REVIEW
published: 04 Decembe 2018
doi: 10.3389/ immu.2018.02837
F on ie s in Immunology | www. on ie sin.o g 1Decembe 2018 | Volume 9 | A icle 2837
Edi ed by:
Guido Moll,
Cha i é Uni e si ä smedizin Be lin,
Ge many
Re iewed by:
Fede ica Casi aghi,
Is i u o Di Rice che Fa macologiche
Ma io Neg i, I aly
En ico Luca elli,
Is i u o O opedico Rizzoli (IRCCS),
I aly
Ma cella F anquesa,
Ins i u d’In es igació en Ciències de
la Salu Ge mans T ias i Pujol (IGTP),
Spain
*Co espondence:
Raquel M. Gonçal es
[email p o ec ed]
†P esen Add ess:
G aciosa Q. Teixei a,
Ins i u e o O hopaedic Resea ch and
Biomechanics, Uni e si y o Ulm, Ulm,
Ge many
Special y sec ion:
This a icle was submi ed o
Alloimmuni y and T ansplan a ion,
a sec ion o he jou nal
F on ie s in Immunology
Recei ed: 19 Sep embe 2018
Accep ed: 16 No embe 2018
Published: 04 Decembe 2018
Ci a ion:
Fe ei a JR, Teixei a GQ, San os SG,
Ba bosa MA, Almeida-Po ada G and
Gonçal es RM (2018) Mesenchymal
S omal Cell Sec e ome: In luencing
The apeu ic Po en ial by Cellula
P e-condi ioning.
F on . Immunol. 9:2837.
doi: 10.3389/ immu.2018.02837
Mesenchymal S omal Cell
Sec e ome: In luencing The apeu ic
Po en ial by Cellula P e-condi ioning
Joana R. Fe ei a1,2,3, G aciosa Q. Teixei a1,2†, Susana G. San os1,2, Má io A. Ba bosa1,2,3,
G aça Almeida-Po ada4and Raquel M. Gonçal es1,2,3*
1Ins i u o de In es igação e Ino ação em Saúde (i3S), Uni e sidade do Po o, Po o, Po ugal, 2Ins i u o de Engenha ia
Biomédica, Uni e sidade do Po o, Po o, Po ugal, 3Ins i u o de Ciências Biomédicas Abel Salaza , Uni e sidade do Po o,
Po o, Po ugal, 4Wake Fo es Ins i u e o Regene a i e Medicine, Wins on-Salem, NC, Uni ed S a es
Mesenchymal s omal cells (MSCs) a e sel - enewing, cul u e-expandable adul
s em cells ha ha e been isola ed om a a ie y o issues, and possess
mul ipo en di e en ia ion capaci y, immunomodula o y p ope ies, and a e ela i ely
non-immunogenic. Due o his unique se o cha ac e is ics, hese cells ha e a ac ed
g ea in e es in he ield o egene a i e medicine and ha e been shown o possess
p onounced he apeu ic po en ial in many di e en pa hologies. MSCs’ mode o ac ion
in ol es a s ong pa ac ine componen esul ing om he high le els o bioac i e
molecules hey sec e e in esponse o he local mic oen i onmen . Fo his eason,
MSCs’ sec e ome is cu en ly being explo ed in se e al clinical con ex s, ei he using
MSC-condi ioned media (CM) o pu i ied MSC-de i ed ex acellula esicles (EVs) o
modula e issue esponse o a wide a ay o inju ies. Ra he han being a cons an mix u e
o molecula ac o s, MSCs’ sec e ome is known o be dependen on he di e se s imuli
p esen in he mic oen i onmen ha MSCs encoun e . As such, he composi ion o he
MSCs’ sec e ome can be modula ed by p econdi ioning he MSCs du ing in i o cul u e.
This manusc ip e iews he exis en li e a u e on how p econdi ioning o MSCs a ec s
he he apeu ic po en ial o hei sec e ome, ocusing on MSCs’ immunomodula o y and
egene a i e ea u es, he eby p o iding new insigh s o he he apeu ic use o MSCs’
sec e ome.
Keywo ds: MSCs (Mesenchymal S omal Cells), p e-condi ioning, egene a ion, immunomodula ion, he apeu ic
po en ial, sec e ome
INTRODUCTION
Mesenchymal s omal cells (MSCs), de ined by he In e na ional Socie y o S em Cell Resea ch
(ISSCR) as ib oblas -like non-hema opoie ic cells, ha e been explo ed in ecen yea s due o he
clinical p omise hey hold o issue epai in egene a i e medicine (1,2). They p esen a capaci y
o di e en ia e in o mul iple lineages, which was on he basis o he high numbe o clinical ials
using MSCs. By 2015, 493 MSC-based clinical ials we e epo ed (2), a numbe ha g ea ly
inc eased in he nex 2 yea s, eaching a o al o 861 ials in 2018 acco ding o he o icial da abase
o he US Na ional Ins i u es o Heal h. In an e o o add ess his as -inc easing knowledge base,
Fe ei a e al. P e-condi ioning Mesenchymal S omal Cells Sec e ome
se e al e iews ha e been published o p o ide a ho ough
analysis o he e olu ion o MSC-based clinical ials (3,4).
Pe haps one o he bes documen ed p ope ies o hese cells
is hei abili y o p omo e egene a ion in a a ie y o issues
and o be a majo con ibu o o he posi i e esul s achie ed
in many published pape s (5,6). Indeed, up o 2015 mos o he
s udies wi h MSCs had ocused on hei use o ea diso de s o
he musculoskele al sys em, namely in hei applica ion o epai
bone o ca ilage (2). Looking beyond hei po en ial in issue
epai and egene a ion, MSCs ha e also been used ex ensi ely
o hei immunomodula o y p ope ies, o example o ea
g a - s.-hos disease (GVHD) (7) and au o-immune diseases
such as lupus (8,9), o C ohn’s disease (10). Fu he mo e,
MSCs’ clinical po en ial has been ex ended o ea myoca dial
in a c ion (11,12), s oke (13), mul iple scle osis (14,15), li e
ci hosis (16,17), diabe es (18,19), lung inju ies (20), among
o he s. MSCs a e known as ela i ely immune-ine cells (21), bu
depending on he con ex can ha e immunosupp essi e (22–24),
o immune-s imula ing capaci y (25,26) (see Figu e 1).
Despi e his g ea p omise, howe e , hei he apeu ic bene i s
a e no limi ed solely o hei egene a i e abili ies. MSCs
ha e also been e e ed o as ophic “ ac o ies” due o he
la ge numbe o bioac i e molecules hey sec e e in esponse
o he local en i onmen , which hen exe pa ac ine e ec s
upon neighbo ing cells and issues (27). Indeed, an inc easing
numbe o au ho s ha e come o conside hese pa ac ine
o ophic p ope ies o be he p ima y means by which
MSCs conduc many o hei he apeu ic e ec s (28–30). This
conclusion has been u he ed by he obse a ion ha , in
many cases, he numbe o di e en ia ed cells is a oo
small o explain he obse ed esponse (27). Ne e heless,
his pa ac ine ac ion is known o be in luenced by he
mic oen i onmen su ounding he cells (31). The e o e, he e’s
a need o unde s and how in i o cul u e condi ions a ec
he egene a i e and immunomodula o y po en ial o MSCs’
sec e ome, wi h he ul ima e goal o de ining an op imal
“cock ail” o p econdi ion MSCs o a gi en he apeu ic
applica ion. While he as pace o esea ch in his ield
is p o iding a la ge amoun o da a ela ed o MSCs’
he apeu ic po en ial, an in eg a ed in es iga ion in o how
p econdi ioning can speci ically in luence he MSC sec e ome
is lacking. To add ess his de iciency, we pe o med a
comp ehensi e li e a u e sea ch on he ollowing da abases:
clinical ials.go , Google Schola , Scopus, and PubMed, using
ei he di ec wo d-co espondence sea ch o MESH in eg a ed
sea ch, wi h se e al combina ions o he ollowing wo ds:
mesenchymal s em cells, hypoxia, in lamma o y, p e ea men ,
p econdi ioning, s imula ion, s imulus, p iming, egene a ion,
immunomodula ion, sec e ome, condi ioned medium (CM),
pa ac ine, he apeu ic, b ain, ne ous sys em, bone, ca ilage,
kidney, li e , lung, panc eas, cance , umo , diabe es, skin,
hea , ca dio ascula , and in e e eb al disc. The compila ion
o da abase ou pu s (∼20,000 pape s) was analyzed acco ding
o he ocus o he s udy and ele ance o he esul s ob ained.
F om hese esul s, a icles ound wi hin e e ence lis s we e also
sc eened and included when ele an o his a icle, conside ing
he ocus on MSCs p econdi ioning.
MSCS SECRETOME: PRECLINICAL AND
CLINICAL EVIDENCES OF ITS
THERAPEUTIC POTENTIAL
The MSCs-de i ed cell- ee sec e ome appea s o be able
o ecapi ula e many o he p ope ies/e ec s ha ha e
been desc ibed o he MSCs hemsel es. MSCs sec e ome
is en iched in se e al soluble ac o s including cy okines,
chemokines, immunomodula o y molecules, and g ow h ac o s
(32). Addi ionally, pa ac ine ac o s p oduced by cells can be
ound encapsula ed in cell-sec e ed esicles. These Ex acellula
Vesicles (EV) a e usually di ided acco ding o hei size and
o igin in he cell in o exosomes, mic o esicles and apop o ic
bodies. The smalle nanosized esicle popula ions ha e dese ed
he mos a en ion. Mic o esicles (100–1,000 nm) o igina e on
he plasma memb ane, and exosomes (30–120 nm) ha a e
o med in he mul i esicula endosomes, ha e o e lapping
size anges and when hei sepa a ion canno be comple ely
asce ained a e collec i ely designa ed EV (33,34). EV con en is
hough o mimic ha o he cells (35). The exac composi ion
o MSCs’ sec e ome has been in es iga ed o iden i y he key
molecules esponsible o MSCs he apeu ic po en ial, wi h he
inal goal being he subs i u ion o a cell- ee p oduc o achie e
he desi ed he apeu ic e ec (see Table 1) (32,36–38,40–43).
P o- egene a i e e ec s o MSCs sec e ome ha e been obse ed
in many di e en sys ems, ac ing by modula ing he immune
sys em (44), inhibi ing cell dea h and ib osis (45,46), s imula ing
ascula iza ion (44), p omo ing issue emodeling, and ec ui ing
o he cells (47).
P eclinical E idence
P eclinical e idence o he egene a i e po en ial o MSCs
sec e ome will be b ie ly desc ibed. ASC (adipose issue-de i ed
MSCs)-CM was able o egene a e/ epai mandible lesions in
abbi s. In he ASC-CM ob ained om 24 h cul u e in se um-
ee medium unde hypoxic condi ions, he au ho s de ec ed 43
angiogenic ac o s, 11 o which also appea ed o be in ol ed
in bone egene a ion: IGF-1, TGF-β1, VEGF, Angiogenin, IL-
6, PDGF-BB, basic FGF (bFGF), EGF, RANTES, MCP-1, and
MCP3 (38). This epe oi e o sec e ed ac o s seemed o be
in acco dance wi h he BM-de i ed MSCs-CM composi ion
epo ed by o he au ho s (36,41,43,48), wi h he ema kable
excep ion ha he BM-de i ed MSCs-CM also con ained HGF
(41) and BMP-1 (36). HGF in pa icula seems o be a key
ac o in MSCs-media ed e e sal o hepa ic ib osis (49). O he
s udies explo ing he e ec o locally adminis e ed MSCs o
degene a ed issues ound e idence o suppo he no ion ha
he soluble ac o s p oduced in esponse o he inju y played a
decisi e ole in he obse ed bene i s o MSCs adminis a ion
(50–52).
In he con ex o in e e eb al disc (IVD) inju y, MSCs
also seem o ac ia a pa ac ine ole h ough c oss alk wi h
IVD cells (53–55). In an ex- i o bo ine model o p o-
in lamma o y/degene a ed IVDs, MSCs in co-cul u e we e able
o immunomodula e he in lamma o y eac ion media ed by he
nucleus pulposus (NP), e en hough ew cells we e ound o
F on ie s in Immunology | www. on ie sin.o g 2Decembe 2018 | Volume 9 | A icle 2837
Fe ei a e al. P e-condi ioning Mesenchymal S omal Cells Sec e ome
FIGURE 1 | MSCs pheno ype, di e en ia ion po en ial, and immunological p ope ies. Schema ic ep esen a ion o MSCs pheno ype and immunological p o ile. (A)
MSCs capaci y o di e en ia ion in o os eogenic, chond ogenic and adipogenic lineages. (B) MSCs pheno ype acco dingly wi h he In e na ional Socie y o S em Cell
Resea ch (ISSCR). (C) MSCs immunological p o ile. (D) Soluble ac o s amilies p oduced by MSCs and p o ile o in e ac ion wi h immune cells.
ha e ac ually mig a ed o he disc (56). Zheng e al. u he
analyzed MSCs-CM e ec on he gene exp ession o NP-like
cells, and ound an up egula ion o KRT19 and down egula ion
o MMP12 and MGP (57). As MMP12, KRT19, and MGP ha e
been associa ed wi h IVD degene a ion, he au ho s sugges ed
ha a heal hy NP-like pheno ype could be es o ed by MSCs-
CM. In ac , i was u he p oposed ha he MSCs’ sec e ome
was s imula ing IVD p ogeni o cells ac i i y (54) and he
communica ion mechanism be ween MSCs and NP cells was a
leas pa ially ia sec e ion o mic o esicles (58).
E idence o he pi o al ole o MSCs pa ac ine ac i i y in
inju ed issues con inues o a ise in many di e en sys ems
and pa hologic condi ions. In 2007, Dai e al. obse ed ha ,
in myoca dial in a c ion, using MSCs-CM had a simila ,
albei less in ense, e ec o wha had been epo ed ea lie
o MSCs pe se, indica ing ha a leas pa o he e ec
ha had been obse ed ollowing MSCs injec ion could be
a ibu ed o soluble ac o s (59). In he con ex o neu onal
damage, a local injec ion o MSCs o he lesion si e in
a s oke model imp o ed coo dina ed unc ion, inhibi ed
sca issue o ma ion and cell apop osis, and s imula ed
angiogenesis (60). Despi e hese ma ked imp o emen s, no
neu al di e en ia ion o he ansplan ed MSCs was obse ed,
ein o cing he key ole o hei pa ac ine mode o ac ion.
Mo eo e , i has been es ablished ha he p esence o BDNF,
Glial Cell Line-de i ed Neu o ophic Fac o (GDNF), Ne e
G ow h Fac o (NGF), and IGF in he MSCs sec e ome is
necessa y o obse e he MSCs-induced neu onal su i al
and di e en ia ion bo h in i o and in i o (61). O he
models in which MSCs-CM has demons a ed he apeu ic
e icacy include ch onic kidney disease, in which adminis a ion
o MSCs-CM pa ially escued kidney unc ion, mainly by
a ac ing endo helial cells, which led o neo-angiogenesis and
s imula ed wound closu e (62). In his s udy, he au ho s
concluded ha he enal-p o ec i e pa ac ine ac o s p esen
wi hin he MSCs-CM we e likely o be VEGF, HGF, and
IGF.
MSCs-de i ed EV, pa icula ly exosomes, ha e been
inc easingly shown o con ibu e o o e en comple ely
eplica e he he apeu ic e ec s obse ed wi h he use o he
en i e sec e ome (63). They we e shown o imp o e ca diac
unc ion a e a po cine myoca dial in a c ion, educing in a c
size and main aining he sys olic and dias olic pe o mance,
as a esul o inducing neo- e ascula iza ion and modula ing
he in lamma o y esponse (64). Simila ly, hBM-MSCs-
de i ed exosomes injec ed locally 24 h a e an induced ocal
F on ie s in Immunology | www. on ie sin.o g 3Decembe 2018 | Volume 9 | A icle 2837
Fe ei a e al. P e-condi ioning Mesenchymal S omal Cells Sec e ome
TABLE 1 | Main ac o s de ec ed in he MSCs sec e ome.
Fac o s Re e ences
BMP (36)
CCL5/RANTES (37,38)
EGF (38)
FGF (38)
G-CSF (39)
GM-CSF (39)
HGF (40,41)
ICAM (37)
IDO (37)
IGF (38,40–43)
IL-10 (37)
IL-6 (38,39,42)
IL-8 (39,42)
LIF (42)
MCP-1 (38,39,42)
MMP-1 (36)
MMP-2 (36)
MMP-3 (36)
MMP-7 (36)
PDGF (38)
PGE2 (37)
TGF-β(37–39,41)
TIMP-1 (36,42)
TIMP-2 (36,42)
VEGF (38,40–43)
ce eb al ischemia we e able o educe he esul ing unc ional
impai men s h ough an inc ease o angioneu ogenesis and
he modula ion o he pe iphe al immune esponse (65).
Addi ionally, he ea men seemed o also induce long- e m
neu op o ec ion. O he s udies epo ed ha MSCs-de i ed
exosomes could media e he ans e o he mic o RNA
(miRNA)-133b o neu onal cells, which induced neu i e
ou g ow h and unc ional eco e y a e s oke (66), hin ing o
he impo ance o his mechanism in he neu onal p o ec i e
capaci y exhibi ed by MSCs. These e ec s we e also obse ed
by o he s in di e en models o ischemic inju y (67,68),
e en hough hei abili y o modula e he local in lamma o y
eac ion has no been obse ed by all (67). In ano he s udy, a
single adminis a ion o MSCs-de i ed mic o esicles inhibi ed
apop osis and s imula ed ubula epi helial cell p oli e a ion,
hus p o ec ing animals om acu e kidney inju y (69). B uno
e al. has demons a ed as well ha he ea men o acu e
kidney inju y wi h MSC-EVs leads o unc ional imp o emen s
and educed mo ali y h ough an inhibi ion o he apop o ic
cascade (70). Mo eo e , ea men wi h mul iple adminis a ions
was shown o be signi ican ly mo e e ec i e han a single
adminis a ion o he EVs. In a simila ashion, MSCs-de i ed
exosomes we e shown o p o ec hepa ocy es and educe bo h
hepa ic in lamma ion and collagen deposi ion (45). Indeed,
MSCs-de i ed esicles ha e consis en ly been epo ed o play a
key ole in he pa ac ine ac i i y o hese cells.
Clinical T ials
While he p eclinical e idence showing he egene a i e and
immunomodula o y po en ial o he MSCs sec e ome con inues
o expand apidly, he clinical s udies e ol ing a ound his
hypo hesis a e s ill sca ce. E en so, he ew clinical ials
pe o med using he p oduc o he MSCs pa ac ine ac i i y
seem o ha e al eady es ablished he sa e y and easibili y o his
me hod, as none o hem epo ed ela ed ad e se e ec s (71–
75). Fu he mo e, he use o he sec e ome seemed o be e ec i e
in imp o ing he clinical ou comes o he in ol ed pa ien s. In
he case o al eola bone egene a ion, condi ioned media om
comme cially a ailable BM-MSCs was adminis e ed o 8 pa ien s
su e ing om se e e al eola bone a ophy and needing bone
augmen a ion (75). These pa ien s ecei ed ei he po ous pu e
be a- icalcium phospha e (β-TCP) o shell-shaped a elocollagen
sponge (ACS) sca old g a s soaked in he CM. A e he su ge y,
mino in lamma ion o he local issues was obse ed wi h less
in il a ion o in lamma o y cells eco ded. The sca old was
g adually eplaced by newly o med bone, wi h no eco ds o
bone eso p ion in any o he cases and ea ly mine aliza ion
obse ed in he augmen ed bone. IGF-1, VEGF, TGF-β, and
HGF we e p esen in he CM, e en hough molecules ypically
in ol ed in bone homeos asis, like BMP-2, we e no de ec ed by
he me hodology used.
Clinical ials add essing alopecia (73) and Female Pa e n
Hai Loss (74) we e able o inc ease hai densi y a e
injec ing pa ien s no wi h he MSCs sec e ome bu wi h
a comme cially a ailable p oduc con aining i s p o ein
componen s. Fu he mo e, he ea men o one ea men -
e ac o y GVHD pa ien wi h MSCs-de i ed exossomes
yielded a p onounced clinical imp o emen sho ly a e he
adminis a ion wi h a dec ease in mo e han 50% o he IL-
1β/INF-γ/TNF-α-p oducing pe iphe al blood mononuclea cells
(PBMCs) (72). A e 4 mon hs, he clinical condi ion o he
pa ien was s ill s able, indica ing a long-las ing he apeu ic e ec
o he exossomes. Cu en ly, allogeneic MSC-de i ed exosomes,
en iched o miR-124, a e epo ed in a egis e ed clinical ial,
di ec ed o s oke pa ien s (h p://clinical ials.go ).
THE EFFECT OF PRE-CONDITIONING ON
MSCs SECRETOME
Al hough MSCs ha e an inna e po en ial o induce and/o
con ibu e o egene a ion, his po en ial is now known o be
g ea ly in luenced by di e se ex insic ac o s such as he issue
sou ce o he MSCs, he heal h s a us and age o he MSCs dono ,
he ba ch/lo o se um used o he in i o cul u e o he MSCs,
passage numbe , oxygen concen a ion, and he p esence/absence
o a p o-in lamma o y en i onmen when he MSCs a e in used
(76–82). Thus, in i o p econdi ioning o MSCs wi h a a ie y
o di e en ac o s has been explo ed o enhance he he apeu ic
capaci y/po en ial o MSCs, which included: 3D cul u e (83–85),
pha macological compounds (86–88), in lamma o y cy okines
(89,90), and hypoxia (91,92) (Table 2). Conside ing MSCs main
mechanism o ac ion upon ansplan a ion migh be ia pa ac ine
signaling, i is somewha su p ising ha only a ew g oups ha e
F on ie s in Immunology | www. on ie sin.o g 4Decembe 2018 | Volume 9 | A icle 2837
Fe ei a e al. P e-condi ioning Mesenchymal S omal Cells Sec e ome
s udied how p econdi ioning o MSCs a ec s hei sec e o y
p o ile. This is pa icula ly ele an when he MSCs’ sec e ome
may ul ima ely p o e o be an ex emely aluable he apeu ic
ool. The in luence o hese ac o s on MSCs’ sec e ome will be
e iewed in he ollowing sec ion.
Hypoxia
No moxic oxygen ension, as used o s anda d cell cul u e, is
he a mosphe ic p essu e (21% O2). The e m hypoxia, when
employed in he con ex o cell cul u e is ou inely used o e e o
oxygen ensions anging om 0 o 10% (108). The physiological
oxygen ension in issues can a y om 1% in ca ilage and bone
ma ow o 12% in pe iphe al blood (109). Thus, he 21% O2
ou inely used o MSCs cul u e is a highe han he oxygen
ound physiologically.
In gene al, hypoxic p econdi ioning enhances MSCs’
egene a i e and cy op o ec i e e ec s (82,91–98). Mo eo e ,
cul u ing MSCs in hypoxic condi ions has been shown o
main ain MSCs’ mul ipo ency (110), enhance MSCs p oli e a ion
(111), and inc ease hei le els o cy op o ec i e molecules (98)
(Table 3), he eby imp o ing he abili y o MSCs o su i e
in he ha sh en i onmen ound wi hin inju y si es upon
ansplan a ion. The bene icial e ec s o hypoxic cul u e
p econdi ioning can likely be explained by he ac ha MSCs
exis in i o in hypoxic en i onmen s (131) and hence ha e
he abili y o espond o a hypoxic mic oen i onmen h ough
he up egula ion o he ansc ip ion ac o HIF-1α(132).
When s abilized due o he lack o oxygen, and dependen
upon he inc ease o phospho yla ed Ak and p38 mi ogen-
ac i a ed p o ein kinase (p38MAPK), his ac o binds o he
p omo e egions o genes esponsi e o hypoxia, leading o
an inc ease in a ailable glucose (109). As MSCs a e capable
o swi ching om ae obic o anae obic me abolic pa hways,
hey a e hen able o endu e e y low oxygen ension alues in
hei mic oen i onmen (133). The e o e, using hese cul u e
condi ions o p econdi ion MSCs enhances hei capaci y o
su i al o longe pe iods, inc eases hei p oli e a ion a e,
and main ains hem in an undi e en ia ed s a e (109,131).
Small di e ences, howe e , in he oxygen ension used o cul u e
MSCs, and in he cul u e p o ocol i sel , can in luence bo h hei
abili y o di e en ia e in o each o he di e en mesenchymal
lineages (134) and hei pa ac ine p oduc ion (109). This
ex eme sensi i i y o oxygen ension is an impo an ac o o
bea in mind when analyzing esul s om s udies using di e en
p econdi ioning p o ocols. The a ious s udies o-da e ha
ha e used hypoxia as a means o p econdi ioning MSCs used a
concen a ion up o 2% O2, o a ime pe iod o 4–72 h (Table 4).
Un o una ely, a high deg ee o a iabili y exis s be ween he
p o ocols ha ha e been employed, and his mus be conside ed
when assessing he MSCs’ he apeu ic unc ion.
HIF-1αac i a ion due o p econdi ioning MSCs wi h hypoxia
leads o he induc ion o ac o s such as VEGF and Angio ensin,
p omo e s o ascula iza ion (136,137). As neo ascula iza ion
is a key ac o in he egene a i e p ocess o damaged issues,
his may accoun , in i sel , o he be e he apeu ic capaci y
ha has been seen wi h MSCs p e ea ed wi h hypoxia. This
hypo hesis is suppo ed by a g owing numbe o publica ions
iden i ying VEGF as a c ucial molecule o he obse ed p o-
egene a i e e ec s o MSCs (47,121,138,139). Liu e al.
desc ibed a di ec impac o he hypoxia-p econdi ioned MSCs
ea men on endo helial cell p oli e a ion wi h a simul aneous
educ ion in apop osis (139). In addi ion, in usion o hypoxia-
p econdi ioned BM-MSCs in o he po al ein o a s subjec o
hepa ec omy p omo ed hepa ocy e p oli e a ion and su i al and
imp o ed se um albumin le els a e su ge y h ough a TGF-
βdependen mechanism (95). Again, inc eased p oduc ion o
VEGF was obse ed. Hypoxia-p econdi ioning induced MSCs
o exp ess highe le els o HIF-1α, and he g ow h ac o s
GDNF, BDNF, VEGF, Ang-1, and SDF-1, as well as i s ecep o
CXCR4, all o which ha e been linked o neo ascula iza ion,
as well as EPO and i s ecep o EPOR, a neu op o ec i e and
p o-angiogenic molecule (120). Also, when using speci ically
hypoxia-p econdi ioned MSCs-de i ed EVs o ea acu e
myoca dial in a c ion, au ho s epo ed he impo ance o he
inc eased ascula iza ion in he he apeu ic e ec s. Bian e al.
obse ed ha EVs de i ed om BM-MSCs p econdi ioned wi h
hypoxia o 72 h we e able o signi ican ly imp o e ca diac
unc ion a e acu e myoca dial in a c ion, mainly h ough he
p omo ion o angiogenesis (140). Indeed, a comp ehensi e
p o eomic analysis o exosomes de i ed om hypoxia-exposed
MSCs showed ha hese exosomes induce angiogenesis in
endo helial cells ia he ac i a ion o he NFκB pa hway (141).
Howe e , in ano he s udy exosomes de i ed om hypoxia-
p econdi ioned MSCs con ibu ed o he a enua ion o he
inju y esul ing om an ischemia/ epe usion episode ia he
Wn signaling pa hway (142). Beyond ha , hypoxia seems o
inc ease exosome sec e ion in gene al (141). Also, in a a
g a model, co- ansplan a ion o exosomes om hypoxia p e-
condi ioned adipose-de i ed MSC imp o ed ascula iza ion and
g a su i al (143) (see Table 5).
Ne e heless, o he g ow h ac o s a e also up egula ed in
esponse o his s imulus (43,46,147) (Table 3), and hese ac o s
likely con ibu e o he speci ici y o issue egene a ion in a
a ie y o scena ios. An analysis o he hypoxia-p econdi ioned
MSCs’ CM used o ea wounds in diabe ic a s, e ealed
highe le els o VEGF, IGF-1, and bFGF (94), while ano he
s udy epo ed inc eased p oduc ion o VEGF-1αand Bcl-2,
wi h up egula ion o HIF-1α, HGF, bFGF, MMP9, and PDGF in
MSCs p e ea ed wi h hypoxia (101). In ag eemen wi h hese
a o emen ioned s udies, Zhang and colleagues obse ed ha p e-
ea men wi h hypoxia led o inc eased le els o VEGF, bFGF,
and Ak ha we e implica ed in he enhancemen o MSCs’
an i-oxida i e, an i-apop o ic, and p o-angiogenic e ec s in a a
model o acu e kidney inju y (96). Addi ionally, o he s udies
showed ha bo h hypoxia and, o an e en g ea e deg ee, o ced
o e exp ession o Ak , up egula ed exp ession o VEGF, bFGF,
HGF, IGF, and TB4, molecules associa ed wi h issue epai and
egene a ion (113). The Ak signaling pa hway was also epo ed
o play a ole in he enhanced wound healing obse ed in mice
ea ed wi h he sec e ome om hypoxia-p econdi ioned MSCs
(121). The e ec o his sec e ome was ela ed o inc eased
le els o ib onec in, AKT, PI3K, and SMAD2 in he inju ed
issue; molecules ha a e all in ol ed in cell p oli e a ion and
mig a ion. The hypoxia-p econdi ioned MSCs sec e ome was
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Fe ei a e al. P e-condi ioning Mesenchymal S omal Cells Sec e ome
TABLE 2 | MSCs p econdi ioning pa ame e s di e si y.
P e-condi ioning ea men T ea men condi ions MSCs Sou ces Re e ences
Hypoxia Anoxia o 2% O2, 4−72 h Placen a, Gingi a, Bone ma ow, Adipose
issue, Umbilical Co d Blood
(82,91–98)
Cy okines, g ow h ac o s and
ho mones
SDF-1, TGF-α, Angio ensin II, INF-γ,
TNF-α, Mela onin, Oxy ocin 30 min−7
days
Bone ma ow, Umbilical blood co d (89,90,99–101)
3D Agg ega es/sphe oids, 24 h−4 days Bone ma ow, Adipose issue, Syno ium (76,83–85,102–104)
Pha macological agen s A o as a in, Diazoxide, LPS, Pacli axel,
Cu cumin, S1P, Valp oa e, Li hium, 30
min−48 h
Bone ma ow, Adipose issue, Cell line (86–88,101,105–107)
also shown o con ain highe le els o VEGF and TGF-β, which
led o inc eased cell p oli e a ion and mig a ion o de mal
ib oblas s, ia he TGF-β/SMAD2 and PI3K/AKT signaling
pa hways (121). These esul s we e u he alida ed by Chen
e al. who demons a ed ha he hypoxia-p econdi ioned MSCs
sec e ome signi ican ly inc eased p oli e a ion and mig a ion o
ke a inocy es, ib oblas s, endo helial cells, and monocy es in
i o, and ha skin wound con ac ion was accele a ed in an in
i o mouse model (47). The sec e ome p oduced by hypoxia-
p econdi ioned placen a-de i ed MSCs was also shown o educe
sca o ma ion and inhibi p oli e a ion and mig a ion o skin
ib oblas s in i o (126). In his case, IL-10 was iden i ied
as he key playe in he p ocess. In ag eemen wi h all hese
esul s, Lan and colleagues epo ed inc eased exp ession o an i-
apop o ic (HGF, Bcl-2), an i-oxida i e (ca alase, HO-1), and p o-
angiogenic (VEGF) ac o s in hypoxia- ea ed BM-MSCs in used
wi h he goal o imp o ing he espi a o y unc ion o mice
su e ing om pulmona y ib osis (98). Chen e al. obse ed
an inc ease in he MSCs p oduc ion le els o no only VEGF-
A and bFGF, bu also IL-6 and IL-8 (molecules in ol ed in he
in lamma o y esponse) unde hypoxic condi ions (47).
The cy op o ec i e e ec o he hypoxia-p e ea men o
MSCs, along wi h changes in me abolism and main enance
o hei di e en ia ion po en ial, ha e now been epea edly
demons a ed by a a ie y o au ho s, despi e di e ences in
he hypoxic condi ions used (138,148). F om hese s udies,
i has been concluded ha hypoxia-p econdi ioning inc eases
MSCs’ su i al in ha sh en i onmen s (148) and enhances hei
angiogenic capaci y, which oge he boos MSCs’ egene a i e
and immunomodula o y abili ies, con ibu ing o he egula ion
o excessi e ib osis and cell dea h due o uncon olled
in lamma ion (96,98,101).
Cy okines, G ow h Fac o s, and Ho mones
When conside ing a signi ican amoun o expe imen al
da a ega ding MSCs p econdi ioning wi h in lamma o y
cy okines, i is eadily appa en ha such a s imulus seems
o p edominan ly p omo e an inc ease in he p oduc ion o
ac o s in ol ed in he egula ion o he immune esponse
(see Table 3). This includes chemoa ac ion o mos immune
cells, modula ion o in lamma ion, and e en enhancing
mig a ion and homing o ansplan ed MSCs o si es wi h
highe concen a ions o such in lamma o y molecules. Thei
immuno egula o y abili ies encompass he inhibi ion o he
complemen sys em ac i a ion, he inhibi ion o NK cells, he
guidance o monocy e di e en ia ion owa d an i-in lamma o y
mac ophages (M2 pheno ype), he supp ession o cy o oxic T
cell p oli e a ion, and he inc ease in he numbe s o egula o y
T cells (149). Many o hese ou comes a e explained by he la ge
numbe o chemokines p oduced by he MSCs ha e ec i ely
a ac nume ous immune cells o esol e an in lamma o y
esponse (150). Speci ically, IL-6, PGE2, and IDO all seem o
be majo e ec o molecules in he immuno egula o y e ec s
MSCs media e (123,151,152). The p oduc ion o his po en
iad o immunomodula o y molecules is s imula ed by he
p esence o p o-in lamma o y ac o s such as IL-1β, TNF-α,
IFN-γ, and LPS (22,42,123,153,154), ha induce MSCs
o adop an immunomodula o y pheno ype and o igge
he p oduc ion o a cock ail o g ow h ac o s. These s udies
hus collec i ely indica e he close ela ionship ha exis s
be ween in lamma ion and egene a ion. In ag eemen wi h his
supposi ion, he he apeu ic e ec s ha we e obse ed wi h TNF-
α ea ed MSCs in a wound closu e model, mainly media ed
by inc eased angiogenesis and immune cells in il a ion,
we e obse ed o be dependen on inc eased le els o IL-6
and IL-8 (116). Indeed, a ecen publica ion ea u ing an
ex ensi e p o eomic analysis o he sec e ome om BM-MSCs
p econdi ioned wi h p o-in lamma o y ac o s (IL-1β, IL-6,
and TNF-α) clea ly demons a es how a p o-in lamma o y
s imulus mainly inc eases MSCs p oduc ion o p o eins
in ol ed in in lamma ion and angiogenesis (155). Mo eo e , he
au ho s also explo e he idea ha MSCs ole in egula ing he
p o eoly ic ac i i y in issues is key o he egula ion o hese
p ocesses.
S ill, he mechanism by which hese ac o s seem o
in luence MSCs is s ill la gely unde ined. The e is e idence ha
MSCs immunomodula o y abili ies a e media ed by bo h cell-
o-cell con ac -de i ed mechanisms (156–158) and pa ac ine
communica ion (159–161). Also, some au ho s belie e ha
MSCs a e no na u ally immunosupp essi e and hus, need
licensing a he si e o in lamma ion o become so (162–165).
This heo y is suppo ed by esul s demons a ing ha molecules
such as IFN-γ, TNF-α, o IL-1βa e necessa y o ac i a e
he MSCs immunomodula o y ac i i y (166,167). One s udy
explo ing he e ec o he p econdi ioning wi h TNF-αon MSCs-
de i ed exosomes demons a ed ha he e ec he s imula o y
F on ie s in Immunology | www. on ie sin.o g 6Decembe 2018 | Volume 9 | A icle 2837
Fe ei a e al. P e-condi ioning Mesenchymal S omal Cells Sec e ome
TABLE 3 | Dynamics o MSCs sec e ome composi ion wi h cells p e-condi ioning.
Molecule P econdi ioning ac o s Re e ences
Hypoxia In lamma o y
s imuli
3D cul u e
Adhesion Gal-9 +(32)
VCAM-1 +(112)
ICAM-1 +(112)
ICAM-4 +(112)
An ioxida ion Ca alase +(98)
HO-1 +(98)
Apop osis IL-24 +(32)
TRAIL +(32)
CD82 +(32)
Cell p oli e a ion
and di e en ia ion
IGF +++(32,38,40–43,46,85,101,
113)
EGF + + (38)
G-CSF +(114)
TB4 +(113)
Chemoa ac ion CCL2 (MCP-1 ) +++(37,38,48,114)
CCL5 (RANTES) + + (37,38,48,112)
CCL7 (MCP-3) + + (38,48,114)
CCL20 +(112)
CXCL1 +(112)
CXCL2 +(115)
CXCL3 +(112)
CXCL5 +(112,115)
CXCL6 +(112,115)
CXCL8 (IL-8) + + (42,47,112,115,116)
CXCL9 +(117)
CXCL10 +(112,115,117)
CXCL11 +(112,115,117)
CXCL12 (SDF-1) + + (41,43,114)
CXCR4 +++(32,93,118–120)
CXCR7 +(118)
Immuno egula ion TGF-β+++(32,37,38,41,42,114,121)
IDO + + (32,37,117,122–124)
Fac o H +(32,125)
IL-10 –/+(37,126,127)
PD-L1 +(117)
HLA-G +(117)
IL-1Ra +(114)
PD-L2 +(117)
TSG-6 +(32,85)
In lamma ion IL-6 +++(37,38,42,47,48,112,
114–116,119,128)
PGE2+ + (32,37,122–124)
PTX3 +(115)
Complemen ac o B +(115)
Complemen ac o D +(115)
COX-2 +(119)
TNF-α+(112)
IL-23 +(112)
IL-16 +(114)
(Con inued)
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Fe ei a e al. P e-condi ioning Mesenchymal S omal Cells Sec e ome
TABLE 3 | Con inued
Molecule P econdi ioning ac o s Re e ences
Hypoxia In lamma o y
s imuli
3D cul u e
IL-7 +(114)
IL-11 +(129)
IL-2Rα+(114,129)
Me abolism STC-1 +(32)
Ca hepsin L1 +(115)
P oca hepsin B +(129)
Mig a ion MMP-1 +(36,115)
MMP12 +(130)
Mig a ion Inhibi ion PAI-1 +(115)
PAI-2 +(115)
Neu op o ec ion BDNF +(46,120)
GDNF +(46)
Os eogenesis BMP + + (32,36,41,43,129)
Plu ipo ency Oc 4 +(32)
Rex1 +(32)
LIF +(42,114)
Su i al HGF +++(32,40,41,85,98,113,130)
Bcl-2 + + (85,98,101)
Ak + + (89,91,98,120)
HIF-1α+(93,101,120)
Vascula iza ion Angiogenin + + (38,93,120,129)
FGF + + (32,38,41–43,47,85,101,
113,120,129,130)
PDGF +(38,41,43)
VEGF +++(32,37,38,40–
43,47,85,90,93–
95,101,113,114,120,
121,129,130)
EPO +(93,120)
EPOR +(93)
e ec s hese esicles had on human os eoblas s was po en ia ed
h ough inc ease o Wn -3a con en in ASC-exosomes (168).
Con e sely, IFN-γp iming o MSC be o e EV isola ion was
epo ed no o in luence he immunomodula o y capaci y o
exosomes o mic opa icles, which displayed dose-dependen
immunomodula o y e ec s in in lamma o y animal models
(169). Addi ionally, TLRs (Toll-Like Recep o ) ha e also been
implica ed as impo an media o s o his ac i a ion. Op iz e al.
epo ed ha ac i a ion o TLR3 and TLR4 lead o he induc ion
o IDO which, in u n, media ed he immunosupp essi e ac ions
o he MSCs (170). Ac i a ion o TLR-2 was shown o cause
an inc ease in he p oduc ion o galec in-3 by MSCs and, hus,
po en ia e hei capaci y o supp ess T-cell ac i a ion (171).
None heless, con adic o y epo s ha e also been published.
Lio a e al. demons a ed ha TLR3 and TLR4 ac i a ion no
only inc eased he p oduc ion o p o-in lamma o y molecules
bu also educed hei inhibi o y e ec on he p oli e a ion o
T-cells (172). Fu he mo e, hey obse ed ha he ac i a ion
o hese TLRs didn’ ha e any e ec on le els o IDO. Mo e
ecen ly, along wi h he demons a ion ha p iming wi h IFN-
γenhanced MSCs immunosupp essi e abili ies, mainly h ough
he induc ion o IDO, i was also shown ha TLR3 ac i a ion
did no a ec IDO le els and did no in luence he cells
immunosupp essi e ac i i y (165).
P econdi ioning wi h a my iad o o he soluble ac o s, such
as g ow h ac o s o ho mones, seems o also po en ia e MSCs
egene a i e capaci y, mainly by s imula ing angiogenesis and
inhibi ing ib osis. Fo example, in aca diac ansplan a ion
o SDF-1-p econdi ioned MSCs inc eased angiogenesis and
educed ib osis in he ischemic a ea o a pos -in a c hea (89).
The e ec s obse ed we e a ibu ed o he ac i a ion o he Ak
signaling pa hway, simila ly o wha was desc ibed o hypoxia-
p econdi ioned MSCs. TGF-α-p econdi ioned MSCs enhanced
ca diac unc ion mainly h ough inc eased VEGF p oduc ion
ia a p38 MAPK-dependen mechanism (90). TNF-αo hypoxia
we e hen combined wi h he TGF-αdu ing p es imula ion,
and his led o a u he imp o emen in ca diac unc ion.
Once mo e, VEGF seemed o play a key ole in MSCs’ mode
F on ie s in Immunology | www. on ie sin.o g 8Decembe 2018 | Volume 9 | A icle 2837
Fe ei a e al. P e-condi ioning Mesenchymal S omal Cells Sec e ome
TABLE 4 | E ec o p econdi ioning on he apeu ic po en ial o MSCs sec e ome.
P e-condi ioning
ea men
Animal S udy model MSCs
sou ce
T ea men
condi ions
Main iden i ied media o s Majo conclusions Re e ences
Hypoxia Ra In i o ischemic
hea
BM 0.5% O2 o 12 h CM Cy op o ec ion o ARVCs o hypoxia (135)
Mouse Acu e kidney inju y AT 0.5% O2 o 48 h CM Enhancemen o issue egene a ion
and enal unc ion. Dec ease in le els
o IL-1βand IL-6
(130)
Mouse Scald skin wound Placen a 1–5% O2 o 72 h CM (IL-10) Reduc ion in sca o ma ion. Inhibi ion
o p oli e a ion and mig a ion o skin
ib oblas s
(126)
Mouse Excisional skin
wound
AT 1/5% O2 o 72 h CM (VEGF, TGF-β1, ia
TGF-β/SMAD and PI3K/Ak )
Inc ease in MSCs and skin ib oblas s
p oli e a ion. Accele a ion o wound
closu e
(121)
Mouse Excisional skin
wound
BM 2% O2 o 48 h CM (bFGF, VEGF, IL-6, IL-8) Enhancemen e o p oli e a ion/
mig a ion o ib oblas s, ke a inocy es
and en helial cells. Neo ascula iza ion
and ec ui men o mac ophages.
Accele a ion o wound con ac ion
(47)
Cy okines, g ow h
ac o s and
ho mones
Ra Cu aneous wound AT TNF-α(10 ng/mL) o
48 h
CM (IL-6, IL-8) Accele a ion o wound closu e.
Inc ease in angiogenesis and
in il a ion o immune cells in o he
wound
(116)
TABLE 5 | E ec o p econdi ioning on he apeu ic po en ial o MSCs-de i ed exosomes.
P e-condi ioning
ea men
Animal S udy model MSCs
sou ce
T ea men
condi ions
Main iden i ied
media o s
Majo conclusions Re e ences
Hypoxia Ra Acu e myoca dial
in a c ion
BM 1%O2 o 72 h EVs Inc eased angiogenesis and imp o ed
ca diac unc ion
(140)
Ra I/R ca diac inju y ? ? EVs
(miRNA26a)
A enua ion o he inju ed a ea and
a y hmias
(142)
Mouse Acu e myoca dial
in a c ion
BM Anoxia +
eoxygena ion
EVs
(miRNA-22)
Reduc ion o pos -in a c ion ib osus (144)
Cy okines, G ow h
Fac o s and Ho mones
Ra Kidney
ischemia/ epe usion
inju y
UCB IFN-γ(100 ng/mL)
o 24–48 h
E s Loss o cy op o ec i e e ec . Loss o
complemen ac o s and lipid binding
p o eins and gain o e aspanins, a mo e
comple e p o easome complex and MHCI
(145)
Pha macological
agen s
Ra Local ce eb al ischemia Cell line BYHWD (2,4
g’mL) o 48 h
E s
(VEGF)
A enua ion o ischemic inju y by an
inc ease in ascula iza ion
(146)
o ac ion. P econdi ioning MSCs wi h a cock ail o g ow h-
ac o s (FGF-2, IGF-1, and BMP-2) was also a emp ed, and his
was ound o yield p o ec i e e ec s on ca diomyocy es and o
imp o e le en icula sys olic unc ion in a a myoca dial
in a c ion model (173). Ano he soluble molecule ha has
been used o p econdi ion MSCs is mela onin, which ac i a es
he ERK 1/2 signaling pa hway, and consequen ly enhances
cell su i al unde oxida i e s ess (100). Thus, mela onin-
p econdi ioned MSCs inc eased angiogenesis and neu ogenesis,
educed in a c size, and imp o ed neu obeha io al ou come in
a a ce eb al ischemia model, and once mo e his seems o
ha e been ela ed o inc eased VEGF le els (100). Mela onin-
p econdi ioned MSCs also exhibi ed signi ican ly highe su i al
a es a e in apa enchymal injec ion in a a kidney ischemia
model (99). This e ec was a ibu ed o an up egula ion o he
enzymes ca alase and supe oxide dismu ase-1 ha imbued MSCs
wi h g ea e an ioxidan capaci y. Las ly, H2O2has been used
o p econdi ion MSCs whose exosomes we e used o ea and
ischemia/ epe usion inju y in a a model (174). The ea men
lead o inc eased ascula iza ion, which led o highe su i al
a es, and a educed in lamma o y eac ion.
3-Dimensional (3D) Cul u e
MSCs cul u e in a 3-dimensional (3D) en i onmen is ano he
ype o p econdi ioning ha aims o mo e closely mimic he
physiological condi ions which he cells would see in i o. 3D
cul u e o MSCs, namely as sphe oids, induces an inc ease
in he p oduc ion o ac o s associa ed wi h cell su i al and
p oli e a ion and ascula iza ion (129,175) (Table 3). This, in
u n, has been shown o inc ease hese cells’ immunomodula o y,
angiogenic, an i- ib o ic, and an i-apop o ic ac i i ies (83,85,
104,176). MSCs sphe oids dec eased neu ophil ac i i y,
he le els o he p o-in lamma o y molecules TNF-α, IL-1β,
CXCL2/MIP-2, PGE2, and plasmin ac i i y in a mouse pe i oni is
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Fe ei a e al. P e-condi ioning Mesenchymal S omal Cells Sec e ome
inc eases IL-10 sec e ion. J Biomed Bio echnol. (2012) 2012:295167.
doi: 10.1155/2012/295167
129. Po apo a IA, Gaude e GR, B ink PR, Robinson RB, Rosen MR, Cohen
IS, e al. Mesenchymal s em cells suppo mig a ion, ex acellula ma ix
in asion, p oli e a ion, and su i al o endo helial cells in i o S em Cells
(2007) 25:1761–8. doi: 10.1634/s emcells.2007-0022
130. O e a h JM, Gaue S, Obe mulle N, Schube R, Scha e R, Geige
H, e al. Sho - e m p econdi ioning enhances he he apeu ic
po en ial o adipose-de i ed s omal/s em cell-condi ioned medium in
cispla in-induced acu e kidney inju y. Exp Cell Res. (2016) 342:175–83.
doi: 10.1016/j.yexc .2016.03.002
131. Ma T, G ayson WL, F ohlich M, Vunjak-No ako ic G. Hypoxia and s em
cell-based enginee ing o mesenchymal issues. Bio echnol P og. (2009)
25:32–42. doi: 10.1002/b p .128
132. Kiani AA, Kazemi A, Halabian R, Mohammadipou M, Jahanian-Naja abadi
A, Roudkena MH. HIF-1αcon e s esis ance o induced s ess in bone
ma ow-de i ed mesenchymal s em cells. A ch Med Res. (2013) 44:185–93.
doi: 10.1016/j.a cmed.2013.03.006
133. Descheppe M, Oudina K, Da id B, My il V, Colle C, Bensidhoum M, e al.
Su i al and unc ion o mesenchymal s em cells (MSCs) depend on glucose
o o e come exposu e o long- e m, se e e and con inuous hypoxia. J Cellu
Mol Med. (2011) 15:1505–14. doi: 10.1111/j.1582-4934.2010.01138.x
134. Cicione C, Muinos-Lopez E, He mida-Gomez T, Fuen es-Boque e I, Diaz-
P ado S, Blanco FJ. E ec s o se e e hypoxia on bone ma ow mesenchymal
s em cells di e en ia ion po en ial. S em Cells In . (2013) 2013:232896.
doi: 10.1155/2013/232896
135. Gnecchi M, He H, Liang OD, Melo LG, Mo ello F, Mu H, e al.
Pa ac ine ac ion accoun s o ma ked p o ec ion o ischemic hea
by Ak -modi ied mesenchymal s em cells Na Med. (2005) 11:367–8.
doi: 10.1038/nm0405-367
136. Im iyaz HZ, Simon MC. Hypoxia-inducible ac o s as essen ial egula o s
o in lamma ion. Cu Top Mic obiol Immunol. (2010) 345:105–20.
doi: 10.1007/82_2010_74
137. Ahluwalia A, Ta nawski AS. C i ical ole o hypoxia senso - HIF-1αin VEGF
gene ac i a ion. Implica ions o angiogenesis and issue inju y healing. Cu
Med Chem. (2005) 19:90–7. doi: 10.2174/092986712803413944
138. Bade AM, Klose K, Bieback K, Ko in h D, Schneide M, Sei e M, e al.
Hypoxic p econdi ioning inc eases su i al and p o-angiogenic capaci y o
human co d blood mesenchymal s omal cells in i o.PLoS ONE (2015)
10:e0138477. doi: 10.1371/jou nal.pone.0138477
139. Liu J, Hao H, Xia L, Ti D, Huang H, Dong L, e al. Hypoxia p e ea men o
bone ma ow mesenchymal s em cells acili a es angiogenesis by imp o ing
he unc ion o endo helial cells in diabe ic a s wi h lowe ischemia. PLoS
ONE (2015) 10:e0126715. doi: 10.1371/jou nal.pone.0126715
140. Bian S, Zhang L, Duan L, Wang X, Min Y, Yu H. Ex acellula esicles de i ed
om human bone ma ow mesenchymal s em cells p omo e angiogenesis
in a a myoca dial in a c ion model. J Mol Med. (2014) 92:387–97.
doi: 10.1007/s00109-013-1110-5
141. Ande son JD, Johansson HJ, G aham CS, Ves e lund M, Pham MT, B amle
CS, e al. Comp ehensi e p o eomic analysis o mesenchymal s em cell
exosomes e eals modula ion o angiogenesis ia nuclea ac o -kappab
signaling. S em Cells (2016) 34:601–13. doi: 10.1002/s em.2298
142. Pa k H, Pa k H, Mun D, Kang J, Kim H, Kim M, e al. Ex acellula esicles
de i ed om hypoxic human mesenchymal s em cells a enua e GSK3be a
exp ession ia mi na-26a in an ischemia- epe usion inju y model. Yonsei
Med J. (2018) 59:736–45. doi: 10.3349/ymj.2018.59.6.736
143. Han YD, Bai Y, Yan XL, Ren J, Zeng Q, Li XD, e al. Co- ansplan a ion o
exosomes de i ed om hypoxia-p econdi ioned adipose mesenchymal s em
cells p omo es neo ascula iza ion and g a su i al in a g a ing Biochem
Biophys Res Commun. (2018) 497:305–12. doi: 10.1016/j.bb c.2018.02.076
144. Feng Y, Huang W, Wani M, Yu X, Ash a M. Ischemic p econdi ioning
po en ia es he p o ec i e e ec o s em cells h ough sec e ion o
exosomes by a ge ing Mecp2 ia miR-22. PLoS ONE (2014) 9:e88685.
doi: 10.1371/jou nal.pone.0088685
145. Kilpinen L, Impola U, Sankkila L, Ri amo I, Aa onen M, Kilpinen S,
e al. Ex acellula memb ane esicles om umbilical co d blood-de i ed
MSC p o ec agains ischemic acu e kidney inju y, a ea u e ha is
los a e in lamma o y condi ioning. J Ex acell Vesicles (2013) 2, 1–15.
doi: 10.3402/je . 2i0.21927
146. Yang J, Gao F, Zhang Y, Liu Y, Zhang D. Buyang huanwu decoc ion
(BYHWD) enhances angiogenic e ec o mesenchymal s em cell by
up egula ing eg exp ession a e ocal ce eb al ischemia. J Mol Neu osci.
(2015) 56:898–906. doi: 10.1007/s12031-015-0539-0
147. C isos omo PR, Wang Y, Ma kel TA, Wang M, Lahm T, Meld um
DR. Human mesenchymal s em cells s imula ed by TNF-α, LPS,
o hypoxia p oduce g ow h ac o s by an NFκB- bu no JNK-
dependen mechanism. Am J Physiol Cell Physiol. (2008) 294:C675–82.
doi: 10.1152/ajpcell.00437.2007
148. Beegle J, Laka os K, Kalomoi is S, S ewa H, Isse o R, Nol a RJA, e al.
Hypoxic p econdi ioning o mesenchymal s omal cells induces me abolic
changes, enhances su i al, and p omo es cell e en ion in i o.S em Cells
(2015) 33:1818–28. doi: 10.1002/s em.1976
149. Sapa o A, Ogay V, Nu gozhin T, Jumabay M, Chen WCW. P econdi ioning
o human mesenchymal s em cells o enhance hei egula ion o he immune
esponse. S em Cells In . (2016) 2016:3924858. doi: 10.1155/2016/3924858
150. Wang Y, Chen X, Cao W, Shi Y. Plas ici y o mesenchymal s em cells
in immunomodula ion: pa hological and he apeu ic implica ions. Na
Immunol. (2014) 15:1009–16. doi: 10.1038/ni.3002
151. Pa k CW, Kim KS, Bae S, Son HK, Myung PK, Hong HJ, e al. Cy okine
sec e ion p o iling o human mesenchymal s em cells by an ibody a ay
in e na ional J S em Cells (2009) 2:59–68.
152. Hsu WT, Lin CH, Chiang BL, Jui HY, Wu KK, Lee CM. P os aglandin E2
po en ia es mesenchymal s em cell-induced IL-10+IFN-γ+CD4+ egula o y
T cells o con ol ansplan a e ioscle osis. J Immunol. (2013) 190:2372–80.
doi: 10.4049/jimmunol.1202996
153. Néme h K, Leelaha anichkul A, Yuen PST, Maye B, Pa melee A, Doi
K, e al. Bone ma ow s omal cells a enua e sepsis ia p os aglandin
E(2)-dependen ep og amming o hos mac ophages o inc ease hei
in e leukin-10 p oduc ion. Na Med. (2009) 15:42–9. doi: 10.1038/nm.1905
154. Chen K, Wang D, Du WT, Han ZB, Ren H, Chi Y, e al. Human umbilical co d
mesenchymal s em cells hUC-MSCs exe immunosupp essi e ac i i ies
h ough a PGE2-dependen mechanism. Clin Immunol. (2010) 135:448–58.
doi: 10.1016/j.clim.2010.01.015
155. Ma ioli E, Nonnis S, Angioni R, San aga a F, Cali B, Zano i L, e al.
P o eomic analysis o he sec e ome o human bone ma ow-de i ed
mesenchymal s em cells p imed by p o-in lamma o y cy okines. J P o eomics
(2017) 166:115–26. doi: 10.1016/j.jp o .2017.07.012
156. Augello A, Tasso R, Neg ini SM, Ama eis A, Indi e i F, Cancedda R, e al.
Bone ma ow mesenchymal p ogeni o cells inhibi lymphocy e p oli e a ion
by ac i a ion o he p og ammed dea h 1 pa hway. Eu J Immunol. (2005)
35:1482–90. doi: 10.1002/eji.200425405
157. Ren G, Zhang L, Zhao X, Xu G, Zhang Y, Robe s AI, e al.
Mesenchymal s em cell-media ed immunosupp ession occu s ia conce ed
ac ion o chemokines and ni ic oxide. Cell S em Cell (2008) 2:141–50.
doi: 10.1016/j.s em.2007.11.014
158. Akiyama K, Chen C, Wang D, Xu X, Qu C, Yamaza T, e al.
Mesenchymal-s em-cell-induced immuno egula ion in ol es FAS-ligand-
/FAS-media ed T cell apop osis. Cell S em Cell (2012) 10:544–55.
doi: 10.1016/j.s em.2012.03.007
159. Di Nicola M, Ca lo-S ella C, Magni M, Milanesi M, Longoni P, Ma eucci DP,
e al. Human bone ma ow s omal cells supp ess T-lymphocy e p oli e a ion
induced by cellula o nonspeci ic mi ogenic s imuli. Blood (2002) 99:3838–
43. doi: 10.1182/blood.V99.10.3838
160. Lee RH, Pulin AA, Seo MJ, Ko a DJ, Ylos alo J, La son BL, e al. In a enous
hMSCs imp o e myoca dial in a c ion in mice because cells embolized in
lung a e ac i a ed o sec e e he an i-in lamma o y p o ein TSG-6. Cell S em
Cell. (2009) 5:54–63. doi: 10.1016/j.s em.2009.05.003
161. Roddy GW, Oh JY, Lee RH, Ba osh TJ, Ylos alo J, Coble K, e al.
Ac ion a a dis ance: sys emically adminis e ed adul s em/p ogeni o cells
(MSCs) educe in lamma o y damage o he co nea wi hou eng a men and
p ima ily by sec e ion o TNF-αs imula ed gene/p o ein 6. S em Cells (2011)
29:1572–9. doi: 10.1002/s em.708
162. Ryan JM, Ba y F, Mu phy JM, Mahon BP. In e e on-γdoes no
b eak, bu p omo es he immunosupp essi e capaci y o adul human
F on ie s in Immunology | www. on ie sin.o g 16 Decembe 2018 | Volume 9 | A icle 2837

Fe ei a e al. P e-condi ioning Mesenchymal S omal Cells Sec e ome
mesenchymal s em cells. Clin Exp Immunol. (2007) 149:353–63.
doi: 10.1111/j.1365-2249.2007.03422.x
163. Polche D, Sobinsky J, Douglas GW, Kidd M, Moadsi i A, Reina
E, e al. IFN-γac i a ion o mesenchymal s em cells o ea men
and p e en ion o g a e sus hos disease Eu J Immunol. (2008)
38:1745–55.
164. English K. Mechanisms o mesenchymal s omal cell immunomodula ion.
Immunol Cell Biol. (2013) 91:19–26. doi: 10.1038/icb.2012.56
165. Kim DS, Jang IK, Lee MW, Ko YJ, Lee D-H, Lee JW, e al. Enhanced
immunosupp essi e p ope ies o human mesenchymal s em cells p imed
by in e e on-γ.EBio Med. (2018) 28:261–73. doi: 10.1016/j.ebiom.2018.
01.002
166. K ampe a M, Cosmi L, Angeli R, Pasini A, Lio a F, And eini A, e al.
Role o in e e on-gamma in he immunomodula o y ac i i y o human
bone ma ow mesenchymal s em cells. S em Cells (2006) 24:386–98.
doi: 10.1634/s emcells.2005-0008
167. Ren G, Su J, Zhang L, Zhao X, Ling W, L’Huillie A, e al. Species a ia ion in
he mechanisms o mesenchymal s em cell-media ed immunosupp ession.
S em Cells (2009) 27:1954–62. doi: 10.1002/s em.118
168. Lu Z, Chen Y, Duns an C, Roohani-Es ahani S, Z eiqa H. P iming adipose
s em cells wi h umo nec osis ac o -αp econdi ioning po en ia es hei
exosome e icacy o bone egene a ion. Tissue Eng Pa A (2017) 23:1212–20.
doi: 10.1089/ en. ea.2016.0548
169. Cosenza S, Toupe K, Maumus M, Luz-C aw o d P, Blanc-B ude
O, Jo gensen C, e al. Mesenchymal s em cells-de i ed exosomes
a e mo e immunosupp essi e han mic opa icles in in lamma o y
a h i is. The anos ics (2018) 8:1399–410. doi: 10.7150/ hno.
21072
170. Opi z CA, Li zenbu ge UM, Lu z C, Lanz TV, T i schle I, Koppel A, e al.
Toll-like ecep o engagemen enhances he immunosupp essi e p ope ies
o human bone ma ow-de i ed mesenchymal s em cells by inducing
indoleamine-2,3-dioxygenase-1 ia in e e on-be a and p o ein kinase R.
S em Cells (2009) 27:909–19. doi: 10.1002/s em.7
171. Sioud M, Mobe gslien A, Boudabous A, Floisand Y. E idence o
he in ol emen o galec in-3 in mesenchymal s em cell supp ession
o allogeneic T-cell p oli e a ion. Scand J Immunol. (2010) 71:267–74.
doi: 10.1111/j.1365-3083.2010.02378.x
172. Lio a F, Angeli R, Cosmi L, Fili L, Manuelli C, F osali F, e al.
Toll-like ecep o s 3 and 4 a e exp essed by human bone ma ow-
de i ed mesenchymal s em cells and can inhibi hei T-cell modula o y
ac i i y by impai ing No ch signaling. S em Cells (2008) 26:279–89.
doi: 10.1634/s emcells.2007-0454
173. Hahn JY, Cho HJ, Kang HJ, Kim TS, Kim MH, Chung JH, e al. P e-
ea men o mesenchymal s em cells wi h a combina ion o g ow h ac o s
enhances gap junc ion o ma ion, cy op o ec i e e ec on ca diomyocy es,
and he apeu ic e icacy o myoca dial in a c ion. J Am Coll Ca diol. (2008)
51:933–43. doi: 10.1016/j.jacc.2007.11.040
174. Bai Y, Han YD, Yan XL, Ren J, Zeng Q, Li XD, e al. Adipose
mesenchymal s em cell-de i ed exosomes s imula ed by hyd ogen
pe oxide enhanced skin lap eco e y in ischemia- epe usion inju y.
Biochem Biophys Res Commun. (2018) 500:310–7. doi: 10.1016/j.bb c.2018.
04.065
175. Po apo a IA, B ink PR, Cohen IS, Do onin SV. Cul u ing o human
mesenchymal s em cells as h ee-dimensional agg ega es induces unc ional
exp ession o CXCR4 ha egula es adhesion o endo helial cells. J Biol
Chem. (2008) 283:13100–7. doi: 10.1074/jbc.M800184200
176. Pe enko Y, Syko á E, Kubino á Š. The he apeu ic po en ial o h ee-
dimensional mul ipo en mesenchymal s omal cell sphe oids. S em Cell Res
The . (2017) 8:94. doi: 10.1186/s13287-017-0558-6
177. Cesa z Z, Tamama K. Sphe oid cul u e o mesenchymal s em cells. S em Cells
In . (2016) 2016:9176357. doi: 10.1155/2016/9176357
178. Lee EJ, Pa k SJ, Kang SK, Kim GH, Kang HJ, Lee SW, e al. Sphe ical bulle
o ma ion ia E-cadhe in p omo es he apeu ic po ency o mesenchymal
s em cells de i ed om human umbilical co d blood o myoca dial
in a c ion Mol The . (2012) 20:1424–33. doi: 10.1038/m .2012.58
179. Kim YS, Ahn Y, Kwon JS, Cho YK, Jeong MH, Cho JG, e al. P iming
o mesenchymal s em cells wi h oxy ocin enhances he ca diac epai
in ischemia/ epe usion inju y. Cells Tissues O gans (2012) 195:428–42.
doi: 10.1159/000329234
180. Liu J, Zhu P, Song P, Xiong W, Chen H, Peng W, e al. P e ea men o
adipose de i ed s em cells wi h cu cumin acili a es myoca dial eco e y
ia an iapop osis and angiogenesis. S em Cells In . (2015) 2015:638153.
doi: 10.1155/2015/638153
181. Phan J, Kuma P, Hao D, Gao K, Fa me D, Wang A. Enginee ing
mesenchymal s em cells o imp o e hei exosome e icacy and
yield o cell- ee he apy. J Ex acell Vesicles (2018) 7:1522236.
doi: 10.1080/20013078.2018.1522236
182. Phelps J, Sana i-Nezhad A, Ung in M, Duncan NA, Sen A. biop ocessing o
mesenchymal s em cells and hei de i a i es: owa d cell- ee he apeu ics.
S em Cells In . (2018) 2018:9415367. doi: 10.1155/2018/9415367
183. Tola J, Le Blanc K, Kea ing A, Blaza BR. Hi ing he igh spo
wi h mesenchymal s omal cells (MSCs). S em Cells (2010) 28:1446–55.
doi: 10.1002/s em.459
184. Zhang J, Huang X, Wang H, Liu X, Zhang T, Wang Y, e al. The challenges
and p omises o allogeneic mesenchymal s em cells o use as a cell-based
he apy. S em Cell Res The . (2015) 6:234. doi: 10.1186/s13287-015-0240-9
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