cells
Review

Mesenchymal Stem Cells in the Treatment of COVID-19,
a Promising Future
Daniela Gois Beghini, Samuel Iwao Horita and Andrea Henriques-Pons *Laboratório de Inovações em Terapias, Ensino e Bioprodutos, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz,
Rio de Janeiro 21040-360, Brazil; beghini@ioc.fiocruz.br (D.G.B.); samuelhorita@hotmail.com (S.I.H.)* Correspondence: andreah@ioc.fiocruz.br; Tel.: +55-21-2562-1319

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Citation: Beghini, D.G.; Horita, S.I.;

Abstract: Mesenchymal stem cells (MSCs) are multipotent adult stem cells present in virtually all
tissues; they have a potent self-renewal capacity and can differentiate into multiple cell types. They
also affect the ambient tissue by the paracrine secretion of numerous factors in vivo, including theinduction of other stem cells’ differentiation. In vitro, the culture media supernatant is named secretome and contains soluble molecules and extracellular vesicles that retain potent biological function
in tissue regeneration. MSCs are considered safe for human treatment; their use does not involve
ethical issues, as embryonic stem cells do not require genetic manipulation as induced pluripotent
stem cells, and after intravenous injection, they are mainly found in the lugs. Therefore, these cells
are currently being tested in various preclinical and clinical trials for several diseases, including
COVID-19. Several affected COVID-19 patients develop induced acute respiratory distress syndrome(ARDS) associated with an uncontrolled inflammatory response. This condition causes extensive
damage to the lungs and may leave serious post-COVID-19 sequelae. As the disease may cause
systemic alterations, such as thromboembolism and compromised renal and cardiac function, theintravenous injection of MSCs may be a therapeutic alternative against multiple pathological manifestations. In this work, we reviewed the literature about MSCs biology, focusing on their function inpulmonary regeneration and their use in COVID-19 treatment.

Henriques-Pons, A. Mesenchymal
Stem Cells in the Treatment of

Keywords: mesenchymal stem cells; COVID-19; cell therapy; inflammation; tissue regeneration

COVID-19, a Promising Future. Cells
2021, 10, 2588. https://doi.org/
10.3390/cells10102588

1. Mesenchymal Stem Cells Can Be Isolated from Different Adult Tissues
Academic Editor: Joni H. Ylostalo
Received: 18 August 2021
Accepted: 17 September 2021
Published: 29 September 2021

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Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
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Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Mesenchymal stem cells (MSCs) were described by Friedenstein in 1970 [1] and werefirst isolated from the bone marrow as non-hematopoietic stem cells. In 1991, Caplan
introduced the term “mesenchymal stem cells” because it was believed that these cells
would differentiate into mesodermal (middle germ layer) cells, such as bone, cartilage,
tendon, fat, skin, muscle, and marrow stromal cells [2–4]. However, today we know that
MSCs can also differentiate into ectodermal [5] and endodermal [6] cell lines, characterizingthem as pluripotent stem cells [7,8]. They are adherent spindle-shaped cells found in
virtually all adult tissues as a population of undifferentiated tissue-resident cells thatfacilitate tissue remodeling and repair during life [9]. MSCs generate progeny by selfrenewal and can differentiate into various cell types depending on the particular tissue.
However, in many organs, their pluripotency decreases with age, leading to reduced
regenerative potential.
Although several trials are being conducted using MSCs to regenerate multiple tissues and organs, much remains to be elucidated about their biology and regenerative
capacity. In general, MSCs are perivascular cells [10,11] that can be expanded for long
periods in culture and, as observed in vivo, extended maintenance in vitro may reduce
their pluripotency, limiting their applicability. Moreover, empirical evidence demonstratingthat MSCs have the capacity for asymmetric cell division is still lacking [12], a characteristicof conventional stem cells [13]. Additionally, by definition, stem cells are rare cell populations that undergo self-renewal and yield progenitors to differentiate hierarchically into

Cells 2021, 10, 2588. https://doi.org/10.3390/cells10102588

https://www.mdpi.com/journal/cells

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Cells 2021, 10, 2588

2 of 19characteristic of conventional stem cells [13]. Additionally, by definition, stem cells are
2 of 18
rare cell populations that undergo self-renewal and yield progenitors to differentiate hierarchically into other cell types. However, MSCs appear to affect different cell types andalter the tissue microenvironment via paracrine signaling, inducing differentiation ofotherresident
cell types.
However,
MSCs appear
to affectdifferent cell
types andalteration
alter the oftissueotherstemcells (cooperative
activity),
angiogenesis,
chemotaxis,
themicroenvironment
paracrine
signaling,
inducing
differentiationof otherresident
stem
local
inflammatory via
response
againstpathogensand damage[14], tissue
repair,proliferacells apoptosis(cooperative
activity),tion,
control,
and angiogenesis,
others [15,16].chemotaxis, alteration of the local inflammatory
response
againstpathogensanddamage[14],
tissue repair, proliferation,
apoptosis
control,
Finally, MSCs seem to bea muchmoreheterogeneous
population thaninitially
enviandothers[15,16].
sioned. It is unclear, for example, whether these cells regenerate only in the tissues from
MSCs seembe heterogeneitya much moreallows
heterogeneous
population
whichFinally,
they originate,
or if totheir
the differentiation
intothanotherinitiallytissueenvisioned.
Itisunclear,forexample,whetherthese
cells
regenerate
onlyin
the tissues
parenchymal cell types in vivo [3,9]. Moreover, there are no ethical issues involved,
infrom whichtheyor ifstem
theircells
heterogeneity
allowsthe differentiation
into
contrast
to theuseoriginate,of embryonic
in regenerative
medicine,
and they canbeotherhartissue parenchymal
cell types
in vivo[3,9].from
Moreover,there areandno adipose
ethical issues
vestedin relatively large
numbers,
mainlybone marrow
tissueinvolved,[17]. Allincontrast
to
the
use
of
embryonic
stem
cells
in
regenerative
medicine,
andthey can bethese characteristics contribute significantly to the therapeutic use of MSCs [14].
harvested
relatively
large numbers,
mainly frombone marrow
adiposetissue[17].
Due tointhelack of definitiveand consensualmarkers,
human and
MSCshave been
miniAllthesecharacteristics
contributesignificantly
tothetherapeutic
use
of
MSCs
[14].
mally identified using their capacity to adhere to plastic culture surfaces, and their canonDue to the lack of definitive and consensual markers, human MSCs have been miniical phenotype is based on expressed markers after in vitro expansion [18]. The identificamally identified using their capacity to adhere to plastic culture surfaces, and their canonicaltion of MSCs is usually based on the combined expression of CD73, CD90, and CD105,
phenotype is based on expressed markers after in vitro expansion [18]. The identification ofwith no expression of CD34, CD45, CD14, and HLA-DR (Figure 1A). Moreover, it is also
MSCs is usually based on the combined expression of CD73, CD90, and CD105, with no expresnecessary that they differentiate into three lineage cells: adipocytes, chondrocytes, andsion of CD34, CD45, CD14, and HLA-DR (Figure 1A). Moreover, it is also necessary that theyosteocytes, under inductive culture conditions [18]. So far, bone marrow-derived MSCs
differentiate into three lineage cells: adipocytes, chondrocytes, and osteocytes, under inductive
are the best-characterized cells with multipotent differentiation capacity. These cells haveculture conditions [18]. So far, bone marrow-derived MSCs are the best-characterized cells withfibroblast-like morphology, adhere to plastic, and undergo extensive proliferation andmultipotent differentiation capacity. These cells have fibroblast-like morphology, adhere to plasclonal expansion confirmed by colony-forming unit fibroblast assay (CFU-F). MSCs cantic, and undergo extensive proliferation and clonal expansion confirmed by colony-forming unit
also express various additional surface markers, including CD10, CD73, CD140b, CD146,
fibroblast assay (CFU-F). MSCs can also express various additional surface markers, including
the disialoganglioside GD2, and CD271. Moreover, they may express pluripotency markCD10, CD73, CD140b, CD146, the disialoganglioside GD2, and CD271. Moreover, they may
ers
suchpluripotency
as Oct4, Nanog,
andsuchstage-specificembryonic
antigen-4 (SSEA-4)
thatantigen-4
identify
express
markers
as Oct4, Nanog,
and stage-specificembryonic
differentin thebone
marrow in[19].
(SSEA-4) cell
thatpopulations
identify different
cellpopulations
the bone marrow [19].

Figure 1.1.Mesenchymal
Mesenchymal
stem
cells
(MSCs),
generalcharacteristics
function.
(A)conventional
The conventional
phenotypefor
Figurestem
cells(MSCs),
generalcharacteristics
and andfunction.
(A) Thephenotype
for MSCs
identification
includes
CD73,
CD90,
and
CD105
with
no
CD34,
CD45,
CD14,
and
HLA-DR
expression.
Once
isolated,
MSCs
MSCs identification includes CD73, CD90, and CD105 with no CD34, CD45, CD14, and HLA-DR expression. Once
differentiate
intodifferentiatechondrocytes,
adipocytes,
and osteoblasts
vitro.
(B) MSCs’inbiologicalin treatingfunctions
COVID-19
isolated, MSCs
intochondrocytes,
adipocytes,inand
osteoblasts
vitro. (B)functions
MSCs’ biologicalinpneumonia(andsystemic
disease)includeimmunomodulatory
andantiviral
activity,
angiogenesis,celldifferentiation,
treating COVID-19 pneumonia (and systemic disease) include immunomodulatory and antiviral activity, angiogenesis, cellmesenchymal–epithelialtransition (MET),
environment
to the paracrine
secretionof soluble
factors(sedifferentiation, mesenchymal–epithelialtransition(MET),modulation
environmentdue
modulation
due to theparacrine
secretionof soluble
cretome), and tissue repair.
factors (secretome), and tissue repair.

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The nomenclature “MSC” causes great confusion in the scientific literature and canalso mean “mesenchymal stromal cells”. Thus, the International Society for Cellular
Therapy (ISCT) suggests that the fibroblast-like plastic-adherent cells, regardless of thetissue from which they were isolated, be termed “multipotent mesenchymal stromal cells”.
Then, the term mesenchymal stem cells should beused only for cells that meet specific stem
cell criteria. The widely recognized acronym, MSC, may be used for both cell populations
since researchers define the correct designation in their research [20]. In fact, mesenchymal
stromal cells are present in the stroma of several organs and tissue and are a heterogeneous
population comprising several cell types such as stem cells, progenitor cells, fibroblasts,
and many others [21,22]. Thus, it is the stem cells present in this population that candifferentiate into cells of the mesodermal, ectodermal, and endodermal lineage, at leastin vitro [23]. Therefore, the acronym MSC refers more appropriately to the stem cells
defined by the term multipotent mesenchymal stromal cells.
MSCs isolated from different adult tissues are highly heterogeneous. In fact, a comparison of MSCs isolated from bone marrow, umbilical cord blood, or adipose tissue showed
diverse characteristics in cell differentiation [24]. With the large number of clinical trials
involving MSCs, the scientific community gathered to standardize the studies. This group
aimed to prevent the propagation of questionable stem cell-based results and avoid themisuse and commercialization of uncertain stem cell-based treatments. Thus, a consensus
was reached with the content of a tool named “DOSES” which is based on the reporting offive core items: D—donor (i.e., autologous, allogeneic, xenogeneic); O—origin of tissue;
S—Separation from other cell types/preparation method; E—exhibited cell characteristics
associated with behavior; and S—site of delivery [25,26].
2. MSCs’ Regenerative Capacity in the Lungs
With the onset of the COVID-19 pandemic and the high mortality rate due to pneumonia and thromboembolic events in the lungs, several initiatives using MSCs started
being tested to treat the acute phase of symptomatic patients and pulmonary post-COVID
sequelae. The lung is conditionally renewed, and the renovation of epithelial cells in
the airways, for example, is less than 1% per day under normal healthy conditions [27].
The lung’s regenerative capacity contrasts with continuously renewed tissues, such as skinand the bone marrow, which, considering proliferation, self-renewal, and differentiationof hematopoietic stem cells (HSCs), generates approximately 1 × 109 hematopoietic cells
daily. Similarly to the kidneys and liver, the lungs undergo compensatory growth quickly
by increasing the cellular regeneration rate after injury [27].
Several preclinical and clinical trials indicate that MSC-based therapies are safe totreat lung diseases [28]. In 2007, it was demonstrated that plastic-adherent cells could
be observed in bronchoalveolar lavage (BAL) fluid from patients that underwent a lungtransplant. In this case, clonal proliferation of fibroblast-like cells was observed, andimmunophenotypic analysis showed the expression of vimentin and prolyl-4-hydroxylase,suggesting a mesenchymal phenotype. Multiparametric flow cytometry analysis further
revealed the expression of CD73, CD90, and CD105, and hematopoietic lineage markers
such as CD14, CD34, and CD45 were absent. In addition, they showed multipotency, withthe ability to differentiate into adipocytes, chondrocytes, and osteocytes in vitro [29].
MSCs can differentiate into epithelial cells [30], and a remarkable and reversible
phenomenon named mesenchymal–epithelial transition (EMT) and epithelial–mesenchymaltransition (MET) can occur in embryo development, tumor progression, wound healing,
and infections [31] (Figure 1B). In EMT, the natural polarity of epithelial cells is lost due
to a profound alteration in the cytoskeleton, associated with the loss of cell–cell adhesion
molecules. Moreover, these cells gain migratory and invasive properties and assume an
undifferentiated phenotype [32]. This process is triggered by receptors such as the transforming growth factor-β (TGFbRs) and (neurogenic locus notch homolog) NOTCH, andsignaling pathways as wingless-related integration site (Wnt) and hypoxia response. Then,transcription factors (TFs) such as ZEB1/2, Snail1/2, Twist, and LEF-1 [33] are activated.

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These TFs bind and inhibit the expression of genes encoding adherens, tight, GAP junction
molecules, desmosomes, E-cadherin, ZO-1, claudins, occludin, and others [34]. While
EMT is mainly associated with tumor progression and metastasis, MET leads mostly towound healing and cicatrization. MET is triggered by growth factor receptors, such as
fibroblast growth factor receptors (FGFRs), that bind to FGF10 and -7, for example. This
membrane interaction leads to the upregulation of the transcription repressors Sox2 andOct4 that suppress the EMT mediator Snail1 [35]. Moreover, c-Myc downregulates TGF-b1
and TGF-b receptor 2, and the TF Klf4 activates the epithelial program. These interactions
induce epithelial genes such as E-cadherin, EPCAM, MPZL2, STK17A, CLDN3 (claudin),
FAM3C, and many others.
Still regarding lung epithelial cells, studies in mice indicate that the lungs containmultiple stem and progenitor cell populations residing in distinct regional niches, besides
MSCs [27]. Several types of endogenous airways and alveolar epithelial progenitor cells
are distributed in microenvironments throughout the pulmonary tract. Therefore, many
pulmonary progenitor cells may also be suitable for future studies and clinical trials, withpotential great clinical importance [36,37]. A study used the c-kit (CD117) marker to identify stem cells in the human lung and generated epithelial and mesodermal cell lineages
in culture [38]. In another study, human lung stem cells formed functional and structurally integrated bronchioles, alveoli, and pulmonary vessels after injection into damaged
mouse lungs in vivo [38]. Another putative lung population of human stem cells with the
E-Cadherin+ Lgr6+ phenotype was isolated and expanded in vitro, showing the ability to
self-renew and differentiate in vitro into bronchioalveolar cells. In addition, when the cells
were injected into the kidney’s capsule, they produced fully differentiated bronchioalveolar
tissue [39]. Using sorted murine EpCAM+ epithelial stem/progenitor cells cocultured
with lung mesenchymal EpCAM− Sca-1+ cells, not MSCs, it was demonstrated that themesenchymal cells supported the proliferation and differentiation of lung epithelial cells.
The authors used a lung injury model and showed the importance of microenvironmental
factors, such as FGF10, in epithelial regeneration [40].
Several authors showed that human lung MSCs share common features with bonemarrow MSCs. For example, a murine CD45− cell subset also expressing Sca-1, CD73, andCD105 generated a population of cells capable of adipogenic, osteogenic, and chondrogenic
differentiation in vitro [41]. Based on that phenotype, the cells were sorted and differedfrom unsorted lung adherent cells, exhibiting a pattern of gene expression nearly identicalto bone marrow-derived MSCs. In BAL fluid, the same authors observed that MSCs from ahuman asthmatic patient had identical cell surface markers and differentiation potential
compared with bone marrow-derived cells [41]. Besides the similarities in phenotype
and biological function between MSCs from both compartments, the authors concluded
that allergen sensitization and challenge increased the population of lung MSCs. Hoffman et al. also compared adult lung and bone marrow MSCs [42] and showed that MSCs
from both compartments are phenotypically and biologically similar in vitro and in vivo
after transplantation. However, lung MSCs showed greater survival rates, were moreresistant to anchorage-dependent short-term culture, and had enhanced lung retention
after the intravenous transplantation. The authors discussed that the increased retention
in the lungs was probably due to a higher expression of intercellular adhesion molecule-1(ICAM-1), platelet-derived growth factor receptor alpha (PDGFRα), and integrin α2 [42].
Moreover, lung MSCs were more efficient in healing elastase-induced injury due to theparacrine anti-inflammatory effect of these cells [42]. Finaly, some studies also showed that
MSCs can prevent cell death of alveolar macrophages in vitro by reducing the expressionof Bax, a pro-apoptotic protein, and increasing the anti-apoptotic protein Bcl-2 [43].

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MSCs also have antimicrobial activity (Figure 1B) and intra-pulmonary cell delivery
in a murine model of acute lung injury induced by LPS, improved survival, and mitigated inflammation [44]. MSCs alone have beneficial effects in sepsis induced by cecal
ligation and puncture, where they reduced systemic and pulmonary cytokine levels in
mice, preventing acute lung injury and organ dysfunction [45]. Using an in vivo mousemodel of E. coli-induced pneumonia, intratracheal administration of MSCs reduced bacterial growth in lung homogenates and BAL fluid. The BAL itself from MSC-treated mice
had a more significant antimicrobial activity than the BAL from PBS-treated mice. This
antimicrobial activity was in part mediated by the secretion of the cathelicidin antimicrobial
peptide LL-37 [46].
For human treatment, MSCs are generally administered by the intravenous route.
Then, it is necessary to understand how these cells home to target tissues [47] since theymust interact with endothelial cells and transpose the endothelial barrier to reach damaged
or infected tissues. However, while transplanted MSCs flow throughout the recipient’s
body, it seems that many of these cells are physically trapped within low-caliber biforked
pulmonary vases [48,49]. This possibility may be a partial limitation for MSC-based
regimens to treat other damaged tissues; although local vasodilators could easily avoid it,
it is a positive characteristic to treat COVID-19 pulmonary impairment.
Regarding the repertoire of adhesion molecules required for cell tethering and adhesion do endothelium, it was demonstrated that MSCs behave much like leukocytes [50].
Then, they require interaction with P-selectin expressed by endothelial cells and the engagement of vascular cell adhesion molecule 1 (VCAM-1) with very late antigen 4 (VLA-4
or CD49d) [51]. The adhesion of circulating MSCs to the endothelium also depends on
shear stress due to the flow rate and previous activation of epithelial cells by TNF or IL-1β,
but not intercellular cell adhesion molecule (ICAM-1) expression [52]. Although a matterof debate, endogenous MSCs also circulate in peripheral blood at very low frequencies,
and their number significantly increases after infection or injury [53]. Moreover, in blood,
MSCs may be bound and shielded by platelets and neutrophils, which may affect theircapacity to interact with activated endothelium for transmigration [54–56].
Understanding all characteristics involved in MSCs biology regarding homing, migration, differentiation capacity, paracrine function, and tissue regeneration will improve theoutcome of their clinical use and pave the way for future applications [57], including COVID-19.
3. MSCs Are Being Used in Several Treatments
Bone marrow-derived MSCs have been investigated in several in vivo models of lungdiseases and are more attractive than embryonic stem cells, as indicated in a previous
item [44,58–61]. They are also more suitable than induced pluripotent stem (iPS) cells, asthese cells must be genetically manipulated in a laboratory [62]. MSCs offer great promisefor treating several wasting and as yet incurable lung diseases, including emphysema,
idiopathic pulmonary fibrosis, pulmonary hypertension, and acute respiratory distresssyndrome [63]. One study showed that a population of bone marrow-derived stromal cells
transfected with hepatocyte growth factor (HGF) co-expressed MSCs-associated markers,
such as CD44, CD29, CD105, CD90, and CXCR4 in rats. To understand whether these cells
could have antifibrotic effects, bone marrow MSCs transfected with HGF were injected
into fibrotic areas in the lung of rats treated with bleomycin. In this condition, the authors
observed that the fibrosis was alleviated [64] (Table 1). The mechanism involved in thisbeneficial effect has not yet been fully understood, but experimental evidence points toa paracrine activity for repairing injured tissue cells. In a model of bronchopulmonary
dysplasia, the intratracheal delivery of bone marrow MSCs on post-natal day four improved
survival and exercise tolerance while attenuating alveolar and lung vascular injury andpulmonary hypertension. However, the number of engrafted cells was disproportionately
low to solely account for the therapeutic benefit observed, suggesting a paracrine-mediatedmechanism [65]. Another study showed the effect of MSCs in a mouse model of ovalbumin(OVA)-induced allergic inflammation and evaluated the effects of systemic administration

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6 of 18of human MSCs over allergic inflammation. The authors observed pathology attenuation in
many areas, with mucus production and inflammatory cell infiltration inhibition, improved
lung function, reduced IgE serum levels, and increased Th2-driven cytokines [66] (Table 1).
Table 1. Different pulmonary diseases and the described effects of MSCs treatment.
Disease and Disease Model

MSCs Origin

Pathogeny Improvement

References

Bleomycin-induced usualinterstitial pneumonia
Bronchopulmonary
dysplasia

Rat stromal
cells

Reduced fibrosis

[64]

Rat

Increased mice survival andimproved exercise tolerance[65]

OVA-induced allergy

Human MSCs
intravenously
injected into
mice

Reduce mucus, inflammation,
improved pulmonary function[66]

Bacteria-induced pneumonia

Mouse

Incluenza infection
Sulfur mustard-induced
lung injuty
emphysema
Acute respiratory distresssyndrome (ARDS)

Mouse

Rat

Increased mice suvival andbacterial clearance
Reduced lung injury
Improved pulmonary function,
Altered Th1/Th2 balance
Improved pulmonary function

Human

Reduced mortality

Mouse

[67]
[68–70]
[71]
[72]
[73]

In a murine model of Gram-negative-induced pneumonia, the treatment with MSCs
enhanced mouse survival and promoted bacterial clearance, which was at least partially
due to lipocalin 2 upregulation produced by the MSCs [67]. The application of MSCs was
also efficient in controlling influenza infection, leading to reduced lung injury. This effect
was attributed to attenuated pro-inflammatory cytokines secretion, reduced recruitment
of inflammatory cells, and increased alveolar macrophages [68–70] (Table 1). In sulfur
mustard-induced lung pathology, one of the most lethal chemicals that leads to major
pulmonary complications, the treatment with MSCs was very effective. After intraperitoneal administration of 2-chloroethyl ethyl sulfide (CEES), a sulfur mustard analog, theprogression of histopathological changes in the lungs reduced, and the Th1/Th2 balance
was restored [71] (Table 1).
Progressive pulmonary inflammation and emphysema have been implicated in chronic
obstructive pulmonary disease progression, and the effect of MSCs administration in thepulmonary function was evaluated in rats. The animals were exposed to cigarette smoke
for eleven weeks, followed by the administration of MSCs into the lungs. This procedure
improved emphysema and destructive pulmonary function by suppressing the inflammatory response, excessive protease expression and apoptosis, and by upregulating vascularendothelial growth factor (VEGF) receptor 2 and TGF-β [72]. The transplant of MSCs into
patients with H7N9-induced acute respiratory distress syndrome (ARDS) has already been
conducted, and it significantly reduced the mortality of patients in the experimental group,compared with the control group (17.6% against 54.5%) [73] (Table 1).
4. MSCs and Their Immunomodulatory Capacity
The primary mechanism of MSC-mediated immunosuppression may vary among
different species. For example, immunosuppression of human- or monkey-derived MSCs
is mediated by indoleamine 2,3-dioxygenase 1 (IDO1), whereas mouse MSCs utilize nitricoxide (NO) under the same culture conditions [74]. The beneficial effect of MSC-based
therapies is multifaceted and may vary according to the target tissues/organs and thenature of the damage. They seem to exert therapeutical activity by enabling the damaged
tissues to establish a balanced inflammatory response and regenerative microenvironment.
Therefore, MSCs have high plasticity, adopting immunosuppressive or pro-inflammatory
phenotypes according to the environment (Figure 1B). For example, it has been publishedCells 2021, 10, 2588

is mediated by indoleamine 2,3-dioxygenase 1 (IDO1), whereas mouse MSCs utilize nitricoxide (NO) under the same culture conditions [74]. The beneficial effect of MSC-based
therapies is multifaceted and may vary according to the target tissues/organs and the nature of the damage. They seem to exert therapeutical activity by enabling the damaged
7 of 18
tissues to establish a balanced inflammatory response and regenerative microenvironment. Therefore, MSCs have high plasticity, adopting immunosuppressive or pro-inflammatory phenotypes according to the environment (Figure 1B). For example, it has been
published that in the presence of high levels of pro-inflammatory cytokines or after enthat in the presence of high levels of pro-inflammatory cytokines or after engagement ofgagement of the Toll-like receptor (TLR)-3 [75], MSCs adopt an immunosuppressive phethe Toll-like receptor (TLR)-3 [75], MSCs adopt an immunosuppressive phenotype andnotype and reduce cell proliferation and function of multiple cells of the immune system.
reduce cell proliferation and function of multiple cells of the immune system. On the otherOn the other hand, in the early inflammation phase, with low inflammatory cytokines
hand, in the early inflammation phase, with low inflammatory cytokines levels and after
levels engagement,
and after TLR-4
engagement,
MSCs adopt a pro-inflammatory
phenotypeand enTLR-4
MSCs
adopt a pro-inflammatory
phenotype and enhance
the immune
hance
the
immuneresponse
[75].
These
responses
aremediatedbythereleaseof
growthresponse [75]. These responses are mediated by the release of growth factors, microvesicles,
factors, microvesicles,
complement
components,
immunosupchemokines,
cytokines, chemokines,
complement cytokines,
components,
immunosuppressive
mediators,
andpressive
mediators,
andmanyothers[76].
many others [76].
In mostmost cases,
cases, however,
however,MSCs
MSCsareareinvolved
involvedin
indownregulating
downregulatingthe
theimmune
immuneresponse
responseInbysilencing
multiple
effectorTlymphocyte
subpopulations
andinducing
Treg
cells(Figby silencing multiple effector T lymphocyte subpopulations and inducing Treg cells
ure
2A).
In
addition,theycandirectlyorindirectly
inhibitdisease-associated
Th1-,
Th2-,
(Figure 2A). In addition, they can directly or indirectly inhibit disease-associated Th1-,or Th17-mediatedcell responses
as wellcytotoxic
T lymphocytes
function,but manyTh2-,or Th17-mediatedcell responses
as as
well
as cytotoxic
T lymphocytes
function,but
centralquestionsremain[77].
Indiabetes,itwasdemonstrated
that
MSCs
that
express
ILmany central questions remain [77]. In diabetes, it was demonstrated that MSCs that1Ra,anaturalsolubleinhibitor
of
the
IL1
receptor,protected
thetissuefrom
inflammaexpress IL-1Ra, a natural soluble inhibitor of the IL1 receptor, protected the tissue fromtion-induced injuries andcontrolled
the disease[78].onset
It has[78].
alsoItbeen
proposedthat
inflammation-induced
injuriesand controlled
theonsetdiseasehas also
been proMSCs
induceT-cellanergyasapotential
mechanismof
immunesuppression,as
MSCs
posed that MSCs induce T-cell anergy as a potential mechanism of immune suppression,lack
surfaceof costimulatory
molecules,molecules,
such as CD80
CD86
(B7-2),
andas
MSCs
lackexpression
surface expressionof costimulatory
such(B7-1),
as CD80(B7-1),
CD86
others and[79].others [79].
(B7-2),

FigureFigure2.2.Mesenchymal
Mesenchymal stem
stem cells
cells andandtheirtheirimmunomodulatory
immunomodulatory capacity.
capacity. (A)(A) InIn mostmost cases,
cases, MSCs
MSCs areareinvolved
involvedin
inthe
the
downregulation
downregulationof
ofthe
theimmune
immuneresponse,
response,silencing
silencingcells
cellssuch
suchasasdendritic
dendriticcells
cells(DCs),(DCs),TTand
andBBlymphocytes,
lymphocytes,NK
NKcells,
cells,and
and
neutrophils.
neutrophils.Moreover,Moreover,they
theyareareinvolved
involvedin
inthe
theinduction
inductionof
ofcells
cellssuch
suchasasTregTregandandmacrophages
macrophagespolarization
polarizationtowards
towardsM2
M2
function. Several factors are also secreted from MSCs, such as prostaglandin E2 (PGE2), TGF-β, hepatocyte growth factor(HGF), vascular endothelial growth factor (VEGF), IDO, and others that can inhibit the inflammatory response. (B) The
MSCs are dichotomic and can be immunosuppressive when in a pro-inflammatory environment (upper drawing); when in
the presence of acute infections or exposed to low levels of pro-inflammatory cytokines, they may potentiate the immune
response (lower drawing).

Through the secretion of multiple inflammatory inhibitors such as prostaglandin
E2 (PGE2), membrane molecules such as HLA-G, and cytoplasmic mediators such as
IDO1, MSCs inhibit neutrophils, B and T lymphocytes, NK cells, and others (Figure 2A),
also attenuate the production of IL-2 [80]. Moreover, MSCs secrete or express IL-10 on
the cell membrane [81], produce leukemia inhibitory factor (LIF) [82], galectin-1 andsemaphorin 3A [83], and galectin 3 [84], all of which are critical immunosuppressive
molecules. Interestingly, interferon-gamma (IFN-γ) and tumor necrosis factor (TNF) areCells 2021, 10, 2588

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known to enhance the immunosuppressive properties of MSCs, in agreement with theview that these cells downmodulate ongoing inflammatory responses [85] (Figure 2B).
MSCs can also reduce the differentiation of naïve CD4+ T lymphocytes into Th1
effector cells or attenuate their function by decreasing IFN-γ production and promoting ashift towards a Th2 immune response. In this case, MSCs downregulated the expressionof the natural killer group 2, member D (NKG2D) receptor on CD8+ T lymphocytes, thensuppressing cell activation [86]. The same study showed that MSCs increased PGE2, IDO1,
and TGF-β1 secretion when cocultured with CD8+ T lymphocytes [86]. MSCs can also
suppress the proliferation of NK cells (Figure 2A). At low NK to MSC ratios, MSCs alter thephenotype of NK cells and suppress cell proliferation, cytokine secretion, and cytotoxicity
against HLA-class I-dependent target cells [87]. MSCs have also been shown to inhibit the
expansion of blood invariant natural killer T (iNKT) cells and γδT lymphocytes in bothcell–cell contact and in transwell systems. This inhibition was mainly mediated by PGE2 [88].
Activated MSCs stimulated by LPS or TNF can also mediate M2 macrophage polarization byreleasing PGE2 that acts through the prostaglandin receptors EP2 and EP4, and IL-10 [89].
Melief et al. found that MSCs co-cultured with monocytes induced the secretionof IL-6 and prevented monocyte differentiation into immunogenic antigen-presenting
cells [90]. MSCs also skew the differentiation of monocytes towards anti-inflammatory
IL-10-producing cells [90]. The authors showed that MSCs promoted monocyte survival
and differentiation into CD206+ and CD163+ type 2 macrophages that secreted high levelsof IL-10 and CCL18. Moreover, they observed that MSCs directly induce Treg cells by thesecretion of TGF-β and indirectly by triggering the secretion of CCL18 by macrophagesthat in turn induce more Treg cells [90,91] (Figure 2A).
Among the multiple advantages of using MSCs in cell therapy, their ability to makeallosuppression can be included, thus playing a role in immune tolerance and regulationof anti-donor immune responses after solid organ transplant [92]. Apparently, one of themain mechanisms employed by MSCs for allosupression, and immunosuppression in
general, is the induction of Treg cells. To date, when MSCs are cocultured with antigenspecific T cells, they induce the expansion of Treg cells [93]. It has also been shown that
under inflammatory conditions, MSCs mediate the adhesion of Th17 cells via CCR6 andinduce a Treg phenotype in these cells, thereby exerting an anti-inflammatory effect [94].
Moreover, MSCs express Notch 1 ligands (Jagged1, Jagged2, and Delta-Like (DLL) 1, 3, and4) that activate the Notch-1-dependent signaling pathway in CD4+ T lymphocytes, also
leading to Treg differentiation [95]. Allogeneic MSCs induced the forkhead box P3 (FoxP3)
and CD25 expression (Treg phenotype) by the secretion of prostaglandins and TGF-β andexpression of HLA-G5 in coculture with CD4+ T lymphocytes [96]. Moreover, when MSCs
and B lymphocytes from healthy human donors were cocultured with various B lymphocyte
stimuli, cell proliferation and immunoglobulin production were inhibited by the MSCs [97].
In addition to the immunosuppressive activities of MSCs over T and B lymphocytes, they can also inhibit the differentiation and activation of dendritic cells (DCs). This
inhibitory effect was shown to be ratio-dependent and resulted in phenotypical and functional modifications in the DCs, as demonstrated by reduced expression of costimulatory
molecules and hampered capacity to stimulate naïve T lymphocyte proliferation [98]. MSCs
had a profound inhibitory effect on the generation and function of both CD34+ -derived
and monocyte-derived DCs [98]. It was also demonstrated that the coculture of MSCs
with DCs led to increased production of IL-10 by the JAK1/STAT3 signaling pathway inDCs, thus inhibiting cell maturation [99]. Collectively, these results indicate that MSCs canmodulate immune responses at multiple levels, employing modulatory molecules such as
TGF-β, hepatocyte growth factor (HGF), prostaglandins [93,100], IDO1 [101], and others.
Regarding the balance of MSCs as pro- or anti-inflammatory mediators, they may
require a ‘licensing’ step provided by inflammatory molecules such as IFN-γ, TNF, or
TLR ligands [102]. Probably, the timing of cell exposure to the factors that influence thedecision of a pro- or anti-inflammatory role is also critical. Although the inflammatory
modulation triggered by TLR engagement remains unclear, the concept of MSCs polariza-

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tion provides an attractive model to explain some apparently contradictory roles of MSCs
in inflammation. Indeed, a pro-inflammatory activity of MSCs may be beneficial in theearly phase of inflammation and help mounting a proper immune response [14]. It was
published that LPS-stimulated MSCs express chemokine receptors and acquire higher
mobility. These stimulated cells secreted large amounts of pro-inflammatory cytokines
and recruited neutrophils in an IL-8- and macrophage migration inhibitory factor (MIF)dependent manner [103]. Although the functional importance of these results remains to be
demonstrated in vivo, endogenous MSCs may participate in the early phase of pathogen
exposure [103]. Indeed, the balance between apparent opposite biological functions of
MSCs depending on the environment further supports their use in therapeutic trials [14],which may, at least in part, also be explained by cellular heterogeneity.
5. MSCs Secretome in Tissue Regeneration
As stated before, some main points are considered very important in the biology
of MSCs, making them excellent candidates for the treatment of several diseases. These
include the fact that sentinel resident MSCs and blood MSCs, which selectively home
to injured tissues [51], compose a heterogenous population able to carry out multiple
tasks. Moreover, they differentiate into numerous cell types and trigger the differentiationof other local stem cell populations, leading to general tissue repair and angiogenesis.
Additionally, they promote tissue inflammation and inflammatory control in the earlyphase of pathogenic infection. Moreover, their plasticity and adaptation to different tissues
and organs make them suitable for systemic injection to treat multi-organ diseases. Many ofthese functions rely on their capacity to alter the tissue environment through the paracrine
secretion of several, and possibly tissue-specific, mediators [104].
The secretome is the supernatant of MSCs maintained in culture under normoxic
conditions or in conditioned media and is composed of soluble factors, such as cytokines,
chemokines, growth factors, and extracellular vesicles (EV). EVs can be classified into
exosomes, microvesicles, and apoptotic bodies depending on their size, biogenesis, andcomposition [105]. Many of the regenerative properties previously credited to stem cells
are being shown to be mediated by secreted exosomes (Figure 1B), and MSCs produce
significant amounts of exosomes when compared with other cells [106,107]. The EVs
secreted by MSCs are essential, as they preserve most of their parental cell features such ascell homing, immunomodulatory capacity, and regenerative potentials [108]. It is possible,
for example, that MSCs-derived EVs act at a distance in vivo, maybe supporting stem
cell-based tissue regeneration systemically.
The use of cell-free therapies such as the secretome of MSCs in regenerative medicine
provides advantages over cell-based applications, including immune compatibility, tumorigenicity, embolism, and transmission of intracellular infections. Moreover, the storage of
MSC-derived EVs can be done without potentially toxic cryopreservative agents and retaining the potency of the sample for an extended period. The MSCs secretome is practicaland economical, and samples consisting of either MSCs plus EVs or EVs solely can beobtained from the same cultures. Moreover, the biological composition of secretomes canbe modified for more specific and potent effects in therapeutic application.
It was observed that secretome-based therapies improved the pathological condition
after cerebral ischemia, myocardial infarction, and trauma [107]. Moreover, it promoted
the resolution of LPS-induced lung injury by attenuating lung inflammation and promoting wound healing. It also induced anti-inflammatory M2 macrophages, at least inpart by insulin-like growth factor I (IGF-I) activity [109]. The authors also observed thathuman MSCs secretome reduced the myocardial infarction area in acute patients [110].
Additionally, the secretome contained EVs, HGF, and VEGF, stimulated neurogenesis, andimproved traumatic brain injury recovery in a rat model [111]. It was also shown thathuman adipose-derived MSCs secretome [112] improved tissue reperfusion after ischemia
in mice and significantly enhanced endothelial cell growth, bone marrow cells mobilization,and cell homing to the ischemic region [113].

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MSCs secretome also contains pro-inflammatory cytokines [114], and the balance
between pro- and anti-inflammatory mediators can determine the final effect over thepathogenic condition. Corroborating with this view, some authors showed that MSCs
secretome reduced the severity of murine colitis by lowering the pro-inflammatory mediators IL6, IL8, TNF, and MIP-1α transcription, but increasing IL10, an anti-inflammatory
cytokine [115]. In another example, the treatment of alkali-injured corneas with MSCs conditioned medium improved wound healing and showed a robust bactericidal effect [116].
The neuroprotective and neurotrophic effects of MSC-derived secretome [110,111] werealso demonstrated, with beneficial effects in an animal model of Alzheimer’s disease. Thismedium contained factors involved in multiple neuroregenerative mechanisms, such asneuroprotection, axonal elongation, neurotransmission, inflammation suppression, andmicroglial regulation. Notably, secretomes also attenuated pro-inflammatory responses
induced by β-amyloid plaques and generated an anti-inflammatory/tissue regeneration
environment, accompanied by the induction of anti-inflammatory M2-like microglia [117].
The role of MSCs in angiogenesis is of great interest due to the large number of
diseases related to insufficient or abnormal vessel growth, such as arteriosclerotic diseases.
In this regard, some studies have shown that MSCs secrete factors that induce proliferationand migration of endothelial cells, promoting tube formation and preventing endothelial
cell apoptosis in vitro [118]. Moreover, the secretion of pro- and anti-angiogenic factors canbe modified by hypoxia conditions [119].
Purified exosomes from human MSCs culture reduced the infarcted area in a mousemodel of myocardial ischemia/reperfusion injury [120]. Moreover, microvesicles derived
from human adult MSCs protected from acute kidney injury induced by ischemia-reperfusionand subsequent chronic renal damage [121]. MSC-derived microvesicles also ameliorated lunginflammation, and their therapeutic effects and immunomodulatory properties were partly
mediated by angiopoietin-1 [122]. In another study, bone marrow-derived MSCs microvesicles
played a protective role in acute pancreatitis by reducing the levels of pro-inflammatory
cytokines and regulating the nuclear translocation of NF-κB p65 [123]. MSC-derived exosomes
promoted liver regeneration in drug-induced liver injury models [124], and exosomes derived
from MSCs also exerted a cardioprotective effect after myocardial ischemia/reperfusion injury
through the activation of the Wnt/β-catenin signaling pathway [125].
MSCs have also been suggested for the treatment of tumors, although some contradictory results were obtained. It was observed that MSC-derived microvesicles inhibited thetumor bladder T24 cells growth in vitro and in vivo [126]. The in vivo intra-tumor administration of MSCs microvesicles in already established tumors in SCID mice significantly
inhibited tumor growth [127]. An additional advantage of using exosomes to treat tumors
is that they can be engineered to contain relevant gene information and retain the ability tohome to the tumor site and be internalized by the target cells [107,128].
Recently, exosomes derived from MSCs were proposed as a possible therapy to treat
the “cytokine storm” observed in COVID-19 patients with severe pneumonia [129], but alimited number of human clinical studies using MSCs is available. Furthermore, it was evaluated if MSCs transplantation would improve the outcome of seven enrolled patients with
COVID-19. The pulmonary function and symptoms of those patients were significantly
improved within two days after MSCs transplantation. After three to six days of treatment,
the overactivated inflammatory CXCR3+ CD4+ T cells, CXCR3+ CD8+ T cells, and CXCR3+
NK cells were not observed. In addition, a group of CD14+ CD11c+ CD11bmid regulatory
DC population dramatically increased, and the intravenous transplantation was provensafe and effective, especially for patients critically ill [130]. Currently, ClinicalTrials.gov
and the WHO Clinical Trials Registry Platform (WHO ICTRP) report a combined 28 trials
exploring the potential of MSCs or their products for COVID-19 treatment [131].

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6. MSC As an Alternative Treatment for COVID-19
The COVID-19 pandemic is caused by the virus SARS-CoV-2, which spread quickly
and became a global health emergency. The rapid virus replication in the lungs triggers
a strong inflammatory response that leads to a “cytokine storm”, causing ARDS andrespiratory failure, the leading cause of death in severely affected patients [132]. Although
the lungs are more evidently compromised, the disease affects multiple organs, and patientswith a previous history of cardiac symptoms, obesity, diabetes, or coagulation disorders,for example, have a higher morbimortality risk. Then, considering COVID-19 a systemic
disease, we can assume that multiple affected organs and tissues could benefit from thetreatment with MSCs and/or with their secretome.
In the lungs, the inflammation is caused by the release of cytokines virally triggered,including IL-2, IL-6, IL-7, granulocyte colony-stimulating factor (GSCF), interferon-gammainduced protein 10 (IP10), monocyte chemoattractant protein 1 (MCP1), macrophageinflammatory protein 1α (MIP1α), and TNF. These mediators and others lead to pulmonary
edema, air exchange dysfunction, and acute cardiac injury, for example [133]. Currently,
available therapies include non-specific antivirals, antibiotics to prevent secondary infection
and sepsis, and corticosteroids to reduce inflammation. However, the success rate ofthis therapeutic strategy is low in patients with severely affected pulmonary function.
Therefore, several drugs are being tested for repositioning, including hydroxychloroquine,
Remdesivir, and Ivermectin worldwide [134]. Among cell-based alternatives, MSCs havemore registered clinical trials and possibly more chances to be approved for COVID-19
treatment [135]. Moreover, they have documented safety profiles from FDA-approved
clinical trials. A systematic review of several clinical trials involving over one thousand
participants that received intravascular transplants of MSCs for different diseases andclinical conditions was performed in 2010. In that study, a follow-up was done varying
from two weeks to thirty months and attested to the safety of MSCs therapy [136].
A clinical study of severely affected COVID-19 patients using MSCs derived fromthe human umbilical cord was performed. The patients were divided into the standard
treatment group and the standard treatment group that received the cell infusion. Aftertwenty-eight days, the mortality in the group that received cell infusion was 0% and in theother group was 10.34%. Moreover, the clinical condition improved from the third day aftercell infusion and reached a significant difference on the seventh day. There was also a significant reduction in C-reactive protein and the level of IL-6, and the number of lymphocytes
returned to the normal range significantly faster [137]. A study using MSCs from adipose
tissue was also carried out in thirteen patients with COVID-19 on mechanical ventilation.
There was a clinical improvement in 70% of the patients, on average sixteen days after
the first application. Seven patients were extubated, and four remained intubated (twowith an improvement in their ventilatory and radiological parameters and two in stablecondition). Two patients died, but particularly in those patients with clinical improvement,
there was a decrease in the inflammatory and coagulation parameters, C-reactive protein,
IL-6, ferritin, LDH, and d-dimer, as well as an increase in lymphocytes [138]. Anotherstudy evaluated the allogeneic transplantation of menstrual blood-derived MSCs in two
hospitalized patients. The first patient received MSCs transplantation through intravenous
infusion three times on 5th, 6th, and 8th February 2020. The injection dose of MSCs was
set to one million cells per kg of body weight. The second patient received the same
intravenous infusion three times on 8th, 9th, and 11th February 2020. Then, the fractionof inspired O2 of the two patients gradually decreased while the oxygen saturation andpartial oxygen pressure improved. Furthermore, the patients’ computed tomography (CT)scans showed that bilateral lung exudate lesions were adsorbed after MSCs infusion [139].
In a case study, a 65 year-old patient was diagnosed with COVID-19, and after twelvedays of treatment, the patient’s symptoms and inflammation were still severe. Then,
allogenic human umbilical cord MSCs were given three times (5 × 107 cells each time)with a three day interval, with thymosin α1 and antibiotics daily. After treatment, mostlaboratory indexes and CT images showed inflammation remission [140].

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In another study, twenty-five patients with severe COVID-19 received a clinical grade,and MSCs were given at a dose of 1 × 106 cells per Kg. Promethazine hydrochloride(intramuscular injection, 25 mg) was used before the injection of MSCs to prevent allergies.
Seven individuals received MSCs once, other seven patients received MSCs twice, andeleven individuals were infused with cells three times. After MSCs therapy, sixteen cases(64%) showed CT scan improvement, and all cases gained clinical improvement. No
fatalities occurred during hospitalization, but none of the inflammation indexes changed
significantly after MSCs therapy. In addition, the serum levels of lactate (LAC), cardiac
troponin T (cTnT), and creatine kinase MB (CK-MB) elevated significantly after MSC
therapy. The authors discussed that maybe no immunomodulation was observed because
the optimal detection time frame was lost. Finally, MSCs therapy appears to be a promising
option for treating severe COVID-19 but should be used with caution, especially in patientswith coronary heart disease and metabolic acidosis [141].
As discussed, the importance and immunomodulatory effects of MSC-derived EV
in vitro emerged as a potential tool for treating COVID-19. This is due to a marked antiinflammatory effect and regenerative capacity in various in vitro and in vivo models of
the disease [142,143]. MSC-derived EVs are also promising in treating post-COVID-19
pulmonary fibrosis, described as a complication in COVID-19 patients [144]. This possibility
is reinforced by the observation that MSC-derived EVs can prevent the development offibrosis following experimental lung injury [145].
In one of the studies using MSCs to treat COVID-19, exosomes derived from allogeneic
bone marrow MSCs (ExoFlo) were used in twenty-four SARS-CoV-2 PCR-positive patients.
Patients with moderate-to-severe ARDS received a single 15 mL intravenous dose of ExoFlo
and were evaluated for treatment safety and efficacy for fourteen days. The results werevery encouraging, with no adverse effects, and reached an 83% survival rate. Of the total
patients, 71% recovered, 13% remained critically ill though stable, and 16% died for reasons
unrelated to the treatment. Laboratory results revealed a reduction in blood neutrophils,
CD4+ , and CD8+ lymphocyte numbers in recovering patients. Likewise, acute phasereactants declined, with mean C-reactive protein, ferritin, and d-dimer reduction [146].
7. Conclusions
MSCs have several advantages over other populations of stem cells, including theirmaintenance in vitro for long-term clonal expansion, generation of secretome, and thepossibility for in vitro manipulation to improve cell properties. Moreover, they can migrate
and disperse in different tissues, adapting to the biological demands and necessities locallywith shallow risk after in vivo injection. Then, they may populate different organs and tissues
and affect the environment by the paracrine secretin of local mediators and be affected by
the environment, acquiring specific functions. Much remains to be understood about MSCs
biology, but they pose a promising tool for treating systemic diseases such as COVID-19.
Author Contributions: Conceptualization A.H.-P. and D.G.B.; writing—review and editing, D.G.B.,
A.H.-P. and S.I.H. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by the INOVA program of Fundação Oswaldo Cruz (grant
number VPPCB-005-FIO-20-2-34-52).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.

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