Mesenchymal Stromal Cell Secretome: Influencing Therapeutic Potential by Cellular Pre-conditioning

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 F on ie s in Immunology | www. on ie sin.o g 5Decembe 2018 | Volume 9 | A icle 2837 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) F on ie s in Immunology | www. on ie sin.o g 7Decembe 2018 | Volume 9 | A icle 2837 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). 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Copy igh © 2018 Fe ei a, Teixei a, San os, Ba bosa, Almeida-Po ada and Gonçal es. This is an open-access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License (CC BY). The use, dis ibu ion o ep oduc ion in o he o ums is pe mi ed, p o ided he o iginal au ho (s) and he copy igh owne (s) a e c edi ed and ha he o iginal publica ion in his jou nal is ci ed, in acco dance wi h accep ed academic p ac ice. No use, dis ibu ion o ep oduc ion is pe mi ed which does no comply wi h hese e ms. F on ie s in Immunology | www. on ie sin.o g 17 Decembe 2018 | Volume 9 | A icle 2837