Trends in Pharmacological Sciences

Review

Mesenchymal Stem Cell Immunomodulation:

Mechanisms and Therapeutic Potential
Na Song,1,2,3 Martijn Scholtemeijer,1,2 and Khalid Shah1,2,4,*

Mesenchymal stem/stromal cells (MSCs) are multipotent cells that are emerging Highlights
as the most promising means of allogeneic cell therapy. MSCs have inherent MSCs are multipotent cells that are
immunomodulatory characteristics, trophic activity, high in vitro self-renewal emerging as the most promising means
of allogeneic cell therapy.
ability, and can be readily engineered to enhance their immunomodulatory func-
tions. MSCs affect the functions of most immune effector cells via direct contact MSCs participate in both innate and
with immune cells and local microenvironmental factors. Previous studies have adaptive immunity, and their immuno-
confirmed that the immunomodulatory effects of MSCs are mainly communi- modulatory functions are exerted mainly
via interactions with immune cells
cated via MSC-secreted cytokines; however, apoptotic and metabolically through cell-to-cell contact and para-
inactivated MSCs have more recently been shown to possess immunomodula- crine activity.
tory potential, in which regulatory T cells and monocytes play a key role. We
review the immunomodulatory aspects of naïve and engineered MSCs, and Engineering MSCs to express specific
immunomodulatory agents contributes
discuss strategies for increasing the potential of successfully using MSCs in to MSC capacity and pluripotency, and
clinical settings. also enables them to deliver large
doses of cancer-targeting biologics with
a single dose.
Mesenchymal Stem Cells
MSCs are pluripotent T cells that have self-renewing, differentiation, and immunomodulatory MSC administration has shown potential
properties. Their two most attractive features are plasticity (see Glossary) and tropism. They efficacy in the treatment of several dis-
are distinguished from other cell types by the expression of cell-surface markers including eases that resist standard treatment.
However, there are some challenges in
CD73, CD90, and CD105, and by the lack of expression of CD45, CD34, CD14, CD19, efficiently translating MSC-based thera-
CD11b, and human leukocyte antigen DR isotype (HLA-DR) [1], and play a central role in tissue peutics into the clinic.
repair in addition to their antitumorigenic, antifibrotic, antiapoptotic, anti-inflammatory,
proangiogenic, neuroprotective, antibacterial, and chemoattractive effects [2,3]. This unique set Efficient homing and migration of MSCs
to the target tissue will be essential for
of characteristics makes MSCs attractive for their therapeutic potential in the fields of regenerative the future development of MSC-based
medicine [4], inflammatory disorders [2], and, increasingly, cancer therapy [5,6]. therapies.

Initially, MSCs were mainly used for tissue repair and regeneration [3]. However, they have been
increasingly used for diseases including graft-versus-host disease (GVHD) and autoimmune
1
Center for Stem Cell Therapeutics and
diseases such as lupus and Crohn’s disease [2]. In addition, the clinical potential of MSCs has
Imaging (CSTI), Harvard Medical School,
been extended to the treatment of myocardial infarction, stroke, multiple sclerosis, liver cirrhosis, Boston, MA 02115, USA
2
diabetes, lung injuries, and cancer [2,3]. MSCs have been harvested and expanded from a variety Department of Neurosurgery, Brigham
and Women’s Hospital, Harvard Medical
of adult and perinatal tissues such as bone marrow [7], adipose tissue [7,8], peripheral blood, fetal
School, Boston, MA 02115, USA
tissues [9], dental pulp [8], umbilical cord tissue [7,8], and placental tissues [7]. Most preclinical 3
Department of Medical Oncology, the
studies have been performed with bone marrow-derived MSCs (BM-MSCs); however, adipose First Hospital of China Medical
University, Shenyang 110001, China
tissue-derived MSCs (A-MSCs) and umbilical cord blood-derived MSCs (UC-MSCs) have also 4
Harvard Stem Cell Institute, Harvard
received considerable attention in recent years [9]. University, Cambridge, MA 02138, USA

Immunomodulation by MSCs
MSCs have recently been shown to possess immunomodulatory potential in which interactions
with regulatory T cells (Tregs) and monocytes play a key role [2,10]. Several studies have sug-
gested that A-MSCs exert more potent immunomodulatory effects than BM-MSCs, implying that *Correspondence:
A-MSCs are a better alternative for immunomodulatory therapy [11]. UC-MSCs, on the other kshah@bwh.harvard.edu (K. Shah).

Trends in Pharmacological Sciences, September 2020, Vol. 41, No. 9 https://doi.org/10.1016/j.tips.2020.06.009 653
© 2020 Elsevier Ltd. All rights reserved.
Trends in Pharmacological Sciences

hand, have been suggested to show minimal risk of initiating an allogeneic immune response Glossary
when administered in vivo. This, as well as their ease of collection, makes UC-MSCs suitable ther- Adaptive immunity: a subset of the
apeutic candidates [8]. In the following we focus on immunomodulatory aspects of naïve and immune system that is activated by
engineered MSCs, and synthesize our current understanding of the effects of MSC modulation exposure to pathogens and acts on the
threat using an immunological memory
on immune cells.
to enhance its effect. Cells of the
adaptive immune system include B and
Immunomodulatory Actions through Cell-to-Cell Contacts T cells.
MSCs participate in both innate immunity and adaptive immunity, and their immunomodula- Allogeneic: tissues or cells from
individuals of the same species, but
tory functions are exerted mainly via interactions with immune cells through cell-to-cell contact which are genetically dissimilar and
and paracrine activity involving T cells, B cells, natural killer (NK) cells, macrophages, hence immunologically incompatible.
monocytes, dendritic cells (DCs), and neutrophils [12] (Figure 1). Dendritic cells (DCs): antigen-
presenting cells in the mammalian
immune system. DCs act as
Cell-to-Cell Contact with Immune Cells of Adaptive Immunity
messengers between the innate and the
In vitro, MSCs have been shown to inhibit naïve T cell and memory T cell responses to adaptive immune systems.
communicate with antigen-presenting cells [13] by upregulating intercellular adhesion molecule 1 Eczema area and severity index
(EASI): a clinical scoring system that is
used to measure the extent and severity
of actopic eczema.
Granule polarization: a prelude to the
release of cytotoxic contents in
response to target-cell binding.
Innate immunity: a subset of the
immune system that is activated by the
presence of antigens and their chemical
properties, using nonspecific defense
mechanisms, meaning that anything that
is identified as foreign or non-self is a
target. Cells of the innate immune
system include natural killer (NK) cells,
macrophages, mast cells, neutrophils,
and DCs.
Lymphocyte: a subtype of white blood
cells that include NK cells, T cells, and
B cells.
M1 macrophages: polarized
macrophages that encourage
inflammation.
M2 macrophages: polarized
macrophages that decrease
inflammation and encourage tissue
repair.
Macrophage polarization: refers to
how macrophages have been activated
at a given point in space and time in
response to signals from their
microenvironment.
Membrane particles (MPs): particles
of the membrane of a cell ranging from
63 to 700 nm in diameter.
Trends in Pharmacological Sciences Memory T cells: a subset of infection-
and cancer-fighting T cells. They can
Figure 1. Multifaceted Immunomodulatory Interactions between Mesenchymal Stem Cells (MSCs) and
reproduce to mount a faster and
Immune Cells. MSCs exert immunomodulatory functions mainly via interactions with immune cells, such as T cells, stronger immune response, and
B cells, natural killer (NK) cells, macrophages, monocytes, dendritic cells (DCs), and neutrophils, as well as through cell-to-
recognize foreign invaders, such as
cell contacts (blue arrows) and paracrine activity (shown by secretome). The MSC secretome includes several cytokines,
bacteria or viruses, as well as cancer
growth factors, and chemokines, and their immunomodulatory functions vary depending on the source of the MSCs, the
cells.
target cells, and the microenvironment. Abbreviations: ICAM-1, intercellular adhesion molecule-1; IDO, indoleamine-pyrrole
Monocytes: the largest type of white
2,3-dioxygenase; IFN, interferon; IL, interleukin; PD-L1, programmed death ligand 1; PD-L2, programmed death ligand 2;
blood cells that can differentiate into
PGE2, prostaglandin E2; TGF-β, transforming growth factor-β; TNF-α, tumor necrosis factor-α; VEGF, vascular
macrophages and myeloid-lineage DCs.
endothelial growth factor. Figure drawn with BioRender (https://biorender.com/).

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(ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) that are crucial for T cell activation and Naïve T cells: primary cellular effectors
in the adaptive immune system that play
leukocyte (white blood cell, WBC) recruitment to the site of inflammation [14]. Furthermore,
crucial roles in antigen specificity.
coculture of BM-MSCs with activated T cells has been shown to induce lymphocytes that ex- Natural killer (NK) cells: cytotoxic
press interleukin-17A (IL-17A) [15]. MSCs cocultured with CD4+ T cells activate the Notch1/ lymphocytes that are crucial for the
Forkhead box P3 (FOXP3) pathway and increase the percentage of CD4+CD25 FOXP3+ cells innate immune system and provide rapid
responses to virus-infected cells and
[16]. Furthermore, knockdown of galectin 1, a protein that is abundantly expressed intracellularly tumor cells.
and on the cell surface of MSCs, and that has effects on T lymphocytes and cytokine secretion Neutrophils: the most abundant type
[17], in MSCs results in loss of immunomodulatory properties and restores the proliferation of of white blood cells and an essential part
CD4+ and CD8+ T cells [17]. In addition, BM-MSCs express high levels of Toll-like receptors of the innate immune system.
Plasticity: the capacity to differentiate
(TLRs) 3 and 4, which are responsible for nuclear factor κB (NF-κB) activity and cytokine produc- into other cell types in response to
tion. Expression of TLR3 and TLR4 in MSCs has been shown to restore an efficient T cell response different stimuli.
to infection [18]. Human placenta MSCs (PMSCs) have been shown to express high levels of the Regulatory T cells (Tregs): formerly
known as suppressor T cells, Tregs are
cell adhesion molecules programmed death ligand 1 (PD-L1) and PD-L2, which can inhibit T cell
a subpopulation of T cells that modulate
proliferation by arresting the cell cycle [19]. the immune system to downregulate the
induction and proliferation of effector
In vivo mouse models have also shown interactive immune regulation between MSCs and T cells. T cells.
Secretome: the set of proteins that are
In a syngeneic orthotopic mouse model of ovarian cancer, compact bone-derived MSCs
expressed by an organism and secreted
(CB-MSCs) have antitumor effects in combination with a fusion protein consisting of an anti- into the extracellular space, including
mesothelin single-chain antibody variable region (scFv) genetically fused to Mycobacterium cytokines, growth factors, extracellular
tuberculosis heat-shock protein 70 (Hsp70) (designated VIC-008), which is involved in activating matrix proteins, regulators, and shed
receptors.
CD4+ and CD8+ T cells and inhibiting Tregs in the tumor microenvironment (TME) [20]. In fetal
T helper 17 (Th17) cells: a subset of
abortion models, MSCs have been shown to enhance the suppressive regulation of T cells and proinflammatory T helper cells that are
macrophages [21]. Conversely, MSCs primed by activated T cells derived from IFN-γ null mice defined by the production of interleukin
exhibited a dramatically reduced ability to suppress T cell proliferation, and this supports the cell- 17 (IL-17).
Toll-like receptors (TLRs): receptors
to-cell contact mechanism for the immunosuppressive function of MSCs [22]. that sense invading pathogens or
endogenous damage signals, and
In addition to T cells, MSCs also affect B cells through cell-to-cell contact in vitro. A-MSCs have initiate the innate and adaptive immune
been shown to increase the survival of quiescent B cells via contact-dependent mechanisms and response. TLR3 and TLR4 are highly
expressed in BM-MSCs, and their
to facilitate B cell differentiation independently of T cells [23]. In addition, A-MSCs not only inhibit activation induces nuclear factor κB
caspase 3-mediated apoptosis of B cells by upregulating vascular endothelial growth (NF-κB) activity and cytokine production.
factor (VEGF) [24] but also inhibit cell proliferation by blocking the cell cycle of B lymphocytes in Tropism: the ability to migrate to
damaged or diseased tissues or cells.
G0/G1 phase by activating p38 mitogen-activated protein kinase (MAPK) pathways [25].

Cell-to-Cell Contacts with Immune Cells of Innate Immunity

In addition to the adaptive immune system, MSCs also affect the innate immune system through
cell-to-cell contacts. Tracking studies reveal that infused UC-MSCs briefly reside in the lungs
and are rapidly phagocytosed by monocytes, which subsequently migrate to other body sites.
UC-MSC phagocytosis induces phenotypic and functional changes in monocytes, which in turn
modulate cells of the adaptive immune system and thus play a crucial role in mediating, distributing,
and transferring the immunomodulatory effects of MSCs [26]. Coculture studies of MSCs with
different types of NK cell lines (KHYG-1 and NK-92) have shown that granule polarization is either
suppressed or induced, indicating differential crosstalk between MSCs and cytotoxic NK cells [27].
In addition, A-MSCs are known to switch activated inflammatory M1 macrophages to an M2
macrophage-like phenotype via prostaglandin E2 (PGE2) [28]. MSCs can also prevent neutrophil
death via an ICAM-1-dependent mechanism to further exert tissue-protective effects [29].

Immunomodulatory Actions through Paracrine Activity

Importantly, MSCs also exert their immunomodulatory properties by secreting multifunctional
molecules via paracrine mechanisms [12] (Figure 1). This secretome is a diverse repertoire of
multifaceted cytokines, growth factors, and chemokines, which combine to modulate the

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function of immune and cancer cells. These include transforming growth factor-β1 (TGF-β1),
tumor necrosis factor-α (TNF-α), PGE2, IFN-γ, hepatocyte growth factor (HGF), fibroblast
growth factor (FGF), indoleamine-pyrrole 2,3-dioxygenase (IDO), and nitric oxide, among many
others [30,31]. These paracrine factors are encapsulated in cell-secreted extracellular vesicles
(EVs), which in turn are usually divided into exosomes, microvesicles (MVs), and apoptotic bodies
according to their size and cell of origin [30,32]. Although MSC-derived EVs (MSC-EVs) display
immunoregulatory functions similar to the parent MSCs [33], their paracrine actions vary
according to the source of the MSCs, the target cells, and the microenvironment surrounding
the cells [34].

Paracrine Activity on the Adaptive Immune System

MSCs have been shown to act on the adaptive immune system, particularly T cells, via paracrine
secretion. MSCs inhibit T helper 17 (Th17) cell differentiation by inducing the production of IL-10
and PGE2, and by inhibiting IL-17, IL-22, and IFN-γ [35]. Coculture studies of BM-MSCs with
T cells have shown that both priming with cytokines and the cell ratio of BM-MSCs influence their
cytokine profiles, and suggest that BM-MSCs modulate the Th17 lymphocyte pathway in a complex
manner [15]. However, the mechanisms underlying interactions between MSCs and Th17 lympho-
cytes are not fully understood. Previous studies utilizing knockdown of IL-25 in vitro and in vivo have
shown that MSCs suppress Th17 responses via regulation of the IL-25/STAT3/PD-L1 axis [36].
MSC-secreted IDO induces Tregs that are responsible for kidney allograft tolerance [37]. Moreover,
cocultured exosomes derived from BM-MSCs transfected with a plasmid encoding sh RNA
targeting Fas cell death receptor (shFas) and anti-miR-375 and peripheral blood mononuclear
cells (PBMCs) suppress the immune response in an immunodeficient mouse model by inhibiting
PBMC proliferation and enhancing Treg function [38]. In addition, MSCs secrete PD-1 ligands
(including PD-L1 and PD-L2) and exert immunosuppressive effects directly on T cell behavior by
suppressing the activation of CD4+ T cells [39].

Paracrine Activity on the Innate Immune System

In the innate immune system MSCs are known to interact with NK cells by inhibiting IL-2-induced NK
cell proliferation [40] and by inducing cytotoxic activity or cytokine production via secretion of IDO and
PGE2 [41]. MSCs have also been shown to enhance the ability of IL-12/IL-18-stimulated NK cells to
secrete IFN-γ, which has the potential to improve defense against infections at the site of injury and
also influence tissue regeneration [42]. Moreover, MSC-derived IL-6 has been shown to protect neu-
trophils from apoptosis, thus preserving them in the bone marrow niche [43]. MSC-derived exosomes
have been shown to augment neutrophil viability, whereas MSC-conditioned medium (CM) increases
neutrophil function, demonstrating that both MSC-derived exosomes and CM are useful for increas-
ing immunity by modulating neutrophils [44]. Lipopolysaccharide (LPS)-stimulated MSCs augment
the antimicrobial functions of neutrophils via the release of IL-8 and macrophage migration inhibi-
tory factor (MIF), thus contributing to the resolution of infection and inflammation [45].

MSCs-secreted IL-6 can prevent the differentiation of monocytes towards an anti-inflammatory

IL-10 producing phenotype [46], whereas MSCs-derived PGE2 empower MSCs to suppress
the differentiation of monocytes to mature DCs [47]. MSC-derived EVs have been shown to pre-
vent antigen uptake by immature DCs and attenuate DC maturation, accompanied by downreg-
ulation of markers of mature DCs (CD83, CD38, and CD80) as well as of proinflammatory
cytokines (IL-6 and IL-12p70), and upregulation of the anti-inflammatory cytokine TGF-β [48].
In addition, miR-21-5p, a microRNA that is enriched in MSC-derived EVs, has been shown to
influence DC maturation and function [48]. MSC-EVs also play a crucial role in triggering
macrophage polarization [49] by enhancing the formation of anti-inflammatory M2 macro-
phages over M1-like inflammatory macrophages via downregulation of IL-23 and IL-22 [50].

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BM-MSCs activated by LPS or TNF-α reprogram macrophages by releasing PGE2 that acts on
the macrophages through the prostaglandin EP2 and EP4 receptors [51]. Human PMSCs have
also been shown to transform macrophages from an inflammatory M1 into an anti-
inflammatory M2 phenotype via soluble IL-10, IL-1β, IL-12, and macrophage inflammatory
protein 1α (MIP-1α), as well as via the glucocorticoid and progesterone receptors [52].

Preconditioning MSCs To Increase Their Immunomodulatory Functions and

Therapeutic Efficacy
Preconditioning of MSCs is known to influence their therapeutic efficacy, and modification
with diverse factors and a variety of conditions have therefore been explored to manipulate
the secretory profiles and enhance the therapeutic efficacy of MSCs [53]. Preconditioning
MSCs with hypoxia, oxidative stress, heat shock, starvation, or inflammatory biological
agents has potential to increase their survival and potency [53]. The main methods of pre-
conditioning are hypoxia and priming with immunomodulatory factors [32]. We discuss
these briefly next.

Preconditioning with Hypoxia

Hypoxia-preconditioning has been shown to have a positive influence on the MSC immune phe-
notype. Specifically, it increases paracrine and antioxidative effects, particularly the secretion of
angiogenic factors, as observed in examples of acute kidney injury [54] and bleomycin-induced
pulmonary fibrosis [55]. Hypoxia-preconditioning of dental MSCs overexpressing hypoxia-
inducible factor 1α (HIF-1α) can impair DC differentiation, attract more monocytes, and increase
resistance to NK cell-mediated lysis [56]. Hypoxia-preconditioning of CB-MSCs coupled to pre-
treatment with proinflammatory cytokines IFN-γ, TNF-α, and IL-17A improves suppression of
CD8+ T cells [57]. Hypoxia-conditioning of MSCs results in higher infiltration of endothelial cells
into MSC-containing scaffolds for dermal regeneration [58], positively affecting cell fitness and
the transcriptome, and potentially improving cellular therapies targeting orthopedic disorders
[59] as well as promoting maintenance of stem-like characteristics with IDO upregulation [60].
Furthermore, MSCs primed with hypoxia and calcium ions (HC-MSCs) have been shown to be
resistant to passage-dependent senescence mediated via the monocyte chemoattractant pro-
tein 1 and p53/p21 cascade, and secrete large amounts of proangiogenic and immunomodula-
tory factors, resulting in suppression of T cell proliferation in vitro. Administration of HC-MSCs
significantly attenuated the symptoms of GVHD in a humanized mouse model, resulting in signif-
icantly improved survival [61].

Preconditioning by Priming with Immunomodulatory Factors

Another important method of preconditioning is priming with immunomodulatory factors. IFN-γ is
a primary factor that can activate the transcription and synthesis of IDO-1, HGF, and TGF-β in
MSCs [62]. Single IFN-γ priming induces IDO expression and leads to sustained improvement
in the MSC T cell-suppressive phenotype [63]. MSCs primed by IFN-γ have been shown to
have enhanced immunosuppressive function via the IFN-γ–JAK–STAT1 pathway [64,65]. In
addition, membrane particles (MPs) of MSCs stimulated with IFN-γ can increase mRNA
expression of PD-L1 in monocytes as well as the percentage of anti-inflammatory PDL-1- and
CD90-positive monocytes, thus demonstrating the potential of MSC derived MPs as a novel
cell-free therapy for the treatment of immunological disorders [66]. Priming of MSCs with TNF-α
and IFN-γ has been shown to reverse the proinflammatory effects of palmitate, a potent modulator
of MSC immunosuppressive function that is enriched in type 2 diabetes, and can maintain the abil-
ity to block T cell proliferation and cytokine production [62]. More recently, it has been shown that
modification of BM-MSCs with small heat-shock proteins 27 and 20 (sHsp27 and sHsp20) and E7
oncoprotein significantly enhances E7-specific T cell responses and suppresses tumor growth in

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mice, indicating that MSC-based vaccination is a promising approach for immunotherapy and pro-
tection against HPV-associated cancers [67].

Engineering MSCs To Increase Their Immunomodulatory Functions and

Therapeutic Efficacy
MSCs have an intrinsic ability to evade the immune response temporarily. In the context of using
MSCs for targeted therapy, they are mainly considered as gene delivery vehicles for immunomod-
ulatory molecules such as IFNs and ILs, or are engineered to deliver oncolytic viruses (OVs)
(Figure 2A). Engineering MSCs to express specific immunomodulatory agents contribute to
their immunomodulatory capacity and pluripotency, and also enables them to deliver large
doses of cancer-targeting biologics with a single dose. Some examples of engineered MSCs
expressing diverse immunomodulatory molecules are given in Table 1 and are discussed in the
following section.

We previously showed that encapsulated MSCs engineered to express the cytokine IFN-β can
increase the survival of mice with a highly malignant brain tumor, glioblastoma, by promoting se-
lective postsurgical infiltration of CD8 T cells and inducing cell-cycle arrest in tumor cells (Table 1)
[68]. Likewise, recently, engineered MSCs expressing IFN-γ have also been shown to elicit tumor
growth reduction and increase survival via polarization of murine macrophages to the proinflam-
matory M1 phenotype in vitro and in vivo [69]. Similarly, MSCs engineered to express IL-4 have

Trends in Pharmacological Sciences

Figure 2. Three Strategies To Enhance the Therapeutic Efficacy of Mesenchymal Stem Cells (MSCs). (A) MSCs can be engineered with immunomodulatory
molecules such as interferons (IFNs), interleukins (ILs), and miRNA, or can be engineered to deliver oncolytic viruses. (B) MSCs can also be primed, which can contribute to
their efficient homing and migration to the target tissue. (C) Different methods have been modified and standardized to improve the cryopreservation of MSCs.
Abbreviations: CSF-2, colony-stimulating factor 2; CXCR4, CXC chemokine receptor 4; IFN, interferon; IL, interleukin; IGFBP3, insulin-like growth factor binding protein
3; miRNA, microRNA, SDF-1, stromal cell-derived factor-1. Figure drawn with BioRender (https://biorender.com/).

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Table 1. Engineered MSCs Expressing Diverse Immunomodulatory Moleculesa

Engineered agent Model Mechanism Refs
IFN-β Syngeneic orthotopic glioblastoma mouse model Infiltration of CD8 T cells, cell-cycle arrest [68]
IFN-γ Neuroblastoma murine model Macrophage polarization [69]
IL-4 Osteoarthritis rat model Chondroprotective and anti-inflammatory effects [70]
IL-12 Renal cell carcinoma, melanoma, breast tumor, and Reduction of lymphatic sprouts [71,72]
HCC mouse models Apoptosis induction
IL-15 Pancreatic tumor mouse model Tumor-specific T cell immune memory response [73]
IL-21 B cell lymphoma mouse model Induction of effector T and NK cells [74]
SDF-1 Streptozotocin-induced diabetic rats Increasing nNOS, VEGF, and bFGF [75]
miR-199a HCC mouse model mTOR pathway inhibition [76]
Oncolytic virus Brain metastatic melanoma mouse model Increasing IFN-γ-producing CD8+ tumor-infiltrating T lymphocytes [78]
with PD-L1 blockade
HCC mouse model Inhibiting HCC cell proliferation [79]
Lung adenocarcinoma mouse model TLR9 overexpression and activation of the NF-κB pathway [80]

a
Abbreviations: bFGF, basic fibroblast growth factor; IFN, interferon; GVHD, graft-versus-host disease; IL, interleukin; HCC, hepatocellular carcinoma; mTOR, mammalian
target of rapamycin; NK cell, natural killer cell; nNOS, neuronal nitric oxide synthase; SDF-1, stromal cell-derived factor-1; VEGF, vascular endothelial growth factor.

increased chondroprotective and anti-inflammatory effects in a rat model of osteoarthritis [70],

whereas IL-12-engineered MSCs reduce tumor growth and prolong survival in mouse models
of renal cell carcinoma, melanoma, breast tumor, and hepatocellular carcinoma (HCC) via reduc-
tion of lymphatic sprouts and induction of apoptosis [71,72]. Engineered MSCs expressing mu-
rine IL-15 significantly inhibit pancreatic cancer growth and prolong the survival of tumor-bearing
mice, as well as promoting resistance to pancreatic tumor rechallenge, via the induction of tumor-
specific T cell immune memory response [73]. Moreover, MSCs engineered to express IL-21 in-
duce effector T and NK cells, and can prevent the formation of tumor nodules in a mouse model of
B cell lymphoma [74]. In addition, MSCs engineered to express stromal cell-derived factor 1
(SDF-1) have been shown to alleviate erectile dysfunction in streptozotocin-induced diabetic
rats (Table 1) mediated by increasing the levels of neuronal nitric oxide synthase (nNOS), VEGF,
and basic fibroblast growth factor (bFGF,) and by lowering the levels of the apoptosis factors Bcl2-
associated x (Bax) and caspase 3 [75]. Furthermore, exosomes derived from A-MSCs expressing
miR-199 can promote miR-199a delivery to HCC cells and effectively sensitize cancer cells to che-
motherapeutic agents by targeting the mammalian target of rapamycin (mTOR) pathway [76].

MSCs have also been explored as a potential means of OV delivery because they can shield the
virus from host antiviral immunity and transport viral particles systemically or intratumorally [77].
We discuss some examples where the immunomodulatory function of MSCs has been shown
to be enhanced (Table 1). MSCs loaded with oncolytic herpes simplex virus (oHSV) or other
OVs can successfully deliver viral progeny to established glioblastoma tumors and brain-
metastatic melanoma, eventually decreasing tumor growth and prolonging mouse survival
[77,78]. Another study reported that MSCs loaded with HCC-targeted OVs effectively lyze
HCC cells in vitro, ultimately leading to potent tumor growth inhibition [79]. When OV-loaded
MSCs were cocultured with allogeneic PBMCs, they induced TLR9 overexpression and activa-
tion of the NF-κB pathway, creating a proinflammatory environment and enhancing antitumor ef-
ficacy both in vitro and in vivo in lung adenocarcinoma xenograft tumors [80]. In addition,
combined blockade of MSC-oHSV and PD-L1 increases the number of IFN-γ-producing CD8+
tumor-infiltrating T cells, and further increases the median survival of mice in melanoma brain
metastasis models [78,81].

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MSC-Mediated Immunomodulation in Clinical Studies and Trials

MSC infusion has shown potential efficacy in the treatment of several diseases that resist stan-
dard treatment. For example, in a Phase I/II clinical trial (EuDRA CTi: 2012-003741-15) involving
autologous tumor-specific herpes simplex virus thymidine kinase (HSV-TK), MSCs demonstrated fa-
vorable safety and tolerability in patients with gastrointestinal tumors: the median time to progression
was 1.8 months, and median overall survival was 15.6 months (Table 2) [82,83]. However, the study
was terminated early because of an insufficient number of patients. Another recent Phase I/IIa trial
(clinical trialii NCT02351011) using BM-MSCs in patients with osteoarthritis demonstrated that
autologous BM-MSCs are safe and elicit significant reductions in pain and synovial inflammation
[84]. Similarly, an expanded Phase II/III study (clinical trial UMIN-CTR numberiii UMIN000006719)
demonstrated that BM-MSC infusion improved the overall survival rate in patients with steroid-
resistant GVHD (Table 1) [85]. In a Phase IIa study of secondary progressive multiple sclerosis,
autologous BM-MSCs elicited safe and effective neuroprotection (clinical trial NCT00395200) [86].
For patients with moderate-to-severe atopic dermatitis, subcutaneous administration of human
UC-MSCs brought about marked improvement of disease features without serious adverse events,
and 55% of patients showed a 50% reduction in the eczema area and severity index (EASI)
(Table 1) [87]. In refractory fistulizing Crohn's disease, a pilot study therapy with autologous BM-
MSCs proved feasible, safe, and beneficial, and sustained complete closure was observed in 7/12
patients [88]. Another important Phase III study (clinical trial NCT01541579) has assessed the efficacy
and safety of expanded A-MSCs (Cx601) for the treatment of complex perianal fistulas in patients with
Crohn's disease. Cx601 treatment significantly achieved remission with fewer treatment-related ad-
verse events, suggesting that MSCs may be an effective strategy for clinical applications [89].

Nevertheless, it should be noted that MSC-based treatment might produce some potential unex-
pected side effects. For example, recent preclinical studies suggest that alteration of the brain mi-
croenvironment by acute hypothermia modulates MSC function, resulting in a proinflammatory
environment and increasing long-lasting motor-cognitive deficits in the neonatal hypoxic/ischemic
brain [90].

Promising Preclinical Studies That Have Potential To Increase the Efficacy of

Future MSC Trials
Apart from improving immunomodulatory capacity of MSCs (Figure 2A), there are several ap-
proaches to improve MSC migration or homing mechanisms (Figure 2B). Recent studies have

Table 2. Examples of Clinical Trials Currently Using MSCsa

Disease Origin of MSCs Treatment route Clinical trial number Phase Therapeutic efficacy Refs
Gastrointestinal Autologous BM-MSCs Intravenous infusion EudraCT numberi I/II Favorable safety and tolerability, median [82,83]
adenocarcinoma 2012-003741-15 overall survival is 15.6 months
Osteoarthritis Autologous BM-MSCs Intralesional injection Clinical trial I/II Safe, reduction in pain and symptoms, [84]
numberii and reduced synovial inflammation
NCT02351011
GVHD Allogeneic BM-MSCs Infusion UMIN-CTR II/III Improve the overall survival rate [85]
numberiii
UMIN000006719
Multiple sclerosis Autologous BM-MSCs Intravenous infusion NCT00395200 IIa Safe, effective neuroprotection [86]
Atopic dermatitis Human UC-MSCs Subcutaneous NCT01927705 I/II Marked improvement of features without [87]
serious adverse events
Crohn's disease Allogeneic A-MSCs Intralesional injection NCT01541579 III Increased remission with less [89]
treatment-related adverse events

a
Abbreviations: A-MSCs, adipose tissue-derived MSCs; BM-MSCs, bone marrow-derived MSCs; GVHD, graft-versus-host disease; UC-MSCs, umbilical cord blood-derived
MSCs.

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shown that MSC homing is driven by SDF-1-stimulated endothelial cell production of platelet- Outstanding Questions
derived growth factor (PDGF) via activation of PDGF receptor A (PDGFRA)/PI3K/Akt, PDGFRA/ What external factors influence
MAPK/Grb2, and PDGFRA/JAK2/STAT signaling [91]. In addition, FGF21 also influences the immunomodulation mediated by
MSCs?
homing ability of MSCs to injury sites [92], and colony-stimulating factor 2 (CSF-2) has been
shown to significantly enhance their differentiation and migratory capacity [93]. IL-1β induces ma- How can we minimize variation in MSC
trix metalloproteinase-1 (MMP-1), which subsequently activates the protease-activated receptor paracrine effectiveness and therapeu-
1 (PAR1), and G protein-coupled signaling pathways have been recently shown to promote MSC tic efficacy for clinical translation?
migration [94]. Interestingly, intranasally administered MSC-derived exosomes specifically target
Which new preconditioning techniques
and accumulate in the brain in pathological murine models for up to 96 h, and this long homing can lead to better MSC therapy out-
response is driven by neuroinflammatory signals [95]. Hypoxic preconditioning also promotes comes and further enhance therapeu-
the migration and survival of MSCs through increased expression of lincRNA-p21 via the tic efficacy?

HIF-1α/CXCR4 and CXCR7 pathway [96]. Can MSCs be engineered with multiple
immunomodulators that mediate key
Another way to increase the efficacy of MSCs would be to improve methods for their cryopreser- biological signaling pathways?
vation. Recent studies have indicated that cryopreservation modulates the efficacy and clinical
How can we efficiently translate
application of MSCs. Specifically, cryopreserved and thawed MSCs have impaired functional promising preclinical studies into the
properties and stunted immunosuppressive capabilities [97,98], which have significant implica- clinic?
tions for therapeutic outcomes [98]. Therefore, MSCs need to be standardized for reliability and
robust quality-control purposes, especially in large-scale production of frozen products. A recent
study revealed the feasibility of two freezing steps after a cell culture phase of at least one pas-
sage, without affecting basic cell manufacturing parameters or quality attributes of the final frozen
and thawed product [98]. A successful Phase III clinical trial (NCT01541579) using expanded A-
MSCs (Cx601) to treat patients with Crohn's disease revealed that a recovery period of at least 24
h after cell thawing improved MSC product quality [89]. These MSCs maintained their multipotent
differentiation, immunomodulatory, and anti-inflammatory properties [97]. Therefore, a 24 h accli-
mation period to reactivate thawed cells is crucial to restore their diminished MSC function
(Figure 2C).

Concluding Remarks and Future Perspectives

MSCs have emerged as a promising therapeutic strategy because of their tropism for other cell
types as well as their immunomodulatory functions. The immunomodulatory ability of MSCs is
regulated by different inflammatory cytokines, and the interaction between immune cells and
MSCs could contribute to regeneration as well as to the progression of different inflammatory dis-
eases. The main mechanisms involved in MSC immunomodulation are cell-to-cell contact and
paracrine activity primed by cytokines, chemokines, extracellular vesicles, inflammatory stimuli,
or coculture with other cells. Therefore, priming or licensing of MSCs has made cell-free therapy
a controllable, manageable, and feasible method. However, several issues remain to be solved
(see Outstanding Questions). MSCs are very heterogeneous and change significantly with inflam-
matory or anti-inflammatory stimuli. Therefore, it is difficult to determine how MSC variability influ-
ences their induced immunomodulatory effects. It is possible that viable MSCs provoke more
complex immunomodulatory mechanisms because of their intact secretome. Future work should
explore the influence of other factors and/or chronic inflammation on immunomodulation medi-
ated by MSCs. This has the potential to identify new methods of preconditioning to enhance
MSC efficacy and minimize variation in MSC paracrine effectiveness and therapeutic efficacy, es-
pecially with regards to clinical translatability.

In the context of using MSCs for targeted therapy in the clinic, they are mainly considered as gene
delivery vehicles for immunomodulatory molecules or OVs. Because engineered MSCs releasing
targeted therapeutics are already being used in multiple clinical trials for immune and inflamma-
tory diseases, it is essential to define the targets in the diseased cells/microenvironment to

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Trends in Pharmacological Sciences

develop effective MSC therapies. For example, previous studies have shown that MSCs
engineered to coexpress an antigen-specific domain (VHH) targeting epidermal growth factor re-
ceptor (EGFR) and death receptor (DR4/5) ligand (DRL) target both cell proliferation and death
pathways in tumors [99]. MSCs engineered to express TandAb, a tetravalent bispecific tandem
diabody for CD3 and CD19, were recently found to have enhanced therapeutic efficacy in a
mouse model of B cell lymphoma as compared with targeting CD3 or CD19 alone [100].

A challenge in the field is the discrepancy between in vitro and in vivo findings in MSC research. For
example, in a New Zealand white rabbit osteogenesis model, osteogenic cells differentiated from
MSCs (DOC cells) retained their immunoprivileged and immunomodulatory properties in vitro,
but their immunomodulatory properties were lost following transplantation [101]. In a rhesus ma-
caque model, allogeneic versus autologous MSCs transplanted intracranially revealed that alloge-
neic MSCs, but not autologous MSCs, were weakly immunogenic post-transplantation, thus
negatively affecting the levels of long-term engraftment [102]. These discrepancies underline the
importance of careful monitoring of MSC immunogenicity when moving from an in vitro to an
in vivo model, as well as in further clinical applications, so as to ensure a lasting effect. Ultimately,
by enhancing their immunomodulatory potential and homing mechanisms, and by improving cryo-
preservation techniques, in conjunction with rigorous preclinical and clinical studies, MSCs have
enormous potential for wider application in clinical settings in the future.

Acknowledgments
This work was supported by National Institutes of Health grant R01-NS107857 and Department of Defence grant
CA180698 to K.S.

Disclaimer Statement
K.S. owns equity in and is a member of the Board of D irectors of AMASA Therapeutics, a company developing stem cell-based
therapies for cancer; his interests were reviewed and are managed by Brigham and Women’s Hospital and Partners HealthCare
in accordance with their conflict of interest policies. The other authors declare that they have no competing interests.

Resources
i
https://eudract.ema.europa.eu/
ii
https://clinicaltrials.gov/
iii
https://www.umin.ac.jp/

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