracellular vesicles (EVs) released by almost all eukaryotic cells [11].
In general, their size ranges from 30 nM to 200 nM. Two other subpopulations of EVs are microvesicles
(100–1000 nM) and apoptotic bodies (500–2000 nM) [12–14]. Exosomes derived from stem cells have
attractive therapeutic potential in several aspects [15]. It has been established that the mode of action
(MoA) for therapeutic effects of stem cells is mainly paracrine effects mediated by secreted factors from
stem cells [6,16]. Among parts of the secretome of stem cells, exosomes have been reported to play
the major role in the paracrine effects [16–18]. Mesenchymal stem/stromal cells (MSCs) are the most
preferable source of therapeutic exosomes, since MSCs themselves appear to be safe based on huge
amount of clinical data over the last decade [15]. In addition, MSC-derived exosomes (MSC-exosomes)
can be sterilized by filtration and produced as an off-the-shelf product, while MSCs themselves cannot.
Moreover, MSC-exosomes are considered to be free from the safety issues in the context of cell-based
therapy, such as tumorigenic potential by cell administration [19,20]. Indeed, MSC-exosomes have
been applied as alternatives to MSCs for new cell-free therapeutic strategies in a variety of disease
models including neurological, cardiovascular, immune, renal, musculoskeletal, liver, respiratory, eye,
and skin diseases, as well as cancers [15,17,19,21,22].
2. MSCs as Sources of Exosomes

MSCs have both self-renewal capabilities (i.e., they can generate more MSCs themselves) and
differentiation (into other types of cells) potentials [23]. MSCs can be obtained from a range of
tissues and body fluids, such as adipose tissue, bone marrow (BM), dental pulp, synovial fluid (SF),
amniotic fluid (AF), placenta (PL), umbilical cord (UC), umbilical cord blood (UCB), and Wharton’s
jelly (WJ) [24]. MSCs can also be derived from embryonic stem cells (ESCs) or induced pluripotent
stem cells (iPSCs) [25–27]. MSCs, depending on their origins, are able to differentiate into diverse
types of cells including adipocytes, chondrocytes, osteoblasts, and myocytes [28]. In addition, MSCs
have immunomodulatory properties to regulate various cells involved in immune responses, such
as dendritic cells (DCs), lymphocytes, macrophages, mast cells, neutrophils, and natural killer (NK)
cells [24]. On these bases, MSCs have been spotlighted as potent cell therapeutics for various diseases
over the last decades.
In the reported preclinical studies of MSC-exosomes, MSCs were isolated from various tissues/cells
in the following order: BM (51%), umbilical/placental tissues (23%), adipose tissue (13%), derived from
ESCs or iPSCs (8%), and others (5%) [29]. Since characteristics and functionality of MSCs depend on
their origins, it is obvious that those of MSC-exosomes vary according to the origin of MSCs. However,
comparative studies of MSC-exosomes by their tissue origin are still limited, and only a few reports
have compared different MSC-exosomes within the same study (Table 1) [30–35]: (1) human adipose
tissue-derived MSC (ASC)-exosomes exhibited a higher activity of neprilysin, an amyloid β (Aβ) peptide
degrading enzyme in the brain, than human bone marrow MSC (BM-MSC)-exosomes, suggesting the
therapeutic relevance of ASC-exosomes in Alzheimer’s disease [30]; (2) human BM-MSC-EVs and
Wharton’s jelly MSC (WJ-MSC)-EVs decreased cell proliferation and induced apoptosis, while ASC-EVs
increased cell proliferation and had no apoptotic effect in U87MG glioblastoma cells [31]. However,
the effects of MSC-exosomes on cancer cells are controversial [36]. For example, ASC-exosomes have
been reported to have anti-cancer activity on prostate cancer both in vitro and in vivo [37]; (3) human
menstrual fluid MSC (MenSC)-exosomes and BM-MSC-exosomes promoted neurite growth both
in cortical and sensory neurons, while human chorion MSC-exosomes and UC-MSC-exosomes did
not. This suggests that appropriate selection of MSC sources might be essential for the treatment of
neurodegenerative diseases [32]; (4) human iPSC MSC (iMSC)-exosomes and synovial membrane
MSC (SM-MSC)-exosomes both attenuated osteoarthritis (OA) in a murine model, but iMSC-exosomes
had a superior therapeutic effect compared to SM-MSC-exosomes [33]; (5) a study comparing
Cells 2020, 9, 1157 3 of 45
canine MSCs reported that BM-MSCs released a higher level of secretome, including exosomes,
than ASCs did [34]; and (6) human amniotic fluid MSCs (AF-MSCs) released a higher amount of
exosomes than BM-MSCs [35]. However, it is difficult to directly compare the results between the
above studies, since they were not performed with comparable processes or methods for isolation,
characterization, and efficacy evaluation for exosomes. In addition, variations from different donors or
preparation methods for MSCs remain a prominent challenge [38,39]. Nevertheless, it is suggested
that MSC-exosomes might exhibit different properties and efficacies depending on the origin of MSCs.
Therefore, biological differences such as the origin of MSCs and efficacy of their exosomes should be
considered for specific clinical applications.
Cells 2020, 9, 1157 4 of 45
Table 1. Mesenchymal stem cell (MSC)-exosomes from different sources.
Diseases/Focuses Nomenclature Exosome Isolation MSC Origin Outcome Reference
Human adipose tissue Adipose stem cell (ASC)-exosomes had superior effects compared to
Alzheimer’s disease Exosomes Ultracentrifugation bone marrow (BM)-MSC-exosomes [30]
Human bone marrow Decreased Aβ peptide in the N2a cells
Human bone marrow Decreased U87MG cell proliferation

Extracellular Vesicles
Glioblastoma Ultrafiltration Induced apoptosis in the U87MG cells [31]
(EVs) Human Wharton’s jelly
Increased U87MG cell proliferation
Human adipose tissue
No apoptotic effect
Human menstrual fluid
Promoted neurite outgrowth in cortical and sensory neurons
Neurodegenerative Ultracentrifugation Human bone marrow
Exosomes
disease [32]
Human chorion
No effect
Human umbilical cord
Attenuated OA in a murine model

Human iPSCs Stimulated chondrocyte migration and proliferation
Osteoarthritis (OA) Exosomes Ultrafiltration Induced pluripotent stem cell-derived MSC (iMSC)-exosomes exert [33]
superior therapeutic effects compared to synovial membrane
Human synovial (SM)-MSC-exosomes
membrane
Canine bone marrow
Exosomes Ultracentrifugation BM-MSCs released higher amount of exosome compared to ASCs [34]
Exosome release Canine adipose tissue
Total Exosome Isolation Kit Human amniotic fluid Amniotic fluid (AF)-MSCs released higher amount of exosome
Exosomes [35]
(Invitrogen) compared to BM-MSCs
Human bone marrow
Abbreviations: AF, amniotic fluid; ASC, adipose stem cell; BM, bone marrow; EVs, extracellular vesicles; iMSC, induced pluripotent stem cell-derived MSC; OA, osteoarthritis.
Cells 2020, 9, 1157 5 of 45
3. Quality Control of EVs for Development of Therapeutic EVs

It is of importance to manufacture clinical-grade EVs with a good manufacturing practice
(GMP)-compliant process and quality control (QC) for the development of EV-based therapeutics [40–42].
Appropriate QC is also crucial for reproducible studies in academic settings. Recently, the International
Society for Extracellular Vesicles (ISEV) proposed a series of the Minimal Information for Studies of
Extracellular Vesicles (MISEV), finalized as MISEV2018 [43–45]. The Korea Ministry of Food and Drug
Safety (MFDS) published the world’s first guideline for EV therapy products, entitled the Guideline
on Quality, Non-clinical, and Clinical Assessment of Extracellular Vesicles Therapy Products [46].
As shown in Table 2, most of the criteria in these guidelines are similar [1] and have been already been
applied in GMP settings [42,47,48]. Routine QC criteria include the determination of the quantity, size,
identity, and purity of EVs.
Table 2. Quality control (QC) criteria in the guidelines and good manufacturing practice (GMP) settings.
Examples in Guidelines Examples in GMP Settings

QC Criteria
ISEV Recommendation MFDS Guideline (2018) Pachler et al.
Andriolo et al. [48] Mendt et al. [42]
[43–45] [46] [47]
Particle number by NTA,
Particle number by NTA
high-resolution FCM (ZetaVeiw NTA) (NanoSight NTA) (NanoSight NTA)
or compatible methods 1
RPS, cryo-EM, AFM, etc.
Total protein amount 2 - (BCA assay) (microBCA assay)
Exosome
Quantity Total lipid amount - - - -
Total RNA amount - - - -
Quantification of specific
- - TSG101 ELISA -
molecules
NTA NTA 1
- DLS 1 - - -
RPS RPS 1 - - -
Exosome Size High-resolution FCM - - - -
AF4 - - - -
- - - -
FCS FCS 1 - - -
(FCM: CD47, CD63,
(WB: CD9, CD81, (FCM: CD9, CD63, CD81
Proteins Proteins CD81, CD9, CD29,
TSG101) ELISA: TSG101)
Identity CD90)
Phospholipids Lipids - - -
Nucleic acids RNAs - - -
Ratio of protein:particle - - - -
Ratio of lipids:particle - - - -
Ratio of lipids:protein - - - -
Purity Proteins that are expected Proteins that are expected
not to be enriched not to be enriched (WB: GM130) - -
in exosomes in exosomes
Process impurities
Process impurities (serum
depending on the source - - -
albumin, antibiotics, etc.)
of exosomes
Biological assay, which can Anti-apoptotic activity;
Potency Assays Dose-response assessment - Apoptosis assay
represent MoA Pro-angiogenic activity
Cells 2020, 9, 1157 6 of 45
Table 2. Cont.
Examples in Guidelines Examples in GMP Settings

QC Criteria
ISEV Recommendation MFDS Guideline (2018) Pachler et al.
Andriolo et al. [48] Mendt et al. [42]
[43–45] [46] [47]
Mycoplasma test - - -
Microbiological Control
Sterility test - -
Others Not mentioned for Cellular Products
Endotoxin test - Quantitative LAL test -
Adventitious virus test - - -
1 Since these methods cannot differentiate EVs from non-EV particles, it is recommended to compare results from
these methods with results from TEM, AFM, or other microscopic observation. 2 Comparison with results from
quantification methods such as protein quantification is also recommended. Abbreviations: AF4, multi-angle
light scattering coupled to asymmetric flow field-flow fractionation; AFM, atomic force microscopy; DLS, dynamic
light scattering; FCM, flow cytometry; FCS, florescence correlation spectroscopy; ISEV, International Society for
Extracellular Vesicles; LAL, limulus amebocyte lysate; MoA, mode of action; MFDS, Ministry of Food and Drug
Safety; NTA, nanoparticle tracking analysis; RPS, resistive pulse sensing; WB, Western blotting.
3.1. EV Quantity and Size

Both the MISEV2018 and the MFDS guidelines recommend using at least two different methods
for determining the quantity of EVs [45,46]. Quantification of EVs can be achieved by measuring the
total amounts of proteins, lipids, or RNAs, since EVs consist of all these molecules. These methods,
however, do not provide the information on the number of EV particles. Several methods are
available to measure the number and size of particles, including nanoparticle tracking analysis
(NTA), resistive pulse sensing (RPS), and dynamic light scattering (DLS). The most widely used
method is NTA [42,47–53]. NTA determines the number and size of particles by tracking the
Brownian motion of single particles in an aqueous solution [54]. However, NTA suffers from a low
resolution of poly-dispersed samples and high variations, such as inter-device, inter-assay, and intra-
and inter-individual variations [55–57]. In addition, NTA does not differentiate EVs from other
nanoparticles such as protein aggregates. Recently, instruments for fluorescence NTA have been
introduced to detect fluorescently labeled EVs with specific antibodies [58]. Quantification of EVs,
however, remains extremely challenging. New technologies and instruments have been introduced
annually, especially during the ISEV conference, such as nano flow cytometry [59,60], direct stochastic
optical reconstruction microscopy [61], ExoCounter with the optical disc technology [62], and imaging
flow cytometry [63]. Although it will take some time to develop fully GMP-compatible instruments,
the great strides forward in methodologies for the quantification of EVs are expected to result in the
overcoming of current hurdles in the near future.
3.2. EV Identity
A variety of proteins have been reported to be associated with EV, especially exosomes, including
tetraspanins (CD9, CD63, and CD81), Annexins, Flotillin, ALG-2-interacting protein X (Alix), and tumor
susceptibility gene 101 (TSG101) protein [45,64]. Proteins such as CD9, CD63, CD81, TSG101, and Alix
are recommended as specific markers for exosomes since they are known to be highly enriched in
exosomes compared to the originating cells [45,64–66]. In addition, because Alix and TSG101 are
involved in the formation of multivesicular bodies (MVBs), presence of these proteins is essential to
support the endocytic origin of exosomes [43,45,64]. For QC, at least semi-quantitative methods are
recommended to detect these proteins in exosomes [46]. The enzyme-linked immunosorbent assay
(ELISA) and flow cytometric analysis are each suitable for both GMP-compliant facilities and general
academic labs. Although Western blotting has been widely used in the academic labs, this method is
limited by lack of appropriate quantification and method validation [67].
3.3. EV Purity
Purity of EVs is also a critical criterion for QC. A simple method to monitor purity of EVs is
to determine the particle-to-protein, protein-to-lipid, or RNA-to-particle ratios [45]. The absence of
Cells 2020, 9, 1157 7 of 45
intracellular proteins, such as histones, lamin A/C, GRP94 (i.e., HSP90B1), GM130 (i.e., GOLGA2),
and cytochrome C (i.e., CYC1), is another important criterion to determine the purity of EVs or exosomes,
since these proteins are not enriched in exosomes due to their strict cellular localization [43,45].
Impurities from cell culture process including antibiotics and serum should also be analyzed to
monitor the removal of potential hazardous substances [46]. Every batch of EVs should be qualified by
routine QC before being used for therapeutic purposes or functional assays, even in the academic labs,
to ensure reproducibility.
3.4. Potency Assays

Potency assays are the most important QC criterion to predict efficacy of EVs in vivo. Regulatory
authorities such as the US Food and Drug Administration (FDA) recommend using appropriate
potency tests for cellular and gene therapy products [68]. The MISEV2018 and the MFDS guidelines
also recommend including potency assays for EV QC [45,46]. Potency is defined as “the specific ability
or capacity of the product, as indicated by appropriate laboratory tests or by adequately controlled
clinical data obtained through the administration of the product in the manner intended, to effect a
given result” [68]. Many biological and biochemical assays have been reported to demonstrate the
potency of EVs or exosomes [69,70]. Since quantification of EVs remains challenging, establishment
of an appropriate potency assay would be an invaluable tool to monitor batch-to-batch consistency
and determine the dose of EVs [71]. Although ideal potency assays should represent the MoA, it is
difficult to set-up an appropriate potency assay with single biochemical or isolated cell-based assays
due to the difficulty in the identification of single bioactive substances in the complex cargo of EVs.
As an example, it is hard to mimic the complex immune responses in vivo with in vitro cell-based
assays [70–73].
4. Anti-Inflammation and Immunomodulation by MSC-Exosomes

Immune cells secrete soluble factors such as inflammatory cytokines and mediators, which
can contribute in the event of inflammation [74,75]. In particular, pro-inflammatory cytokines,
including tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1β, are mainly produced by
activated macrophages. These cytokines play important roles in the upregulation of inflammatory
responses such as activation of macrophages and recruitment of additional immune cells [74,75].
In contrast, anti-inflammatory cytokines are produced by regulatory T cells (Tregs), helper T (Th)2
cells, alternatively activated macrophages, and monocytes, which control the inflammatory responses
and immunity [75,76]. Major anti-inflammatory cytokines include IL-1 receptor agonist (IL-1RA), IL-4,
IL-10, and transforming growth factor (TGF)-β [76]. These cytokines inhibit the Th1 responses and
production of pro-inflammatory cytokines [76].
Inflammation is a mechanism of innate immunity in response to harmful stimuli, including
pathogens, damaged cells, or irritants, and typically manifests as heat, pain, redness, swelling,
and loss of function [77]. Uncontrolled chronic inflammatory responses are associated with diverse
inflammatory diseases such as allergy, asthma, autoimmune diseases, inflammatory bowel disease (IBD),
OA, atherosclerosis, and hepatitis [77–79]. In addition, many scientists now consider inflammation as
the root cause of most chronic diseases such as heart attacks, strokes, type 2 diabetes, Alzheimer’s
disease, and even cancer [80,81]. Therefore, regulation of inflammation is an important therapeutic
target to treat inflammatory diseases. It has been demonstrated that MSCs have property of intrinsic
immunosuppressive capabilities to alleviate inflammation and immune responses [82]. MSC-exosomes
can be an excellent alternative to MSC cell therapy since MSC-exosomes possess similar biological
functions to the originating cells, while they are more stable and have lower immunogenicity compared
to their originating cells [83]. In fact, anti-inflammatory and immunomodulatory functions of
MSC-exosomes have been extensively reported (Table 3) [21,84–151].
Cells 2020, 9, 1157 8 of 45
Table 3. Anti-inflammatory and immunomodulatory effects of MSC-exosomes.
Category Exosome Source Nomenclature Exosome Isolation Related Exosomal Cargo Secreted Factors or Expressed Genes Affected Immunomodulatory Effects Reference
Human jaw bone Ultracentrifugation Accelerated wound healing in mice
TNF-α ↓
marrow (JM-MSCs) Exosomes ExoQuick miR-223 Induced M2 macrophage polarization [85]
IL-10 ↑
Human BM-MSCs (System Biosciences) (CD206+ macrophage ↑)
Collagen, Il-6, Ccl2, Cd206, Ccl7, Ccl17, Tnfα,
Human JM-MSCs Reduced BPD through macrophage M22
Exosomes Ultracentrifugation - Retnia ↓ [86]
Human BM-MSCs polarization
Arg1 ↑
Alleviated inflammation and enhanced
Human umbilical cord TLR4, p-p65, iNOS ↓ diabetic cutaneous wound healing in rats
Exosomes Ultracentrifugation let-7b [87]
(UC)-MSCs p-STAT3, p-AKT, ARG1 ↑ Induced M2 macrophage polarization
Inhibited TLR4 signaling pathway
Reduced burn-induced inflammation in rats
PureExo TNF-α, IL-1β, TLR4, p65, p-p65 ↓ Reduced neutrophil and macrophage
Human UC-MSCs Exosomes miR-181c [88]
(101Bio) IL-10 ↑ infiltration (MPO+ cell, CD68+ cell ↓)
Inhibited TLR4 signaling pathway
Resolved inflammation and ameliorate
Human menstrual blood iNOS ↓ cutaneous non-healing wounds in
Exosomes Ultracentrifugation - [89]
derived MSCs (MenSCs) ARG1, VEGF ↑ diabetic mice
Macrophage Induced M2 macrophage polarization
polarization Attenuated atherosclerosis in mice
Mouse BM-MSCs Exosomes HPLC let-7 HMGA2, IGF2BP1 ↓ Reduced area of atherosclerotic plaques [90]
Promoted M2 macrophage polarization
Reduced myocardial ischemic-reperfusion
IL-6, iNOS, IL-1 β, IL-6, TNF-α ↓ injury in mice
Mouse BM-MSCs Exosomes Ultracentrifugation miR-182 [91]
ARG1, IL-10, TGF-β ↑ Reduced infarct size and inflammation
Promoted M2 macrophage polarization
IFN-γ, IL-1β, IL-6, TNF-α ↓ Reduced IBD by polarizing M2 macrophage
Human BM-MSCs Exosomes Ultracentrifugation MT2A [92]
IL-10, Lyz1, Defa20, Defa29, Ang4 ↑ in mice
S1P, SphK1, S1PR1 ↑ Reduced cardiac damage in rats
Rat ASCs Exosomes Ultracentrifugation - AGR1, Ym1, TGF-β1, IL-10 ↑ Reduced fibrosis and apoptosis [93]
IL-1β, IL-6, TNF-α, IFN-γ, p65 ↓ Promoted M2 macrophage polarization
Exosome Isolation Kit Increased the expression of M2 macrophage
Human ASCs Exosomes - CD163, ARG1, CD206, STAT6, MafB ↑ [94]
(System Biosciences) markers
Induced M2 macrophage polarization in
ARG1, IL-10, tyrosine hydroxylase ↑ obese mice
Mouse ASCs Exosomes Ultrafiltration STAT3 [95]
TNF-α, IL-12 ↓ ASC-exosome-educated M2 macrophage
promoted WAT beiging
Cells 2020, 9, 1157 9 of 45
Table 3. Cont.
Induced conversion of Th1 into Th2
Reduced differentiation of Th17
ExoQuick TNF-α, IL-1β ↓
Human BM-MSCs Exosomes - Increased the level of Tregs [96]
(System Biosciences) TGF-β ↑
Induced apoptosis of PBMCs and CD3+
T cells
Promoted proliferation and
Human BM-MSCs Exosomes Ultracentrifugation - IL-10, TGF-β ↑ [97]
immune-suppression capacity of Tregs
Induced an increase of Tregs in PBMCs
Human UC-MSCs Exosomes PEG6000 precipitation - IL-10, IDO ↑ [98]
Inhibited proliferation of PBMCs
Induced Tregs through activation of APCs in
Human embryonic stem Tangential flow filtration + TNF-α, IL-1β, IL-6, IL-12p40 ↓
Exosomes EDA-FN the MyD88-dependent manner [99]
cell (ES)-MSCs HPLC IL-10 ↑
T cell regulation Enhanced allogeneic skin graft
IL-17, IFN-γ ↓ Ameliorated autoimmune type 1 diabetes
Mouse ASCs Exosomes Ultracentrifugation - [100]
IL-4, IL-10, TGF-β ↑ mellitus by increasing Tregs in mice
Improved motor skill in the MS mouse
experimental autoimmune
IL-6, IL-12p70, IL-22, IL-17AF ↓
Human BM-MSCs Exosomes Ultracentrifugation - encephalomyelitis model [101]
IDO ↑
Increased Tregs and decreased infiltration
and proliferation of pro-inflammatory T cells
Decreased aminotransferase (ALT), liver
necrotic areas, and apoptosis in Con
Mouse BM-MSCs Exosomes Ultracentrifugation - IL-1, IL-2, IL-4, IL-10, TNF-α, IFN-γ ↓ [102]
A-induced liver injury in mice
Increased Tregs
Size exclusion
UC-MSCs EVs - - Suppressed T cell proliferation [105]
chromatography
MZB1, CXCL8 ↑
B cell regulation Human BM-MSCs Exosomes Ultracentrifugation - Reduced proliferation of T and B cells [106]
IgM ↓
TNF-α, IL-1β ↓ Reduced photoaging of skin in mice
Photoaging Human BM-MSCs Exosomes Ultrafiltration - [107]
TGF-β, CTLA4 ↑ Ameliorated inflammation
Enhanced neovascularization and survival
Skin flap Human ASCs Exosomes Ultracentrifugation - - of the skin flap in rats [108]
Reduced inflammation and apoptosis
Reduced pathological symptoms of AD in
mice
Atopic IgE, IL-4, IL-5, IL-13, IL-17, IL-23, IL-31, IFN-γ, Reduced mast cell infiltration
Human ASCs Exosomes Tangential flow filtration - [20,109]
dermatitis (AD) TNF-α, TSLP ↓ Reduced inflammatory dendritic epidermal
cells
(CD86+/CD206+ cells↓)
Cells 2020, 9, 1157 10 of 45
Table 3. Cont.
Decreased histopathological score of kidney
injury in rats
Reduced the levels of blood urea nitrogen
MDA, HIF1α, NOX2, Caspase 3, BAX, PARP1,
(BUN) and creatinine
MPO, ICAM1, IL-1β, NF-κB ↓
Rat BM-MSCs Exosomes Ultracentrifugation - Reduced the level of oxidative stress [110]
SOD, CAT, GPX, HO-1, BCL2, IL-10, bFGF, HGF,
Increased anti-oxidant status
SOX9, VEGF ↑
Reduced apoptosis and inflammation
Improved regeneration and enhanced
Renal injury angiogenesis
Reduced BUN and creatinine in the mouse
Mouse BM-MSCs Exosomes Ultracentrifugation CCR2 TNF-α, IL-6, IL-1β ↓ IR model [111]
Reduced infiltration of macrophages
PCNA, BCL-XL, BCL2, IL-1β, 4E-BP1 ↑
Reduced cisplatin-induced AKI in rats
Human UC-MSCs Exosomes Ultracentrifugation - Bax, cytochrome C, Caspase-3, p65, TNF-α, IL-6, [112]
Reduced BUN and creatinine
IL-1β, p-mTOR ↓-
Reduced experimental autoimmune uveitis
in rats
Uveitis Human UC-MSCs Exosomes Ultracentrifugation - - [113]
Reduced infiltration of Gr-1+, CD161+,
CD68+ and CD4+ cells in retina
Duchenne
TGF-β, creatine kinase, collagen I, collagen IV, Reduced DMD in mice
muscular
Human Placenta MSCs Exosomes Ultracentrifugation miR-29c TNF-α, IL-6 ↓ Decreased the tissue fibrosis and [114]
dystrophy
Utrophin ↑ inflammation
(DMD)
Improved pathology of lung, cardiac and
Bronchopulmonary brain in neonatal mice with BPD
Human UC-MSCs Exosomes Ultracentrifugation TSG-6 Neutrophil ↓ [115]
dysplasia (BPD) Reduced pulmonary inflammation and
alveolar-capillary leak
Improved cognitive function in transgenic
Alzheimer’s TNF-α, IL-1β, IL-6 ↓ APP/PS1 mice
Mouse BM-MSCs Exosomes Ultracentrifugation - [116]
disease IL-10, IL-4, IL-13 ↑ Reduced plaque deposition and Aβ levels
Reduced activation of astrocytes
Improved neurological impairment (motor
coordination) and long-term neuroprotection
(neuronal survival and cell proliferation) in
Post-stroke stroke mice
Human BM-MSCs EVs PEG6000 precipitation - Dcx, NeuN, CD31 ↑ [117]
neuroregeneration Reduced post-ischemic immunosuppression
and lymphopenia
Stimulated post-ischemic neurogenesis and
angiogenesis
Cells 2020, 9, 1157 11 of 45
Table 3. Cont.
Decreased the threshold for thermal and
Diabetic miR-17 mechanical stimuli in mice
TNF-α, IL-1β, iNOS, TLR4, IRAK1, p65 ↓
peripheral Mouse BM-MSCs Exosomes Ultracentrifugation miR-23a Increased nerve conduction velocity, the [118]
ARG1, IL-10, TGF-β ↑
neuropathy miR-125b number of intraepidermal nerve fibers,
myelin thickness, and axonal diameters
Increased chondrocytes viability under
p-p38, p-ERK ↓
Rabbit BM-MSCs Exosomes Ultracentrifugation - IL-1β-induced inflammatory status through [119]
p-AKT ↑
activating AKT pathway
Promoted repair and regeneration of
α-SMA, MMP-13, IL-1β, iNOS ↓
Human ES-MSCs Exosomes Tangential flow filtration CD73 temporomandibular joint OA in rats through [120]
PCNA, s-GAG ↑
OA the AKT/ERK/AMPK-dependent manner
ExoQuick PTGS, Bcl-2, IL-6, TNF-α, IL-8, IL-1β ↓ Alleviated OA damage in rats treated with
Human BM-MSCs Exosomes miR-26a-5p [121]
(System Biosciences) Bax, caspase-3 ↑ pentobarbital
Induced cartilage repair through the
TNF-α, IL-1β ↓
Human ES-MSCs Exosomes Tangential flow filtration CD73 CD73-mediated activation of AKT and ERK [122]
PCNA ↑
pathway
Intervertebral
Caspase-9/3, iNOS, MMP-3/13, caspase-1, IL-1β, Prevented progression of IVDD in rabbit
disc
Mouse BM-MSCs Exosomes Ultrafiltration - TXNIP, NLRP3 ↓ Suppressed activation of NLRP3 [123]
degeneration
COL2A, SOX9 ↑ inflammasome
(IVDD)
Demonstrated anti-inflammatory and
Human UC-MSCs EVs Ultracentrifugation IL-1β, IL-6 ↓ anti-scarring activities in the spinal cord [124]
parenchyma in rats
Reduced spinal cord injury-induced A1
Rat BM-MSCs Exosomes Ultracentrifugation - C3, GFAP, TNF-α, IL-1α, IL-1β, p-p65, p-IκBα ↓ [125]
Spinal cord astrocytes in rats
injury
Improved functional behavioral recovery in
rats
NO, Bax, caspase-3, TNF-α, Attenuated neuronal cells apoptosis,
BM-MSCs Exosomes Ultrafiltration - IL-1β, IL-6 ↓ suppressed glial scar formation [126]
Bcl2, VEGF, NF200 ↑ Suppressed activation of microglia, A1
neurotoxic reactive astrocytes and
neuroinflammation
Cells 2020, 9, 1157 12 of 45
Table 3. Cont.
Total Exosome Isolation Inhibited cardiac fibrosis, inflammation, and
Rat BM-MSCs Exosomes Kit miR-29, miR-24 - improved cardiac function in rat myocardial [127]
Myocardial (Invitrogen) infarction model
infarction Improved microenvironment of infarcted
ExoQuick NO, Bax, caspase-3/9 ↓
Rat BM-MSCs Exosomes - myocardium in rats through angiogenesis [128]
(System Biosciences) Bcl2 ↑
and anti-inflammation
Exosome extractant P2X7, TNF-α, IL-6, IL-8 ↓
Rat BM-MSCs Exosomes miR-124-3p Increased survival rate of rats [129]
(Ribobio Co., Ltd.) GSH, SOD ↑
Acute lung TNF-α, IL-1β, IL-6, MMP-9 ↓
injury (ALI) Rat BM-MSCs Exosomes Ultracentrifugation Attenuated phosgene-induced ALI in rats [130]
IL-10, SP-C ↑
Attenuated ischemia repurfusion
Rat BM-MSCs Exosomes Ultracentrifugation - Caspase-3, TNF-α, IL-1β, IL-6, TLR4, NF-κB ↓ (IR)-induced lung injury in rats [131]
Decreased apoptosis and inflammation
Induced
Reduce bleomycin-induced IPF in mice
pulmonary Human BM-MSCs Exosomes Ultracentrifugation - CCL2, ARG1 ↓ [132]
Reduced collagen deposition and apoptosis
fibrosis (IPF)
Suppressed hepatocyte necrosis and
TNF-α, IL-6, HMGB1, caspase-3, Bax ↓
Hepatic IR injury Human iMSCs Exosomes Ultrafiltration sinusoidal congestion [133]
GSH, GSH-Px, SOD ↑
Reduced the AST and ALT
Alleviated hepatic inflammation and
AST ↑
Liver fibrosis Human UC-MSCs Exosomes Ultrafiltration - collagen deposition in the CCl4 -induced [134]
Collagen I/III, TGF-β 1, p-Smad2 ↓
fibrotic liver of mice
Ameliorated acute liver failure by reducing
Total Exosome Isolation
Acute liver TNF-α, IFN-γ, IL-1β, IL-6, IL-18, TXNIP, NLRP3, ALT and AST in mice
Mouse ASCs Exosomes Kit miR-17 [135]
failure ASC, caspase-1 ↓ Reduced activation of TXNIP/NLRP3
(Invitrogen)
inflammasome in macrophages
Intestinal bowel TNF-α, IFN-γ, IL-1β, IL-6, IL-17 ↓
Human UC-MSCs Exosomes Ultracentrifugation - Ameliorated DSS-induced IBD in mice [136]
disease (IBD) TGF-β 1, IL-10 ↑
Necrotizing
PureExo Reduced incidence and severity of NEC in
enterocolitis Mouse BM-MSCs Exosomes - - [137]
(101Bio) premature newborn rats
(NEC)
Reduced inflammation and macrophage
Abdominal IL-6, IL-17, IFN-γ, IL-23, RANTES, KC, MCP-1,
Human UC-MSCs EVs Ultracentrifugation miR-147 activation in a mouse abdominal aortic [138]
aortic aneurysm MIP-1α, HMGB1 ↓
aneurysm model
Cells 2020, 9, 1157 13 of 45
Table 3. Cont.
Human Wharton’s jelly TNF-α, IL-6, IL-1β, CXCL10, IκBα, p-ERK1/2, Reduced neuroinflammation in rats with
Exosomes Ultracentrifugation - [139]
Perinatal brain (WJ)-MSCs p-JNK, p-p38 ↓ perinatal brain injury
injury
Reduced neuron-specific cell death in rats
Human WJ-MSCs Exosomes Ultracentrifugation - Mbp, Map 2 ↑ [140]
with perinatal brain injury
ExoQuick
Rat BM-MSCs Exosomes - GFAP ↑ Improved spatial learning in rats with TBI [141]
Traumatic brain (System Biosciences)
injury (TBI)
ExoQuick
Human ASCs Exosomes MALAT1 TNF-α, IL-1β, IFN-γ ↓ Improved motor behavior in rats with TBI [142]
(System Biosciences)
Improved function of brain by reducing the
Hypoxic-ischemic
Human BM-MSCs EVs PEG6000 precipitation - - total number and duration of seizures in [143]
brain injury
sheep
α-SMA, collagen I/III, IL-6, IL-1β, IRAK1,
Urethral stricture Human UC-MSCs Exosomes Ultracentrifugation miR-146a Reduced urethral fibrosis and stricture in rats [144]
TRAF6, NF-κB ↓
Reduced pilocarpine-induced SE in mice
Anion exchange TNF-α, IL-1β, MCP-1, SCF, MIP-1a, GM-CSF ↓ Reduced loss of glutamatergic and
Human BM-MSCs Exosomes - [145]
Status chromatography IL-10, PDGF-B, IL-6, IL-2 ↑ GABAergic neurons
epilepticus (SE) Reduced inflammation in hippocampus
Ameliorated SE-induced learning and
Human UC-MScs Exosomes Ultracentrifugation - GFAP, TNF-α, IL-1β ↓ [146]
memory impairment in mice
ExoQuick
Retinal IR injury Human BM-MSCs EVs - TNF-α, IL-6, caspase-3 ↓ Reduced neuro-inflammation and apoptosis [147]
Laser-induced Mouse ASCs Reduced damage, inhibited apoptosis, and
Exosomes Ultracentrifugation MCP-1 ↓ [148]
retinal injury Human UC-MSCs suppressed inflammatory responses in mice
Protected cardiomyocytes from cecal ligation
and puncture-induced sepsis in mice
Sepsis Mouse BM-MSCs Exosomes Ultracentrifugation miR-223 TNF-α, IL-1β, IL-6 ↓ [149]
through downregulation of SEMA3A and
STAT3
Prevented acute GvHD in a mouse model of
Graft versus IL-2, TNF-α, IFN-γ ↓
Human UC-MSCs EVs Ultracentrifugation - allogeneic hematopoietic stem cell [150]
Host Disease IL-10 ↑
transplantation
(GvHD)
Human
Exosomes PEG6000 precipitation - TNF-α, IL-1β, IFN-γ ↓ Modulated the patient’s immune cells [151]
BM-MSCs
Abbreviations: AD, atopic dermatitis; ALI, acute lung injury; BPD, bronchopulmonary dysplasia; DMD, Duchenne muscular dystrophy; ES, embryonic stem cell; IBD, intestinal bowel
disease; IPF, induced pulmonary fibrosis; IR, ischemia reperfusion; IVDD, intervertebral disc degeneration; JM, jaw bone marrow; MenSCs, menstrual blood derived MSCs; SE, status
epilepticus; UC, umbilical cord; WJ, Wharton’s jelly.
Cells 2020, 9, 1157 14 of 45
4.1. Macrophage Polarization

There is accumulating evidence that MSC-exosomes promote macrophage polarization from
M1 toward M2. M1 macrophages are characterized by the expression of a broad spectrum of
pro-inflammatory cytokines and chemokines, such as IL-1β, IL-12, and TNF-α. By contrast, the M2
macrophage phenotype is induced by Th2 cytokines and leads to secretion of anti-inflammatory
factors, such as IL-10 and TGF-β, and M2 markers such as IL-1RA, CD163, and C-C motif chemokine
22 (CCL22) [152]. It has been reported that human BM-MSC-exosomes and jaw bone marrow MSC
(JM-MSC)-exosomes promote cutaneous wound healing [86], and ameliorate bronchopulmonary
dysplasia (BPD) [86] through macrophage M2 polarization. The miR-223 contained in exosomes
alleviated inflammation and accelerated wound healing by inducing macrophage M2 polarization.
Co-culture with BM-MSC-exosomes increased the expression of miR-223 and decreased the expression
of PBX/knotted homeobox 1 (PKNOX1) protein, an important regulator of macrophage polarization, in
macrophages isolated from peripheral blood mononucleated cells (PBMCs). Besides, after co-culture
with BM-MSC-exosomes CD206-positive macrophages were elevated, and miR-223 inhibitors reversed
this elevation [85]. In a high-fat diet (HFD) mouse model, miR-223 deficiency enhanced infiltration of
M1 macrophage, and increased production of pro-inflammatory cytokines, but decreased M2-associated
biomarkers including peroxisome proliferator-activated receptor γ (PPARγ) and arginase 1 (ARG1) [153].
Another study elucidated that human UC-MSC-exosomes also promote M2 macrophage activation
and regulate diabetic cutaneous wound healing [87]. Compared to those from unconditioned
UC-MSCs, exosomes from LPS-preconditioned UC-MSCs contained a high level of let-7b, ameliorated
inflammation, and promoted wound healing more intensely. UC-MSC-exosomes decreased toll-like
receptor 4 (TLR4) and phospho (p)-p65 proteins regardless of LPS preconditioning. After treatment
of LPS-preconditioned UC-MSC-exosomes, ARG1, an M2 macrophage marker, was increased,
and inducible nitric oxide synthase (iNOS), an M1 macrophage marker, was decreased [88]. The let-7b
targets TLR4, activation of which leads to activation of nuclear factor κB (NF-κB). Additionally, let-7b
downregulates the expression of cyclooxygenase-2 (COX-2) and cyclin D1 proteins [154]. It was revealed
that UC-MSC-exosomes suppress inflammation and promote wound healing by inducing secretion
of cytokines from M2 macrophages in rats with severe burn-induced skin inflammation through
downregulation of TLR4, NF-κB, and p-p65 expression [89]. A higher level of miR-181c was observed
in UC-MSC-exosomes compared to in human dermal fibroblast (HDF)-exosomes. The expression level
of miR-181c was decreased by burn injury and was increased after treatment of UC-MSC-exosomes
in the cutaneous wound. In addition, treatment of UC-MSC-exosomes reduced the expression of
TNF-α and IL-1β and increased the expression of IL-10. These effects were strengthened by exosomes
derived from miR-181c-overexpressed UC-MSCs [88]. In an experiment conducted in mouse astrocytes,
the expression level of miR-181c was decreased by LPS, a TLR4 receptor ligand. Overexpression
of miR-181c increased IL-10 secretion induced by LPS [155]. In primary microglia, oxygen-glucose
deprivation (OGD) upregulated TLR4, while miR-181c reversed this upregulation. The miR-181c also
downregulated NF-κB and pro-inflammatory cytokines such as TNF-α, IL-1β, and iNOS induced
by OGD [156]. In addition, it was found that human MenSC-exosomes induced macrophage M2
polarization, which was confirmed by the increased ARG1/iNOS ratio, which led to the alleviation of
inflammation in the diabetic cutaneous wound [89].
Moreover, exosomes derived from various MSCs also play an important role in promoting
activation of M2 macrophages in other inflammatory diseases as well as cutaneous wounds. It was
found that mouse BM-MSC-exosomes relieve inflammation in atherosclerosis via macrophage M2
polarization in vivo through the let-7/high mobility group AT-Hook 2 (HMGA2)/NF-κB pathway [90].
An enrichment of the let-7 family was found in BM-MSC-exosomes, and treatment of BM-MSC-exosomes
upregulated the let-7 level in ApoE–/– mice [90]. Zhao et al. revealed that mouse BM-MSC-exosomes
also attenuated myocardial ischemia-reperfusion (IR) injury through polarizing macrophages toward
M2 phenotypes (iNOS-CD206+), and increasing IL-10 and ARG1, which are regulated by miR-182
targeting TLR4 [91]. Human BM-MSC-exosomes have been reported to reduce dextran sodium sulfate
Cells 2020, 9, 1157 15 of 45
(DSS)-induced IBD in mice through the polarization of M2b macrophages in a metallothionein-2

(MT2A)-dependent manner [92]. Another report revealed that mouse ESC-exosomes improved
cardiomyopathy by increasing M2 macrophages and IL-10 release [157]. Additionally, it was reported
that rat ASC-exosomes ameliorated myocardial infarction by promoting M2 macrophage polarization,
which is regulated by increasing sphingosine-1-phosphate receptor 1 (S1PR1) [93]. The importance of
the sphingosine 1-phosphate (S1P)/sphingosine kinase 1 (SphK1)/S1PR axis was further confirmed by
silencing of S1PR1, which abolished the decrease of hypoxia-induced apoptosis by ASC-exosomes in
H9c2 cells. Similarly, human ASC-exosomes induced M2 macrophage markers in human PBMCs [94].
Heo et al. revealed that human ASC-exosomes also induce M2 macrophage phenotype by confirming
the increased level of transcription factors (e.g., signal transducer and activator of transcription 6
(STAT6), MAF BZIP transcription factor B (MafB), etc.), which led to regulating immunomodulatory
and anti-inflammatory effects such as increased Tregs and anti-inflammatory cytokines (e.g., IL-10
and TNF-α-stimulated gene-6 (TSG-6)) [94]. Mouse ASC-exosomes also induced M2 macrophage
polarization and reduced inflammation of white adipose tissues (WAT) in obese mice [96]. These effects
are dependent on a transcription factor, STAT3, in ASC-exosomes. Furthermore, ASC-exosome-educated
M2 macrophages induced proliferation of ASCs themselves and production of lactate from ASCs, which
further promoted WAT beiging [95]. However, further studies are needed to understand the detailed
underlying molecular mechanism for the regulation of M2 macrophage polarization by MSC-exosomes.
4.2. T Cell Regulation

MSC-exosomes also modulate functions or activities of T cells (Table 3). BM-MSC-exosomes
were reported to convert Th1 to Th2, and reduce Th17 differentiation in PBMCs [97]. More
importantly, BM-MSC-exosomes increased the level of Tregs in PBMCs. These effects might be
mediated by suppression of pro-inflammatory cytokines such as TNF-α and IL-1β, and an increase of
anti-inflammatory cytokine TGF-β [9]. Another report also revealed that BM-MSC-exosomes modulate
immune reactions in PBMCs from asthmatic patients [98]. The proliferation and immune-suppression
capacity of Tregs was promoted by BM-MSC-exosomes through upregulation of IL-10 and TGF-β1 in
PBMCs. Tregs was also induced by exosomes derived from TGF-β/IFN-γ-stimulated UC-MSCs [99].
The proposed mechanism of this Treg regulation is an antigen presenting cell (APC)- but not CD4+
T cell-dependent pathway [97]. A previous report demonstrated that differentiation of Tregs is
mediated by activated APCs, which is induced by ESC-MSC-exosomes in a myeloid differentiation
primary response 88 (MYD88)-dependent manner [100]. It has been also reported that mouse
ASC-exosomes induce the increase of Tregs population in the splenic mononuclear cells from mice
with streptozotocin-induced autoimmune type 1 diabetes mellitus [100]. Upregulation of Tregs has
been also reported in a multiple sclerosis (MS) mouse experimental autoimmune encephalomyelitis
model by human BM-MSC-exosomes [101], and a concanavalin A (Con A)-induced mouse liver injury
model by mouse BM-MSC-exosomes [102]. Downregulation of proliferation of activated T and B
lymphocytes by BM-MSC-exosomes has been also reported [103]. Of note, studies by Del Fattore et al.
and Di Trapani et al. have shown that EVs from BM-MSC suppress T cell proliferation indirectly by
induction of Treg differentiation, unlike MSCs, which directly suppress T cell proliferation [103,104].
In addition, UC-MSC-EVs purified by size exclusion chromatography only showed an inhibitory effect
on T cell proliferation and did not induce cytokine response and monocyte polarization [105]. Further
studies are needed to elucidate the molecular mechanism of these regulations by MSC-exosomes.
4.3. Inflammation in Skin

It was reported that human BM-MSC-exosomes reduce photoaging and inflammation in mice,
which might be helpful to prevent and treat cutaneous aging [107]. Human ASC-exosomes were
reported to enhance neovascularization and the survival of the skin flap in a rat IR injury of the
flap transplantation model by reducing inflammation and apoptosis [108]. In this experimental
setting, ASC-exosomes derived from H2 O2 -preconditioned ASCs had better outcomes compared
Cells 2020, 9, 1157 16 of 45
to those from unconditioned ASCs. Regulation of inflammation is also important to treat atopic
dermatitis (AD), a representative skin inflammatory disease. It has been demonstrated that human
ASC-exosomes can ameliorate AD in two distinct mouse models via reducing pathological symptoms
and expression of multiple cytokines such as IL-4, IL-5, IL-13, IL-17, IL-23, IL-31, TNF-α, IFN-γ, and
thymic stromal lymphopoietin (TSLP) [20,109]. Th2 cytokines, such as IL-4, IL-5, IL-13, and IL-31,
mainly produced by activated Th2 cells, are crucial contributing factors in the development of
allergic inflammation in the skin [158,159]. Notably, Th2 cytokines including IL-4, IL-13, and IL-31
are therapeutic targets for AD [160]. Additionally, ASC-exosomes also reduced the infiltration
of inflammatory dendritic epidermal cells (IDECs, CD86+, and CD206+), which led to release of
pro-inflammatory cytokines in lesional skin of AD [20]. Taken together, MSC-exosomes are key players
in skin regeneration by promoting macrophage M2 polarization with anti-inflammatory properties
and reducing pro-inflammatory cytokine-releasing cells such as M1 macrophages and IDECs.
4.4. Immunomodulation in Other Inflammatory Diseases

Immunomodulation by MSC-exosomes was also reported in various inflammatory disease models.
Examples are as follows: (1) Exosomes from melatonin-preconditioned rat BM-MSCs reduced the kidney
injury in a rat renal IR injury model by decreasing oxidative stress and apoptosis, increasing anti-oxidant
and anti-apoptotic proteins, and enhancing angiogenesis [110]. In addition, mouse BM-MSC-exosomes
reduced the renal IR injury in a CCR2-dependent manner [111]. Human UC-MSC-exosomes have been
also reported to reduce cisplatin-induced acute kidney injury (AKI) in rats in an autophagy-dependent
manner [112]; (2) Human UC-MSC-exosomes reduced the experimental autoimmune uveitis in
rats [113]; (3) Human placenta-derived MSC (PL-MSC)-exosomes reduced the tissue fibrosis and
inflammation in a mouse Duchenne muscular dystrophy (DMD) model partly through the delivery
of miR-29c [114]; (4) Human UC-MSC-exosomes improved the pathology of lung, cardiac, and brain
in neonatal mice with BPD by reducing the pulmonary inflammation and alveolar-capillary leak
potentially through the delivery of TSG-6 [115] or macrophage M2 polarization [86]; (5) Targeted
delivery of mouse BM-MSC-exosomes by rabies viral glycoprotein (RVG) peptide improved the
cognitive function of transgenic APP/PS1 mice by reducing plaque deposition, the level of Aβ,
activation of astrocytes, and the expression of pro-inflammatory cytokines TNF-α, IL-β, and IL-6,
while increasing the levels of IL-10, IL-4, and IL-13 [116]; (6) Human BM-MSC-EVs improved the
neurological impairment and long-term neuroprotection in stoke mice by attenuating the post-ischemic
immunosuppression and lymphopenia, and as well as stimulating neurogenesis and angiogenesis [117];
and (7) Mouse BM-MSC-exosomes decreased the threshold for thermal and mechanical stimuli in a
mouse diabetic peripheral neuropathy model by regulating multiple factors involved in macrophage
polarization through the delivery of miRNAs targeting the TLR4/NF-κB signaling pathway [118].
Other inflammatory diseases, which can be modulated by MSC-exosomes or MSC-EVs, include
OA [119,120], intervertebral disc degeneration (IVDD) [123], spinal cord injury [124–126], myocardial
infarction [127,128], acute lung injury (ALI) [129–131], idiopathic pulmonary fibrosis (IPF) [132], hepatic
IR injury [133], liver fibrosis [134], acute liver failure [135], IBD [92,136], necrotizing enterocolitis [137],
abdominal aortic aneurysm [139], brain injuries [139–143], urethral stricture [144], status epilepticus
(SE) [145,146], retinal injuries [147,148], sepsis [150], and graft-versus-host disease (GvHD) [150].
The immunomodulation of MSC-exosomes was highlighted in their first clinical application in
an allogeneic setting to a patient suffering from steroid refractory GvHD [151]. In this study,
MSC-exosomes modulated the status of the patient’s immune cells. The differentiation of Tregs by
MSC-exosome-mediated APC activation might contribute to suppression of GvHD [99].
In summary, MSC-exosomes or MSC-EVs suppress inflammatory responses in diverse disease
settings by inducing polarization and differentiation of M2 macrophages and Tregs. Although exact
cargo compositions and MoA of exosomes need to be further studied, mounting evidence suggests that
MSC-exosomes have similar anti-inflammatory and immunomodulatory properties of MSCs, which
could be beneficial for the treatment of inflammatory and autoimmune diseases, as well as for skin
Cells 2020, 9, 1157 17 of 45
regeneration. However, MSC-exosomes may also possess distinct immunomodulatory mechanisms

from those of MSCs, which needs to be further elucidated to facilitate application in clinical settings.
5. Anti-Aging Effects of MSC-Exosomes

Aging, defined as irreversible deterioration of physiological processes of organisms over time,
is characterized by nine hallmarks: cellular senescence, mitochondrial dysfunction, deregulated
nutrient sensing, epigenetic alterations, telomere attrition, genomic instability, altered intercellular
communication, and stem cell exhaustion [161,162]. Among these, cellular senescence has recently
been focused on as one of the key factors in the complex aging process as it is interlinked with other
hallmarks [163]. Senescent cells are accumulated in tissues of vertebrates with age. Interestingly,
removal of senescent cells in animals results in the delayed onset of age-associated diseases [164–168].
Senescence is characterized by a stable cell-cycle arrest in the G1 phase and an inflammatory response
called senescence-associated secretory phenotype (SASP), which modifies the microenvironment
around senescent cells [161]. Senescence is induced by intracellular and extracellular stresses, including
replicative stress, DNA damage, oncogene activation, telomere damage or shortening, inflammation,
mitochondrial dysfunction, oxidative stress, and drug insults, to eliminate damaged cells, and prevents
potential malignant cell transformation [161,169]. Components of the SASP include growth factors,
pro-inflammatory cytokines, chemokines, and extracellular matrix remodeling enzymes [170–172].
SASP contributes to inflammaging, a term coined by Franceschi et al. in 2000, which describes
low-grade, controlled, asymptomatic, chronic, and systemic inflammation associated with aging
processes [173]. Indeed, many evidences point out that inflammaging may ultimately lead to
age-related diseases [174–176]. Thus, interventions that suppress SASP and inflammaging processes
may hold potential to alleviate various chronic diseases [177]. In addition, senescent cells display
the expression of senescence-associated β-galactosidase (SA-β-gal), increases of mRNAs/proteins
including p53, p21, p16, and γ-H2AX, and a decrease in cell proliferation [161].
5.1. EVs in Senescence

EVs or exosomes have a role in both transferring the senescence phenotype and alleviating or
even rejuvenating senescence cells, depending on their originating cells. Studies suggest that EVs or
exosomes act as new components of the SASP and age-related disease markers [169–171]. Age-related
changes of EVs or exosomes have been reported to result in the following: (1) an increase in the number
of EVs or exosomes released during senescence of fibroblast, epithelial cells, and cancer cells [178,179];
(2) a decrease in the levels of circulating EVs with age, at least from the 30s to 60s in humans, as well
as in mice and rats [180–182]; and (3) changes of EV or exosome composition (miRNAs, proteins, or
lipids) associated with aging or senescence [171,183–189]. In fact, EVs or exosomes mediate paracrine
senescence, transmitting senescence from senescent or diseased cells to normal cells, in both normal
and disease conditions [169,190–195]. This paracrine senescence is positively correlated with the
uptake of exosomes by target cells and is prevented by inhibition of exosome generation [169].
It has also been reported that various long noncoding RNAs (lncRNAs) are enriched in exosomes
from senescent cells and accumulating evidence shows that these RNAs may contribute to the
progression of age-related diseases such as atherosclerosis, type 2 diabetes, osteoporosis, OA,
rheumatoid arthritis, Parkinson’s disease, and multiple sclerosis [196]. It has also been reported
that various long noncoding RNAs (lncRNAs) are enriched in exosomes from senescent cells,
and accumulating evidence shows that these RNAs may contribute to the progression of age-related
diseases such as atherosclerosis, type 2 diabetes, osteoporosis, OA, rheumatoid arthritis, Parkinson’s
disease, and multiple sclerosis. For instance, in atherosclerosis, monocytes exposed to oxidized
low-density lipoprotein (oxLDL) drives progression of the disease. A study by Chen et al. has shown
that THP-1, a monocyte cell line, treated with oxLDL shows significant upregulation of exosomal
lncRNA GAS5, and these exosomes cause apoptosis of endothelial cells [197]. The role of exosomal
lncRNA was also highlighted by Ruan et al. In this study, it was found that exosomal lncRNA-p3134
Cells 2020, 9, 1157 18 of 45
contents in diabetic patients were higher than those in non-diabetic subjects [198]. Senescent cells
also exert effects by transferring protein cargo. For instance, exosomes from drug-induced senescent
multiple myeloma cells promote activation and proliferation of NK cells by transferring IL-15RA and
IL-15 [199]. Taken together, EVs from senescent cells may serve as disease markers.
5.2. Anti-Aging Effects

It has been elusive that circulating mediators are responsible for rejuvenating multiple tissues of
old organisms by parabiosis of young organisms [200]. Very recently, it was demonstrated that EVs from
young mice plasma extend the lifespan of old mice by delaying aging through exosomal nicotinamide
phosphoribosyl transferase (eNAMPT) [201]. Another study also reported that exosomes from young
mice could transfer miR-126b-5p to tissue of old mice, and reverse the expression of aging-associated
molecules such as p16, mTOR, IGF-1R, and telomerase-related genes including Men1, Mre11a, Tep1,
Terf2, Tert, and Tnks, in aged mice [202]. Another report revealed that EVs derived from serum of young
mice attenuated inflammaging in old mice by partially rejuvenating aged T-cell immunotolerance [203].
Implantation of hypothalamic stem/progenitor cells, which were genetically engineered to survive from
aging-related hypothalamic inflammation, was reported to induce retardation of aging and extension
of lifespan in mid-aged mice [204].
More importantly, growing evidence suggests that cellular senescence can be alleviated or reversed
by EVs or exosomes derived from stem cells (Table 4) [205–214]. Human ASC-exosomes reduced
the high glucose-induced premature senescence of endothelial progenitor cells (EPCs) and enhanced
wound healing in diabetic rats [205]. In the same study, overexpression of nuclear factor erythroid
2-related factor 2 (NRF2) in human ASC-exosomes further reduced premature senescence of EPCs,
and promoted wound healing in diabetic rats by modulating the expression of various proteins [205].
Since high glucose in diabetic patients induces reactive oxygen species (ROS) and inflammation, which
promotes senescence and impairs function of EPCs, reduced senescence of EPCs by ASC-exosomes
may be beneficial for the treatment of diabetic foot ulcers [205]. It has also been reported that human
ASC-exosomes contain lnRNA MALAT1 and recover function of motor behavior with reduction of
cortical brain injury in a rat traumatic brain injury model [142]. Regarding this, a study revealed that
the MALAT1 expression is reduced in aged mice and that treatment of human UC-MSC-exosomes
containing MALAT1 prevents aging in D-galactose (gal)-treated mice and senescence in H2 O2 -treated
H9C2 cardiomyocytes [206]. MALAT1 is one of the candidates for anti-aging effects in stem cell-derived
exosomes, since MALAT1-knockdown in UC-MSCs abolished these effects of UM-MSC-exosomes.
Similarly, exosomal miR-146a was known to negatively regulate senescence of MSCs by targeting the
NF-κB signaling [191]. Recently, miR-146a in AF-MSC-exosomes was reported to reduce LPS-induced
inflammation in the human trophoblast cells [215]. The miR-146a is also known to be enriched in
human UC-MSC-exosomes by TNF-α-pre-conditioning, and mediate anti-inflammatory effects in a rat
urethral stricture model [145]. Antioxidant enzymes peroxiredoxins (PRDXs) were reported as being
highly enriched in iPSC-EVs and BM-MSC-EVs [208]. Transferring of PRDXs by these EVs resulted
in alleviation of cellular aging phenotypes such as increases of SA-β-gal, p21, p53, IL-1α, IL-6, and
γ-H2AX in both replicative and genetically induced senescent MSCs [209]. Interestingly, proteomic
analysis revealed that ASC-exosomes also contain PRDXs such as PRDX1, PRDX4, and PRDX6 [109].
Human ASC-exosomes were also reported to reduce IL-1β-induced senescence in osteoblasts from OA
patients [209]. In this study, ASC-exosomes reduced not only the levels of SA-β-gal, γ-H2AX, and IL-6
protein, but also the levels of prostaglandin E2, oxidative stress, and mitochondrial membrane potential.
It has been reported that miR-214 in exosomes prevents senescence of endothelial cells by repressing
the expression of ataxia telangiectasia mutated (ATM) protein by targeting the 3’-untranslated region
(UTR) of its mRNA [216]. Interestingly, the next generation sequencing (NGS) analysis revealed that
ASC-exosomes also contain miR-214 (Ha et al. unpublished observation).
Cells 2020, 9, 1157 19 of 45
Table 4. Anti-senescence effects of exosomes derived from stem cells.
Exosome Source Nomenclature Exosome Isolation Potential MoA Senescent Cells In Vitro Effects In Vivo Effects Reference
Cell viability, Tube formation ↑
ExoQuick HG-induced senescent
Human ASCs Exosomes NFR2 SMP30, p-VEGFR2 ↑ Wound healing in diabetic rat [205]
(System Biosciences) EPCs
NOX1, NOX4, IL-6, IL-1β, TNF-α ↓
Reducing NF-κB/TNFα SA-β-gal ↓ Improvement cardiac
Total exosome isolation kit
Human UC-MSCs Exosomes signaling by lncRNA H2 O2 -treated H9C2 NF-κB activation, p21, TNFα ↓ function in D-gal-induced [206]
(Invitrogen)
MALAT1 Cell proliferation ↑ aged mouse
TGF-β1 downregulation by Perfusion in ischemic
Human UC-MSCs Exosome Ultracentrifugation H2 O2 -treated H9C2 SA-β-gal, p21, TGF-β1 ↓ [207]
miR-675 hindlimb
RS MSCs Cell growth ↑
Human BM-MSCs Size exclusion Reduction of ROS by PRDXs
EVs Progerin-induced senescent SA-β-gal, IL-1A, IL-6, γ-H2AX↓ ↓ ND [208]
Human iPSCs chromatography enriched in exosomes
MSCs p21, p53 mRNAs ↓
SA-β-gal, γ-H2AX ↓
IL-1β-treated OA IL-6 and Prostaglandin E2 ↓
Human ASCs Exosomes Ultracentrifugation Unknown ND [209]
osteoblasts Oxidative stress, Mitochondrial membrane
potential ↓
Oxidative stress ↓ Attenuating
Activation of Wnt/β-catenin
Rat BM-MSCs Exosomes Ultracentrifugation Irradiated rat BM-MSCs γ-H2AX, Rb, p53, p21, p16 ↓ radiation-induced bone loss [210]
signaling
SOD1/2, Catalase ↑ in rat
ExoQuick TGF-β Receptor 2 inhibition
RS HDFs SA-β-gal ↓
Mouse ESCs Exosome (System Biosciences) by mouse miR-291a-3p ND [211]
AS HDFs Cell proliferation, migration ↑
or Ultracentrifugation (human miR-371a-3p
SA-β-gal, p16, p21 ↓
KEAP1 downregulation by ROS ↓ Pressure ulcer healing in
Human ESCs Exosome Ultracentrifugation D-gal-induced HUVECs [212]
miR-200a Cell proliferation, migration, D-gal-induced aged mouse
tube formation ↑
ExoQuick RS HDFs SA-β-gal, MMP-1/3 ↓
Human iPSCs Exosomes Unknown ND [213]
(System Biosciences) Photoaged HDFs Collagen Type I ↑
Human iPSCs Exosomes Ultracentrifugation Unknown HG-injured HUVECs SA-β-gal ↓Cell viability, Tube formation↑ ND [214]
Abbreviations: AS, adriamycin-induced cellular senescence; HG, high glucose; ND, not determined; IRS, ionizing radiation-induced senescence; RS, replicative senescence.
Cells 2020, 9, 1157 20 of 45
Mouse miR-291a-3p was identified to target TGF-β2 receptor and as a cargo of mouse
ESC-exosomes [211]. Treatment of mouse ESC-exosomes reduced the SA-β-gal expression and
promoted cell proliferation and migration of replicative or adriamycin-induced senescent HDFs [211].
It was reported that human ESC-exosomes inhibited D-gal-induced senescence of human vascular
endothelial cells (HUVECs) [212]. Treatment of ESC-exosomes resulted in a decrease in SA-β-gal
activity, p16 and p21 protein levels, and ROS in HUVECs, and an increase in cell proliferation,
migration, and tube formation of HUVECs. The miR-200a in ESC-exosomes reduced the level of
Kelch-like ECH-associated protein 1 (KEAP1) by targeting the 3’-UTR of KEAP1 mRNA. As a result,
the level of NRF2, a master regulator of anti-oxidative responses [217], was increased to induce the
expression of its downstream targets such as heme oxygenase 1 (HO1), superoxide dismutase (SOD),
and catalase (CAT) [213]. ESC-exosomes promoted pressure ulcer healing in D-gal-induced aged mice
by reducing endothelial senescence and increasing angiogenesis [212]. Human iPSC-exosomes were
reported to protect HDFs from UVB damage, reduce the senescence-associated MMP-1/3 expression,
and induce synthesis of collagen type I in both UVB-damaged and senescent HDFs [214]. Human
iPSC-exosomes were also reported to reduce SA-β-gal and increase cell viability and tube formation
of high glucose-injured HUVECs with unknown mechanism [214]. Exosomes from various cells are
also useful as a delivery vehicle of biomolecules to suppress senescence. The miR-675 was discovered
as a candidate marker for aging [207]. Delivery of miR-675 through UC-MSC-exosomes reduced the
SA-β-gal expression, and the levels of p21 and TGF-β1 proteins in H2 O2 -induced senescent H9C2
cells by targeted downregulation of TGF-β1. Additionally, miR-675-UC-MCS- exosomes promoted
perfusion in ischemic hindlimb by inhibiting the expression of both mRNAs and proteins of p21 and
TGF-β1 [207]. Another study reported that exosomes derived from Wnt4-overexpressed mouse thymic
epithelial cells (TECs) inhibited dexamethasone-induced aging phenotypes in TECs [218].
Taken together, MSC-exosomes confer anti-senescence effects through their unique miRNA,
lnRNA, and enzyme contents. By inducing proliferation and reducing SASP in senescent cells, they
hold great potential to reduce senescent cells in tissues. Since removal of senescent cells from tissues
was reported to create a pro-regenerative environment [168] and tissue homeostasis [166], application of
MSC-exosomes to remove the senescent cells may be a preferable approach to induce the regeneration
or rejuvenation of tissues.
6. Cutaneous Wound Healing by MSC-Exosomes

A wound is a type of injury in skin. An open wound is caused by a tear, cut, or puncture, and a
closed wound is caused by blunt trauma [219]. Cutaneous wounds can be classified into acute and
chronic wounds [220]. Acute wounds are highly prevalent from a loss of dermis and epidermis
caused by mechanical, chemical, biological, or thermal injuries. Chronic wounds, on the other hand,
are common comorbidities of complex diseases such as obesity, diabetes, and vascular disorders. Four
categories of chronic wounds include pressure ulcers, diabetic ulcers, venous ulcers, and arterial
insufficiency ulcers according to the Wound Healing Society [221]. Since chronic wounds do not
heal within three months, they are considered as non-healing wounds [222,223]. Another major
medical issue is pathological wound healing and scar formation, which cause both physiological
and psychological challenges [224]. The annual Medicare cost for the treatment of acute and chronic
wounds was estimated at from $28.1 to $96.8 billion [225]. In addition, the annual product market for
wound care is estimated to reach $15 to $22 billion by 2024 [225].
Cutaneous wound healing is the complex process of restoring the injured skin. It consists
of four phases: the homeostasis, inflammatory, proliferative, and remodeling phases [226–228].
Responses in these phases are tightly coordinated to secure vital skin barrier functions [224]. However,
the mechanism of cutaneous wound healing and the interplays between a variety of cells during the
wound healing process have been only partly delineated [229]. Many cell types interact with each
other in a highly sophisticated sequence during the cutaneous wound healing process as follows [230]:
(1) the platelets initiate the formation of the blood clots, which consist of platelets, red blood cells,
Cells 2020, 9, 1157 21 of 45
and extracellular matrix molecules in the first homeostasis phase; (2) neutrophils, monocytes, as well
as macrophages are major players during the inflammatory phase. Chemotactic factors released by
neutrophils attract monocytes and cytokines from macrophages and stimulate migration of fibroblasts
to enter the injured site from the surrounding normal tissues; (3) angiogenesis and vascularization of
endothelial cells provide oxygen supply to support proliferation of migrated cells in the wound site
during the proliferative phase. Fibroblasts also differentiate into myofibroblasts to generate a tensile
strength in the wound. In addition, fibroblasts secrete growth factors, which activate migration and
proliferation of keratinocytes. Reepithelialization is completed by stopping migration of cells by contact
inhibition [230]; and (4) remodeling through apoptosis of fibroblasts, myofibroblasts, and other cells,
and degradation of extracellular matrix occur during the wound scar remodeling phase, which spans
months to years. Adverse scarring, caused by aberrant wound healing, includes chronic non-healing
wounds and pathological scarring such as hypertrophic scars and keloids, and it affects millions of
people globally since currently no effective treatment option is available [224]. The prevention or
reduction of scars is also an important issue to solve in the regenerative aesthetics [231].
MSC-EVs or MSC-exosomes orchestrate all phases of skin wound healing because of their ability
to modulate inflammation, activate migration and proliferation of various cells including immune
cells, fibroblasts, and keratinocytes, and even ameliorate scarring (Table 5) [85,87,88,205,226,231–245].
As an example, complete reepithelialization was reported in a rabbit cutaneous wound healing model
by EVs from rabbit ASCs and BM-MSCs with an unknown mechanism [232]. Human ASC-EVs were
also reported to enhance cutaneous wound healing in rat [233].
Table 5. Wound healing effects of MSC-exosomes.
Related Animal for In Vivo

Exosome Source Nomenclature Exosome Isolation Factors Affected Reference
Exosomal Cargo Study
Ultracentrifugation
Human JM-MSCs TNF-α ↓
Exosomes ExoQuick miR-223 Mouse [85]
Human BM-MSCs IL-10 ↑
TLR4, p-p65, iNOS ↓
Human UC-MSCs Exosomes Ultracentrifugation let-7b Rat [87]
p-STAT3, p-AKT, ARG1 ↑
TNF-α, IL-1β, TLR4, p65,
PureExo
Human UC-MSCs Exosomes miR-181c p-p65 ↓ Rat [88]
(101Bio)
IL-10 ↑
NOX1, NOX4, IL-6, IL-1β,
ExoQuick
Human ASCs Exosomes - TNF-α ↓ Rat [205]
SMP30, p-VEGFR2 ↑
Rabbit ASCs
EVs Ultracentrifugation - - Rabbit [232]
Rabbit BM-MSCs
Human ASCs EVs Ultracentrifugation - - Rat [233]
ExoQuick N-cadherin, cyclin 1, PCNA,
Human ASCs Exosomes - Mouse [234]
(System Biosciences) collagen I/III, elastin ↑
Collagen I/II, TGF-β1/3,
ExoQuick
Human ASCs Exosomes - MMP1/3 Mouse [235]
α-SMA ↓
Human fetal ExoQuick Collagen I/III, elastin,
Exosomes Jagged 1 Mouse [236]
dermal MSCs (System Biosciences) fibronectin mRNA ↑
Human UC-MSCs Exosomes Ultracentrifugation Wnt4 CK19, PCNA, collagen I ↑ Rat [237]
Human UC
Exosomes Ultracentrifugation - Ang, Ang1, HFG, VEGF ↑ Rat [238]
blood-MSCs
β-catenin, N-cadherin,
Human UC-MSCs Exosomes Ultracentrifugation Wnt4 Rat [239]
PCNA, Cyclin D3 ↑
Human
Exosomes Ultracentrifugation - Collagen I/III, elastin, ↑ Rat [240]
iPSC-MSCs
Human UC-MSCs Exosomes Ultracentrifugation - α-SMA, collagen I ↓ Mouse [241]
Human gingival Size exclusion
Exosomes - Collagen ↑ Rat [242]
MSCs chromatography
Dog BM-MSCs Exosomes Ultracentrifugation - α-SMA ↓ Dog [243]
Cells 2020, 9, 1157 22 of 45
6.1. Homeostasis Phase

During the homeostasis phase, the formation of blood clots by platelets protects the injured site.
Up to now, no direct evidence has been available that shows the involvement of MSC-exosomes in blood
clotting during wound healing. A recent result might suggest the potential benefit of MSC-exosomes on
blood clotting in the wound healing process; human UC-MSC-EVs have been reported to induce blood
coagulation in vitro [244]. However, further studies are required to analyze the effects of MSC-EVs or
MSC-exosomes in blood clotting in both healthy and disease conditions.
6.2. Inflammatory Phase

Regulation of inflammation is also important in skin regeneration during the wound healing
process. Although inflammation is one phase of the normal skin repair cascade, the prolonged
inflammation is harmful and may cause excessive scarring [245]. The prolonged inflammation
happens mainly in chronic or burn wounds [226,246] and it is of importance to appropriately
transit from inflammatory to proliferative phases in normal wound healing [247]. Macrophages
are crucial in the wound healing process, which should appropriately transition from M1 to M2
macrophages [248,249]. M2 macrophages have anti-inflammatory properties, which are promoted
in order to repair wounds in the latter phases of skin wound healing [248,249]. As mentioned
earlier, MSC-exosomes promote the polarization of macrophages from M1 to M2 in cutaneous wound
healing models (see 4. Anti-inflammation and immunomodulation by MSC-exosomes): (1) human
BM-MSC-exosomes and JM-MSC-exosomes promote cutaneous wound healing in mice by transferring
miR-223 [85]; (2) human UC-MSC-exosomes promoted diabetic cutaneous wound healing in rats by
delivering let-7b [88]; and (3) human UC-MSC-exosomes enhanced the wound healing in rats with
severe burn injury through miR-181c transfer [88].
6.3. Proliferative Phase

During the proliferative phage, fibroblasts from surrounding normal tissues migrate into the
injured site. These fibroblasts produce various matrix proteins including collagen I and III to strengthen
the newly formed scar tissue. MSC-exosomes affect these dermal fibroblasts to promote migration and
proliferation, and produce collagen, elastin, and fibronectin: (1) human ASC-EVs or ASC-exosomes
induced migration and proliferation of dermal fibroblasts or keratinocyte in vitro [234,235]; (2) human
ASC-exosomes induced collagen I/III and elastin in HDFs, and they enhanced cutaneous wound
healing in mice [234,235]; (3) human fetal dermal (FD)-MSC-exosomes induced the expression of
collagen I/III, elastin, and fibronectin mRNAs by activating the Notch pathway through delivering
Jagged 1 protein [236]; and (4) human UC-MSC-exosomes were shown to contain Wnt4 and accelerated
reepithelialization of burn skin in rats [237]. The wound healing effects were inhibited when the Wnt4
expression in UC-MSC-exosomes was knock-downed by siRNA. Furthermore, human MSC-exosomes
were reported to induce proliferation and migration of fibroblasts in vitro from diabetic wound
patients [250]. The positive effects of MSC-exosomes on keratinocytes were also reported as follows:
(1) human UC-MSC-exosomes protect the immortalized human keratinocytes HaCaT from heat-induced
apoptosis by activating the AKT pathway [237]; and (2) human WJ-MSC- and iMSC-exosomes increased
the secretion of collagen in HaCaT [251].
As mentioned above, angiogenesis is of importance to support the oxygen needed for the
proliferation of fibroblasts or other cells in the injured site [229]. It has been also reported that
MSC-exosomes induce angiogenic activity of endothelial cells. Human ASC-exosomes induced tube
formation of HUVECs by delivery of miR-125a, which suppresses the expression of angiogenic inhibitor
delta-like 4 (DLL4) [252]. Human BM-MSC-EVs or rat BM-MSC-exosomes were also reported to
enhance angiogenesis in stroke mice [118] or in rats with renal IR injury [110], respectively. Exosomes
from human endometrial MSCs were reported to increase proliferation, migration, and angiogenesis
of HUVECs with increased expression levels of angiogenic markers including Tie2, angiopoietin
Cells 2020, 9, 1157 23 of 45
1 (Ang1), Ang2, and vascular endothelial growth factor (VEGF) [253]. In addition, the following
pro-angiogenic effects of MSC-exosomes have been confirmed in vivo: (1) human umbilical cord blood
(UCB)-MSC-exosomes with thrombin preconditioning accelerated cutaneous wound healing in rats
with full-thickness wounds. Human UCB-MSC-exosomes increased the angiogenic factors such as
angiogenin (Ang), Ang1, hepatocyte growth factor (HGF), and VEGF, while reducing TNF-α and
IL-6 [238]; (2) human UC-MSC-exosomes enhanced angiogenesis in rats through the Wnt4/β-catenin
pathway. The pro-angiogenic effects of human UC-MSC-exosomes was abolished when the Wnt4
expression was knock-downed by shRNA [239]; and (3) human iMSC-exosomes accelerated both the
formation and maturation of new vessels in the wound sites with unknown mechanism [241].
6.4. Remodeling Phase

MSC-exosomes might be beneficial to further reduce scar formation. Uncontrolled accumulation
of myofibroblasts in the wound sites causes scar formation. Recently, human UC-MSC-exosomes have
been reported to reduce scar formation by inhibiting accumulation of myofibroblasts in mice [242].
A variety of proteases such as matrix metalloproteinases (MMPs) are necessary for all phases of
the cutaneous wound healing process [254]. During the remodeling phase, controlled release of
MMPs by fibroblasts, macrophages, epidermal cells, and endothelial cells contributes to degrading the
majority of collagen III fibers [255]. Regulation of extracellular matrix remodeling by ASC-exosomes
has been reported [235]. In this study, it was demonstrated that ASC-exosomes promoted scarless
cutaneous wound repair by regulating the ratios of collagen I-to-collagen III, TGF-β3-to-TGF-β1,
and MMP3-to-MMP1.
6.5. Proteolytic Environment

Uncontrolled protease activities are known to be associated with impaired wound healing [256].
Additionally, prolonged high levels of protease activities have been suggested to be associated
with delayed wound healing in chronic wounds [254–257]. In fact, elevated levels and activities of
collagenase (MMP-1 and MMP-8) and gelatinases (MMP-2 and MMP-9) are characteristics of chronic
wounds [255]. This highly proteolytic environment is not favorable for advanced biologicals such
as growth factors [258]. In fact, the use of platelet-derived growth factor (PDGF) for treatment of
chronic wounds has been reported with modest effect [259]. Based on this, a clinical study for the
treatment of chronic wounds with a combination of topical growth factors and proteinase inhibitors
was recently initiated [260]. The proteolytic environment of chronic wounds might be also unfavorable
for the treatment of MSC-exosomes, since the surface proteins on the exosomes are susceptible to
proteolysis, which may change the interaction between exosomes and recipient cells [261]. Therefore,
a protease-resistant formulation of MSC-exosomes would be necessary for the maximum efficacy,
especially for topical applications, as reported for PDGF [262,263]. Recently, human gingival MSC
(GMSC)-exosomes with chitosan/silk hydrogel showed enhanced wound healing in diabetic rats
with appropriate swelling and moisture retention capacity suggested as effects of this hydrogel [242].
This hydrogel may also provide protection of exosomes from proteases in the wound site.
6.6. Animal Models

Most of the animal studies for wound healing with MSC-exosomes have been performed in rodents,
except for two studies with rabbit and dog [232,243] (Table 5). However, the structure and physiology of
rodent skin do not reflect those of human skin. Pigs are the most optimal preclinical models for wound
healing because of the highest similarities between pig and human skin including skin architecture,
hair density, and physiology of the wound healing process [264–268]. It is necessary to confirm the
effects of MSC-exosomes on cutaneous wound healing in pig models for better understanding of MoA
and clinical applications.
Cells 2020, 9, 1157 24 of 45
6.7. ASC-Exosomes
The beneficial effects of fat graft on wound repair are widely accepted, while the underlying
mechanism remains unknown [269]. These effects might be related to exosomes from the subcutaneous
fat layer. Recently, it has been revealed that human ASC-exosomes induce proliferation and migration
of HDFs, and the expression of N-cadherin, cyclin 1, PCNA, collagen I/III, and elastin in HDFs in vitro,
which results in reduced scar formation in mice by regulating extracellular matrix remodeling [234,235].
No direct evidence that shows an advantage of ASC-exosomes over exosomes from other MSCs is
available. ASCs, however, are distinct in immunomodulation compared to BM-MSCs. BM-MSCs enter
the wound site through the blood supply to initiate the first phase of wound healing [270]. In the
injured site, BM-MSCs prolong and enhance the inflammation by increasing survival and function
of neutrophils [271]. Under hypoxic conditions, which induces the activation of TRL4, BM-MSCs
secreted pro-inflammatory factors and decreased the polarization of macrophage from M1 to M2
phenotype [272,273]. Therefore BM-MSCs in the wound site might not induce the anti-inflammatory
M2 macrophages without enough oxygen supply by neovascularization. On the contrary, phenotype
and secretome of ASCs were largely unaffected by prolonged hypoxia [274], and the CM from
ASCs showed better inducing effects of the anti-inflammatory M2 macrophage phenotype than
the CM from BM-MSCs [275]. These results suggest that ASC-exosomes might be more beneficial
than BM-MSC-exosomes to induce appropriate wound healing processes. In summary, MSC-EVs
or MSC-exosomes contribute to each phase of wound healing by inducing M2 polarization and
stimulating dermal fibroblasts to produce structural proteins and proteases necessary for remodeling
of the extracellular matrix.
7. MSC-Exosome-Induced Hair Growth

Hair follicle cycling is a dynamic and complex process involving alternating phases of rapid
growth (anagen), regression (catagen), and quiescence (telogen) [276]. Hair follicles, which reside in
the dermal layer of the skin, are made up of various cell types including dermal papilla (DP) cells
and outer root sheath (ORS) keratinocytes, each having distinct roles [277]. In addition to these cells,
ASCs located in the adipose tissue below dermis may also affect hair cycling as ASCs differentiate
into mature adipocytes and surround hair follicles during the telogen to anagen transition [278].
Although a direct relationship between dermal papilla cells and ASCs has not been elucidated,
it can be anticipated that ASCs exert effects on hair growth, as numerous studies have shown that
transplantation of ASCs and CM from ASCs enhance proliferation of DP cells in vitro and promote
hair growth in mice and human [279–281]. Indeed, interactions between these cell types through
various mediators lead to transition from the telogen to anagen phase. Activation of the Wnt/ß-catenin
signaling is one of the main pathways involved in the hair follicle development. Previous studies have
shown that dermal Wnt ligands regulate hair-inducing activity of DP cells by maintaining the anagen
phase [282,283]. In addition, growth factors such as fibroblast growth factor-5 (FGF-5) produced by
ORS cells or insulin-like growth factor-1 (IGF-1) produced by DP cells increase proliferation of hair
follicle cells [284,285]. Thus, the Wnt/ß-catenin signaling and secretion of growth factors are crucial for
hair growth.
Dysregulation of hair cycling caused by various factors such as environmental, genetic, hormonal,
and aging, results in hair loss [286–288]. Currently, finasteride and minoxidil are the mainstay
treatments for alopecia, although they are not fundamental treatments that induce hair growth, not to
mention having various side effects associated with them [289,290]. Hair transplantation is frequently
utilized as a fundamental treatment of hair loss but it is an invasive procedure and graft survival rate
largely depends on the surgeon [291]. There is a strong unmet need of a minimally invasive treatment
that not only retards hair loss but also promotes hair growth.
Cells 2020, 9, 1157 25 of 45
7.1. The Effects of DP-Exosomes on Hair Cells

As DP cells are the key player in hair follicle cycling as they secrete growth factors, activate
the Wnt signaling, and promote differentiation of hair follicle stem cells, it can be anticipated that
exosomes derived from DP cells can also modulate hair follicle cycling. Indeed, studies have shown that
exosomes derived from DP cells (DP-exosomes) promote hair growth. Cutaneous injection of human
DP-exosomes increased the anagen to catagen ratio in mice and stimulated proliferation and ß-catenin
expression of ORS cells [292]. Exosomes derived from 3D culture of human DP cells increased the
percentage of Ki67-positive cells in cultured hair follicles and induced hair follicles in mice implanted
with human DP spheres by activating the Wnt and bone morphogenic protein (BMP) signaling [293].
A study by Yan et al. identified 34 differentially expressed miRNAs that are involved in proliferation
and differentiation of hair follicle stem cells by goat DP-exosomes [294].
7.2. The Effects of MSC-Exosomes on Hair Growth

Similar to DP-exosomes, MSC-exosomes are also known to carry a myriad of growth factors
and Wnt activators in their cargo. For instance, human UC-MSC-exosomes were found to transport
Wnt4 and Wnt11 and subsequently activate the Wnt signaling and promote cell proliferation in target
cells [237,239,295]. Therefore, MSC-exosomes are attractive treatment options for hair growth as well.
However, to date, there is only one publication reporting the effects of MSC-EVs on hair growth [296].
The authors showed that mouse BM-MSC-EVs promoted proliferation of human DP cells and induced
secretion of growth factors such as VEGF and IGF-1, which are essential for hair growth [285,297,298].
In addition, when mice were intradermally injected with BM-MSC-EVs, an increased anagen to
telogen ratio was evident in C57BL/6 mice, along with elevated Wnt protein levels in the dorsal skin.
These results suggest that MSC-EVs or MSC-exosomes might have the potential to promote hair
growth. Further studies will be necessary to elucidate the potential of various MSC-exosomes on hair
follicle cycling.
8. Repair and Regeneration of Skin barrier by MSC-Exosomes

The skin is the largest organ in the human body, comprising about 15% of the total body weight,
and is well known as the barrier between the external environment and the human body, preventing loss
of moisture and protecting the body from UV light, pathogens, chemicals, and mechanical injuries [299].
The skin is composed of three layers: epidermis, dermis, and hypodermis. The epidermis is the outermost
layer of skin and functions as the waterproof barrier. The dermis is a layer below the epidermis, consisting
of tough connective tissues, hair follicles, sebaceous glands, apocrine glands, lymphatic vessels, blood
vessels, and sweat glands. The hypodermis (also known as subcutaneous tissue) is the deepest layer of
skin and is composed of fat and connective tissue [300,301].
8.1. Skin Barrier

The skin barrier is commonly divided into three distinct functional barriers: microbiome, chemical,
and physical barriers [302]. The microbiome barrier comprises the outer side of the skin barrier and
is composed of diverse microbial communities such as bacteria, fungi, and viruses [295]. The skin
microbiome can protect the body against exogenous exposure and invasion of pathogens and can
affect immune cell maturation in the skin development. It also functions as the skin immune mediator,
which cross-talks between skin cells and the skin immune system [302]. In some cases, altered microbial
states result in skin disease [303]. As an example, the increased abundance of Gemella and Streptococcus
species are observed in AD [304].
The chemical barrier provides the acidic surface pH, which is the key factor of desquamation
and regeneration of the skin barrier [303]. It also provides the lipid barrier of ceramides, cholesterol,
and free fatty acids, consisting of a molar ratio of 1:1:1 [305]. The lipids prevent loss of moisture
from skin and invasion of environmental substances. Furthermore, free fatty acids contribute to the
Cells 2020, 9, 1157 26 of 45
homeostasis of the barrier function, maintaining acidic pH in skin [306]. In addition, the chemical
barrier, especially the biochemical barrier, provides antimicrobial peptides. Antimicrobial peptides are
a major factor of the innate immune systems and build the first line of defense against bacteria and
viruses [307].
The physical barrier consists of stratum corneum (SC) and tight junction (TJ). The SC is the
outermost layer of epidermis consisting of dead keratinocytes (corneocytes) [308]. Living keratinocytes
are transformed into non-living corneocytes during cornification. Cornification is completed by the
replacement of the cell membrane with a layer of ceramides covalently linked to the cornified envelope.
This ceramide–corneocyte complex in SC contributes to the skin’s barrier function [309]. The epidermal
TJ not only anchors cells to the neighboring cells, but also prevents the escape of moisture between
cells [310]. If TJ is damaged, the Langerhans or dendritic cells, which are located below the TJ network,
stretch their dendrites to the upper side of TJ, and then are activated by allergens and lead to allergic
responses [301,311].
Dysfunction and damage of the skin barrier leads to several diseases such as AD [310],
psoriasis [310], rosacea [312], and acne vulgaris [313]. Up to now, most of the therapeutic approaches
for these diseases have targeted inflammation: (1) dupilumab, a dual inhibitor of IL-4 and IL-13,
was recently approved to treat AD [314]; (2) monoclonal antibodies inhibiting IL-12, IL-23, or IL-17 are
being developed for the treatment of psoriasis [315]; (3) a topical drug, ivermectin, for the treatment of
mild-to-moderate rosacea has an anti-inflammatory effect [316]; and (4) anti-inflammatory drugs are
also used to treat acne vulgaris, although the first line treatment of acne vulgaris is antibiotics [317].
Moisturizers, used to reduce xerosis or dryness, could prove to be toxic to individuals with compromised
skin while being harmless to those with normal skin [318]. Physiologic lipid-based barrier creams,
containing three essential lipids including ceramides, cholesterol, and free fatty acids, have been
reported to improve barrier function and reduce pruritus as well [318]. However, currently no treatment
option is available to repair or regenerate skin barrier functions.
8.2. The Effects of ASC-Exosomes on Skin Barrier

Recently, human ASC-exosomes have been reported to promote epidermal barrier repair in a
mouse AD model [109]. Repeated exposures of oxazolone to hairless mice induced AD-like symptoms
including inflammation and skin barrier abnormalities [319]. Subcutaneous injection of ASC-exosomes
induced restoration of the skin barrier by production of ceramides and dihydroceramides with long acyl
chains in a dose-dependent manner. ASC-exosomes also induced synthesis of sphingoids including
sphingosine and S1P, increased SphK1 activity, and reduced S1P lyase (S1P1) activity in the injured
skin. As mentioned previously, the S1P/Sphk1/S1PR axis is of importance for inducing M2 macrophage
polarization by ASC-exosomes, which reduce inflammation and promote cutaneous wound healing [94].
Further study will be needed to elucidate the role of M2 macrophage polarization by ASC-exosomes in
skin barrier repair. In addition, ASC-exosomes increased the number of epidermal lamellar bodies and
formation of the lamellar layer at the interface of SC and stratum granulosum. Transcriptome analysis
of diseased skins revealed that ASC-exosomes reversed the abnormal expression of genes involved in
skin barrier maintenance, lipid metabolism, the cell cycle, and inflammatory responses induced by
repeated oxazolone exposures. These results suggest that ASC-exosomes could be a promising cell-free
treatment for the regeneration of the skin barrier in various diseases with skin barrier defects.
9. Application of MSC-Exosomes for Regenerative Aesthetics

Physical changes in skin over time produce psychosocial impacts that significantly affect social
interactions [320]. With the global increase of older individuals over 65, there is an expanding
demand for repair or rejuvenating products and procedures for aged skin [300,320]. Stem cell
conditioned media (CM), mostly from the culture of MSCs, have been used as a skin care
product for anti-aging, anti-wrinkle, and skin and hair care [321]. MSC-CM contain beneficial
secretomes, including secreted growth factors as well as exosomes. However, MSC-CM also contain
Cells 2020, 9, 1157 27 of 45
unintended ingredients including media components and additives, and cellular waste such as
lactate and ammonia, both of which are restricted in cosmetics [322,323]. On the contrary, isolated
MSC-exosomes avoid these potential harmful components. Currently, the tangential flow filtration
(TFF) method is recommended as a suitable industrial-scale method to isolate exosomes among
various techniques [40,324]. The TFF method can markedly reduce the levels of lactate and ammonia
from exosome preparation (Ha et al. unpublished observation). Recently, it has been demonstrated
that human ASC-exosomes isolated by the ExoSCRT™ technology, a TFF-based exosome isolation
method, are safe, showing no adverse effects in GLP toxicological tests including skin sensitization,
in vitro photosensitization, eye and skin irritation, or acute oral toxicity in accordance with OECD
guidelines [325]. In addition, the commercial product ASCE™ (the trademark of ExoCoBio), the
ASC-exosome isolated by the ExoSCRT™ technology, was firstly registered as a cosmetic ingredient
in the International Cosmetic Ingredient Dictionary (ICID). The TFF-isolated ASC-exosomes have
multiple effects on the skin: (1) inducing regeneration of epidermal skin barrier by increasing
synthesis of ceramides, dihydroceramides, sphingosine, and S1P [110]; (2) reducing inflammation
through downregulation of multiple cytokine levels [20,109,325]; (3) reducing the level of TSLP,
a pruritus-causing cytokine [110]; (4) inducing synthesis of collagen and elastin in HDFs [325]; and (5)
inducing proliferation of HDFs and HDPs (Ha et al. unpublished observation). Recently, a potential
effect of ASC-exosomes on subcutaneous fat has been also suggested. Mouse ASC-exosomes promoted
WAT beiging through induction of M2 macrophage polarization in WAT of obese mice [95]. Under the
same condition, ASC-exosomes induced proliferation of ASCs themselves. Further studies are needed
to decipher the effects of human ASC-exosomes on subcutaneous fat in normal physiological conditions.
The safety and efficacy of secretomes from different cells were analyzed for skin and wound
care products, and it was found that secretome from ASCs is safer and more effective than that
from BM-MSCs in many aspects: (1) lack of expression of major histocompatibility complex (MHC)
class II on ASCs; (2) induction of higher levels of anti-inflammatory M2 macrophages by ASC-CM
than by BM-MSC-CM; and (3) suppression of cancer growth by ASC-exosomes both in vivo and
in vitro [326,327]. ASC-exosomes could be a preferable regenerative aesthetic ingredient since an
important function of ASCs in skin is signaling to surrounding cells to induce the differentiation of
dermal fibroblasts and keratinocytes, and activate epidermal stem cells including hair follicles [326].
A pioneering cosmeceutical product, the ASCE+™ lyophilized human ASC-exosomes (ASCE+ is the
trademark of ExoCoBio), showed various beneficial effects including anti-inflammation and reduction
of downtime after ablative skin treatments such as laser therapies (unpublished observation). Taken
together, ASC-exosomes could be a next-generation product for the regenerative aesthetics, which
affects multiple layers of skin including the epidermis (keratinocytes), dermis (fibroblast, inflammatory
cells, and hair follicle), and potentially the hypodermis (subcutaneous fat) (Figure 1).
after ablative skin treatments such as laser therapies (unpublished observation). Taken together, ASC-exosome
next-generation product for the regenerative aesthetics which affects multiple layers of skin including epidermis (k
dermis (fibroblast, inflammatory cells, and hair follicle), and potentially hypodermis (subcutaneous fat) (Figure 1).
Cells 2020, 9, 1157 28 of 45
Figure 1. Effects of ASC-exosomes on skin.

Figure 1. Effects of ASC-exosomes on skin.
10. Conclusions
10.
With the
With the recent
recent burst
burst of
ofresearch,
research,MSC-exosomes
MSC-exosomes are are now
nowwidely
widely accepted
accepted as asnext-generation
next-generation
cell-free therapeutics for intractable diseases. Many challenges in industrialization
cell-free therapeutics for intractable diseases. Many challenges in industrialization of exosomes of exosomes are
are still
stillthere
out out there
such as such as large-scale
large-scale culture
culture of MSCs,ofcontinuous
MSCs, continuous
supply of supply
MSCs withof MSCs with comparable
comparable therapeutic
effects, and accurate determination of quantity and quality of exosomes. However, technical However,
therapeutic effects, and accurate determination of quantity and quality of exosomes. advances
technical
in the MSC advances in the
cell therapy MSC
field, cellthe
with therapy field,first
expected with the expected
marketing first marketing
approval approval
by the US FDA in thebynear
the
US FDA
future in the
[328], arenear
alsofuture
able to[329], are also able
be integrated to be
in the integrated
exosome in the exosome
industry soon. The industry soon. The use
use of immortalized
of immortalized MSCs, with similar functionalities and safety profile
MSCs, with similar functionalities and safety profile compared to naïve MSCs, might compared to naïve MSCs, might
be also an
be also anstrategy
alternative alternative strategy
for stable for stable
production production [329,330].
of MSC-exosomes of MSC-exosomes
Successful [330,331]. Successful
commercialization of
commercialization
MSC-exosomes mayof MSC-exosomes
provide a completely maynewprovide a completely
therapeutic paradigmnew therapeutic
for human paradigm for
healthcare.
human healthcare.
Author Contributions: Conceptualization, D.H.H., J.H.L., Y.W.Y., and B.S.C.; investigation, D.H.H., H.-k.K., J.H.L.,
and Y.W.Y.;
Author data curation,
Contributions: D.H.H., H.-k.K., and
Conceptualization, Y.W.Y.;
D.H.H., writing—original
J.H.L., draft preparation,
Y.W.Y., and B.S.C.; D.H.H.
investigation, and H.K.K.,
D.H.H., Y.W.Y.;
writing—review and editing, D.H.H., H.-k.K., J.L., H.H.K., G.-H.P., S.H.Y., J.Y.J., and H.C.; visualization, D.H.H.,
J.H.L., and
H.-k.K., S.S.,Y.W.Y.; data supervision,
and Y.W.Y.; curation, D.H.H.,
Y.W.Y.H.K.K., and project
and B.S.C.; Y.W.Y.;administration,
writing – original draftfunding
Y.W.Y.; preparation, D.H.H.
acquisition, and
B.S.C.
All authors have read and agreed to the published version of the manuscript.
Funding: This review was funded by ExoCoBio Inc.
Conflicts of Interest: Y.W.Y. and B.S.C. are founders and stockholders of ExoCoBio Inc. D.H.H., H.-k.K., J.H.L.,
S.S., Y.W.Y., and B.S.C. are employees of ExoCoBio Inc. Other authors declare no conflict of interest.
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cells

Keywords: anti-aging; anti-inflammation; hair growth; immunomodulation; mesenchymal stem cells

Cells 2020, 9, 1157; doi:10.3390/cells9051157 www.mdpi.com/journal/cells

2. MSCs as Sources of Exosomes

Table 1. Mesenchymal stem cell (MSC)-exosomes from different sources.

Diseases/Focuses Nomenclature Exosome Isolation MSC Origin Outcome Reference

Human bone marrow Decreased U87MG cell proliferation

Attenuated OA in a murine model

3. Quality Control of EVs for Development of Therapeutic EVs

Examples in Guidelines Examples in GMP Settings

Examples in Guidelines Examples in GMP Settings

3.1. EV Quantity and Size

3.4. Potency Assays

4. Anti-Inflammation and Immunomodulation by MSC-Exosomes

Table 3. Anti-inflammatory and immunomodulatory effects of MSC-exosomes.

4.1. Macrophage Polarization

(DSS)-induced IBD in mice through the polarization of M2b macrophages in a metallothionein-2

4.2. T Cell Regulation

4.3. Inflammation in Skin

4.4. Immunomodulation in Other Inflammatory Diseases

regeneration. However, MSC-exosomes may also possess distinct immunomodulatory mechanisms

5. Anti-Aging Effects of MSC-Exosomes

5.1. EVs in Senescence

5.2. Anti-Aging Effects

Table 4. Anti-senescence effects of exosomes derived from stem cells.

6. Cutaneous Wound Healing by MSC-Exosomes

Table 5. Wound healing effects of MSC-exosomes.

Related Animal for In Vivo

6.1. Homeostasis Phase

6.2. Inflammatory Phase

6.3. Proliferative Phase

6.4. Remodeling Phase

6.5. Proteolytic Environment

6.6. Animal Models

7. MSC-Exosome-Induced Hair Growth

7.1. The Effects of DP-Exosomes on Hair Cells

7.2. The Effects of MSC-Exosomes on Hair Growth

8. Repair and Regeneration of Skin barrier by MSC-Exosomes

8.1. Skin Barrier

8.2. The Effects of ASC-Exosomes on Skin Barrier

9. Application of MSC-Exosomes for Regenerative Aesthetics

Cells 2020, 9, 1157 28 of 45

Figure 1. Effects of ASC-exosomes on skin.

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