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Research ArticleOsteodifferentiated Mesenchymal Stem Cells fromBone Marrow and Adipose Tissue Express HLA-G and DisplayImmunomodulatory Properties in HLA-Mismatched SettingsImplications in Bone Repair Therapy
Florent Montespan12 Freacutedeacuteric Deschaseaux3 Luc Senseacutebeacute3
Edgardo D Carosella12 and Nathalie Rouas-Freiss12
1 CEA Institut des Maladies Emergentes et des Therapies Innovantes (IMETI) Service de Recherche en Hemato-Immunologie (SRHI)Hopital Saint-Louis IUH 1 avenue Claude Vellefaux 75010 Paris France
2Universite Paris Diderot Sorbonne Paris Cite IUH Hopital Saint-Louis UMR E5 IUH 1 avenue Claude Vellefaux75010 Paris France
3 Stromalab UMR UPSCNRS 5273 EFS-Pyrenees-Mediterranee U1031 Inserm 31432 Toulouse France
Correspondence should be addressed to Nathalie Rouas-Freiss nathalierouas-freissceafr
Received 10 January 2014 Accepted 10 March 2014 Published 30 April 2014
Academic Editor Fabio Morandi
Copyright copy 2014 Florent Montespan et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited
Mesenchymal stem cells (MSCs) are multipotent cells that can be obtained from several sources such as bone marrow and adiposetissue Depending on the culture conditions they can differentiate into osteoblasts chondroblasts adipocytes or neurons In thisregard they constitute promising candidates for cell-based therapy aimed at repairing damaged tissues In addition MSCs displayimmunomodulatory properties through the expression of soluble factors including HLA-GWe here analyse both immunogenicityand immunosuppressive capacity of MSCs derived from bone marrow and adipose tissue before and after osteodifferentiationResults show that HLA-G expression is maintained after osteodifferentiation and can be boosted in inflammatory conditionsmimicked by the addition of IFN-120574 and TNF-120572 Both MSCs and osteodifferentiated MSCs are hypoimmunogenic and exertimmunomodulatory properties in HLA-mismatched settings as they suppress T cell alloproliferation in mixed lymphocytereactions Finally addition of biomaterials that stimulate bone tissue formation did not modify MSC immune properties As MSCscombine both abilities of osteoregeneration and immunomodulation theymay be considered as allogenic sources for the treatmentof bone defects
1 Introduction
Bone is among the most frequently transplanted tissues withabout 1 million procedures annually in Europe Despite theirconsiderable disadvantages including the risk of diseasetransfer and immunologic rejection limited supply of bonecosts and complications allografts and autografts accountfor more than 80 of total graft volume Significant growthopportunities exist for synthetic bone grafts in associationwith mesenchymal stem cells (MSCs) from autologous orallogenic sources as alternatives to biological bone grafts in
orthopaedic and maxillofacial surgery [1 2] In a classicalapproach bone tissue engineering consists of harvesting bonemarrow from a patient isolating MSCs by their adherenceto tissue culture plastic expanding and differentiating thosecells in culture to a sufficient number and then seeding themonto a suitable synthetic scaffold prior to implantation intothe same patient [3]
MSCs can be isolated from different tissuesincluding bone marrow (BM) adipose tissue (AT)and perinatal sources [4] Many reports highlighted theimmunomodulatory properties of MSCs relying on three
Hindawi Publishing CorporationJournal of Immunology ResearchVolume 2014 Article ID 230346 10 pageshttpdxdoiorg1011552014230346
2 Journal of Immunology Research
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Figure 1 BM-derived MSCs express HLA-G and are hypoimmunogenic (a) Expression of HLA-DR molecules was evaluated by flowcytometry analysis on BM-derived MSCs pretreated with IFN120574 and TNF120572 (MSC-BM IFN120574TNF120572) or not (MSC-BM) IgG1 was used asisotype control Ab (b) Expression of HLA-G5 was evaluated by intracellular flow cytometry analysis on BM-derived MSCs pretreated withIFN120574 and TNF120572 (MSC-BM IFN120574TNF120572) or not (MSC-BM) Tubulin was used as positive control of cell permeabilization (c) PBMC fromhealthy individual (15) were used as responder cells towards BM-derived MSCs pretreated with IFN120574 and TNF120572 (MSC-BM IFN120574TNF120572)or not (MSC-BM) as stimulating cells at various responder stimulator ratios Irradiated LCLlowast were used as positive control of T cellalloproliferation Results are given as mean cpm plusmn sem one representative experiment is shown
main mechanisms (1) cell cycle arrest of immune cells atthe G1 phase (2) direct interaction with immune cells and(3) paracrine effect through secretion of various factorsincluding HLA-G prostaglandin E2 cytokines (TGF120573IL6 IL10 HGF VEGF etc) and enzymes (indoleamine23-dioxygenase and inducible nitric oxide synthase) [5ndash8]Based on these tolerogenic properties allogenic MSCs arecurrently tested in various clinical trials [9 10]
HLA-G molecules expressed by mesenchymal stem cellsfulfill an important function since blockade of HLA-G usingHLA-G neutralizing antibodies could reverse MSC ability to(i) generate in vitro the expansion of CD4+CD25+ FoxP3+regulatory T cells (ii) inhibit the alloproliferative T cellresponse and (iii) suppress the cytotoxic function of NKcells These results show that HLA-G molecules mainly sol-uble HLA-G5 actively contribute to the immunosuppressiveproperties exerted by MSCs [11 12]
In clinical trials aimed at repairing bone defects themainobjective is to develop new biomaterials that simulate bone
issue formation in combination with MSCs In this contextourwork entailed assessing from an immunological perspec-tive whether allogenic MSCs could be used without a risk ofrejection instead of autologous MSCs The results obtainedin vitro validate this hypothesis since the MSCs proved tobe hypoimmunogenic and immunosuppressive in allogenicconditions Moreover following infusion in bone MSCs mayundergo osteodifferentiation process under the influence of119894119899 119907119894119907119900 osteogenic factors We thus evaluated whether (1)allogenic MSCs committed to osteodifferentiation processcan be rejected or not due to histoincompatibility and(2) combination with biomaterials modifies MSCs immuneproperties
2 Materials and Methods
21 Isolation of PBMC PBMC were isolated from bloodof healthy volunteer donors (after informed consent) fromthe French Blood Establishment (EFS Saint-Louis Hospital
Journal of Immunology Research 3
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310
232
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(CPM
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LCLlowast
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Figure 2 AT-derived MSCs express HLA-G and are hypoimmunogenic (a) Expression of HLA-DR molecules was evaluated by flowcytometry analysis on AT-derivedMSCs pretreated with IFN120574 and TNF120572 (MSC-AT IFN120574TNF120572) or not (MSC-AT) IgG1 was used as isotypecontrol Ab (b) Expression of HLA-G5 was evaluated by intracellular flow cytometry analysis on AT-derived MSCs pretreated with IFN120574 andTNF120572 (MSC-AT IFN120574TNF120572) or not (MSC-AT) (c) PBMC from healthy individual (59-) were used as responder cells towards AT-derivedMSCs pretreated with IFN120574 and TNF120572 (MSC-AT IFN120574TNF120572) or not (MSC-AT) as stimulating cells at various responder stimulator ratiosIrradiated LCLlowast were used as positive control of T cell alloproliferation Results are given asmean cpmplusmn sem one representative experimentis shown
Paris France) by density-gradient centrifugation over Ficoll-Paque PLUS (GE Healthcare) These cells were used as HLA-mismatched responding cells in MLR
22 In Vitro Osteodifferentiation MSCs from BM orAT were obtained from Reborne consortium center(httpwwwreborneorg) Before experiments MSCswere thawed and expanded through seeding 1000 cellscm2in T75 flasks When cultures reach 60ndash70 confluencecells were harvested and seeded for immunological assaysThe osteodifferentiated MSCs used as stimulating cells inMLR were obtained as previously described [13] BrieflyMSCs were cultured in osteoblastic differentiation mediumconsisting of 120572MEM FBS ascorbic acid NaH
2PO4 and
BMP-4 during 14 to 23 days
23 Preparation of MSC-Biomaterial Complex For Immuno-logical Tests 24-well (ultralow attachment) plates containingdisks of MBCP+ (macroporous biphasic calcium phosphate)granules with the same diameter of each well bottomwere prepared in collaboration with Biomatlante (France)
to achieve a complete adherence of seeded MSCs to thebiomaterial and then a complete induction of osteoblasticdifferentiation of MSCs The disks of MBCP+ were washedfor 48 hours before use to avoid any toxic effect on MSCsThen MSCs were seeded on MBCP discs during 5 daysin order to colonize homogenously the particles ThenMSC-biomaterial complex was tested in mixed lymphocytereactions
24 Mixed Lymphocyte Reaction (MLR) Using MSC asEither Stimulating Cells Or Third-Party Cells Facing HLA-Mismatched PBMC as Responder Cells PBMC from differentdonors were used as responder cells andMSCs or osteodiffer-entiated MSCs from BM or AT were used as either allogenicstimulating cells or third-party cells at various ratios TheHLA class II+ human B-lymphoblastoid cell line LCL 721221(ATCC) was used as positive control to stimulate HLA-mismatched PBMCWhen LCL cells were used as stimulatorcells a 75Gy dose irradiation was given and responderstimulator ratio was of 1 05 with a final concentrationof PBMC responder cells of 105 cellswell in a 96-well flatbottomed plate
4 Journal of Immunology Research
IgG1ALPtubulin
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(c)
Figure 3 BM-derived MSCs committed to osteodifferentiation process express HLA-G and are hypoimmunogenic (a and b) Expressionof ALP and HLA-G5 was evaluated by intracellular flow cytometry analysis on BM-derived MSCs committed to 14-day osteodifferentiationprocess and pretreated with IFN120574 and TNF120572 (Os-MSC-BM IFN120574TNF120572) or not (Os-MSC-BM) IgG1 was used as isotype control Ab Tubulinwas used as positive control of cell permeabilization (c) PBMC from healthy individual (00) were used as responder cells towards BM-derived MSCs committed to osteodifferentiation process and pretreated with IFN120574 and TNF120572 (Os-MSC-BM IFN120574TNF120572) or not (Os-MSC-BM) as stimulating cells at various responder stimulator ratios Irradiated LCLlowast were used as positive control of T cell alloproliferationResults are given as mean cpm plusmn sem one representative experiment is shown
Particularly in MBCP experiments MLR were per-formed in 24-well bottomed plates with a final concentrationof PBMC responder cells of 106 cellswell Experiments com-paring the effects of standard culture condition (no MBCP)and three-dimensional culture setting using MBCP discswere both performed in 24-well bottomed plates
Cultures were incubated at 37∘C in a humidified 5CO2air atmosphere PBMC proliferation was measured at
day 6 by [3H]-thymidine incorporation (1120583Ciwell PerkinElmer) during the last 18 hours of culture Cells were thenharvested on filtermats A and thymidine incorporation intoDNA was quantified using a beta counter (Wallac 1450Pharmacia) All samples were run in triplicate The influenceofMSC licensing with inflammatory cytokines such as IFN-120574at 10 ngmL (Peprotech) and plus TNF-120572 at 15 ngmL (RampDsystems) was analyzed by adding these cytokines in cultures48 hours before MLR [14]
25 Flow Cytometry The cytokine (IFN-120574 and TNF-120572) treat-ment efficiency was checked by cytofluorometry analysis
through the upregulation of HLA-DR expression on MSCBriefly cells were washed in PBS and stained with theanti-HLA-DR conjugated with PE (Beckman Coulter) inPBS 2 heat-inactivated fetal calf serum for 30 minutes at4∘C Control aliquots were stained with an isotype-matchedantibody to evaluate nonspecific binding to target cellsFluorescence was detected by using the Epics XL4 flowcytometer (Beckman Coulter Brea CA USA)
The expression of the HLA-G5 soluble isoform by MSCswas assessed by using the 2A12 mAb (Exbio) after cell per-meabilization The osteodifferentiation process was verifiedby cytofluorometry analysis through the induction of alkalinephosphatase (ALP) expression in osteodifferentiated MSCafter cell permeabilization Briefly cells were first permeabi-lized by using saponin (Sigma) and then stained with 2A12or anti-ALP (RampD systems) for 30 minutes at 4∘C Afterwashing cells were subsequently stained with an F(ab1015840)2goat anti-mouse IgG antibody conjugated with PE (BeckmanCoulter) for 30 minutes at 4∘C Control aliquots were stainedwith an isotype-matched antibody to evaluate nonspecific
Journal of Immunology Research 5
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0102030405060708090 Osteodifferentiated adipose tissue-derived MSC
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Figure 4 AT-derived MSCs committed to osteodifferentiation process express HLA-G and are hypoimmunogenic (a) Expression of HLA-DRmolecules was evaluated by flow cytometry analysis onAT-derivedMSCs committed to 14-day osteodifferentiation process and pretreatedwith IFN120574 and TNF120572 (Os-MSC-AT IFN120574TNF120572) or not (Os-MSC-AT) (b) Expression of ALP and HLA-G5 was evaluated by intracellularflow cytometry analysis on AT-derived MSCs committed to 14-day osteodifferentiation process and pretreated with IFN120574 and TNF120572 (Os-MSC-AT IFN120574TNF120572) or not (Os-MSC-AT) IgG1 was used as isotype control Ab (c) PBMC from healthy individual (00) were used asresponder cells towards AT-derived MSCs committed to osteodifferentiation process and pretreated with IFN120574 and TNF120572 (Os-MSC-ATIFN120574TNF120572) or not (Os-MSC-AT) as stimulating cells at various responder stimulator ratios Irradiated LCLlowast were used as positive controlof T cell alloproliferation Results are given as mean cpm plusmn sem one representative experiment is shown
binding to target cells Fluorescencewas detected by using theEpics XL4 flow cytometer
26 Statistical Analysis Significance was assessed by usinga nonparametric Mann-Whitney test assuming a 119875 value lt005 as significant and was marked with lowast in the figures
3 Results and Discussion
Twomain questions were addressed in the present study rely-ing on whether immune regulatory properties of MSCs aremodified by (1) MSC differentiation towards the osteoblasticcell lineage or (2) the addition of synthetic biomaterial (ieMBCP+ granules) In this regard we analyzed both theimmunogenic and immunosuppressive properties of MSCsfrom BM or AT in allogenic conditions that is facing HLA-mismatched PBMC To identify low immunogenic MSCtypes we studied their ability to be recognized as allogenic
cells by HLA-mismatched PBMC in MLR using MSCs asstimulating cells and PBMC from various healthy donorsas responder cells To examine their immunosuppressiveproperties we studied their ability to modulate T cell allo-proliferation as third-party cells in a classical MLR All thefunctional experiments were performed by considering thedifferentiation status of MSC either immature or osteodiffer-entiated and seeded onto biomaterial or not
No PBMC alloproliferation was observed in response tovarious doses of allogenic MSCs derived from bone marrow(Figure 1(c)) or adipose tissue (Figure 2(c)) even after licens-ing with IFN-120574 and TNF-120572 (Figures 1(c) and 2(c)) Tables 1and 2 summarize the results obtained with PBMC from dis-tinct healthy donorsThe efficiency of cytokine treatment wasattested by the induction of HLA-DR expression on MSCs(Figures 1(a) and 2(a)) In order to evaluate the influenceof osteodifferentiation process on the immunogenicity ofMSCs similar functional assays were performed using BM-derived and AT-derived MSCs committed to preosteoblastic
6 Journal of Immunology Research
0
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IFN120574TNF120572 IFN120574TNF120572
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Figure 5 Both BM- andAT-derived MSCs when combined to MBCP biomaterial keep their hypoimmunogenicity PBMC from healthyindividual (713) were used as responder cells towards either BM-derived MSCs (MSC-BM) or AT-derived MSCs (MSC-AT) that werepretreated with IFN120574 and TNF120572 (IFN120574TNF120572) or not and used as stimulating cells at the responder stimulator ratio of 1 02 MSCs werecombined toMBCP biomaterial (MBCP) or not (NoMBCP) Irradiated LCLlowast were used as positive control of T cell alloproliferation Resultsare given as mean cpm plusmn sem one representative experiment is shown
MSCs as stimulating cells The osteodifferentiation processwas validated through the upregulation of ALP expression inosteodifferentiated MSCs (Figures 3(a) 3(b) 4(a) and 4(b))Results show that both BM-derived and AT-derived MSCscommitted to osteodifferentiation are still hypoimmunogenicwhether they are pretreated or not with IFN-120574 and TNF-120572(Figures 3(c) and 4(c) and Tables 1 and 2) Then we lookedat whether combination of biomaterial (ie MBCP) withMSCs alters their immunogenicityNodifferenceswere foundbetween standard 2D-coculture conditions (MSC + PBMC)and 3D-coculture conditions (MSC + MBCP + PBMC) Onerepresentative allogenic combination is shown (Figure 5) forwhich the mean percentage of T cell alloproliferation ispresented in Table 3 (119899 = 3 healthy donors) It is of notethat the addition of MBCP to BM-derived MSCs treated or
not with cytokines modifies slightly their immunogenicityalthough no statistical difference was observed betweenboth conditions (119875 gt 01) (Table 3) Consequently wecan conclude that both the osteodifferentiation process andthe presence of biomaterial (MBCP) did not abrogate thehypoimmunogenicity of MSCs
Notably expression of the tolerogenic soluble HLA-G5protein was observed in MSCs derived from BM and ATand could be enhanced following treatment with IFN-120574 andTNF-120572 (Figures 1(b) and 2(b)) Such enhanced expressionof HLA-G by IFN-120574 treatment was previously reported forother cell types such as monocytes [15] bronchial epithelialcells [16] thymic epithelial cells [17] and various tumor cells[18] Nevertheless HLA-G expression levels varied amongthe various batches of MSCs tested (data not shown) In
Journal of Immunology Research 7
MSC-BM 954 885 667 339 165888 812 647 310 153
0
50
100
Allo
prol
ifera
tion
inhi
bitio
n (
)
Bone marrow-derived mesenchymal stem cells
1 05 05 1 05 025 1 05 0125 1 05 006 1 05 003
Responder stimulator MSC ratioMSC-BM IFN120574TNF120572
(a)
MSC-AT 878 824 879 859 823883 831 741 662 572
0
50
100
Allo
prol
ifera
tion
inhi
bitio
n (
)Adipose tissue-derived mesenchymal stem cells
lowast lowast lowast
MSC-AT IFN120574TNF120572Responder stimulator MSC ratio
1 05 05 1 05 025 1 05 0125 1 05 006 1 05 003
(b)
Figure 6 Both BM- andAT-derived MSCs display immunosuppressive properties in a dose-dependent manner PBMC from healthyindividuals were used as responder cells towards irradiated LCLlowast used as stimulating cells in presence of either (a) BM-derivedMSCs (MSC-BM) or (b) AT-derived MSCs (MSC-AT) that were pretreated with IFN120574 and TNF120572 (IFN120574TNF120572) or not and used as third-party cells atvarious responder stimulator MSC ratios Results are given as mean percentage of alloproliferation inhibition plusmn sem according to themaximal alloproliferation observed with PBMC+LCLlowast using PBMC from 4 and 6 distinct healthy donors in BM- and AT-derived MSCsexperiments respectively
Table 1 MSC from BM after osteodifferentiation and licensing byIFN-120574 and TNF-120572 do not induce PBMC proliferation in allogenicconditions
R Saratio MSC-BM MSC-BM
IFN-120574TNF-120572 Os-MSC-BM Os-MSC-BMIFN-120574TNF-120572
1 05 43 plusmn 21b 11 plusmn 03 33 plusmn 03 17 plusmn 02
1 025 45 plusmn 14 10 plusmn 03 31 plusmn 01 18 plusmn 05
1 012 53 plusmn 19 11 plusmn 04 32 plusmn 10 23 plusmn 12
1 006 54 plusmn 24 14 plusmn 05 26 plusmn 11 39 plusmn 31
1 003 43 plusmn 17 14 plusmn 05 22 plusmn 02 21 plusmn 12
aPBMC from healthy individuals were used as responder cells towards BM-derived MSCs pretreated with IFN-120574 and TNF-120572 (MSC-BM IFN-120574TNF-120572) or not (MSC-BM) as stimulating cells at various responder stimulator(R S) ratios Similar experiments were performed with BM-derived MSCscommitted to osteodifferentiation process and pretreated with IFN-120574 andTNF-120572 (Os-MSC-BM IFN-120574TNF-120572) or not (Os-MSC-BM) as stimulatingcellsbData are mean plusmn sem of alloproliferation percentage obtained with 5and 3 distinct healthy donors for MSC-BM and Os-MSC-BM experimentsrespectively This percentage is calculated considering PBMC stimulatedwith LCLlowast as 100 alloproliferation
agreement with recent findings we observed enhancedHLA-Gexpression byMSCs following osteodifferentiation (Figures3(a) 3(b) and 4(b)) [13]
Table 2 MSC from AT after osteodifferentiation and licensing byIFN-120574 and TNF-120572 do not induce PBMC proliferation in allogenicconditions
R Saratio MSC-AT MSC-AT
IFN-120574TNF-120572 Os-MSC-AT Os-MSC-ATIFN-120574TNF-120572
1 05 56 plusmn 10b 72 plusmn 31 11 plusmn 02 09 plusmn 06
1 025 55 plusmn 15 52 plusmn 26 13 plusmn 03 08 plusmn 05
1 012 53 plusmn 15 47 plusmn 29 10 plusmn 03 11 plusmn 08
1 006 53 plusmn 20 55 plusmn 30 10 plusmn 02 13 plusmn 10
1 003 46 plusmn 11 39 plusmn 20 14 plusmn 03 18 plusmn 13
aPBMC from healthy individuals were used as responder cells towards AT-derived MSCs pretreated with IFN-120574 and TNF-120572 (MSC-AT IFN-120574TNF-120572) or not (MSC-AT) as stimulating cells at various responder stimulator(R S) ratios Similar experiments were performed with AT-derived MSCscommitted to osteodifferentiation process and pretreated with IFN-120574 andTNF-120572 (Os-MSC-AT IFN-120574TNF-120572) or not (Os-MSC-AT) as stimulatingcellsbData are mean plusmn sem of alloproliferation percentage obtained with 4and 3 distinct healthy donors for MSC-AT and Os-MSC-AT experimentsrespectively This percentage is calculated considering PBMC stimulatedwith LCLlowast as 100 alloproliferation
Then we investigated the immunomodulatory propertiesof MSCs as third-party cells in MLR Results showed thatbothBM-derived andAT-derivedMSCsdisplay immunosup-pressive properties in a dose-responsemanner (Figure 6) It is
8 Journal of Immunology Research
MSC-BM 940 911 864938 896 929
Allo
prol
ifera
tion
inhi
bitio
n (
)A
llopr
olife
ratio
n in
hibi
tion
()
Allo
prol
ifera
tion
inhi
bitio
n (
)A
llopr
olife
ratio
n in
hibi
tion
()
461 628 750379 676 796
0
50
100
850 803 893600 725 895
0
50
100
0
50
100
919 879 879909 867 887
0
50
100
Bone marrow-derived mesenchymal stem cells
Adipose tissue-derived mesenchymal stem cells
No MBCP
Bone marrow-derived mesenchymal stem cells
Adipose tissue-derived mesenchymal stem cells
MBCP
MSC-BM IFN120574TNF120572
MSC-BMMSC-BM IFN120574TNF120572
MSC-ATMSC-AT IFN120574TNF120572
MSC-ATMSC-AT IFN120574TNF120572
1 05 04 1 05 02 1 05 01 1 05 04 1 05 02 1 05 01
1 05 04 1 05 02 1 05 011 05 04 1 05 02 1 05 01
Responder stimulator MSC ratio Responder stimulator MSC ratio
Responder stimulator MSC ratio Responder stimulator MSC ratio
Figure 7 Both BM- andAT-derived MSCs when combined to MBCP biomaterial keep their immunosuppressive properties PBMC fromhealthy individuals were used as responder cells towards irradiated LCLlowast used as stimulating cells in presence of either BM-derived MSCs(MSC-BM) or AT-derived MSCs (MSC-AT) that were pretreated with IFN120574 and TNF120572 (IFN120574TNF120572) or not and used as third-party cellsat various responder stimulator MSC ratios MSCs were combined to MBCP biomaterial (MBCP) or not (No MBCP) Results are given asmean percentage of alloproliferation inhibition plusmn sem when compared to PBMC+LCLlowast using PBMC from 3 distinct healthy donors
Table 3 Immunogenicity of MSC from bone marrow and adiposetissue combinedwithMBCPbiomaterial and licensing by IFN-120574 andTNF-120572
MSC-BM MSC-BMIFN-120574TNF-120572 MSC-AT MSC-AT
IFN-120574TNF-120572No MBCPa 75 plusmn 22b 98 plusmn 40 139 plusmn 39 230 plusmn 52
MBCP 519 plusmn 141 411 plusmn 116 81 plusmn 08 110 plusmn 08
aPBMC from healthy individuals were used as responder cells towards BM-or AT-derivedMSCs pretreated with IFN-120574 and TNF-120572 or not as stimulatingcells at 1 02 responder stimulator ratiobData are mean plusmn sem of alloproliferation percentage obtained with 3distinct healthy donors This percentage is calculated considering PBMCstimulated with LCLlowast as 100 alloproliferation
of note that (i) AT-derivedMSCs aremore potent at low dosescompared to BM-derived MSCs (119875 lt 005) (Figures 6(a) and6(b)) and (ii) licensing with cytokines reduces significantlythe immunosuppressive properties of AT-derived MSCs(119875 lt 005) (Figure 6(b)) When combined to biomaterial(MBCP) both BM-derived and AT-derived MSCs still exertimmunosuppressive properties as they greatly inhibit T cell
alloproliferation with or without being seeded with MBCP(Figure 7) Although not statistically significant addition ofMBCP reduces the immunomodulatory properties of BM-derived MSCs at high responder stimulator MSC ratiosThis could be due to steric hindrance when high numbersof cells are used Indeed such MBCP effect is no longerobserved at low ratios (Figure 7) Also both BM-derivedand AT-derived MSCs when committed to preosteoblasticMSCs inhibit T cell alloproliferation and remain thus able toinduce a tolerogenicmicroenvironment (Figure 8) Licensingwith IFN-120574 and TNF-120572 did not modify such MSC-derivedimmunosuppression (Figures 7 and 8) It is of note thatonce osteodifferentiated AT-derived MSCs are more potentat low doses compared to BM-derived MSCs as they displayhigher immunosuppressive effects (119875 lt 005) (Figure 8)Such higher immunomodulatory capacity of adipose tissue-derived multipotent stromal cells compared to their bonemarrow-derived counterparts has been previously reported[19]
Our present results are in agreement with previousreports showing that differentiation of stem cells does not
Journal of Immunology Research 9
894 889 942 649 528956 925 976 678 367
0
50
100
Allo
prol
ifera
tion
inhi
bitio
n (
)
Osteodifferentiated bone marrow-derived MSC
MSC-BMOs-MSC-BM IFN120574TNF120572
1 05 05 1 05 025 1 05 0125 1 05 006 1 05 003
Responder stimulator MSC ratio
(a)
888 926 917 895 854936 935 913 859 780
0
50
100
Allo
prol
ifera
tion
inhi
bitio
n (
)Osteodifferentiated adipose tissue-derived MSC
Responder stimulator MSC ratio
MSC-ATMSC-AT IFN120574TNF120572
1 05 05 1 05 025 1 05 0125 1 05 006 1 05 003
(b)
Figure 8 Both BM- andAT-derived MSCs when committed to osteodifferentiation process keep their immunosuppressive propertiesPBMC from healthy individuals were used as responder cells towards irradiated LCLlowast used as stimulating cells in presence of eitherosteodifferentiated BM-derived MSCs (Os-MSC-BM) or AT-derived MSCs (Os-MSC-AT) that were pretreated with IFN120574 and TNF120572(IFN120574TNF120572) or not and used as third-party cells at various responder stimulator MSC ratios Results are given as mean percentage ofalloproliferation inhibition plusmn sem when compared to PBMC+LCLlowast using PBMC from 3 and 5 distinct healthy donors in BM- and AT-derived MSCs experiments respectively
alter their low immunogenicity and immunomodulatoryproperties For instance human amniotic epithelial cellswhich have stem cell-like properties retain their immuno-suppressive functions after differentiation into hepatocyte-like cells [20] Also human Whartonrsquos jelly-derived MSCsmaintain the expression of immunomodulatory moleculessuch as HLA-G when subjected to osteogenic differentiationin vitro [21]
In conclusion MSCs from BM or AT display tolerogenicproperties which are maintained following osteodifferenti-ation process or addition of biomaterial and may thus beconsidered as allogenic sources for regenerating bone defectsin orthopaedic and maxillofacial surgery [22]
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This project has received funding from the European UnionrsquosSeventh Programme for research technological develop-ment and demonstration underGrant agreement no 241879
References
[1] R Langer and J P Vacanti ldquoTissue engineeringrdquo Science vol260 no 5110 pp 920ndash926 1993
[2] C Laurencin Y Khan and S F El-Amin ldquoBone graft substi-tutesrdquo Expert Review of Medical Devices vol 3 no 1 pp 49ndash572006
[3] H Petite V ViateauW Bensaıd et al ldquoTissue-engineered boneregenerationrdquo Nature Biotechnology vol 18 no 9 pp 959ndash9632000
[4] M Di Trapani G Bassi M Ricciardi et al ldquoComparative studyof immune regulatory properties of stem cells derived fromdifferent tissuesrdquo StemCells andDevelopment vol 22 pp 2990ndash3002 2013
[5] AUccelli V Pistoia andLMoretta ldquoMesenchymal stemcells anew strategy for immunosuppressionrdquo Trends in Immunologyvol 28 no 5 pp 219ndash226 2007
[6] M Najar G Raicevic H Fayyad-Kazan et al ldquoImmune-relatedantigens surface molecules and regulatory factors in human-derived mesenchymal stromal cells the expression and impactof inflammatory primingrdquo Stem Cell Reviews vol 8 pp 1188ndash1198 2012
[7] R Rizzo D Campioni M Stignani et al ldquoA functional role forsoluble HLA-G antigens in immune modulation mediated bymesenchymal stromal cellsrdquoCytotherapy vol 10 no 4 pp 364ndash375 2008
10 Journal of Immunology Research
[8] F Morandi L Raffaghello G Bianchi et al ldquoImmunogenicityof human mesenchymal stem cells in HLA-class I-restricted T-cell responses against viral or tumor-associated antigensrdquo StemCells vol 26 no 5 pp 1275ndash1287 2008
[9] A Naji N Rouas-Freiss A Durrbach E D Carosella LSensebe and F Deschaseaux ldquoConcise review combininghuman leukocyte antigen G and mesenchymal stem cells forimmunosuppressant biotherapyrdquo Stem Cells vol 31 pp 2296ndash2303 2013
[10] G La Rocca and R Anzalone ldquoPerinatal stem cells revisiteddirections and indications at the crossroads between tissueregeneration and repairrdquoCurrent Stem Cell Research ampTherapyvol 8 pp 2ndash5 2013
[11] Z Selmani ANaji EGaiffe et al ldquoHLA-G is a crucial immuno-suppressive molecule secreted by adult human mesenchymalstem cellsrdquo Transplantation vol 87 no 9 supplement pp S62ndashS66 2009
[12] Z Selmani A Naji I Zidi et al ldquoHuman leukocyte antigen-G5 secretion by human mesenchymal stem cells is required tosuppress T lymphocyte andnatural killer function and to induceCD4+ CD25ℎ119894119892ℎFOXP3+ regulatory T cellsrdquo Stem Cells vol 26no 1 pp 212ndash222 2008
[13] F Deschaseaux J Gaillard A Langonne et al ldquoRegulationand function of immunosuppressivemolecule human leukocyteantigen G5 in human bone tissuerdquo The FASEB Journal vol 27pp 2977ndash2987 2013
[14] M Krampera ldquoMesenchymal stromal cell licensing a multistepprocessrdquo Leukemia vol 25 no 9 pp 1408ndash1414 2011
[15] P Moreau F Adrian-Cabestre C Menier et al ldquoIL-10 selec-tively induces HLA-G expression in human trophoblasts andmonocytesrdquo International Immunology vol 11 no 5 pp 803ndash811 1999
[16] O Brugiere GThabutM Pretolani et al ldquoImmunohistochem-ical study of HLA-G expression in lung transplant recipientsrdquoAmerican Journal of Transplantation vol 9 no 6 pp 1427ndash14382009
[17] S Lefebvre S Berrih-Aknin F Adrian et al ldquoA specificinterferon (IFN)-stimulated response element of the distalHLA-G promoter binds IFN-regulatory factor 1 and mediatesenhancement of this nonclassical class I gene by IFN-betardquoTheJournal of Biological Chemistry vol 276 no 9 pp 6133ndash61392001
[18] N Rouas-Freiss S Bruel C Menier C Marcou P Moreauand E D Carosella ldquoSwitch of HLA-G alternative splicing ina melanoma cell line causes loss of HLA-G1 expression andsensitivity to NK lysisrdquo International Journal of Cancer vol 117no 1 pp 114ndash122 2005
[19] S M Melief J J Zwaginga W E Fibbe and H RoelofsldquoAdipose tissue-derived multipotent stromal cells have a higherimmunomodulatory capacity than their bone marrow-derivedcounterpartsrdquo StemCells TranslationalMedicine vol 2 pp 455ndash463 2013
[20] J Y Tee V Vaghjiani Y H Liu P Murthi J Chan and UManuelpillai ldquoImmunogenicity and immunomodulatory prop-erties of hepatocyte-like cells derived from human amnioticepithelial cellsrdquo Current Stem Cell Research amp Therapy vol 8pp 91ndash99 2013
[21] G La Rocca M Lo Iacono T Corsello S Corrao F Farina andR Anzalone ldquoHuman Whartonrsquos jelly mesenchymal stem cellsmaintain the expression of key immunomodulatory moleculeswhen subjected to osteogenic adipogenic and chondrogenic
differentiation in vitro new perspectives for cellular therapyrdquoCurrent Stem Cell Research ampTherapy vol 8 pp 100ndash113 2013
[22] C Menard and K Tarte ldquoImmunoregulatory properties of clin-ical grade mesenchymal stromal cells evidence uncertaintiesand clinical applicationrdquo Stem Cell Research amp Therapy vol 4article 64 2013
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
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Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
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Diabetes ResearchJournal of
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Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
2 Journal of Immunology Research
100 101 102 103 104
Cou
nts
FL 2
R11 R12
IgG1HLA-DR
MSC-BM
100 101 102 103 104
Cou
nts
FL 2
R11 R12
MSC-BM IFN120574TNF120572
(a)
Cou
nts
Cou
nts
100 101 102 103 104 100 101 102 103 104
R11 R12
MSC-BM
R13 R14
IgG1HLA-G5tubulin
MSC-BM IFN120574TNF120572
FL 2 FL 2
(b)
020406080
100120140160 Controls
020406080
100120140160
MSC-BM
LCLlowast
MSC
-BM
(CPM
times10
minus3)
(CPM
times10
minus3)
Bone marrow-derived mesenchymal stem cells
PBM
C15
MSC
-BM
IFN120574
TN
F120572
PBM
C15
LCL
lowast
Responder stimulator ratio1
05
1 025
1 012
1 006
1 003
MSC-BM IFN120574TNF120572
3H
-thym
idin
e inc
orpo
ratio
n
3H
-thym
idin
e inc
orpo
ratio
n
(c)
Figure 1 BM-derived MSCs express HLA-G and are hypoimmunogenic (a) Expression of HLA-DR molecules was evaluated by flowcytometry analysis on BM-derived MSCs pretreated with IFN120574 and TNF120572 (MSC-BM IFN120574TNF120572) or not (MSC-BM) IgG1 was used asisotype control Ab (b) Expression of HLA-G5 was evaluated by intracellular flow cytometry analysis on BM-derived MSCs pretreated withIFN120574 and TNF120572 (MSC-BM IFN120574TNF120572) or not (MSC-BM) Tubulin was used as positive control of cell permeabilization (c) PBMC fromhealthy individual (15) were used as responder cells towards BM-derived MSCs pretreated with IFN120574 and TNF120572 (MSC-BM IFN120574TNF120572)or not (MSC-BM) as stimulating cells at various responder stimulator ratios Irradiated LCLlowast were used as positive control of T cellalloproliferation Results are given as mean cpm plusmn sem one representative experiment is shown
main mechanisms (1) cell cycle arrest of immune cells atthe G1 phase (2) direct interaction with immune cells and(3) paracrine effect through secretion of various factorsincluding HLA-G prostaglandin E2 cytokines (TGF120573IL6 IL10 HGF VEGF etc) and enzymes (indoleamine23-dioxygenase and inducible nitric oxide synthase) [5ndash8]Based on these tolerogenic properties allogenic MSCs arecurrently tested in various clinical trials [9 10]
HLA-G molecules expressed by mesenchymal stem cellsfulfill an important function since blockade of HLA-G usingHLA-G neutralizing antibodies could reverse MSC ability to(i) generate in vitro the expansion of CD4+CD25+ FoxP3+regulatory T cells (ii) inhibit the alloproliferative T cellresponse and (iii) suppress the cytotoxic function of NKcells These results show that HLA-G molecules mainly sol-uble HLA-G5 actively contribute to the immunosuppressiveproperties exerted by MSCs [11 12]
In clinical trials aimed at repairing bone defects themainobjective is to develop new biomaterials that simulate bone
issue formation in combination with MSCs In this contextourwork entailed assessing from an immunological perspec-tive whether allogenic MSCs could be used without a risk ofrejection instead of autologous MSCs The results obtainedin vitro validate this hypothesis since the MSCs proved tobe hypoimmunogenic and immunosuppressive in allogenicconditions Moreover following infusion in bone MSCs mayundergo osteodifferentiation process under the influence of119894119899 119907119894119907119900 osteogenic factors We thus evaluated whether (1)allogenic MSCs committed to osteodifferentiation processcan be rejected or not due to histoincompatibility and(2) combination with biomaterials modifies MSCs immuneproperties
2 Materials and Methods
21 Isolation of PBMC PBMC were isolated from bloodof healthy volunteer donors (after informed consent) fromthe French Blood Establishment (EFS Saint-Louis Hospital
Journal of Immunology Research 3
100 101 102 103 104
FL 2
100 101 102 103 104
FL 2
Cou
nts
Cou
nts
R11 R12
IgG1HLA-DR
R13 R14
MSC-AT MSC-AT IFN120574TNF120572
(a)
310
232
155
77
0
100 101 102 103 104
FL 2
100 101 102 103 104
FL 2
100 101 102 103 104
FL 2
Cou
nts
Cou
nts
Cou
nts
R11 R12
IgG1HLA-G5
R13 R14
MSC-ATMSC-AT
MSC-AT HLA-G5MSC-AT IFN120574TNF120572
IFN120574TNF120572
(b)
0102030405060708090
100Controls
0102030405060708090
100
(CPM
times10
minus3)
(CPM
times10
minus3)
Adipose tissue-derived mesenchymal stem cells
LCLlowast
MSC
-AT
PBM
C59-
MSC-AT
lowastPB
MC
59
- LC
L
MSC
-AT
IFN120574
TN
F120572
Responder stimulator ratio1
05
1 025
1 012
1 006
1 003
MSC-AT IFN120574TNF120572
3H
-thym
idin
e inc
orpo
ratio
n
3H
-thym
idin
e inc
orpo
ratio
n
(c)
Figure 2 AT-derived MSCs express HLA-G and are hypoimmunogenic (a) Expression of HLA-DR molecules was evaluated by flowcytometry analysis on AT-derivedMSCs pretreated with IFN120574 and TNF120572 (MSC-AT IFN120574TNF120572) or not (MSC-AT) IgG1 was used as isotypecontrol Ab (b) Expression of HLA-G5 was evaluated by intracellular flow cytometry analysis on AT-derived MSCs pretreated with IFN120574 andTNF120572 (MSC-AT IFN120574TNF120572) or not (MSC-AT) (c) PBMC from healthy individual (59-) were used as responder cells towards AT-derivedMSCs pretreated with IFN120574 and TNF120572 (MSC-AT IFN120574TNF120572) or not (MSC-AT) as stimulating cells at various responder stimulator ratiosIrradiated LCLlowast were used as positive control of T cell alloproliferation Results are given asmean cpmplusmn sem one representative experimentis shown
Paris France) by density-gradient centrifugation over Ficoll-Paque PLUS (GE Healthcare) These cells were used as HLA-mismatched responding cells in MLR
22 In Vitro Osteodifferentiation MSCs from BM orAT were obtained from Reborne consortium center(httpwwwreborneorg) Before experiments MSCswere thawed and expanded through seeding 1000 cellscm2in T75 flasks When cultures reach 60ndash70 confluencecells were harvested and seeded for immunological assaysThe osteodifferentiated MSCs used as stimulating cells inMLR were obtained as previously described [13] BrieflyMSCs were cultured in osteoblastic differentiation mediumconsisting of 120572MEM FBS ascorbic acid NaH
2PO4 and
BMP-4 during 14 to 23 days
23 Preparation of MSC-Biomaterial Complex For Immuno-logical Tests 24-well (ultralow attachment) plates containingdisks of MBCP+ (macroporous biphasic calcium phosphate)granules with the same diameter of each well bottomwere prepared in collaboration with Biomatlante (France)
to achieve a complete adherence of seeded MSCs to thebiomaterial and then a complete induction of osteoblasticdifferentiation of MSCs The disks of MBCP+ were washedfor 48 hours before use to avoid any toxic effect on MSCsThen MSCs were seeded on MBCP discs during 5 daysin order to colonize homogenously the particles ThenMSC-biomaterial complex was tested in mixed lymphocytereactions
24 Mixed Lymphocyte Reaction (MLR) Using MSC asEither Stimulating Cells Or Third-Party Cells Facing HLA-Mismatched PBMC as Responder Cells PBMC from differentdonors were used as responder cells andMSCs or osteodiffer-entiated MSCs from BM or AT were used as either allogenicstimulating cells or third-party cells at various ratios TheHLA class II+ human B-lymphoblastoid cell line LCL 721221(ATCC) was used as positive control to stimulate HLA-mismatched PBMCWhen LCL cells were used as stimulatorcells a 75Gy dose irradiation was given and responderstimulator ratio was of 1 05 with a final concentrationof PBMC responder cells of 105 cellswell in a 96-well flatbottomed plate
4 Journal of Immunology Research
IgG1ALPtubulin
100 101 102 103 104
FL 2
100 101 102 103 104
FL 2
R11 R12
Cou
nts
Cou
nts
R13 R14
IgG1HLA-G5MSC-BM MSC-BM
(a)
IgG1ALPtubulin
100 101 102 103 104
FL 2
100 101 102 103 104
FL 2
R11 R12
Cou
nts
Cou
nts
R13 R14
IgG1HLA-G5Os-MSC-BM Os-MSC-BM IFN120574TNF120572
(b)
0102030405060708090
100 Controls
0102030405060708090
100
Os-MSC-BM
Osteodiferentiated bone marrow-derived MSC
(CPM
times10
minus3)
(CPM
times10
minus3)
LCLlowast
Os-
MSC
-BM
PBM
C00
Os-
MSC
-BM
IFN120574
TN
F120572 lowastPB
MC
00
LCL
Responder stimulator ratio1
05
1 025
1 012
1 006
1 003
Os-MSC-BM IFN120574TNF120572
3H
-thym
idin
e inc
orpo
ratio
n
3H
-thym
idin
e inc
orpo
ratio
n
(c)
Figure 3 BM-derived MSCs committed to osteodifferentiation process express HLA-G and are hypoimmunogenic (a and b) Expressionof ALP and HLA-G5 was evaluated by intracellular flow cytometry analysis on BM-derived MSCs committed to 14-day osteodifferentiationprocess and pretreated with IFN120574 and TNF120572 (Os-MSC-BM IFN120574TNF120572) or not (Os-MSC-BM) IgG1 was used as isotype control Ab Tubulinwas used as positive control of cell permeabilization (c) PBMC from healthy individual (00) were used as responder cells towards BM-derived MSCs committed to osteodifferentiation process and pretreated with IFN120574 and TNF120572 (Os-MSC-BM IFN120574TNF120572) or not (Os-MSC-BM) as stimulating cells at various responder stimulator ratios Irradiated LCLlowast were used as positive control of T cell alloproliferationResults are given as mean cpm plusmn sem one representative experiment is shown
Particularly in MBCP experiments MLR were per-formed in 24-well bottomed plates with a final concentrationof PBMC responder cells of 106 cellswell Experiments com-paring the effects of standard culture condition (no MBCP)and three-dimensional culture setting using MBCP discswere both performed in 24-well bottomed plates
Cultures were incubated at 37∘C in a humidified 5CO2air atmosphere PBMC proliferation was measured at
day 6 by [3H]-thymidine incorporation (1120583Ciwell PerkinElmer) during the last 18 hours of culture Cells were thenharvested on filtermats A and thymidine incorporation intoDNA was quantified using a beta counter (Wallac 1450Pharmacia) All samples were run in triplicate The influenceofMSC licensing with inflammatory cytokines such as IFN-120574at 10 ngmL (Peprotech) and plus TNF-120572 at 15 ngmL (RampDsystems) was analyzed by adding these cytokines in cultures48 hours before MLR [14]
25 Flow Cytometry The cytokine (IFN-120574 and TNF-120572) treat-ment efficiency was checked by cytofluorometry analysis
through the upregulation of HLA-DR expression on MSCBriefly cells were washed in PBS and stained with theanti-HLA-DR conjugated with PE (Beckman Coulter) inPBS 2 heat-inactivated fetal calf serum for 30 minutes at4∘C Control aliquots were stained with an isotype-matchedantibody to evaluate nonspecific binding to target cellsFluorescence was detected by using the Epics XL4 flowcytometer (Beckman Coulter Brea CA USA)
The expression of the HLA-G5 soluble isoform by MSCswas assessed by using the 2A12 mAb (Exbio) after cell per-meabilization The osteodifferentiation process was verifiedby cytofluorometry analysis through the induction of alkalinephosphatase (ALP) expression in osteodifferentiated MSCafter cell permeabilization Briefly cells were first permeabi-lized by using saponin (Sigma) and then stained with 2A12or anti-ALP (RampD systems) for 30 minutes at 4∘C Afterwashing cells were subsequently stained with an F(ab1015840)2goat anti-mouse IgG antibody conjugated with PE (BeckmanCoulter) for 30 minutes at 4∘C Control aliquots were stainedwith an isotype-matched antibody to evaluate nonspecific
Journal of Immunology Research 5
100 101 102 103 104
FL 2
100 101 102 103 104
FL 2
R12R11 R13 R14
Os-MSC-AT
IgG1HLA-DRC
ount
s
Cou
nts
Os-MSC-AT IFN120574TNF120572
(a)
100 101 102 103 104
FL 2
100 101 102 103 104
FL 2
R12R11 R13 R14
Os-MSC-AT
IgG1ALP IgG1ALPHLA-G5
Cou
nts
Cou
nts
Os-MSC-AT IFN120574TNF120572
(b)
0102030405060708090
Controls
0102030405060708090 Osteodifferentiated adipose tissue-derived MSC
Os-MSC-AT
(CPM
times10
minus3)
(CPM
times10
minus3)
LCLlowast
Os-
MSC
-BM
PBM
C00
Os-
MSC
-AT
IFN120574
TN
F120572 lowastPB
MC
00
LCL
Responder stimulator ratio1
05
1 025
1 012
1 006
1 003
Os-MSC-AT IFN120574TNF120572
3H
-thym
idin
e inc
orpo
ratio
n
3H
-thym
idin
e inc
orpo
ratio
n
(c)
Figure 4 AT-derived MSCs committed to osteodifferentiation process express HLA-G and are hypoimmunogenic (a) Expression of HLA-DRmolecules was evaluated by flow cytometry analysis onAT-derivedMSCs committed to 14-day osteodifferentiation process and pretreatedwith IFN120574 and TNF120572 (Os-MSC-AT IFN120574TNF120572) or not (Os-MSC-AT) (b) Expression of ALP and HLA-G5 was evaluated by intracellularflow cytometry analysis on AT-derived MSCs committed to 14-day osteodifferentiation process and pretreated with IFN120574 and TNF120572 (Os-MSC-AT IFN120574TNF120572) or not (Os-MSC-AT) IgG1 was used as isotype control Ab (c) PBMC from healthy individual (00) were used asresponder cells towards AT-derived MSCs committed to osteodifferentiation process and pretreated with IFN120574 and TNF120572 (Os-MSC-ATIFN120574TNF120572) or not (Os-MSC-AT) as stimulating cells at various responder stimulator ratios Irradiated LCLlowast were used as positive controlof T cell alloproliferation Results are given as mean cpm plusmn sem one representative experiment is shown
binding to target cells Fluorescencewas detected by using theEpics XL4 flow cytometer
26 Statistical Analysis Significance was assessed by usinga nonparametric Mann-Whitney test assuming a 119875 value lt005 as significant and was marked with lowast in the figures
3 Results and Discussion
Twomain questions were addressed in the present study rely-ing on whether immune regulatory properties of MSCs aremodified by (1) MSC differentiation towards the osteoblasticcell lineage or (2) the addition of synthetic biomaterial (ieMBCP+ granules) In this regard we analyzed both theimmunogenic and immunosuppressive properties of MSCsfrom BM or AT in allogenic conditions that is facing HLA-mismatched PBMC To identify low immunogenic MSCtypes we studied their ability to be recognized as allogenic
cells by HLA-mismatched PBMC in MLR using MSCs asstimulating cells and PBMC from various healthy donorsas responder cells To examine their immunosuppressiveproperties we studied their ability to modulate T cell allo-proliferation as third-party cells in a classical MLR All thefunctional experiments were performed by considering thedifferentiation status of MSC either immature or osteodiffer-entiated and seeded onto biomaterial or not
No PBMC alloproliferation was observed in response tovarious doses of allogenic MSCs derived from bone marrow(Figure 1(c)) or adipose tissue (Figure 2(c)) even after licens-ing with IFN-120574 and TNF-120572 (Figures 1(c) and 2(c)) Tables 1and 2 summarize the results obtained with PBMC from dis-tinct healthy donorsThe efficiency of cytokine treatment wasattested by the induction of HLA-DR expression on MSCs(Figures 1(a) and 2(a)) In order to evaluate the influenceof osteodifferentiation process on the immunogenicity ofMSCs similar functional assays were performed using BM-derived and AT-derived MSCs committed to preosteoblastic
6 Journal of Immunology Research
0
10
20
30
40
50
Bone marrow-derived mesenchymal stem cells(C
PMtimes10
minus3)
LCLlowast
MSC
-BM
0
10
20
30
40
50
Adipose tissue-derived mesenchymal stem cells
(CPM
times10
minus3)
LCLlowast
MSC
-AT
0
10
20
30
40
50Bone marrow-derived mesenchymal stem cells
(CPM
times10
minus3)
+
0
10
20
30
40
50Adipose tissue-derived mesenchymal stem cells
(CPM
times10
minus3)
+
No MBCP
MBCP
PBM
C713
lowastPB
MC
713
LCL
PBM
C713
MSC
-BM
PBM
C713
MSC
-BM
PBM
C713
lowastPB
MC
713
LCL
PBM
C713
MSC
-AT
PBM
C713
MSC
-AT
LCLlowast
MSC
-BM
PBM
C713
lowastPB
MC
713
LCL
PBM
C713
MSC
-BM
PBM
C713
MSC
-BM
LCLlowast
MSC
-AT
PBM
C713
lowastPB
MC
713
LCL
PBM
C713
MSC
-AT
PBM
C713
MSC
-AT
IFN120574TNF120572 IFN120574TNF120572
3H
-thym
idin
e inc
orpo
ratio
n
3H
-thym
idin
e inc
orpo
ratio
n
3H
-thym
idin
e inc
orpo
ratio
n
3H
-thym
idin
e inc
orpo
ratio
n
Figure 5 Both BM- andAT-derived MSCs when combined to MBCP biomaterial keep their hypoimmunogenicity PBMC from healthyindividual (713) were used as responder cells towards either BM-derived MSCs (MSC-BM) or AT-derived MSCs (MSC-AT) that werepretreated with IFN120574 and TNF120572 (IFN120574TNF120572) or not and used as stimulating cells at the responder stimulator ratio of 1 02 MSCs werecombined toMBCP biomaterial (MBCP) or not (NoMBCP) Irradiated LCLlowast were used as positive control of T cell alloproliferation Resultsare given as mean cpm plusmn sem one representative experiment is shown
MSCs as stimulating cells The osteodifferentiation processwas validated through the upregulation of ALP expression inosteodifferentiated MSCs (Figures 3(a) 3(b) 4(a) and 4(b))Results show that both BM-derived and AT-derived MSCscommitted to osteodifferentiation are still hypoimmunogenicwhether they are pretreated or not with IFN-120574 and TNF-120572(Figures 3(c) and 4(c) and Tables 1 and 2) Then we lookedat whether combination of biomaterial (ie MBCP) withMSCs alters their immunogenicityNodifferenceswere foundbetween standard 2D-coculture conditions (MSC + PBMC)and 3D-coculture conditions (MSC + MBCP + PBMC) Onerepresentative allogenic combination is shown (Figure 5) forwhich the mean percentage of T cell alloproliferation ispresented in Table 3 (119899 = 3 healthy donors) It is of notethat the addition of MBCP to BM-derived MSCs treated or
not with cytokines modifies slightly their immunogenicityalthough no statistical difference was observed betweenboth conditions (119875 gt 01) (Table 3) Consequently wecan conclude that both the osteodifferentiation process andthe presence of biomaterial (MBCP) did not abrogate thehypoimmunogenicity of MSCs
Notably expression of the tolerogenic soluble HLA-G5protein was observed in MSCs derived from BM and ATand could be enhanced following treatment with IFN-120574 andTNF-120572 (Figures 1(b) and 2(b)) Such enhanced expressionof HLA-G by IFN-120574 treatment was previously reported forother cell types such as monocytes [15] bronchial epithelialcells [16] thymic epithelial cells [17] and various tumor cells[18] Nevertheless HLA-G expression levels varied amongthe various batches of MSCs tested (data not shown) In
Journal of Immunology Research 7
MSC-BM 954 885 667 339 165888 812 647 310 153
0
50
100
Allo
prol
ifera
tion
inhi
bitio
n (
)
Bone marrow-derived mesenchymal stem cells
1 05 05 1 05 025 1 05 0125 1 05 006 1 05 003
Responder stimulator MSC ratioMSC-BM IFN120574TNF120572
(a)
MSC-AT 878 824 879 859 823883 831 741 662 572
0
50
100
Allo
prol
ifera
tion
inhi
bitio
n (
)Adipose tissue-derived mesenchymal stem cells
lowast lowast lowast
MSC-AT IFN120574TNF120572Responder stimulator MSC ratio
1 05 05 1 05 025 1 05 0125 1 05 006 1 05 003
(b)
Figure 6 Both BM- andAT-derived MSCs display immunosuppressive properties in a dose-dependent manner PBMC from healthyindividuals were used as responder cells towards irradiated LCLlowast used as stimulating cells in presence of either (a) BM-derivedMSCs (MSC-BM) or (b) AT-derived MSCs (MSC-AT) that were pretreated with IFN120574 and TNF120572 (IFN120574TNF120572) or not and used as third-party cells atvarious responder stimulator MSC ratios Results are given as mean percentage of alloproliferation inhibition plusmn sem according to themaximal alloproliferation observed with PBMC+LCLlowast using PBMC from 4 and 6 distinct healthy donors in BM- and AT-derived MSCsexperiments respectively
Table 1 MSC from BM after osteodifferentiation and licensing byIFN-120574 and TNF-120572 do not induce PBMC proliferation in allogenicconditions
R Saratio MSC-BM MSC-BM
IFN-120574TNF-120572 Os-MSC-BM Os-MSC-BMIFN-120574TNF-120572
1 05 43 plusmn 21b 11 plusmn 03 33 plusmn 03 17 plusmn 02
1 025 45 plusmn 14 10 plusmn 03 31 plusmn 01 18 plusmn 05
1 012 53 plusmn 19 11 plusmn 04 32 plusmn 10 23 plusmn 12
1 006 54 plusmn 24 14 plusmn 05 26 plusmn 11 39 plusmn 31
1 003 43 plusmn 17 14 plusmn 05 22 plusmn 02 21 plusmn 12
aPBMC from healthy individuals were used as responder cells towards BM-derived MSCs pretreated with IFN-120574 and TNF-120572 (MSC-BM IFN-120574TNF-120572) or not (MSC-BM) as stimulating cells at various responder stimulator(R S) ratios Similar experiments were performed with BM-derived MSCscommitted to osteodifferentiation process and pretreated with IFN-120574 andTNF-120572 (Os-MSC-BM IFN-120574TNF-120572) or not (Os-MSC-BM) as stimulatingcellsbData are mean plusmn sem of alloproliferation percentage obtained with 5and 3 distinct healthy donors for MSC-BM and Os-MSC-BM experimentsrespectively This percentage is calculated considering PBMC stimulatedwith LCLlowast as 100 alloproliferation
agreement with recent findings we observed enhancedHLA-Gexpression byMSCs following osteodifferentiation (Figures3(a) 3(b) and 4(b)) [13]
Table 2 MSC from AT after osteodifferentiation and licensing byIFN-120574 and TNF-120572 do not induce PBMC proliferation in allogenicconditions
R Saratio MSC-AT MSC-AT
IFN-120574TNF-120572 Os-MSC-AT Os-MSC-ATIFN-120574TNF-120572
1 05 56 plusmn 10b 72 plusmn 31 11 plusmn 02 09 plusmn 06
1 025 55 plusmn 15 52 plusmn 26 13 plusmn 03 08 plusmn 05
1 012 53 plusmn 15 47 plusmn 29 10 plusmn 03 11 plusmn 08
1 006 53 plusmn 20 55 plusmn 30 10 plusmn 02 13 plusmn 10
1 003 46 plusmn 11 39 plusmn 20 14 plusmn 03 18 plusmn 13
aPBMC from healthy individuals were used as responder cells towards AT-derived MSCs pretreated with IFN-120574 and TNF-120572 (MSC-AT IFN-120574TNF-120572) or not (MSC-AT) as stimulating cells at various responder stimulator(R S) ratios Similar experiments were performed with AT-derived MSCscommitted to osteodifferentiation process and pretreated with IFN-120574 andTNF-120572 (Os-MSC-AT IFN-120574TNF-120572) or not (Os-MSC-AT) as stimulatingcellsbData are mean plusmn sem of alloproliferation percentage obtained with 4and 3 distinct healthy donors for MSC-AT and Os-MSC-AT experimentsrespectively This percentage is calculated considering PBMC stimulatedwith LCLlowast as 100 alloproliferation
Then we investigated the immunomodulatory propertiesof MSCs as third-party cells in MLR Results showed thatbothBM-derived andAT-derivedMSCsdisplay immunosup-pressive properties in a dose-responsemanner (Figure 6) It is
8 Journal of Immunology Research
MSC-BM 940 911 864938 896 929
Allo
prol
ifera
tion
inhi
bitio
n (
)A
llopr
olife
ratio
n in
hibi
tion
()
Allo
prol
ifera
tion
inhi
bitio
n (
)A
llopr
olife
ratio
n in
hibi
tion
()
461 628 750379 676 796
0
50
100
850 803 893600 725 895
0
50
100
0
50
100
919 879 879909 867 887
0
50
100
Bone marrow-derived mesenchymal stem cells
Adipose tissue-derived mesenchymal stem cells
No MBCP
Bone marrow-derived mesenchymal stem cells
Adipose tissue-derived mesenchymal stem cells
MBCP
MSC-BM IFN120574TNF120572
MSC-BMMSC-BM IFN120574TNF120572
MSC-ATMSC-AT IFN120574TNF120572
MSC-ATMSC-AT IFN120574TNF120572
1 05 04 1 05 02 1 05 01 1 05 04 1 05 02 1 05 01
1 05 04 1 05 02 1 05 011 05 04 1 05 02 1 05 01
Responder stimulator MSC ratio Responder stimulator MSC ratio
Responder stimulator MSC ratio Responder stimulator MSC ratio
Figure 7 Both BM- andAT-derived MSCs when combined to MBCP biomaterial keep their immunosuppressive properties PBMC fromhealthy individuals were used as responder cells towards irradiated LCLlowast used as stimulating cells in presence of either BM-derived MSCs(MSC-BM) or AT-derived MSCs (MSC-AT) that were pretreated with IFN120574 and TNF120572 (IFN120574TNF120572) or not and used as third-party cellsat various responder stimulator MSC ratios MSCs were combined to MBCP biomaterial (MBCP) or not (No MBCP) Results are given asmean percentage of alloproliferation inhibition plusmn sem when compared to PBMC+LCLlowast using PBMC from 3 distinct healthy donors
Table 3 Immunogenicity of MSC from bone marrow and adiposetissue combinedwithMBCPbiomaterial and licensing by IFN-120574 andTNF-120572
MSC-BM MSC-BMIFN-120574TNF-120572 MSC-AT MSC-AT
IFN-120574TNF-120572No MBCPa 75 plusmn 22b 98 plusmn 40 139 plusmn 39 230 plusmn 52
MBCP 519 plusmn 141 411 plusmn 116 81 plusmn 08 110 plusmn 08
aPBMC from healthy individuals were used as responder cells towards BM-or AT-derivedMSCs pretreated with IFN-120574 and TNF-120572 or not as stimulatingcells at 1 02 responder stimulator ratiobData are mean plusmn sem of alloproliferation percentage obtained with 3distinct healthy donors This percentage is calculated considering PBMCstimulated with LCLlowast as 100 alloproliferation
of note that (i) AT-derivedMSCs aremore potent at low dosescompared to BM-derived MSCs (119875 lt 005) (Figures 6(a) and6(b)) and (ii) licensing with cytokines reduces significantlythe immunosuppressive properties of AT-derived MSCs(119875 lt 005) (Figure 6(b)) When combined to biomaterial(MBCP) both BM-derived and AT-derived MSCs still exertimmunosuppressive properties as they greatly inhibit T cell
alloproliferation with or without being seeded with MBCP(Figure 7) Although not statistically significant addition ofMBCP reduces the immunomodulatory properties of BM-derived MSCs at high responder stimulator MSC ratiosThis could be due to steric hindrance when high numbersof cells are used Indeed such MBCP effect is no longerobserved at low ratios (Figure 7) Also both BM-derivedand AT-derived MSCs when committed to preosteoblasticMSCs inhibit T cell alloproliferation and remain thus able toinduce a tolerogenicmicroenvironment (Figure 8) Licensingwith IFN-120574 and TNF-120572 did not modify such MSC-derivedimmunosuppression (Figures 7 and 8) It is of note thatonce osteodifferentiated AT-derived MSCs are more potentat low doses compared to BM-derived MSCs as they displayhigher immunosuppressive effects (119875 lt 005) (Figure 8)Such higher immunomodulatory capacity of adipose tissue-derived multipotent stromal cells compared to their bonemarrow-derived counterparts has been previously reported[19]
Our present results are in agreement with previousreports showing that differentiation of stem cells does not
Journal of Immunology Research 9
894 889 942 649 528956 925 976 678 367
0
50
100
Allo
prol
ifera
tion
inhi
bitio
n (
)
Osteodifferentiated bone marrow-derived MSC
MSC-BMOs-MSC-BM IFN120574TNF120572
1 05 05 1 05 025 1 05 0125 1 05 006 1 05 003
Responder stimulator MSC ratio
(a)
888 926 917 895 854936 935 913 859 780
0
50
100
Allo
prol
ifera
tion
inhi
bitio
n (
)Osteodifferentiated adipose tissue-derived MSC
Responder stimulator MSC ratio
MSC-ATMSC-AT IFN120574TNF120572
1 05 05 1 05 025 1 05 0125 1 05 006 1 05 003
(b)
Figure 8 Both BM- andAT-derived MSCs when committed to osteodifferentiation process keep their immunosuppressive propertiesPBMC from healthy individuals were used as responder cells towards irradiated LCLlowast used as stimulating cells in presence of eitherosteodifferentiated BM-derived MSCs (Os-MSC-BM) or AT-derived MSCs (Os-MSC-AT) that were pretreated with IFN120574 and TNF120572(IFN120574TNF120572) or not and used as third-party cells at various responder stimulator MSC ratios Results are given as mean percentage ofalloproliferation inhibition plusmn sem when compared to PBMC+LCLlowast using PBMC from 3 and 5 distinct healthy donors in BM- and AT-derived MSCs experiments respectively
alter their low immunogenicity and immunomodulatoryproperties For instance human amniotic epithelial cellswhich have stem cell-like properties retain their immuno-suppressive functions after differentiation into hepatocyte-like cells [20] Also human Whartonrsquos jelly-derived MSCsmaintain the expression of immunomodulatory moleculessuch as HLA-G when subjected to osteogenic differentiationin vitro [21]
In conclusion MSCs from BM or AT display tolerogenicproperties which are maintained following osteodifferenti-ation process or addition of biomaterial and may thus beconsidered as allogenic sources for regenerating bone defectsin orthopaedic and maxillofacial surgery [22]
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This project has received funding from the European UnionrsquosSeventh Programme for research technological develop-ment and demonstration underGrant agreement no 241879
References
[1] R Langer and J P Vacanti ldquoTissue engineeringrdquo Science vol260 no 5110 pp 920ndash926 1993
[2] C Laurencin Y Khan and S F El-Amin ldquoBone graft substi-tutesrdquo Expert Review of Medical Devices vol 3 no 1 pp 49ndash572006
[3] H Petite V ViateauW Bensaıd et al ldquoTissue-engineered boneregenerationrdquo Nature Biotechnology vol 18 no 9 pp 959ndash9632000
[4] M Di Trapani G Bassi M Ricciardi et al ldquoComparative studyof immune regulatory properties of stem cells derived fromdifferent tissuesrdquo StemCells andDevelopment vol 22 pp 2990ndash3002 2013
[5] AUccelli V Pistoia andLMoretta ldquoMesenchymal stemcells anew strategy for immunosuppressionrdquo Trends in Immunologyvol 28 no 5 pp 219ndash226 2007
[6] M Najar G Raicevic H Fayyad-Kazan et al ldquoImmune-relatedantigens surface molecules and regulatory factors in human-derived mesenchymal stromal cells the expression and impactof inflammatory primingrdquo Stem Cell Reviews vol 8 pp 1188ndash1198 2012
[7] R Rizzo D Campioni M Stignani et al ldquoA functional role forsoluble HLA-G antigens in immune modulation mediated bymesenchymal stromal cellsrdquoCytotherapy vol 10 no 4 pp 364ndash375 2008
10 Journal of Immunology Research
[8] F Morandi L Raffaghello G Bianchi et al ldquoImmunogenicityof human mesenchymal stem cells in HLA-class I-restricted T-cell responses against viral or tumor-associated antigensrdquo StemCells vol 26 no 5 pp 1275ndash1287 2008
[9] A Naji N Rouas-Freiss A Durrbach E D Carosella LSensebe and F Deschaseaux ldquoConcise review combininghuman leukocyte antigen G and mesenchymal stem cells forimmunosuppressant biotherapyrdquo Stem Cells vol 31 pp 2296ndash2303 2013
[10] G La Rocca and R Anzalone ldquoPerinatal stem cells revisiteddirections and indications at the crossroads between tissueregeneration and repairrdquoCurrent Stem Cell Research ampTherapyvol 8 pp 2ndash5 2013
[11] Z Selmani ANaji EGaiffe et al ldquoHLA-G is a crucial immuno-suppressive molecule secreted by adult human mesenchymalstem cellsrdquo Transplantation vol 87 no 9 supplement pp S62ndashS66 2009
[12] Z Selmani A Naji I Zidi et al ldquoHuman leukocyte antigen-G5 secretion by human mesenchymal stem cells is required tosuppress T lymphocyte andnatural killer function and to induceCD4+ CD25ℎ119894119892ℎFOXP3+ regulatory T cellsrdquo Stem Cells vol 26no 1 pp 212ndash222 2008
[13] F Deschaseaux J Gaillard A Langonne et al ldquoRegulationand function of immunosuppressivemolecule human leukocyteantigen G5 in human bone tissuerdquo The FASEB Journal vol 27pp 2977ndash2987 2013
[14] M Krampera ldquoMesenchymal stromal cell licensing a multistepprocessrdquo Leukemia vol 25 no 9 pp 1408ndash1414 2011
[15] P Moreau F Adrian-Cabestre C Menier et al ldquoIL-10 selec-tively induces HLA-G expression in human trophoblasts andmonocytesrdquo International Immunology vol 11 no 5 pp 803ndash811 1999
[16] O Brugiere GThabutM Pretolani et al ldquoImmunohistochem-ical study of HLA-G expression in lung transplant recipientsrdquoAmerican Journal of Transplantation vol 9 no 6 pp 1427ndash14382009
[17] S Lefebvre S Berrih-Aknin F Adrian et al ldquoA specificinterferon (IFN)-stimulated response element of the distalHLA-G promoter binds IFN-regulatory factor 1 and mediatesenhancement of this nonclassical class I gene by IFN-betardquoTheJournal of Biological Chemistry vol 276 no 9 pp 6133ndash61392001
[18] N Rouas-Freiss S Bruel C Menier C Marcou P Moreauand E D Carosella ldquoSwitch of HLA-G alternative splicing ina melanoma cell line causes loss of HLA-G1 expression andsensitivity to NK lysisrdquo International Journal of Cancer vol 117no 1 pp 114ndash122 2005
[19] S M Melief J J Zwaginga W E Fibbe and H RoelofsldquoAdipose tissue-derived multipotent stromal cells have a higherimmunomodulatory capacity than their bone marrow-derivedcounterpartsrdquo StemCells TranslationalMedicine vol 2 pp 455ndash463 2013
[20] J Y Tee V Vaghjiani Y H Liu P Murthi J Chan and UManuelpillai ldquoImmunogenicity and immunomodulatory prop-erties of hepatocyte-like cells derived from human amnioticepithelial cellsrdquo Current Stem Cell Research amp Therapy vol 8pp 91ndash99 2013
[21] G La Rocca M Lo Iacono T Corsello S Corrao F Farina andR Anzalone ldquoHuman Whartonrsquos jelly mesenchymal stem cellsmaintain the expression of key immunomodulatory moleculeswhen subjected to osteogenic adipogenic and chondrogenic
differentiation in vitro new perspectives for cellular therapyrdquoCurrent Stem Cell Research ampTherapy vol 8 pp 100ndash113 2013
[22] C Menard and K Tarte ldquoImmunoregulatory properties of clin-ical grade mesenchymal stromal cells evidence uncertaintiesand clinical applicationrdquo Stem Cell Research amp Therapy vol 4article 64 2013
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
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Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Journal of Immunology Research 3
100 101 102 103 104
FL 2
100 101 102 103 104
FL 2
Cou
nts
Cou
nts
R11 R12
IgG1HLA-DR
R13 R14
MSC-AT MSC-AT IFN120574TNF120572
(a)
310
232
155
77
0
100 101 102 103 104
FL 2
100 101 102 103 104
FL 2
100 101 102 103 104
FL 2
Cou
nts
Cou
nts
Cou
nts
R11 R12
IgG1HLA-G5
R13 R14
MSC-ATMSC-AT
MSC-AT HLA-G5MSC-AT IFN120574TNF120572
IFN120574TNF120572
(b)
0102030405060708090
100Controls
0102030405060708090
100
(CPM
times10
minus3)
(CPM
times10
minus3)
Adipose tissue-derived mesenchymal stem cells
LCLlowast
MSC
-AT
PBM
C59-
MSC-AT
lowastPB
MC
59
- LC
L
MSC
-AT
IFN120574
TN
F120572
Responder stimulator ratio1
05
1 025
1 012
1 006
1 003
MSC-AT IFN120574TNF120572
3H
-thym
idin
e inc
orpo
ratio
n
3H
-thym
idin
e inc
orpo
ratio
n
(c)
Figure 2 AT-derived MSCs express HLA-G and are hypoimmunogenic (a) Expression of HLA-DR molecules was evaluated by flowcytometry analysis on AT-derivedMSCs pretreated with IFN120574 and TNF120572 (MSC-AT IFN120574TNF120572) or not (MSC-AT) IgG1 was used as isotypecontrol Ab (b) Expression of HLA-G5 was evaluated by intracellular flow cytometry analysis on AT-derived MSCs pretreated with IFN120574 andTNF120572 (MSC-AT IFN120574TNF120572) or not (MSC-AT) (c) PBMC from healthy individual (59-) were used as responder cells towards AT-derivedMSCs pretreated with IFN120574 and TNF120572 (MSC-AT IFN120574TNF120572) or not (MSC-AT) as stimulating cells at various responder stimulator ratiosIrradiated LCLlowast were used as positive control of T cell alloproliferation Results are given asmean cpmplusmn sem one representative experimentis shown
Paris France) by density-gradient centrifugation over Ficoll-Paque PLUS (GE Healthcare) These cells were used as HLA-mismatched responding cells in MLR
22 In Vitro Osteodifferentiation MSCs from BM orAT were obtained from Reborne consortium center(httpwwwreborneorg) Before experiments MSCswere thawed and expanded through seeding 1000 cellscm2in T75 flasks When cultures reach 60ndash70 confluencecells were harvested and seeded for immunological assaysThe osteodifferentiated MSCs used as stimulating cells inMLR were obtained as previously described [13] BrieflyMSCs were cultured in osteoblastic differentiation mediumconsisting of 120572MEM FBS ascorbic acid NaH
2PO4 and
BMP-4 during 14 to 23 days
23 Preparation of MSC-Biomaterial Complex For Immuno-logical Tests 24-well (ultralow attachment) plates containingdisks of MBCP+ (macroporous biphasic calcium phosphate)granules with the same diameter of each well bottomwere prepared in collaboration with Biomatlante (France)
to achieve a complete adherence of seeded MSCs to thebiomaterial and then a complete induction of osteoblasticdifferentiation of MSCs The disks of MBCP+ were washedfor 48 hours before use to avoid any toxic effect on MSCsThen MSCs were seeded on MBCP discs during 5 daysin order to colonize homogenously the particles ThenMSC-biomaterial complex was tested in mixed lymphocytereactions
24 Mixed Lymphocyte Reaction (MLR) Using MSC asEither Stimulating Cells Or Third-Party Cells Facing HLA-Mismatched PBMC as Responder Cells PBMC from differentdonors were used as responder cells andMSCs or osteodiffer-entiated MSCs from BM or AT were used as either allogenicstimulating cells or third-party cells at various ratios TheHLA class II+ human B-lymphoblastoid cell line LCL 721221(ATCC) was used as positive control to stimulate HLA-mismatched PBMCWhen LCL cells were used as stimulatorcells a 75Gy dose irradiation was given and responderstimulator ratio was of 1 05 with a final concentrationof PBMC responder cells of 105 cellswell in a 96-well flatbottomed plate
4 Journal of Immunology Research
IgG1ALPtubulin
100 101 102 103 104
FL 2
100 101 102 103 104
FL 2
R11 R12
Cou
nts
Cou
nts
R13 R14
IgG1HLA-G5MSC-BM MSC-BM
(a)
IgG1ALPtubulin
100 101 102 103 104
FL 2
100 101 102 103 104
FL 2
R11 R12
Cou
nts
Cou
nts
R13 R14
IgG1HLA-G5Os-MSC-BM Os-MSC-BM IFN120574TNF120572
(b)
0102030405060708090
100 Controls
0102030405060708090
100
Os-MSC-BM
Osteodiferentiated bone marrow-derived MSC
(CPM
times10
minus3)
(CPM
times10
minus3)
LCLlowast
Os-
MSC
-BM
PBM
C00
Os-
MSC
-BM
IFN120574
TN
F120572 lowastPB
MC
00
LCL
Responder stimulator ratio1
05
1 025
1 012
1 006
1 003
Os-MSC-BM IFN120574TNF120572
3H
-thym
idin
e inc
orpo
ratio
n
3H
-thym
idin
e inc
orpo
ratio
n
(c)
Figure 3 BM-derived MSCs committed to osteodifferentiation process express HLA-G and are hypoimmunogenic (a and b) Expressionof ALP and HLA-G5 was evaluated by intracellular flow cytometry analysis on BM-derived MSCs committed to 14-day osteodifferentiationprocess and pretreated with IFN120574 and TNF120572 (Os-MSC-BM IFN120574TNF120572) or not (Os-MSC-BM) IgG1 was used as isotype control Ab Tubulinwas used as positive control of cell permeabilization (c) PBMC from healthy individual (00) were used as responder cells towards BM-derived MSCs committed to osteodifferentiation process and pretreated with IFN120574 and TNF120572 (Os-MSC-BM IFN120574TNF120572) or not (Os-MSC-BM) as stimulating cells at various responder stimulator ratios Irradiated LCLlowast were used as positive control of T cell alloproliferationResults are given as mean cpm plusmn sem one representative experiment is shown
Particularly in MBCP experiments MLR were per-formed in 24-well bottomed plates with a final concentrationof PBMC responder cells of 106 cellswell Experiments com-paring the effects of standard culture condition (no MBCP)and three-dimensional culture setting using MBCP discswere both performed in 24-well bottomed plates
Cultures were incubated at 37∘C in a humidified 5CO2air atmosphere PBMC proliferation was measured at
day 6 by [3H]-thymidine incorporation (1120583Ciwell PerkinElmer) during the last 18 hours of culture Cells were thenharvested on filtermats A and thymidine incorporation intoDNA was quantified using a beta counter (Wallac 1450Pharmacia) All samples were run in triplicate The influenceofMSC licensing with inflammatory cytokines such as IFN-120574at 10 ngmL (Peprotech) and plus TNF-120572 at 15 ngmL (RampDsystems) was analyzed by adding these cytokines in cultures48 hours before MLR [14]
25 Flow Cytometry The cytokine (IFN-120574 and TNF-120572) treat-ment efficiency was checked by cytofluorometry analysis
through the upregulation of HLA-DR expression on MSCBriefly cells were washed in PBS and stained with theanti-HLA-DR conjugated with PE (Beckman Coulter) inPBS 2 heat-inactivated fetal calf serum for 30 minutes at4∘C Control aliquots were stained with an isotype-matchedantibody to evaluate nonspecific binding to target cellsFluorescence was detected by using the Epics XL4 flowcytometer (Beckman Coulter Brea CA USA)
The expression of the HLA-G5 soluble isoform by MSCswas assessed by using the 2A12 mAb (Exbio) after cell per-meabilization The osteodifferentiation process was verifiedby cytofluorometry analysis through the induction of alkalinephosphatase (ALP) expression in osteodifferentiated MSCafter cell permeabilization Briefly cells were first permeabi-lized by using saponin (Sigma) and then stained with 2A12or anti-ALP (RampD systems) for 30 minutes at 4∘C Afterwashing cells were subsequently stained with an F(ab1015840)2goat anti-mouse IgG antibody conjugated with PE (BeckmanCoulter) for 30 minutes at 4∘C Control aliquots were stainedwith an isotype-matched antibody to evaluate nonspecific
Journal of Immunology Research 5
100 101 102 103 104
FL 2
100 101 102 103 104
FL 2
R12R11 R13 R14
Os-MSC-AT
IgG1HLA-DRC
ount
s
Cou
nts
Os-MSC-AT IFN120574TNF120572
(a)
100 101 102 103 104
FL 2
100 101 102 103 104
FL 2
R12R11 R13 R14
Os-MSC-AT
IgG1ALP IgG1ALPHLA-G5
Cou
nts
Cou
nts
Os-MSC-AT IFN120574TNF120572
(b)
0102030405060708090
Controls
0102030405060708090 Osteodifferentiated adipose tissue-derived MSC
Os-MSC-AT
(CPM
times10
minus3)
(CPM
times10
minus3)
LCLlowast
Os-
MSC
-BM
PBM
C00
Os-
MSC
-AT
IFN120574
TN
F120572 lowastPB
MC
00
LCL
Responder stimulator ratio1
05
1 025
1 012
1 006
1 003
Os-MSC-AT IFN120574TNF120572
3H
-thym
idin
e inc
orpo
ratio
n
3H
-thym
idin
e inc
orpo
ratio
n
(c)
Figure 4 AT-derived MSCs committed to osteodifferentiation process express HLA-G and are hypoimmunogenic (a) Expression of HLA-DRmolecules was evaluated by flow cytometry analysis onAT-derivedMSCs committed to 14-day osteodifferentiation process and pretreatedwith IFN120574 and TNF120572 (Os-MSC-AT IFN120574TNF120572) or not (Os-MSC-AT) (b) Expression of ALP and HLA-G5 was evaluated by intracellularflow cytometry analysis on AT-derived MSCs committed to 14-day osteodifferentiation process and pretreated with IFN120574 and TNF120572 (Os-MSC-AT IFN120574TNF120572) or not (Os-MSC-AT) IgG1 was used as isotype control Ab (c) PBMC from healthy individual (00) were used asresponder cells towards AT-derived MSCs committed to osteodifferentiation process and pretreated with IFN120574 and TNF120572 (Os-MSC-ATIFN120574TNF120572) or not (Os-MSC-AT) as stimulating cells at various responder stimulator ratios Irradiated LCLlowast were used as positive controlof T cell alloproliferation Results are given as mean cpm plusmn sem one representative experiment is shown
binding to target cells Fluorescencewas detected by using theEpics XL4 flow cytometer
26 Statistical Analysis Significance was assessed by usinga nonparametric Mann-Whitney test assuming a 119875 value lt005 as significant and was marked with lowast in the figures
3 Results and Discussion
Twomain questions were addressed in the present study rely-ing on whether immune regulatory properties of MSCs aremodified by (1) MSC differentiation towards the osteoblasticcell lineage or (2) the addition of synthetic biomaterial (ieMBCP+ granules) In this regard we analyzed both theimmunogenic and immunosuppressive properties of MSCsfrom BM or AT in allogenic conditions that is facing HLA-mismatched PBMC To identify low immunogenic MSCtypes we studied their ability to be recognized as allogenic
cells by HLA-mismatched PBMC in MLR using MSCs asstimulating cells and PBMC from various healthy donorsas responder cells To examine their immunosuppressiveproperties we studied their ability to modulate T cell allo-proliferation as third-party cells in a classical MLR All thefunctional experiments were performed by considering thedifferentiation status of MSC either immature or osteodiffer-entiated and seeded onto biomaterial or not
No PBMC alloproliferation was observed in response tovarious doses of allogenic MSCs derived from bone marrow(Figure 1(c)) or adipose tissue (Figure 2(c)) even after licens-ing with IFN-120574 and TNF-120572 (Figures 1(c) and 2(c)) Tables 1and 2 summarize the results obtained with PBMC from dis-tinct healthy donorsThe efficiency of cytokine treatment wasattested by the induction of HLA-DR expression on MSCs(Figures 1(a) and 2(a)) In order to evaluate the influenceof osteodifferentiation process on the immunogenicity ofMSCs similar functional assays were performed using BM-derived and AT-derived MSCs committed to preosteoblastic
6 Journal of Immunology Research
0
10
20
30
40
50
Bone marrow-derived mesenchymal stem cells(C
PMtimes10
minus3)
LCLlowast
MSC
-BM
0
10
20
30
40
50
Adipose tissue-derived mesenchymal stem cells
(CPM
times10
minus3)
LCLlowast
MSC
-AT
0
10
20
30
40
50Bone marrow-derived mesenchymal stem cells
(CPM
times10
minus3)
+
0
10
20
30
40
50Adipose tissue-derived mesenchymal stem cells
(CPM
times10
minus3)
+
No MBCP
MBCP
PBM
C713
lowastPB
MC
713
LCL
PBM
C713
MSC
-BM
PBM
C713
MSC
-BM
PBM
C713
lowastPB
MC
713
LCL
PBM
C713
MSC
-AT
PBM
C713
MSC
-AT
LCLlowast
MSC
-BM
PBM
C713
lowastPB
MC
713
LCL
PBM
C713
MSC
-BM
PBM
C713
MSC
-BM
LCLlowast
MSC
-AT
PBM
C713
lowastPB
MC
713
LCL
PBM
C713
MSC
-AT
PBM
C713
MSC
-AT
IFN120574TNF120572 IFN120574TNF120572
3H
-thym
idin
e inc
orpo
ratio
n
3H
-thym
idin
e inc
orpo
ratio
n
3H
-thym
idin
e inc
orpo
ratio
n
3H
-thym
idin
e inc
orpo
ratio
n
Figure 5 Both BM- andAT-derived MSCs when combined to MBCP biomaterial keep their hypoimmunogenicity PBMC from healthyindividual (713) were used as responder cells towards either BM-derived MSCs (MSC-BM) or AT-derived MSCs (MSC-AT) that werepretreated with IFN120574 and TNF120572 (IFN120574TNF120572) or not and used as stimulating cells at the responder stimulator ratio of 1 02 MSCs werecombined toMBCP biomaterial (MBCP) or not (NoMBCP) Irradiated LCLlowast were used as positive control of T cell alloproliferation Resultsare given as mean cpm plusmn sem one representative experiment is shown
MSCs as stimulating cells The osteodifferentiation processwas validated through the upregulation of ALP expression inosteodifferentiated MSCs (Figures 3(a) 3(b) 4(a) and 4(b))Results show that both BM-derived and AT-derived MSCscommitted to osteodifferentiation are still hypoimmunogenicwhether they are pretreated or not with IFN-120574 and TNF-120572(Figures 3(c) and 4(c) and Tables 1 and 2) Then we lookedat whether combination of biomaterial (ie MBCP) withMSCs alters their immunogenicityNodifferenceswere foundbetween standard 2D-coculture conditions (MSC + PBMC)and 3D-coculture conditions (MSC + MBCP + PBMC) Onerepresentative allogenic combination is shown (Figure 5) forwhich the mean percentage of T cell alloproliferation ispresented in Table 3 (119899 = 3 healthy donors) It is of notethat the addition of MBCP to BM-derived MSCs treated or
not with cytokines modifies slightly their immunogenicityalthough no statistical difference was observed betweenboth conditions (119875 gt 01) (Table 3) Consequently wecan conclude that both the osteodifferentiation process andthe presence of biomaterial (MBCP) did not abrogate thehypoimmunogenicity of MSCs
Notably expression of the tolerogenic soluble HLA-G5protein was observed in MSCs derived from BM and ATand could be enhanced following treatment with IFN-120574 andTNF-120572 (Figures 1(b) and 2(b)) Such enhanced expressionof HLA-G by IFN-120574 treatment was previously reported forother cell types such as monocytes [15] bronchial epithelialcells [16] thymic epithelial cells [17] and various tumor cells[18] Nevertheless HLA-G expression levels varied amongthe various batches of MSCs tested (data not shown) In
Journal of Immunology Research 7
MSC-BM 954 885 667 339 165888 812 647 310 153
0
50
100
Allo
prol
ifera
tion
inhi
bitio
n (
)
Bone marrow-derived mesenchymal stem cells
1 05 05 1 05 025 1 05 0125 1 05 006 1 05 003
Responder stimulator MSC ratioMSC-BM IFN120574TNF120572
(a)
MSC-AT 878 824 879 859 823883 831 741 662 572
0
50
100
Allo
prol
ifera
tion
inhi
bitio
n (
)Adipose tissue-derived mesenchymal stem cells
lowast lowast lowast
MSC-AT IFN120574TNF120572Responder stimulator MSC ratio
1 05 05 1 05 025 1 05 0125 1 05 006 1 05 003
(b)
Figure 6 Both BM- andAT-derived MSCs display immunosuppressive properties in a dose-dependent manner PBMC from healthyindividuals were used as responder cells towards irradiated LCLlowast used as stimulating cells in presence of either (a) BM-derivedMSCs (MSC-BM) or (b) AT-derived MSCs (MSC-AT) that were pretreated with IFN120574 and TNF120572 (IFN120574TNF120572) or not and used as third-party cells atvarious responder stimulator MSC ratios Results are given as mean percentage of alloproliferation inhibition plusmn sem according to themaximal alloproliferation observed with PBMC+LCLlowast using PBMC from 4 and 6 distinct healthy donors in BM- and AT-derived MSCsexperiments respectively
Table 1 MSC from BM after osteodifferentiation and licensing byIFN-120574 and TNF-120572 do not induce PBMC proliferation in allogenicconditions
R Saratio MSC-BM MSC-BM
IFN-120574TNF-120572 Os-MSC-BM Os-MSC-BMIFN-120574TNF-120572
1 05 43 plusmn 21b 11 plusmn 03 33 plusmn 03 17 plusmn 02
1 025 45 plusmn 14 10 plusmn 03 31 plusmn 01 18 plusmn 05
1 012 53 plusmn 19 11 plusmn 04 32 plusmn 10 23 plusmn 12
1 006 54 plusmn 24 14 plusmn 05 26 plusmn 11 39 plusmn 31
1 003 43 plusmn 17 14 plusmn 05 22 plusmn 02 21 plusmn 12
aPBMC from healthy individuals were used as responder cells towards BM-derived MSCs pretreated with IFN-120574 and TNF-120572 (MSC-BM IFN-120574TNF-120572) or not (MSC-BM) as stimulating cells at various responder stimulator(R S) ratios Similar experiments were performed with BM-derived MSCscommitted to osteodifferentiation process and pretreated with IFN-120574 andTNF-120572 (Os-MSC-BM IFN-120574TNF-120572) or not (Os-MSC-BM) as stimulatingcellsbData are mean plusmn sem of alloproliferation percentage obtained with 5and 3 distinct healthy donors for MSC-BM and Os-MSC-BM experimentsrespectively This percentage is calculated considering PBMC stimulatedwith LCLlowast as 100 alloproliferation
agreement with recent findings we observed enhancedHLA-Gexpression byMSCs following osteodifferentiation (Figures3(a) 3(b) and 4(b)) [13]
Table 2 MSC from AT after osteodifferentiation and licensing byIFN-120574 and TNF-120572 do not induce PBMC proliferation in allogenicconditions
R Saratio MSC-AT MSC-AT
IFN-120574TNF-120572 Os-MSC-AT Os-MSC-ATIFN-120574TNF-120572
1 05 56 plusmn 10b 72 plusmn 31 11 plusmn 02 09 plusmn 06
1 025 55 plusmn 15 52 plusmn 26 13 plusmn 03 08 plusmn 05
1 012 53 plusmn 15 47 plusmn 29 10 plusmn 03 11 plusmn 08
1 006 53 plusmn 20 55 plusmn 30 10 plusmn 02 13 plusmn 10
1 003 46 plusmn 11 39 plusmn 20 14 plusmn 03 18 plusmn 13
aPBMC from healthy individuals were used as responder cells towards AT-derived MSCs pretreated with IFN-120574 and TNF-120572 (MSC-AT IFN-120574TNF-120572) or not (MSC-AT) as stimulating cells at various responder stimulator(R S) ratios Similar experiments were performed with AT-derived MSCscommitted to osteodifferentiation process and pretreated with IFN-120574 andTNF-120572 (Os-MSC-AT IFN-120574TNF-120572) or not (Os-MSC-AT) as stimulatingcellsbData are mean plusmn sem of alloproliferation percentage obtained with 4and 3 distinct healthy donors for MSC-AT and Os-MSC-AT experimentsrespectively This percentage is calculated considering PBMC stimulatedwith LCLlowast as 100 alloproliferation
Then we investigated the immunomodulatory propertiesof MSCs as third-party cells in MLR Results showed thatbothBM-derived andAT-derivedMSCsdisplay immunosup-pressive properties in a dose-responsemanner (Figure 6) It is
8 Journal of Immunology Research
MSC-BM 940 911 864938 896 929
Allo
prol
ifera
tion
inhi
bitio
n (
)A
llopr
olife
ratio
n in
hibi
tion
()
Allo
prol
ifera
tion
inhi
bitio
n (
)A
llopr
olife
ratio
n in
hibi
tion
()
461 628 750379 676 796
0
50
100
850 803 893600 725 895
0
50
100
0
50
100
919 879 879909 867 887
0
50
100
Bone marrow-derived mesenchymal stem cells
Adipose tissue-derived mesenchymal stem cells
No MBCP
Bone marrow-derived mesenchymal stem cells
Adipose tissue-derived mesenchymal stem cells
MBCP
MSC-BM IFN120574TNF120572
MSC-BMMSC-BM IFN120574TNF120572
MSC-ATMSC-AT IFN120574TNF120572
MSC-ATMSC-AT IFN120574TNF120572
1 05 04 1 05 02 1 05 01 1 05 04 1 05 02 1 05 01
1 05 04 1 05 02 1 05 011 05 04 1 05 02 1 05 01
Responder stimulator MSC ratio Responder stimulator MSC ratio
Responder stimulator MSC ratio Responder stimulator MSC ratio
Figure 7 Both BM- andAT-derived MSCs when combined to MBCP biomaterial keep their immunosuppressive properties PBMC fromhealthy individuals were used as responder cells towards irradiated LCLlowast used as stimulating cells in presence of either BM-derived MSCs(MSC-BM) or AT-derived MSCs (MSC-AT) that were pretreated with IFN120574 and TNF120572 (IFN120574TNF120572) or not and used as third-party cellsat various responder stimulator MSC ratios MSCs were combined to MBCP biomaterial (MBCP) or not (No MBCP) Results are given asmean percentage of alloproliferation inhibition plusmn sem when compared to PBMC+LCLlowast using PBMC from 3 distinct healthy donors
Table 3 Immunogenicity of MSC from bone marrow and adiposetissue combinedwithMBCPbiomaterial and licensing by IFN-120574 andTNF-120572
MSC-BM MSC-BMIFN-120574TNF-120572 MSC-AT MSC-AT
IFN-120574TNF-120572No MBCPa 75 plusmn 22b 98 plusmn 40 139 plusmn 39 230 plusmn 52
MBCP 519 plusmn 141 411 plusmn 116 81 plusmn 08 110 plusmn 08
aPBMC from healthy individuals were used as responder cells towards BM-or AT-derivedMSCs pretreated with IFN-120574 and TNF-120572 or not as stimulatingcells at 1 02 responder stimulator ratiobData are mean plusmn sem of alloproliferation percentage obtained with 3distinct healthy donors This percentage is calculated considering PBMCstimulated with LCLlowast as 100 alloproliferation
of note that (i) AT-derivedMSCs aremore potent at low dosescompared to BM-derived MSCs (119875 lt 005) (Figures 6(a) and6(b)) and (ii) licensing with cytokines reduces significantlythe immunosuppressive properties of AT-derived MSCs(119875 lt 005) (Figure 6(b)) When combined to biomaterial(MBCP) both BM-derived and AT-derived MSCs still exertimmunosuppressive properties as they greatly inhibit T cell
alloproliferation with or without being seeded with MBCP(Figure 7) Although not statistically significant addition ofMBCP reduces the immunomodulatory properties of BM-derived MSCs at high responder stimulator MSC ratiosThis could be due to steric hindrance when high numbersof cells are used Indeed such MBCP effect is no longerobserved at low ratios (Figure 7) Also both BM-derivedand AT-derived MSCs when committed to preosteoblasticMSCs inhibit T cell alloproliferation and remain thus able toinduce a tolerogenicmicroenvironment (Figure 8) Licensingwith IFN-120574 and TNF-120572 did not modify such MSC-derivedimmunosuppression (Figures 7 and 8) It is of note thatonce osteodifferentiated AT-derived MSCs are more potentat low doses compared to BM-derived MSCs as they displayhigher immunosuppressive effects (119875 lt 005) (Figure 8)Such higher immunomodulatory capacity of adipose tissue-derived multipotent stromal cells compared to their bonemarrow-derived counterparts has been previously reported[19]
Our present results are in agreement with previousreports showing that differentiation of stem cells does not
Journal of Immunology Research 9
894 889 942 649 528956 925 976 678 367
0
50
100
Allo
prol
ifera
tion
inhi
bitio
n (
)
Osteodifferentiated bone marrow-derived MSC
MSC-BMOs-MSC-BM IFN120574TNF120572
1 05 05 1 05 025 1 05 0125 1 05 006 1 05 003
Responder stimulator MSC ratio
(a)
888 926 917 895 854936 935 913 859 780
0
50
100
Allo
prol
ifera
tion
inhi
bitio
n (
)Osteodifferentiated adipose tissue-derived MSC
Responder stimulator MSC ratio
MSC-ATMSC-AT IFN120574TNF120572
1 05 05 1 05 025 1 05 0125 1 05 006 1 05 003
(b)
Figure 8 Both BM- andAT-derived MSCs when committed to osteodifferentiation process keep their immunosuppressive propertiesPBMC from healthy individuals were used as responder cells towards irradiated LCLlowast used as stimulating cells in presence of eitherosteodifferentiated BM-derived MSCs (Os-MSC-BM) or AT-derived MSCs (Os-MSC-AT) that were pretreated with IFN120574 and TNF120572(IFN120574TNF120572) or not and used as third-party cells at various responder stimulator MSC ratios Results are given as mean percentage ofalloproliferation inhibition plusmn sem when compared to PBMC+LCLlowast using PBMC from 3 and 5 distinct healthy donors in BM- and AT-derived MSCs experiments respectively
alter their low immunogenicity and immunomodulatoryproperties For instance human amniotic epithelial cellswhich have stem cell-like properties retain their immuno-suppressive functions after differentiation into hepatocyte-like cells [20] Also human Whartonrsquos jelly-derived MSCsmaintain the expression of immunomodulatory moleculessuch as HLA-G when subjected to osteogenic differentiationin vitro [21]
In conclusion MSCs from BM or AT display tolerogenicproperties which are maintained following osteodifferenti-ation process or addition of biomaterial and may thus beconsidered as allogenic sources for regenerating bone defectsin orthopaedic and maxillofacial surgery [22]
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This project has received funding from the European UnionrsquosSeventh Programme for research technological develop-ment and demonstration underGrant agreement no 241879
References
[1] R Langer and J P Vacanti ldquoTissue engineeringrdquo Science vol260 no 5110 pp 920ndash926 1993
[2] C Laurencin Y Khan and S F El-Amin ldquoBone graft substi-tutesrdquo Expert Review of Medical Devices vol 3 no 1 pp 49ndash572006
[3] H Petite V ViateauW Bensaıd et al ldquoTissue-engineered boneregenerationrdquo Nature Biotechnology vol 18 no 9 pp 959ndash9632000
[4] M Di Trapani G Bassi M Ricciardi et al ldquoComparative studyof immune regulatory properties of stem cells derived fromdifferent tissuesrdquo StemCells andDevelopment vol 22 pp 2990ndash3002 2013
[5] AUccelli V Pistoia andLMoretta ldquoMesenchymal stemcells anew strategy for immunosuppressionrdquo Trends in Immunologyvol 28 no 5 pp 219ndash226 2007
[6] M Najar G Raicevic H Fayyad-Kazan et al ldquoImmune-relatedantigens surface molecules and regulatory factors in human-derived mesenchymal stromal cells the expression and impactof inflammatory primingrdquo Stem Cell Reviews vol 8 pp 1188ndash1198 2012
[7] R Rizzo D Campioni M Stignani et al ldquoA functional role forsoluble HLA-G antigens in immune modulation mediated bymesenchymal stromal cellsrdquoCytotherapy vol 10 no 4 pp 364ndash375 2008
10 Journal of Immunology Research
[8] F Morandi L Raffaghello G Bianchi et al ldquoImmunogenicityof human mesenchymal stem cells in HLA-class I-restricted T-cell responses against viral or tumor-associated antigensrdquo StemCells vol 26 no 5 pp 1275ndash1287 2008
[9] A Naji N Rouas-Freiss A Durrbach E D Carosella LSensebe and F Deschaseaux ldquoConcise review combininghuman leukocyte antigen G and mesenchymal stem cells forimmunosuppressant biotherapyrdquo Stem Cells vol 31 pp 2296ndash2303 2013
[10] G La Rocca and R Anzalone ldquoPerinatal stem cells revisiteddirections and indications at the crossroads between tissueregeneration and repairrdquoCurrent Stem Cell Research ampTherapyvol 8 pp 2ndash5 2013
[11] Z Selmani ANaji EGaiffe et al ldquoHLA-G is a crucial immuno-suppressive molecule secreted by adult human mesenchymalstem cellsrdquo Transplantation vol 87 no 9 supplement pp S62ndashS66 2009
[12] Z Selmani A Naji I Zidi et al ldquoHuman leukocyte antigen-G5 secretion by human mesenchymal stem cells is required tosuppress T lymphocyte andnatural killer function and to induceCD4+ CD25ℎ119894119892ℎFOXP3+ regulatory T cellsrdquo Stem Cells vol 26no 1 pp 212ndash222 2008
[13] F Deschaseaux J Gaillard A Langonne et al ldquoRegulationand function of immunosuppressivemolecule human leukocyteantigen G5 in human bone tissuerdquo The FASEB Journal vol 27pp 2977ndash2987 2013
[14] M Krampera ldquoMesenchymal stromal cell licensing a multistepprocessrdquo Leukemia vol 25 no 9 pp 1408ndash1414 2011
[15] P Moreau F Adrian-Cabestre C Menier et al ldquoIL-10 selec-tively induces HLA-G expression in human trophoblasts andmonocytesrdquo International Immunology vol 11 no 5 pp 803ndash811 1999
[16] O Brugiere GThabutM Pretolani et al ldquoImmunohistochem-ical study of HLA-G expression in lung transplant recipientsrdquoAmerican Journal of Transplantation vol 9 no 6 pp 1427ndash14382009
[17] S Lefebvre S Berrih-Aknin F Adrian et al ldquoA specificinterferon (IFN)-stimulated response element of the distalHLA-G promoter binds IFN-regulatory factor 1 and mediatesenhancement of this nonclassical class I gene by IFN-betardquoTheJournal of Biological Chemistry vol 276 no 9 pp 6133ndash61392001
[18] N Rouas-Freiss S Bruel C Menier C Marcou P Moreauand E D Carosella ldquoSwitch of HLA-G alternative splicing ina melanoma cell line causes loss of HLA-G1 expression andsensitivity to NK lysisrdquo International Journal of Cancer vol 117no 1 pp 114ndash122 2005
[19] S M Melief J J Zwaginga W E Fibbe and H RoelofsldquoAdipose tissue-derived multipotent stromal cells have a higherimmunomodulatory capacity than their bone marrow-derivedcounterpartsrdquo StemCells TranslationalMedicine vol 2 pp 455ndash463 2013
[20] J Y Tee V Vaghjiani Y H Liu P Murthi J Chan and UManuelpillai ldquoImmunogenicity and immunomodulatory prop-erties of hepatocyte-like cells derived from human amnioticepithelial cellsrdquo Current Stem Cell Research amp Therapy vol 8pp 91ndash99 2013
[21] G La Rocca M Lo Iacono T Corsello S Corrao F Farina andR Anzalone ldquoHuman Whartonrsquos jelly mesenchymal stem cellsmaintain the expression of key immunomodulatory moleculeswhen subjected to osteogenic adipogenic and chondrogenic
differentiation in vitro new perspectives for cellular therapyrdquoCurrent Stem Cell Research ampTherapy vol 8 pp 100ndash113 2013
[22] C Menard and K Tarte ldquoImmunoregulatory properties of clin-ical grade mesenchymal stromal cells evidence uncertaintiesand clinical applicationrdquo Stem Cell Research amp Therapy vol 4article 64 2013
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Disease Markers
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OncologyJournal of
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Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
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Research and TreatmentAIDS
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Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
4 Journal of Immunology Research
IgG1ALPtubulin
100 101 102 103 104
FL 2
100 101 102 103 104
FL 2
R11 R12
Cou
nts
Cou
nts
R13 R14
IgG1HLA-G5MSC-BM MSC-BM
(a)
IgG1ALPtubulin
100 101 102 103 104
FL 2
100 101 102 103 104
FL 2
R11 R12
Cou
nts
Cou
nts
R13 R14
IgG1HLA-G5Os-MSC-BM Os-MSC-BM IFN120574TNF120572
(b)
0102030405060708090
100 Controls
0102030405060708090
100
Os-MSC-BM
Osteodiferentiated bone marrow-derived MSC
(CPM
times10
minus3)
(CPM
times10
minus3)
LCLlowast
Os-
MSC
-BM
PBM
C00
Os-
MSC
-BM
IFN120574
TN
F120572 lowastPB
MC
00
LCL
Responder stimulator ratio1
05
1 025
1 012
1 006
1 003
Os-MSC-BM IFN120574TNF120572
3H
-thym
idin
e inc
orpo
ratio
n
3H
-thym
idin
e inc
orpo
ratio
n
(c)
Figure 3 BM-derived MSCs committed to osteodifferentiation process express HLA-G and are hypoimmunogenic (a and b) Expressionof ALP and HLA-G5 was evaluated by intracellular flow cytometry analysis on BM-derived MSCs committed to 14-day osteodifferentiationprocess and pretreated with IFN120574 and TNF120572 (Os-MSC-BM IFN120574TNF120572) or not (Os-MSC-BM) IgG1 was used as isotype control Ab Tubulinwas used as positive control of cell permeabilization (c) PBMC from healthy individual (00) were used as responder cells towards BM-derived MSCs committed to osteodifferentiation process and pretreated with IFN120574 and TNF120572 (Os-MSC-BM IFN120574TNF120572) or not (Os-MSC-BM) as stimulating cells at various responder stimulator ratios Irradiated LCLlowast were used as positive control of T cell alloproliferationResults are given as mean cpm plusmn sem one representative experiment is shown
Particularly in MBCP experiments MLR were per-formed in 24-well bottomed plates with a final concentrationof PBMC responder cells of 106 cellswell Experiments com-paring the effects of standard culture condition (no MBCP)and three-dimensional culture setting using MBCP discswere both performed in 24-well bottomed plates
Cultures were incubated at 37∘C in a humidified 5CO2air atmosphere PBMC proliferation was measured at
day 6 by [3H]-thymidine incorporation (1120583Ciwell PerkinElmer) during the last 18 hours of culture Cells were thenharvested on filtermats A and thymidine incorporation intoDNA was quantified using a beta counter (Wallac 1450Pharmacia) All samples were run in triplicate The influenceofMSC licensing with inflammatory cytokines such as IFN-120574at 10 ngmL (Peprotech) and plus TNF-120572 at 15 ngmL (RampDsystems) was analyzed by adding these cytokines in cultures48 hours before MLR [14]
25 Flow Cytometry The cytokine (IFN-120574 and TNF-120572) treat-ment efficiency was checked by cytofluorometry analysis
through the upregulation of HLA-DR expression on MSCBriefly cells were washed in PBS and stained with theanti-HLA-DR conjugated with PE (Beckman Coulter) inPBS 2 heat-inactivated fetal calf serum for 30 minutes at4∘C Control aliquots were stained with an isotype-matchedantibody to evaluate nonspecific binding to target cellsFluorescence was detected by using the Epics XL4 flowcytometer (Beckman Coulter Brea CA USA)
The expression of the HLA-G5 soluble isoform by MSCswas assessed by using the 2A12 mAb (Exbio) after cell per-meabilization The osteodifferentiation process was verifiedby cytofluorometry analysis through the induction of alkalinephosphatase (ALP) expression in osteodifferentiated MSCafter cell permeabilization Briefly cells were first permeabi-lized by using saponin (Sigma) and then stained with 2A12or anti-ALP (RampD systems) for 30 minutes at 4∘C Afterwashing cells were subsequently stained with an F(ab1015840)2goat anti-mouse IgG antibody conjugated with PE (BeckmanCoulter) for 30 minutes at 4∘C Control aliquots were stainedwith an isotype-matched antibody to evaluate nonspecific
Journal of Immunology Research 5
100 101 102 103 104
FL 2
100 101 102 103 104
FL 2
R12R11 R13 R14
Os-MSC-AT
IgG1HLA-DRC
ount
s
Cou
nts
Os-MSC-AT IFN120574TNF120572
(a)
100 101 102 103 104
FL 2
100 101 102 103 104
FL 2
R12R11 R13 R14
Os-MSC-AT
IgG1ALP IgG1ALPHLA-G5
Cou
nts
Cou
nts
Os-MSC-AT IFN120574TNF120572
(b)
0102030405060708090
Controls
0102030405060708090 Osteodifferentiated adipose tissue-derived MSC
Os-MSC-AT
(CPM
times10
minus3)
(CPM
times10
minus3)
LCLlowast
Os-
MSC
-BM
PBM
C00
Os-
MSC
-AT
IFN120574
TN
F120572 lowastPB
MC
00
LCL
Responder stimulator ratio1
05
1 025
1 012
1 006
1 003
Os-MSC-AT IFN120574TNF120572
3H
-thym
idin
e inc
orpo
ratio
n
3H
-thym
idin
e inc
orpo
ratio
n
(c)
Figure 4 AT-derived MSCs committed to osteodifferentiation process express HLA-G and are hypoimmunogenic (a) Expression of HLA-DRmolecules was evaluated by flow cytometry analysis onAT-derivedMSCs committed to 14-day osteodifferentiation process and pretreatedwith IFN120574 and TNF120572 (Os-MSC-AT IFN120574TNF120572) or not (Os-MSC-AT) (b) Expression of ALP and HLA-G5 was evaluated by intracellularflow cytometry analysis on AT-derived MSCs committed to 14-day osteodifferentiation process and pretreated with IFN120574 and TNF120572 (Os-MSC-AT IFN120574TNF120572) or not (Os-MSC-AT) IgG1 was used as isotype control Ab (c) PBMC from healthy individual (00) were used asresponder cells towards AT-derived MSCs committed to osteodifferentiation process and pretreated with IFN120574 and TNF120572 (Os-MSC-ATIFN120574TNF120572) or not (Os-MSC-AT) as stimulating cells at various responder stimulator ratios Irradiated LCLlowast were used as positive controlof T cell alloproliferation Results are given as mean cpm plusmn sem one representative experiment is shown
binding to target cells Fluorescencewas detected by using theEpics XL4 flow cytometer
26 Statistical Analysis Significance was assessed by usinga nonparametric Mann-Whitney test assuming a 119875 value lt005 as significant and was marked with lowast in the figures
3 Results and Discussion
Twomain questions were addressed in the present study rely-ing on whether immune regulatory properties of MSCs aremodified by (1) MSC differentiation towards the osteoblasticcell lineage or (2) the addition of synthetic biomaterial (ieMBCP+ granules) In this regard we analyzed both theimmunogenic and immunosuppressive properties of MSCsfrom BM or AT in allogenic conditions that is facing HLA-mismatched PBMC To identify low immunogenic MSCtypes we studied their ability to be recognized as allogenic
cells by HLA-mismatched PBMC in MLR using MSCs asstimulating cells and PBMC from various healthy donorsas responder cells To examine their immunosuppressiveproperties we studied their ability to modulate T cell allo-proliferation as third-party cells in a classical MLR All thefunctional experiments were performed by considering thedifferentiation status of MSC either immature or osteodiffer-entiated and seeded onto biomaterial or not
No PBMC alloproliferation was observed in response tovarious doses of allogenic MSCs derived from bone marrow(Figure 1(c)) or adipose tissue (Figure 2(c)) even after licens-ing with IFN-120574 and TNF-120572 (Figures 1(c) and 2(c)) Tables 1and 2 summarize the results obtained with PBMC from dis-tinct healthy donorsThe efficiency of cytokine treatment wasattested by the induction of HLA-DR expression on MSCs(Figures 1(a) and 2(a)) In order to evaluate the influenceof osteodifferentiation process on the immunogenicity ofMSCs similar functional assays were performed using BM-derived and AT-derived MSCs committed to preosteoblastic
6 Journal of Immunology Research
0
10
20
30
40
50
Bone marrow-derived mesenchymal stem cells(C
PMtimes10
minus3)
LCLlowast
MSC
-BM
0
10
20
30
40
50
Adipose tissue-derived mesenchymal stem cells
(CPM
times10
minus3)
LCLlowast
MSC
-AT
0
10
20
30
40
50Bone marrow-derived mesenchymal stem cells
(CPM
times10
minus3)
+
0
10
20
30
40
50Adipose tissue-derived mesenchymal stem cells
(CPM
times10
minus3)
+
No MBCP
MBCP
PBM
C713
lowastPB
MC
713
LCL
PBM
C713
MSC
-BM
PBM
C713
MSC
-BM
PBM
C713
lowastPB
MC
713
LCL
PBM
C713
MSC
-AT
PBM
C713
MSC
-AT
LCLlowast
MSC
-BM
PBM
C713
lowastPB
MC
713
LCL
PBM
C713
MSC
-BM
PBM
C713
MSC
-BM
LCLlowast
MSC
-AT
PBM
C713
lowastPB
MC
713
LCL
PBM
C713
MSC
-AT
PBM
C713
MSC
-AT
IFN120574TNF120572 IFN120574TNF120572
3H
-thym
idin
e inc
orpo
ratio
n
3H
-thym
idin
e inc
orpo
ratio
n
3H
-thym
idin
e inc
orpo
ratio
n
3H
-thym
idin
e inc
orpo
ratio
n
Figure 5 Both BM- andAT-derived MSCs when combined to MBCP biomaterial keep their hypoimmunogenicity PBMC from healthyindividual (713) were used as responder cells towards either BM-derived MSCs (MSC-BM) or AT-derived MSCs (MSC-AT) that werepretreated with IFN120574 and TNF120572 (IFN120574TNF120572) or not and used as stimulating cells at the responder stimulator ratio of 1 02 MSCs werecombined toMBCP biomaterial (MBCP) or not (NoMBCP) Irradiated LCLlowast were used as positive control of T cell alloproliferation Resultsare given as mean cpm plusmn sem one representative experiment is shown
MSCs as stimulating cells The osteodifferentiation processwas validated through the upregulation of ALP expression inosteodifferentiated MSCs (Figures 3(a) 3(b) 4(a) and 4(b))Results show that both BM-derived and AT-derived MSCscommitted to osteodifferentiation are still hypoimmunogenicwhether they are pretreated or not with IFN-120574 and TNF-120572(Figures 3(c) and 4(c) and Tables 1 and 2) Then we lookedat whether combination of biomaterial (ie MBCP) withMSCs alters their immunogenicityNodifferenceswere foundbetween standard 2D-coculture conditions (MSC + PBMC)and 3D-coculture conditions (MSC + MBCP + PBMC) Onerepresentative allogenic combination is shown (Figure 5) forwhich the mean percentage of T cell alloproliferation ispresented in Table 3 (119899 = 3 healthy donors) It is of notethat the addition of MBCP to BM-derived MSCs treated or
not with cytokines modifies slightly their immunogenicityalthough no statistical difference was observed betweenboth conditions (119875 gt 01) (Table 3) Consequently wecan conclude that both the osteodifferentiation process andthe presence of biomaterial (MBCP) did not abrogate thehypoimmunogenicity of MSCs
Notably expression of the tolerogenic soluble HLA-G5protein was observed in MSCs derived from BM and ATand could be enhanced following treatment with IFN-120574 andTNF-120572 (Figures 1(b) and 2(b)) Such enhanced expressionof HLA-G by IFN-120574 treatment was previously reported forother cell types such as monocytes [15] bronchial epithelialcells [16] thymic epithelial cells [17] and various tumor cells[18] Nevertheless HLA-G expression levels varied amongthe various batches of MSCs tested (data not shown) In
Journal of Immunology Research 7
MSC-BM 954 885 667 339 165888 812 647 310 153
0
50
100
Allo
prol
ifera
tion
inhi
bitio
n (
)
Bone marrow-derived mesenchymal stem cells
1 05 05 1 05 025 1 05 0125 1 05 006 1 05 003
Responder stimulator MSC ratioMSC-BM IFN120574TNF120572
(a)
MSC-AT 878 824 879 859 823883 831 741 662 572
0
50
100
Allo
prol
ifera
tion
inhi
bitio
n (
)Adipose tissue-derived mesenchymal stem cells
lowast lowast lowast
MSC-AT IFN120574TNF120572Responder stimulator MSC ratio
1 05 05 1 05 025 1 05 0125 1 05 006 1 05 003
(b)
Figure 6 Both BM- andAT-derived MSCs display immunosuppressive properties in a dose-dependent manner PBMC from healthyindividuals were used as responder cells towards irradiated LCLlowast used as stimulating cells in presence of either (a) BM-derivedMSCs (MSC-BM) or (b) AT-derived MSCs (MSC-AT) that were pretreated with IFN120574 and TNF120572 (IFN120574TNF120572) or not and used as third-party cells atvarious responder stimulator MSC ratios Results are given as mean percentage of alloproliferation inhibition plusmn sem according to themaximal alloproliferation observed with PBMC+LCLlowast using PBMC from 4 and 6 distinct healthy donors in BM- and AT-derived MSCsexperiments respectively
Table 1 MSC from BM after osteodifferentiation and licensing byIFN-120574 and TNF-120572 do not induce PBMC proliferation in allogenicconditions
R Saratio MSC-BM MSC-BM
IFN-120574TNF-120572 Os-MSC-BM Os-MSC-BMIFN-120574TNF-120572
1 05 43 plusmn 21b 11 plusmn 03 33 plusmn 03 17 plusmn 02
1 025 45 plusmn 14 10 plusmn 03 31 plusmn 01 18 plusmn 05
1 012 53 plusmn 19 11 plusmn 04 32 plusmn 10 23 plusmn 12
1 006 54 plusmn 24 14 plusmn 05 26 plusmn 11 39 plusmn 31
1 003 43 plusmn 17 14 plusmn 05 22 plusmn 02 21 plusmn 12
aPBMC from healthy individuals were used as responder cells towards BM-derived MSCs pretreated with IFN-120574 and TNF-120572 (MSC-BM IFN-120574TNF-120572) or not (MSC-BM) as stimulating cells at various responder stimulator(R S) ratios Similar experiments were performed with BM-derived MSCscommitted to osteodifferentiation process and pretreated with IFN-120574 andTNF-120572 (Os-MSC-BM IFN-120574TNF-120572) or not (Os-MSC-BM) as stimulatingcellsbData are mean plusmn sem of alloproliferation percentage obtained with 5and 3 distinct healthy donors for MSC-BM and Os-MSC-BM experimentsrespectively This percentage is calculated considering PBMC stimulatedwith LCLlowast as 100 alloproliferation
agreement with recent findings we observed enhancedHLA-Gexpression byMSCs following osteodifferentiation (Figures3(a) 3(b) and 4(b)) [13]
Table 2 MSC from AT after osteodifferentiation and licensing byIFN-120574 and TNF-120572 do not induce PBMC proliferation in allogenicconditions
R Saratio MSC-AT MSC-AT
IFN-120574TNF-120572 Os-MSC-AT Os-MSC-ATIFN-120574TNF-120572
1 05 56 plusmn 10b 72 plusmn 31 11 plusmn 02 09 plusmn 06
1 025 55 plusmn 15 52 plusmn 26 13 plusmn 03 08 plusmn 05
1 012 53 plusmn 15 47 plusmn 29 10 plusmn 03 11 plusmn 08
1 006 53 plusmn 20 55 plusmn 30 10 plusmn 02 13 plusmn 10
1 003 46 plusmn 11 39 plusmn 20 14 plusmn 03 18 plusmn 13
aPBMC from healthy individuals were used as responder cells towards AT-derived MSCs pretreated with IFN-120574 and TNF-120572 (MSC-AT IFN-120574TNF-120572) or not (MSC-AT) as stimulating cells at various responder stimulator(R S) ratios Similar experiments were performed with AT-derived MSCscommitted to osteodifferentiation process and pretreated with IFN-120574 andTNF-120572 (Os-MSC-AT IFN-120574TNF-120572) or not (Os-MSC-AT) as stimulatingcellsbData are mean plusmn sem of alloproliferation percentage obtained with 4and 3 distinct healthy donors for MSC-AT and Os-MSC-AT experimentsrespectively This percentage is calculated considering PBMC stimulatedwith LCLlowast as 100 alloproliferation
Then we investigated the immunomodulatory propertiesof MSCs as third-party cells in MLR Results showed thatbothBM-derived andAT-derivedMSCsdisplay immunosup-pressive properties in a dose-responsemanner (Figure 6) It is
8 Journal of Immunology Research
MSC-BM 940 911 864938 896 929
Allo
prol
ifera
tion
inhi
bitio
n (
)A
llopr
olife
ratio
n in
hibi
tion
()
Allo
prol
ifera
tion
inhi
bitio
n (
)A
llopr
olife
ratio
n in
hibi
tion
()
461 628 750379 676 796
0
50
100
850 803 893600 725 895
0
50
100
0
50
100
919 879 879909 867 887
0
50
100
Bone marrow-derived mesenchymal stem cells
Adipose tissue-derived mesenchymal stem cells
No MBCP
Bone marrow-derived mesenchymal stem cells
Adipose tissue-derived mesenchymal stem cells
MBCP
MSC-BM IFN120574TNF120572
MSC-BMMSC-BM IFN120574TNF120572
MSC-ATMSC-AT IFN120574TNF120572
MSC-ATMSC-AT IFN120574TNF120572
1 05 04 1 05 02 1 05 01 1 05 04 1 05 02 1 05 01
1 05 04 1 05 02 1 05 011 05 04 1 05 02 1 05 01
Responder stimulator MSC ratio Responder stimulator MSC ratio
Responder stimulator MSC ratio Responder stimulator MSC ratio
Figure 7 Both BM- andAT-derived MSCs when combined to MBCP biomaterial keep their immunosuppressive properties PBMC fromhealthy individuals were used as responder cells towards irradiated LCLlowast used as stimulating cells in presence of either BM-derived MSCs(MSC-BM) or AT-derived MSCs (MSC-AT) that were pretreated with IFN120574 and TNF120572 (IFN120574TNF120572) or not and used as third-party cellsat various responder stimulator MSC ratios MSCs were combined to MBCP biomaterial (MBCP) or not (No MBCP) Results are given asmean percentage of alloproliferation inhibition plusmn sem when compared to PBMC+LCLlowast using PBMC from 3 distinct healthy donors
Table 3 Immunogenicity of MSC from bone marrow and adiposetissue combinedwithMBCPbiomaterial and licensing by IFN-120574 andTNF-120572
MSC-BM MSC-BMIFN-120574TNF-120572 MSC-AT MSC-AT
IFN-120574TNF-120572No MBCPa 75 plusmn 22b 98 plusmn 40 139 plusmn 39 230 plusmn 52
MBCP 519 plusmn 141 411 plusmn 116 81 plusmn 08 110 plusmn 08
aPBMC from healthy individuals were used as responder cells towards BM-or AT-derivedMSCs pretreated with IFN-120574 and TNF-120572 or not as stimulatingcells at 1 02 responder stimulator ratiobData are mean plusmn sem of alloproliferation percentage obtained with 3distinct healthy donors This percentage is calculated considering PBMCstimulated with LCLlowast as 100 alloproliferation
of note that (i) AT-derivedMSCs aremore potent at low dosescompared to BM-derived MSCs (119875 lt 005) (Figures 6(a) and6(b)) and (ii) licensing with cytokines reduces significantlythe immunosuppressive properties of AT-derived MSCs(119875 lt 005) (Figure 6(b)) When combined to biomaterial(MBCP) both BM-derived and AT-derived MSCs still exertimmunosuppressive properties as they greatly inhibit T cell
alloproliferation with or without being seeded with MBCP(Figure 7) Although not statistically significant addition ofMBCP reduces the immunomodulatory properties of BM-derived MSCs at high responder stimulator MSC ratiosThis could be due to steric hindrance when high numbersof cells are used Indeed such MBCP effect is no longerobserved at low ratios (Figure 7) Also both BM-derivedand AT-derived MSCs when committed to preosteoblasticMSCs inhibit T cell alloproliferation and remain thus able toinduce a tolerogenicmicroenvironment (Figure 8) Licensingwith IFN-120574 and TNF-120572 did not modify such MSC-derivedimmunosuppression (Figures 7 and 8) It is of note thatonce osteodifferentiated AT-derived MSCs are more potentat low doses compared to BM-derived MSCs as they displayhigher immunosuppressive effects (119875 lt 005) (Figure 8)Such higher immunomodulatory capacity of adipose tissue-derived multipotent stromal cells compared to their bonemarrow-derived counterparts has been previously reported[19]
Our present results are in agreement with previousreports showing that differentiation of stem cells does not
Journal of Immunology Research 9
894 889 942 649 528956 925 976 678 367
0
50
100
Allo
prol
ifera
tion
inhi
bitio
n (
)
Osteodifferentiated bone marrow-derived MSC
MSC-BMOs-MSC-BM IFN120574TNF120572
1 05 05 1 05 025 1 05 0125 1 05 006 1 05 003
Responder stimulator MSC ratio
(a)
888 926 917 895 854936 935 913 859 780
0
50
100
Allo
prol
ifera
tion
inhi
bitio
n (
)Osteodifferentiated adipose tissue-derived MSC
Responder stimulator MSC ratio
MSC-ATMSC-AT IFN120574TNF120572
1 05 05 1 05 025 1 05 0125 1 05 006 1 05 003
(b)
Figure 8 Both BM- andAT-derived MSCs when committed to osteodifferentiation process keep their immunosuppressive propertiesPBMC from healthy individuals were used as responder cells towards irradiated LCLlowast used as stimulating cells in presence of eitherosteodifferentiated BM-derived MSCs (Os-MSC-BM) or AT-derived MSCs (Os-MSC-AT) that were pretreated with IFN120574 and TNF120572(IFN120574TNF120572) or not and used as third-party cells at various responder stimulator MSC ratios Results are given as mean percentage ofalloproliferation inhibition plusmn sem when compared to PBMC+LCLlowast using PBMC from 3 and 5 distinct healthy donors in BM- and AT-derived MSCs experiments respectively
alter their low immunogenicity and immunomodulatoryproperties For instance human amniotic epithelial cellswhich have stem cell-like properties retain their immuno-suppressive functions after differentiation into hepatocyte-like cells [20] Also human Whartonrsquos jelly-derived MSCsmaintain the expression of immunomodulatory moleculessuch as HLA-G when subjected to osteogenic differentiationin vitro [21]
In conclusion MSCs from BM or AT display tolerogenicproperties which are maintained following osteodifferenti-ation process or addition of biomaterial and may thus beconsidered as allogenic sources for regenerating bone defectsin orthopaedic and maxillofacial surgery [22]
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This project has received funding from the European UnionrsquosSeventh Programme for research technological develop-ment and demonstration underGrant agreement no 241879
References
[1] R Langer and J P Vacanti ldquoTissue engineeringrdquo Science vol260 no 5110 pp 920ndash926 1993
[2] C Laurencin Y Khan and S F El-Amin ldquoBone graft substi-tutesrdquo Expert Review of Medical Devices vol 3 no 1 pp 49ndash572006
[3] H Petite V ViateauW Bensaıd et al ldquoTissue-engineered boneregenerationrdquo Nature Biotechnology vol 18 no 9 pp 959ndash9632000
[4] M Di Trapani G Bassi M Ricciardi et al ldquoComparative studyof immune regulatory properties of stem cells derived fromdifferent tissuesrdquo StemCells andDevelopment vol 22 pp 2990ndash3002 2013
[5] AUccelli V Pistoia andLMoretta ldquoMesenchymal stemcells anew strategy for immunosuppressionrdquo Trends in Immunologyvol 28 no 5 pp 219ndash226 2007
[6] M Najar G Raicevic H Fayyad-Kazan et al ldquoImmune-relatedantigens surface molecules and regulatory factors in human-derived mesenchymal stromal cells the expression and impactof inflammatory primingrdquo Stem Cell Reviews vol 8 pp 1188ndash1198 2012
[7] R Rizzo D Campioni M Stignani et al ldquoA functional role forsoluble HLA-G antigens in immune modulation mediated bymesenchymal stromal cellsrdquoCytotherapy vol 10 no 4 pp 364ndash375 2008
10 Journal of Immunology Research
[8] F Morandi L Raffaghello G Bianchi et al ldquoImmunogenicityof human mesenchymal stem cells in HLA-class I-restricted T-cell responses against viral or tumor-associated antigensrdquo StemCells vol 26 no 5 pp 1275ndash1287 2008
[9] A Naji N Rouas-Freiss A Durrbach E D Carosella LSensebe and F Deschaseaux ldquoConcise review combininghuman leukocyte antigen G and mesenchymal stem cells forimmunosuppressant biotherapyrdquo Stem Cells vol 31 pp 2296ndash2303 2013
[10] G La Rocca and R Anzalone ldquoPerinatal stem cells revisiteddirections and indications at the crossroads between tissueregeneration and repairrdquoCurrent Stem Cell Research ampTherapyvol 8 pp 2ndash5 2013
[11] Z Selmani ANaji EGaiffe et al ldquoHLA-G is a crucial immuno-suppressive molecule secreted by adult human mesenchymalstem cellsrdquo Transplantation vol 87 no 9 supplement pp S62ndashS66 2009
[12] Z Selmani A Naji I Zidi et al ldquoHuman leukocyte antigen-G5 secretion by human mesenchymal stem cells is required tosuppress T lymphocyte andnatural killer function and to induceCD4+ CD25ℎ119894119892ℎFOXP3+ regulatory T cellsrdquo Stem Cells vol 26no 1 pp 212ndash222 2008
[13] F Deschaseaux J Gaillard A Langonne et al ldquoRegulationand function of immunosuppressivemolecule human leukocyteantigen G5 in human bone tissuerdquo The FASEB Journal vol 27pp 2977ndash2987 2013
[14] M Krampera ldquoMesenchymal stromal cell licensing a multistepprocessrdquo Leukemia vol 25 no 9 pp 1408ndash1414 2011
[15] P Moreau F Adrian-Cabestre C Menier et al ldquoIL-10 selec-tively induces HLA-G expression in human trophoblasts andmonocytesrdquo International Immunology vol 11 no 5 pp 803ndash811 1999
[16] O Brugiere GThabutM Pretolani et al ldquoImmunohistochem-ical study of HLA-G expression in lung transplant recipientsrdquoAmerican Journal of Transplantation vol 9 no 6 pp 1427ndash14382009
[17] S Lefebvre S Berrih-Aknin F Adrian et al ldquoA specificinterferon (IFN)-stimulated response element of the distalHLA-G promoter binds IFN-regulatory factor 1 and mediatesenhancement of this nonclassical class I gene by IFN-betardquoTheJournal of Biological Chemistry vol 276 no 9 pp 6133ndash61392001
[18] N Rouas-Freiss S Bruel C Menier C Marcou P Moreauand E D Carosella ldquoSwitch of HLA-G alternative splicing ina melanoma cell line causes loss of HLA-G1 expression andsensitivity to NK lysisrdquo International Journal of Cancer vol 117no 1 pp 114ndash122 2005
[19] S M Melief J J Zwaginga W E Fibbe and H RoelofsldquoAdipose tissue-derived multipotent stromal cells have a higherimmunomodulatory capacity than their bone marrow-derivedcounterpartsrdquo StemCells TranslationalMedicine vol 2 pp 455ndash463 2013
[20] J Y Tee V Vaghjiani Y H Liu P Murthi J Chan and UManuelpillai ldquoImmunogenicity and immunomodulatory prop-erties of hepatocyte-like cells derived from human amnioticepithelial cellsrdquo Current Stem Cell Research amp Therapy vol 8pp 91ndash99 2013
[21] G La Rocca M Lo Iacono T Corsello S Corrao F Farina andR Anzalone ldquoHuman Whartonrsquos jelly mesenchymal stem cellsmaintain the expression of key immunomodulatory moleculeswhen subjected to osteogenic adipogenic and chondrogenic
differentiation in vitro new perspectives for cellular therapyrdquoCurrent Stem Cell Research ampTherapy vol 8 pp 100ndash113 2013
[22] C Menard and K Tarte ldquoImmunoregulatory properties of clin-ical grade mesenchymal stromal cells evidence uncertaintiesand clinical applicationrdquo Stem Cell Research amp Therapy vol 4article 64 2013
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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ObesityJournal of
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
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Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Journal of Immunology Research 5
100 101 102 103 104
FL 2
100 101 102 103 104
FL 2
R12R11 R13 R14
Os-MSC-AT
IgG1HLA-DRC
ount
s
Cou
nts
Os-MSC-AT IFN120574TNF120572
(a)
100 101 102 103 104
FL 2
100 101 102 103 104
FL 2
R12R11 R13 R14
Os-MSC-AT
IgG1ALP IgG1ALPHLA-G5
Cou
nts
Cou
nts
Os-MSC-AT IFN120574TNF120572
(b)
0102030405060708090
Controls
0102030405060708090 Osteodifferentiated adipose tissue-derived MSC
Os-MSC-AT
(CPM
times10
minus3)
(CPM
times10
minus3)
LCLlowast
Os-
MSC
-BM
PBM
C00
Os-
MSC
-AT
IFN120574
TN
F120572 lowastPB
MC
00
LCL
Responder stimulator ratio1
05
1 025
1 012
1 006
1 003
Os-MSC-AT IFN120574TNF120572
3H
-thym
idin
e inc
orpo
ratio
n
3H
-thym
idin
e inc
orpo
ratio
n
(c)
Figure 4 AT-derived MSCs committed to osteodifferentiation process express HLA-G and are hypoimmunogenic (a) Expression of HLA-DRmolecules was evaluated by flow cytometry analysis onAT-derivedMSCs committed to 14-day osteodifferentiation process and pretreatedwith IFN120574 and TNF120572 (Os-MSC-AT IFN120574TNF120572) or not (Os-MSC-AT) (b) Expression of ALP and HLA-G5 was evaluated by intracellularflow cytometry analysis on AT-derived MSCs committed to 14-day osteodifferentiation process and pretreated with IFN120574 and TNF120572 (Os-MSC-AT IFN120574TNF120572) or not (Os-MSC-AT) IgG1 was used as isotype control Ab (c) PBMC from healthy individual (00) were used asresponder cells towards AT-derived MSCs committed to osteodifferentiation process and pretreated with IFN120574 and TNF120572 (Os-MSC-ATIFN120574TNF120572) or not (Os-MSC-AT) as stimulating cells at various responder stimulator ratios Irradiated LCLlowast were used as positive controlof T cell alloproliferation Results are given as mean cpm plusmn sem one representative experiment is shown
binding to target cells Fluorescencewas detected by using theEpics XL4 flow cytometer
26 Statistical Analysis Significance was assessed by usinga nonparametric Mann-Whitney test assuming a 119875 value lt005 as significant and was marked with lowast in the figures
3 Results and Discussion
Twomain questions were addressed in the present study rely-ing on whether immune regulatory properties of MSCs aremodified by (1) MSC differentiation towards the osteoblasticcell lineage or (2) the addition of synthetic biomaterial (ieMBCP+ granules) In this regard we analyzed both theimmunogenic and immunosuppressive properties of MSCsfrom BM or AT in allogenic conditions that is facing HLA-mismatched PBMC To identify low immunogenic MSCtypes we studied their ability to be recognized as allogenic
cells by HLA-mismatched PBMC in MLR using MSCs asstimulating cells and PBMC from various healthy donorsas responder cells To examine their immunosuppressiveproperties we studied their ability to modulate T cell allo-proliferation as third-party cells in a classical MLR All thefunctional experiments were performed by considering thedifferentiation status of MSC either immature or osteodiffer-entiated and seeded onto biomaterial or not
No PBMC alloproliferation was observed in response tovarious doses of allogenic MSCs derived from bone marrow(Figure 1(c)) or adipose tissue (Figure 2(c)) even after licens-ing with IFN-120574 and TNF-120572 (Figures 1(c) and 2(c)) Tables 1and 2 summarize the results obtained with PBMC from dis-tinct healthy donorsThe efficiency of cytokine treatment wasattested by the induction of HLA-DR expression on MSCs(Figures 1(a) and 2(a)) In order to evaluate the influenceof osteodifferentiation process on the immunogenicity ofMSCs similar functional assays were performed using BM-derived and AT-derived MSCs committed to preosteoblastic
6 Journal of Immunology Research
0
10
20
30
40
50
Bone marrow-derived mesenchymal stem cells(C
PMtimes10
minus3)
LCLlowast
MSC
-BM
0
10
20
30
40
50
Adipose tissue-derived mesenchymal stem cells
(CPM
times10
minus3)
LCLlowast
MSC
-AT
0
10
20
30
40
50Bone marrow-derived mesenchymal stem cells
(CPM
times10
minus3)
+
0
10
20
30
40
50Adipose tissue-derived mesenchymal stem cells
(CPM
times10
minus3)
+
No MBCP
MBCP
PBM
C713
lowastPB
MC
713
LCL
PBM
C713
MSC
-BM
PBM
C713
MSC
-BM
PBM
C713
lowastPB
MC
713
LCL
PBM
C713
MSC
-AT
PBM
C713
MSC
-AT
LCLlowast
MSC
-BM
PBM
C713
lowastPB
MC
713
LCL
PBM
C713
MSC
-BM
PBM
C713
MSC
-BM
LCLlowast
MSC
-AT
PBM
C713
lowastPB
MC
713
LCL
PBM
C713
MSC
-AT
PBM
C713
MSC
-AT
IFN120574TNF120572 IFN120574TNF120572
3H
-thym
idin
e inc
orpo
ratio
n
3H
-thym
idin
e inc
orpo
ratio
n
3H
-thym
idin
e inc
orpo
ratio
n
3H
-thym
idin
e inc
orpo
ratio
n
Figure 5 Both BM- andAT-derived MSCs when combined to MBCP biomaterial keep their hypoimmunogenicity PBMC from healthyindividual (713) were used as responder cells towards either BM-derived MSCs (MSC-BM) or AT-derived MSCs (MSC-AT) that werepretreated with IFN120574 and TNF120572 (IFN120574TNF120572) or not and used as stimulating cells at the responder stimulator ratio of 1 02 MSCs werecombined toMBCP biomaterial (MBCP) or not (NoMBCP) Irradiated LCLlowast were used as positive control of T cell alloproliferation Resultsare given as mean cpm plusmn sem one representative experiment is shown
MSCs as stimulating cells The osteodifferentiation processwas validated through the upregulation of ALP expression inosteodifferentiated MSCs (Figures 3(a) 3(b) 4(a) and 4(b))Results show that both BM-derived and AT-derived MSCscommitted to osteodifferentiation are still hypoimmunogenicwhether they are pretreated or not with IFN-120574 and TNF-120572(Figures 3(c) and 4(c) and Tables 1 and 2) Then we lookedat whether combination of biomaterial (ie MBCP) withMSCs alters their immunogenicityNodifferenceswere foundbetween standard 2D-coculture conditions (MSC + PBMC)and 3D-coculture conditions (MSC + MBCP + PBMC) Onerepresentative allogenic combination is shown (Figure 5) forwhich the mean percentage of T cell alloproliferation ispresented in Table 3 (119899 = 3 healthy donors) It is of notethat the addition of MBCP to BM-derived MSCs treated or
not with cytokines modifies slightly their immunogenicityalthough no statistical difference was observed betweenboth conditions (119875 gt 01) (Table 3) Consequently wecan conclude that both the osteodifferentiation process andthe presence of biomaterial (MBCP) did not abrogate thehypoimmunogenicity of MSCs
Notably expression of the tolerogenic soluble HLA-G5protein was observed in MSCs derived from BM and ATand could be enhanced following treatment with IFN-120574 andTNF-120572 (Figures 1(b) and 2(b)) Such enhanced expressionof HLA-G by IFN-120574 treatment was previously reported forother cell types such as monocytes [15] bronchial epithelialcells [16] thymic epithelial cells [17] and various tumor cells[18] Nevertheless HLA-G expression levels varied amongthe various batches of MSCs tested (data not shown) In
Journal of Immunology Research 7
MSC-BM 954 885 667 339 165888 812 647 310 153
0
50
100
Allo
prol
ifera
tion
inhi
bitio
n (
)
Bone marrow-derived mesenchymal stem cells
1 05 05 1 05 025 1 05 0125 1 05 006 1 05 003
Responder stimulator MSC ratioMSC-BM IFN120574TNF120572
(a)
MSC-AT 878 824 879 859 823883 831 741 662 572
0
50
100
Allo
prol
ifera
tion
inhi
bitio
n (
)Adipose tissue-derived mesenchymal stem cells
lowast lowast lowast
MSC-AT IFN120574TNF120572Responder stimulator MSC ratio
1 05 05 1 05 025 1 05 0125 1 05 006 1 05 003
(b)
Figure 6 Both BM- andAT-derived MSCs display immunosuppressive properties in a dose-dependent manner PBMC from healthyindividuals were used as responder cells towards irradiated LCLlowast used as stimulating cells in presence of either (a) BM-derivedMSCs (MSC-BM) or (b) AT-derived MSCs (MSC-AT) that were pretreated with IFN120574 and TNF120572 (IFN120574TNF120572) or not and used as third-party cells atvarious responder stimulator MSC ratios Results are given as mean percentage of alloproliferation inhibition plusmn sem according to themaximal alloproliferation observed with PBMC+LCLlowast using PBMC from 4 and 6 distinct healthy donors in BM- and AT-derived MSCsexperiments respectively
Table 1 MSC from BM after osteodifferentiation and licensing byIFN-120574 and TNF-120572 do not induce PBMC proliferation in allogenicconditions
R Saratio MSC-BM MSC-BM
IFN-120574TNF-120572 Os-MSC-BM Os-MSC-BMIFN-120574TNF-120572
1 05 43 plusmn 21b 11 plusmn 03 33 plusmn 03 17 plusmn 02
1 025 45 plusmn 14 10 plusmn 03 31 plusmn 01 18 plusmn 05
1 012 53 plusmn 19 11 plusmn 04 32 plusmn 10 23 plusmn 12
1 006 54 plusmn 24 14 plusmn 05 26 plusmn 11 39 plusmn 31
1 003 43 plusmn 17 14 plusmn 05 22 plusmn 02 21 plusmn 12
aPBMC from healthy individuals were used as responder cells towards BM-derived MSCs pretreated with IFN-120574 and TNF-120572 (MSC-BM IFN-120574TNF-120572) or not (MSC-BM) as stimulating cells at various responder stimulator(R S) ratios Similar experiments were performed with BM-derived MSCscommitted to osteodifferentiation process and pretreated with IFN-120574 andTNF-120572 (Os-MSC-BM IFN-120574TNF-120572) or not (Os-MSC-BM) as stimulatingcellsbData are mean plusmn sem of alloproliferation percentage obtained with 5and 3 distinct healthy donors for MSC-BM and Os-MSC-BM experimentsrespectively This percentage is calculated considering PBMC stimulatedwith LCLlowast as 100 alloproliferation
agreement with recent findings we observed enhancedHLA-Gexpression byMSCs following osteodifferentiation (Figures3(a) 3(b) and 4(b)) [13]
Table 2 MSC from AT after osteodifferentiation and licensing byIFN-120574 and TNF-120572 do not induce PBMC proliferation in allogenicconditions
R Saratio MSC-AT MSC-AT
IFN-120574TNF-120572 Os-MSC-AT Os-MSC-ATIFN-120574TNF-120572
1 05 56 plusmn 10b 72 plusmn 31 11 plusmn 02 09 plusmn 06
1 025 55 plusmn 15 52 plusmn 26 13 plusmn 03 08 plusmn 05
1 012 53 plusmn 15 47 plusmn 29 10 plusmn 03 11 plusmn 08
1 006 53 plusmn 20 55 plusmn 30 10 plusmn 02 13 plusmn 10
1 003 46 plusmn 11 39 plusmn 20 14 plusmn 03 18 plusmn 13
aPBMC from healthy individuals were used as responder cells towards AT-derived MSCs pretreated with IFN-120574 and TNF-120572 (MSC-AT IFN-120574TNF-120572) or not (MSC-AT) as stimulating cells at various responder stimulator(R S) ratios Similar experiments were performed with AT-derived MSCscommitted to osteodifferentiation process and pretreated with IFN-120574 andTNF-120572 (Os-MSC-AT IFN-120574TNF-120572) or not (Os-MSC-AT) as stimulatingcellsbData are mean plusmn sem of alloproliferation percentage obtained with 4and 3 distinct healthy donors for MSC-AT and Os-MSC-AT experimentsrespectively This percentage is calculated considering PBMC stimulatedwith LCLlowast as 100 alloproliferation
Then we investigated the immunomodulatory propertiesof MSCs as third-party cells in MLR Results showed thatbothBM-derived andAT-derivedMSCsdisplay immunosup-pressive properties in a dose-responsemanner (Figure 6) It is
8 Journal of Immunology Research
MSC-BM 940 911 864938 896 929
Allo
prol
ifera
tion
inhi
bitio
n (
)A
llopr
olife
ratio
n in
hibi
tion
()
Allo
prol
ifera
tion
inhi
bitio
n (
)A
llopr
olife
ratio
n in
hibi
tion
()
461 628 750379 676 796
0
50
100
850 803 893600 725 895
0
50
100
0
50
100
919 879 879909 867 887
0
50
100
Bone marrow-derived mesenchymal stem cells
Adipose tissue-derived mesenchymal stem cells
No MBCP
Bone marrow-derived mesenchymal stem cells
Adipose tissue-derived mesenchymal stem cells
MBCP
MSC-BM IFN120574TNF120572
MSC-BMMSC-BM IFN120574TNF120572
MSC-ATMSC-AT IFN120574TNF120572
MSC-ATMSC-AT IFN120574TNF120572
1 05 04 1 05 02 1 05 01 1 05 04 1 05 02 1 05 01
1 05 04 1 05 02 1 05 011 05 04 1 05 02 1 05 01
Responder stimulator MSC ratio Responder stimulator MSC ratio
Responder stimulator MSC ratio Responder stimulator MSC ratio
Figure 7 Both BM- andAT-derived MSCs when combined to MBCP biomaterial keep their immunosuppressive properties PBMC fromhealthy individuals were used as responder cells towards irradiated LCLlowast used as stimulating cells in presence of either BM-derived MSCs(MSC-BM) or AT-derived MSCs (MSC-AT) that were pretreated with IFN120574 and TNF120572 (IFN120574TNF120572) or not and used as third-party cellsat various responder stimulator MSC ratios MSCs were combined to MBCP biomaterial (MBCP) or not (No MBCP) Results are given asmean percentage of alloproliferation inhibition plusmn sem when compared to PBMC+LCLlowast using PBMC from 3 distinct healthy donors
Table 3 Immunogenicity of MSC from bone marrow and adiposetissue combinedwithMBCPbiomaterial and licensing by IFN-120574 andTNF-120572
MSC-BM MSC-BMIFN-120574TNF-120572 MSC-AT MSC-AT
IFN-120574TNF-120572No MBCPa 75 plusmn 22b 98 plusmn 40 139 plusmn 39 230 plusmn 52
MBCP 519 plusmn 141 411 plusmn 116 81 plusmn 08 110 plusmn 08
aPBMC from healthy individuals were used as responder cells towards BM-or AT-derivedMSCs pretreated with IFN-120574 and TNF-120572 or not as stimulatingcells at 1 02 responder stimulator ratiobData are mean plusmn sem of alloproliferation percentage obtained with 3distinct healthy donors This percentage is calculated considering PBMCstimulated with LCLlowast as 100 alloproliferation
of note that (i) AT-derivedMSCs aremore potent at low dosescompared to BM-derived MSCs (119875 lt 005) (Figures 6(a) and6(b)) and (ii) licensing with cytokines reduces significantlythe immunosuppressive properties of AT-derived MSCs(119875 lt 005) (Figure 6(b)) When combined to biomaterial(MBCP) both BM-derived and AT-derived MSCs still exertimmunosuppressive properties as they greatly inhibit T cell
alloproliferation with or without being seeded with MBCP(Figure 7) Although not statistically significant addition ofMBCP reduces the immunomodulatory properties of BM-derived MSCs at high responder stimulator MSC ratiosThis could be due to steric hindrance when high numbersof cells are used Indeed such MBCP effect is no longerobserved at low ratios (Figure 7) Also both BM-derivedand AT-derived MSCs when committed to preosteoblasticMSCs inhibit T cell alloproliferation and remain thus able toinduce a tolerogenicmicroenvironment (Figure 8) Licensingwith IFN-120574 and TNF-120572 did not modify such MSC-derivedimmunosuppression (Figures 7 and 8) It is of note thatonce osteodifferentiated AT-derived MSCs are more potentat low doses compared to BM-derived MSCs as they displayhigher immunosuppressive effects (119875 lt 005) (Figure 8)Such higher immunomodulatory capacity of adipose tissue-derived multipotent stromal cells compared to their bonemarrow-derived counterparts has been previously reported[19]
Our present results are in agreement with previousreports showing that differentiation of stem cells does not
Journal of Immunology Research 9
894 889 942 649 528956 925 976 678 367
0
50
100
Allo
prol
ifera
tion
inhi
bitio
n (
)
Osteodifferentiated bone marrow-derived MSC
MSC-BMOs-MSC-BM IFN120574TNF120572
1 05 05 1 05 025 1 05 0125 1 05 006 1 05 003
Responder stimulator MSC ratio
(a)
888 926 917 895 854936 935 913 859 780
0
50
100
Allo
prol
ifera
tion
inhi
bitio
n (
)Osteodifferentiated adipose tissue-derived MSC
Responder stimulator MSC ratio
MSC-ATMSC-AT IFN120574TNF120572
1 05 05 1 05 025 1 05 0125 1 05 006 1 05 003
(b)
Figure 8 Both BM- andAT-derived MSCs when committed to osteodifferentiation process keep their immunosuppressive propertiesPBMC from healthy individuals were used as responder cells towards irradiated LCLlowast used as stimulating cells in presence of eitherosteodifferentiated BM-derived MSCs (Os-MSC-BM) or AT-derived MSCs (Os-MSC-AT) that were pretreated with IFN120574 and TNF120572(IFN120574TNF120572) or not and used as third-party cells at various responder stimulator MSC ratios Results are given as mean percentage ofalloproliferation inhibition plusmn sem when compared to PBMC+LCLlowast using PBMC from 3 and 5 distinct healthy donors in BM- and AT-derived MSCs experiments respectively
alter their low immunogenicity and immunomodulatoryproperties For instance human amniotic epithelial cellswhich have stem cell-like properties retain their immuno-suppressive functions after differentiation into hepatocyte-like cells [20] Also human Whartonrsquos jelly-derived MSCsmaintain the expression of immunomodulatory moleculessuch as HLA-G when subjected to osteogenic differentiationin vitro [21]
In conclusion MSCs from BM or AT display tolerogenicproperties which are maintained following osteodifferenti-ation process or addition of biomaterial and may thus beconsidered as allogenic sources for regenerating bone defectsin orthopaedic and maxillofacial surgery [22]
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This project has received funding from the European UnionrsquosSeventh Programme for research technological develop-ment and demonstration underGrant agreement no 241879
References
[1] R Langer and J P Vacanti ldquoTissue engineeringrdquo Science vol260 no 5110 pp 920ndash926 1993
[2] C Laurencin Y Khan and S F El-Amin ldquoBone graft substi-tutesrdquo Expert Review of Medical Devices vol 3 no 1 pp 49ndash572006
[3] H Petite V ViateauW Bensaıd et al ldquoTissue-engineered boneregenerationrdquo Nature Biotechnology vol 18 no 9 pp 959ndash9632000
[4] M Di Trapani G Bassi M Ricciardi et al ldquoComparative studyof immune regulatory properties of stem cells derived fromdifferent tissuesrdquo StemCells andDevelopment vol 22 pp 2990ndash3002 2013
[5] AUccelli V Pistoia andLMoretta ldquoMesenchymal stemcells anew strategy for immunosuppressionrdquo Trends in Immunologyvol 28 no 5 pp 219ndash226 2007
[6] M Najar G Raicevic H Fayyad-Kazan et al ldquoImmune-relatedantigens surface molecules and regulatory factors in human-derived mesenchymal stromal cells the expression and impactof inflammatory primingrdquo Stem Cell Reviews vol 8 pp 1188ndash1198 2012
[7] R Rizzo D Campioni M Stignani et al ldquoA functional role forsoluble HLA-G antigens in immune modulation mediated bymesenchymal stromal cellsrdquoCytotherapy vol 10 no 4 pp 364ndash375 2008
10 Journal of Immunology Research
[8] F Morandi L Raffaghello G Bianchi et al ldquoImmunogenicityof human mesenchymal stem cells in HLA-class I-restricted T-cell responses against viral or tumor-associated antigensrdquo StemCells vol 26 no 5 pp 1275ndash1287 2008
[9] A Naji N Rouas-Freiss A Durrbach E D Carosella LSensebe and F Deschaseaux ldquoConcise review combininghuman leukocyte antigen G and mesenchymal stem cells forimmunosuppressant biotherapyrdquo Stem Cells vol 31 pp 2296ndash2303 2013
[10] G La Rocca and R Anzalone ldquoPerinatal stem cells revisiteddirections and indications at the crossroads between tissueregeneration and repairrdquoCurrent Stem Cell Research ampTherapyvol 8 pp 2ndash5 2013
[11] Z Selmani ANaji EGaiffe et al ldquoHLA-G is a crucial immuno-suppressive molecule secreted by adult human mesenchymalstem cellsrdquo Transplantation vol 87 no 9 supplement pp S62ndashS66 2009
[12] Z Selmani A Naji I Zidi et al ldquoHuman leukocyte antigen-G5 secretion by human mesenchymal stem cells is required tosuppress T lymphocyte andnatural killer function and to induceCD4+ CD25ℎ119894119892ℎFOXP3+ regulatory T cellsrdquo Stem Cells vol 26no 1 pp 212ndash222 2008
[13] F Deschaseaux J Gaillard A Langonne et al ldquoRegulationand function of immunosuppressivemolecule human leukocyteantigen G5 in human bone tissuerdquo The FASEB Journal vol 27pp 2977ndash2987 2013
[14] M Krampera ldquoMesenchymal stromal cell licensing a multistepprocessrdquo Leukemia vol 25 no 9 pp 1408ndash1414 2011
[15] P Moreau F Adrian-Cabestre C Menier et al ldquoIL-10 selec-tively induces HLA-G expression in human trophoblasts andmonocytesrdquo International Immunology vol 11 no 5 pp 803ndash811 1999
[16] O Brugiere GThabutM Pretolani et al ldquoImmunohistochem-ical study of HLA-G expression in lung transplant recipientsrdquoAmerican Journal of Transplantation vol 9 no 6 pp 1427ndash14382009
[17] S Lefebvre S Berrih-Aknin F Adrian et al ldquoA specificinterferon (IFN)-stimulated response element of the distalHLA-G promoter binds IFN-regulatory factor 1 and mediatesenhancement of this nonclassical class I gene by IFN-betardquoTheJournal of Biological Chemistry vol 276 no 9 pp 6133ndash61392001
[18] N Rouas-Freiss S Bruel C Menier C Marcou P Moreauand E D Carosella ldquoSwitch of HLA-G alternative splicing ina melanoma cell line causes loss of HLA-G1 expression andsensitivity to NK lysisrdquo International Journal of Cancer vol 117no 1 pp 114ndash122 2005
[19] S M Melief J J Zwaginga W E Fibbe and H RoelofsldquoAdipose tissue-derived multipotent stromal cells have a higherimmunomodulatory capacity than their bone marrow-derivedcounterpartsrdquo StemCells TranslationalMedicine vol 2 pp 455ndash463 2013
[20] J Y Tee V Vaghjiani Y H Liu P Murthi J Chan and UManuelpillai ldquoImmunogenicity and immunomodulatory prop-erties of hepatocyte-like cells derived from human amnioticepithelial cellsrdquo Current Stem Cell Research amp Therapy vol 8pp 91ndash99 2013
[21] G La Rocca M Lo Iacono T Corsello S Corrao F Farina andR Anzalone ldquoHuman Whartonrsquos jelly mesenchymal stem cellsmaintain the expression of key immunomodulatory moleculeswhen subjected to osteogenic adipogenic and chondrogenic
differentiation in vitro new perspectives for cellular therapyrdquoCurrent Stem Cell Research ampTherapy vol 8 pp 100ndash113 2013
[22] C Menard and K Tarte ldquoImmunoregulatory properties of clin-ical grade mesenchymal stromal cells evidence uncertaintiesand clinical applicationrdquo Stem Cell Research amp Therapy vol 4article 64 2013
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
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Diabetes ResearchJournal of
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
6 Journal of Immunology Research
0
10
20
30
40
50
Bone marrow-derived mesenchymal stem cells(C
PMtimes10
minus3)
LCLlowast
MSC
-BM
0
10
20
30
40
50
Adipose tissue-derived mesenchymal stem cells
(CPM
times10
minus3)
LCLlowast
MSC
-AT
0
10
20
30
40
50Bone marrow-derived mesenchymal stem cells
(CPM
times10
minus3)
+
0
10
20
30
40
50Adipose tissue-derived mesenchymal stem cells
(CPM
times10
minus3)
+
No MBCP
MBCP
PBM
C713
lowastPB
MC
713
LCL
PBM
C713
MSC
-BM
PBM
C713
MSC
-BM
PBM
C713
lowastPB
MC
713
LCL
PBM
C713
MSC
-AT
PBM
C713
MSC
-AT
LCLlowast
MSC
-BM
PBM
C713
lowastPB
MC
713
LCL
PBM
C713
MSC
-BM
PBM
C713
MSC
-BM
LCLlowast
MSC
-AT
PBM
C713
lowastPB
MC
713
LCL
PBM
C713
MSC
-AT
PBM
C713
MSC
-AT
IFN120574TNF120572 IFN120574TNF120572
3H
-thym
idin
e inc
orpo
ratio
n
3H
-thym
idin
e inc
orpo
ratio
n
3H
-thym
idin
e inc
orpo
ratio
n
3H
-thym
idin
e inc
orpo
ratio
n
Figure 5 Both BM- andAT-derived MSCs when combined to MBCP biomaterial keep their hypoimmunogenicity PBMC from healthyindividual (713) were used as responder cells towards either BM-derived MSCs (MSC-BM) or AT-derived MSCs (MSC-AT) that werepretreated with IFN120574 and TNF120572 (IFN120574TNF120572) or not and used as stimulating cells at the responder stimulator ratio of 1 02 MSCs werecombined toMBCP biomaterial (MBCP) or not (NoMBCP) Irradiated LCLlowast were used as positive control of T cell alloproliferation Resultsare given as mean cpm plusmn sem one representative experiment is shown
MSCs as stimulating cells The osteodifferentiation processwas validated through the upregulation of ALP expression inosteodifferentiated MSCs (Figures 3(a) 3(b) 4(a) and 4(b))Results show that both BM-derived and AT-derived MSCscommitted to osteodifferentiation are still hypoimmunogenicwhether they are pretreated or not with IFN-120574 and TNF-120572(Figures 3(c) and 4(c) and Tables 1 and 2) Then we lookedat whether combination of biomaterial (ie MBCP) withMSCs alters their immunogenicityNodifferenceswere foundbetween standard 2D-coculture conditions (MSC + PBMC)and 3D-coculture conditions (MSC + MBCP + PBMC) Onerepresentative allogenic combination is shown (Figure 5) forwhich the mean percentage of T cell alloproliferation ispresented in Table 3 (119899 = 3 healthy donors) It is of notethat the addition of MBCP to BM-derived MSCs treated or
not with cytokines modifies slightly their immunogenicityalthough no statistical difference was observed betweenboth conditions (119875 gt 01) (Table 3) Consequently wecan conclude that both the osteodifferentiation process andthe presence of biomaterial (MBCP) did not abrogate thehypoimmunogenicity of MSCs
Notably expression of the tolerogenic soluble HLA-G5protein was observed in MSCs derived from BM and ATand could be enhanced following treatment with IFN-120574 andTNF-120572 (Figures 1(b) and 2(b)) Such enhanced expressionof HLA-G by IFN-120574 treatment was previously reported forother cell types such as monocytes [15] bronchial epithelialcells [16] thymic epithelial cells [17] and various tumor cells[18] Nevertheless HLA-G expression levels varied amongthe various batches of MSCs tested (data not shown) In
Journal of Immunology Research 7
MSC-BM 954 885 667 339 165888 812 647 310 153
0
50
100
Allo
prol
ifera
tion
inhi
bitio
n (
)
Bone marrow-derived mesenchymal stem cells
1 05 05 1 05 025 1 05 0125 1 05 006 1 05 003
Responder stimulator MSC ratioMSC-BM IFN120574TNF120572
(a)
MSC-AT 878 824 879 859 823883 831 741 662 572
0
50
100
Allo
prol
ifera
tion
inhi
bitio
n (
)Adipose tissue-derived mesenchymal stem cells
lowast lowast lowast
MSC-AT IFN120574TNF120572Responder stimulator MSC ratio
1 05 05 1 05 025 1 05 0125 1 05 006 1 05 003
(b)
Figure 6 Both BM- andAT-derived MSCs display immunosuppressive properties in a dose-dependent manner PBMC from healthyindividuals were used as responder cells towards irradiated LCLlowast used as stimulating cells in presence of either (a) BM-derivedMSCs (MSC-BM) or (b) AT-derived MSCs (MSC-AT) that were pretreated with IFN120574 and TNF120572 (IFN120574TNF120572) or not and used as third-party cells atvarious responder stimulator MSC ratios Results are given as mean percentage of alloproliferation inhibition plusmn sem according to themaximal alloproliferation observed with PBMC+LCLlowast using PBMC from 4 and 6 distinct healthy donors in BM- and AT-derived MSCsexperiments respectively
Table 1 MSC from BM after osteodifferentiation and licensing byIFN-120574 and TNF-120572 do not induce PBMC proliferation in allogenicconditions
R Saratio MSC-BM MSC-BM
IFN-120574TNF-120572 Os-MSC-BM Os-MSC-BMIFN-120574TNF-120572
1 05 43 plusmn 21b 11 plusmn 03 33 plusmn 03 17 plusmn 02
1 025 45 plusmn 14 10 plusmn 03 31 plusmn 01 18 plusmn 05
1 012 53 plusmn 19 11 plusmn 04 32 plusmn 10 23 plusmn 12
1 006 54 plusmn 24 14 plusmn 05 26 plusmn 11 39 plusmn 31
1 003 43 plusmn 17 14 plusmn 05 22 plusmn 02 21 plusmn 12
aPBMC from healthy individuals were used as responder cells towards BM-derived MSCs pretreated with IFN-120574 and TNF-120572 (MSC-BM IFN-120574TNF-120572) or not (MSC-BM) as stimulating cells at various responder stimulator(R S) ratios Similar experiments were performed with BM-derived MSCscommitted to osteodifferentiation process and pretreated with IFN-120574 andTNF-120572 (Os-MSC-BM IFN-120574TNF-120572) or not (Os-MSC-BM) as stimulatingcellsbData are mean plusmn sem of alloproliferation percentage obtained with 5and 3 distinct healthy donors for MSC-BM and Os-MSC-BM experimentsrespectively This percentage is calculated considering PBMC stimulatedwith LCLlowast as 100 alloproliferation
agreement with recent findings we observed enhancedHLA-Gexpression byMSCs following osteodifferentiation (Figures3(a) 3(b) and 4(b)) [13]
Table 2 MSC from AT after osteodifferentiation and licensing byIFN-120574 and TNF-120572 do not induce PBMC proliferation in allogenicconditions
R Saratio MSC-AT MSC-AT
IFN-120574TNF-120572 Os-MSC-AT Os-MSC-ATIFN-120574TNF-120572
1 05 56 plusmn 10b 72 plusmn 31 11 plusmn 02 09 plusmn 06
1 025 55 plusmn 15 52 plusmn 26 13 plusmn 03 08 plusmn 05
1 012 53 plusmn 15 47 plusmn 29 10 plusmn 03 11 plusmn 08
1 006 53 plusmn 20 55 plusmn 30 10 plusmn 02 13 plusmn 10
1 003 46 plusmn 11 39 plusmn 20 14 plusmn 03 18 plusmn 13
aPBMC from healthy individuals were used as responder cells towards AT-derived MSCs pretreated with IFN-120574 and TNF-120572 (MSC-AT IFN-120574TNF-120572) or not (MSC-AT) as stimulating cells at various responder stimulator(R S) ratios Similar experiments were performed with AT-derived MSCscommitted to osteodifferentiation process and pretreated with IFN-120574 andTNF-120572 (Os-MSC-AT IFN-120574TNF-120572) or not (Os-MSC-AT) as stimulatingcellsbData are mean plusmn sem of alloproliferation percentage obtained with 4and 3 distinct healthy donors for MSC-AT and Os-MSC-AT experimentsrespectively This percentage is calculated considering PBMC stimulatedwith LCLlowast as 100 alloproliferation
Then we investigated the immunomodulatory propertiesof MSCs as third-party cells in MLR Results showed thatbothBM-derived andAT-derivedMSCsdisplay immunosup-pressive properties in a dose-responsemanner (Figure 6) It is
8 Journal of Immunology Research
MSC-BM 940 911 864938 896 929
Allo
prol
ifera
tion
inhi
bitio
n (
)A
llopr
olife
ratio
n in
hibi
tion
()
Allo
prol
ifera
tion
inhi
bitio
n (
)A
llopr
olife
ratio
n in
hibi
tion
()
461 628 750379 676 796
0
50
100
850 803 893600 725 895
0
50
100
0
50
100
919 879 879909 867 887
0
50
100
Bone marrow-derived mesenchymal stem cells
Adipose tissue-derived mesenchymal stem cells
No MBCP
Bone marrow-derived mesenchymal stem cells
Adipose tissue-derived mesenchymal stem cells
MBCP
MSC-BM IFN120574TNF120572
MSC-BMMSC-BM IFN120574TNF120572
MSC-ATMSC-AT IFN120574TNF120572
MSC-ATMSC-AT IFN120574TNF120572
1 05 04 1 05 02 1 05 01 1 05 04 1 05 02 1 05 01
1 05 04 1 05 02 1 05 011 05 04 1 05 02 1 05 01
Responder stimulator MSC ratio Responder stimulator MSC ratio
Responder stimulator MSC ratio Responder stimulator MSC ratio
Figure 7 Both BM- andAT-derived MSCs when combined to MBCP biomaterial keep their immunosuppressive properties PBMC fromhealthy individuals were used as responder cells towards irradiated LCLlowast used as stimulating cells in presence of either BM-derived MSCs(MSC-BM) or AT-derived MSCs (MSC-AT) that were pretreated with IFN120574 and TNF120572 (IFN120574TNF120572) or not and used as third-party cellsat various responder stimulator MSC ratios MSCs were combined to MBCP biomaterial (MBCP) or not (No MBCP) Results are given asmean percentage of alloproliferation inhibition plusmn sem when compared to PBMC+LCLlowast using PBMC from 3 distinct healthy donors
Table 3 Immunogenicity of MSC from bone marrow and adiposetissue combinedwithMBCPbiomaterial and licensing by IFN-120574 andTNF-120572
MSC-BM MSC-BMIFN-120574TNF-120572 MSC-AT MSC-AT
IFN-120574TNF-120572No MBCPa 75 plusmn 22b 98 plusmn 40 139 plusmn 39 230 plusmn 52
MBCP 519 plusmn 141 411 plusmn 116 81 plusmn 08 110 plusmn 08
aPBMC from healthy individuals were used as responder cells towards BM-or AT-derivedMSCs pretreated with IFN-120574 and TNF-120572 or not as stimulatingcells at 1 02 responder stimulator ratiobData are mean plusmn sem of alloproliferation percentage obtained with 3distinct healthy donors This percentage is calculated considering PBMCstimulated with LCLlowast as 100 alloproliferation
of note that (i) AT-derivedMSCs aremore potent at low dosescompared to BM-derived MSCs (119875 lt 005) (Figures 6(a) and6(b)) and (ii) licensing with cytokines reduces significantlythe immunosuppressive properties of AT-derived MSCs(119875 lt 005) (Figure 6(b)) When combined to biomaterial(MBCP) both BM-derived and AT-derived MSCs still exertimmunosuppressive properties as they greatly inhibit T cell
alloproliferation with or without being seeded with MBCP(Figure 7) Although not statistically significant addition ofMBCP reduces the immunomodulatory properties of BM-derived MSCs at high responder stimulator MSC ratiosThis could be due to steric hindrance when high numbersof cells are used Indeed such MBCP effect is no longerobserved at low ratios (Figure 7) Also both BM-derivedand AT-derived MSCs when committed to preosteoblasticMSCs inhibit T cell alloproliferation and remain thus able toinduce a tolerogenicmicroenvironment (Figure 8) Licensingwith IFN-120574 and TNF-120572 did not modify such MSC-derivedimmunosuppression (Figures 7 and 8) It is of note thatonce osteodifferentiated AT-derived MSCs are more potentat low doses compared to BM-derived MSCs as they displayhigher immunosuppressive effects (119875 lt 005) (Figure 8)Such higher immunomodulatory capacity of adipose tissue-derived multipotent stromal cells compared to their bonemarrow-derived counterparts has been previously reported[19]
Our present results are in agreement with previousreports showing that differentiation of stem cells does not
Journal of Immunology Research 9
894 889 942 649 528956 925 976 678 367
0
50
100
Allo
prol
ifera
tion
inhi
bitio
n (
)
Osteodifferentiated bone marrow-derived MSC
MSC-BMOs-MSC-BM IFN120574TNF120572
1 05 05 1 05 025 1 05 0125 1 05 006 1 05 003
Responder stimulator MSC ratio
(a)
888 926 917 895 854936 935 913 859 780
0
50
100
Allo
prol
ifera
tion
inhi
bitio
n (
)Osteodifferentiated adipose tissue-derived MSC
Responder stimulator MSC ratio
MSC-ATMSC-AT IFN120574TNF120572
1 05 05 1 05 025 1 05 0125 1 05 006 1 05 003
(b)
Figure 8 Both BM- andAT-derived MSCs when committed to osteodifferentiation process keep their immunosuppressive propertiesPBMC from healthy individuals were used as responder cells towards irradiated LCLlowast used as stimulating cells in presence of eitherosteodifferentiated BM-derived MSCs (Os-MSC-BM) or AT-derived MSCs (Os-MSC-AT) that were pretreated with IFN120574 and TNF120572(IFN120574TNF120572) or not and used as third-party cells at various responder stimulator MSC ratios Results are given as mean percentage ofalloproliferation inhibition plusmn sem when compared to PBMC+LCLlowast using PBMC from 3 and 5 distinct healthy donors in BM- and AT-derived MSCs experiments respectively
alter their low immunogenicity and immunomodulatoryproperties For instance human amniotic epithelial cellswhich have stem cell-like properties retain their immuno-suppressive functions after differentiation into hepatocyte-like cells [20] Also human Whartonrsquos jelly-derived MSCsmaintain the expression of immunomodulatory moleculessuch as HLA-G when subjected to osteogenic differentiationin vitro [21]
In conclusion MSCs from BM or AT display tolerogenicproperties which are maintained following osteodifferenti-ation process or addition of biomaterial and may thus beconsidered as allogenic sources for regenerating bone defectsin orthopaedic and maxillofacial surgery [22]
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This project has received funding from the European UnionrsquosSeventh Programme for research technological develop-ment and demonstration underGrant agreement no 241879
References
[1] R Langer and J P Vacanti ldquoTissue engineeringrdquo Science vol260 no 5110 pp 920ndash926 1993
[2] C Laurencin Y Khan and S F El-Amin ldquoBone graft substi-tutesrdquo Expert Review of Medical Devices vol 3 no 1 pp 49ndash572006
[3] H Petite V ViateauW Bensaıd et al ldquoTissue-engineered boneregenerationrdquo Nature Biotechnology vol 18 no 9 pp 959ndash9632000
[4] M Di Trapani G Bassi M Ricciardi et al ldquoComparative studyof immune regulatory properties of stem cells derived fromdifferent tissuesrdquo StemCells andDevelopment vol 22 pp 2990ndash3002 2013
[5] AUccelli V Pistoia andLMoretta ldquoMesenchymal stemcells anew strategy for immunosuppressionrdquo Trends in Immunologyvol 28 no 5 pp 219ndash226 2007
[6] M Najar G Raicevic H Fayyad-Kazan et al ldquoImmune-relatedantigens surface molecules and regulatory factors in human-derived mesenchymal stromal cells the expression and impactof inflammatory primingrdquo Stem Cell Reviews vol 8 pp 1188ndash1198 2012
[7] R Rizzo D Campioni M Stignani et al ldquoA functional role forsoluble HLA-G antigens in immune modulation mediated bymesenchymal stromal cellsrdquoCytotherapy vol 10 no 4 pp 364ndash375 2008
10 Journal of Immunology Research
[8] F Morandi L Raffaghello G Bianchi et al ldquoImmunogenicityof human mesenchymal stem cells in HLA-class I-restricted T-cell responses against viral or tumor-associated antigensrdquo StemCells vol 26 no 5 pp 1275ndash1287 2008
[9] A Naji N Rouas-Freiss A Durrbach E D Carosella LSensebe and F Deschaseaux ldquoConcise review combininghuman leukocyte antigen G and mesenchymal stem cells forimmunosuppressant biotherapyrdquo Stem Cells vol 31 pp 2296ndash2303 2013
[10] G La Rocca and R Anzalone ldquoPerinatal stem cells revisiteddirections and indications at the crossroads between tissueregeneration and repairrdquoCurrent Stem Cell Research ampTherapyvol 8 pp 2ndash5 2013
[11] Z Selmani ANaji EGaiffe et al ldquoHLA-G is a crucial immuno-suppressive molecule secreted by adult human mesenchymalstem cellsrdquo Transplantation vol 87 no 9 supplement pp S62ndashS66 2009
[12] Z Selmani A Naji I Zidi et al ldquoHuman leukocyte antigen-G5 secretion by human mesenchymal stem cells is required tosuppress T lymphocyte andnatural killer function and to induceCD4+ CD25ℎ119894119892ℎFOXP3+ regulatory T cellsrdquo Stem Cells vol 26no 1 pp 212ndash222 2008
[13] F Deschaseaux J Gaillard A Langonne et al ldquoRegulationand function of immunosuppressivemolecule human leukocyteantigen G5 in human bone tissuerdquo The FASEB Journal vol 27pp 2977ndash2987 2013
[14] M Krampera ldquoMesenchymal stromal cell licensing a multistepprocessrdquo Leukemia vol 25 no 9 pp 1408ndash1414 2011
[15] P Moreau F Adrian-Cabestre C Menier et al ldquoIL-10 selec-tively induces HLA-G expression in human trophoblasts andmonocytesrdquo International Immunology vol 11 no 5 pp 803ndash811 1999
[16] O Brugiere GThabutM Pretolani et al ldquoImmunohistochem-ical study of HLA-G expression in lung transplant recipientsrdquoAmerican Journal of Transplantation vol 9 no 6 pp 1427ndash14382009
[17] S Lefebvre S Berrih-Aknin F Adrian et al ldquoA specificinterferon (IFN)-stimulated response element of the distalHLA-G promoter binds IFN-regulatory factor 1 and mediatesenhancement of this nonclassical class I gene by IFN-betardquoTheJournal of Biological Chemistry vol 276 no 9 pp 6133ndash61392001
[18] N Rouas-Freiss S Bruel C Menier C Marcou P Moreauand E D Carosella ldquoSwitch of HLA-G alternative splicing ina melanoma cell line causes loss of HLA-G1 expression andsensitivity to NK lysisrdquo International Journal of Cancer vol 117no 1 pp 114ndash122 2005
[19] S M Melief J J Zwaginga W E Fibbe and H RoelofsldquoAdipose tissue-derived multipotent stromal cells have a higherimmunomodulatory capacity than their bone marrow-derivedcounterpartsrdquo StemCells TranslationalMedicine vol 2 pp 455ndash463 2013
[20] J Y Tee V Vaghjiani Y H Liu P Murthi J Chan and UManuelpillai ldquoImmunogenicity and immunomodulatory prop-erties of hepatocyte-like cells derived from human amnioticepithelial cellsrdquo Current Stem Cell Research amp Therapy vol 8pp 91ndash99 2013
[21] G La Rocca M Lo Iacono T Corsello S Corrao F Farina andR Anzalone ldquoHuman Whartonrsquos jelly mesenchymal stem cellsmaintain the expression of key immunomodulatory moleculeswhen subjected to osteogenic adipogenic and chondrogenic
differentiation in vitro new perspectives for cellular therapyrdquoCurrent Stem Cell Research ampTherapy vol 8 pp 100ndash113 2013
[22] C Menard and K Tarte ldquoImmunoregulatory properties of clin-ical grade mesenchymal stromal cells evidence uncertaintiesand clinical applicationrdquo Stem Cell Research amp Therapy vol 4article 64 2013
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Journal of Immunology Research 7
MSC-BM 954 885 667 339 165888 812 647 310 153
0
50
100
Allo
prol
ifera
tion
inhi
bitio
n (
)
Bone marrow-derived mesenchymal stem cells
1 05 05 1 05 025 1 05 0125 1 05 006 1 05 003
Responder stimulator MSC ratioMSC-BM IFN120574TNF120572
(a)
MSC-AT 878 824 879 859 823883 831 741 662 572
0
50
100
Allo
prol
ifera
tion
inhi
bitio
n (
)Adipose tissue-derived mesenchymal stem cells
lowast lowast lowast
MSC-AT IFN120574TNF120572Responder stimulator MSC ratio
1 05 05 1 05 025 1 05 0125 1 05 006 1 05 003
(b)
Figure 6 Both BM- andAT-derived MSCs display immunosuppressive properties in a dose-dependent manner PBMC from healthyindividuals were used as responder cells towards irradiated LCLlowast used as stimulating cells in presence of either (a) BM-derivedMSCs (MSC-BM) or (b) AT-derived MSCs (MSC-AT) that were pretreated with IFN120574 and TNF120572 (IFN120574TNF120572) or not and used as third-party cells atvarious responder stimulator MSC ratios Results are given as mean percentage of alloproliferation inhibition plusmn sem according to themaximal alloproliferation observed with PBMC+LCLlowast using PBMC from 4 and 6 distinct healthy donors in BM- and AT-derived MSCsexperiments respectively
Table 1 MSC from BM after osteodifferentiation and licensing byIFN-120574 and TNF-120572 do not induce PBMC proliferation in allogenicconditions
R Saratio MSC-BM MSC-BM
IFN-120574TNF-120572 Os-MSC-BM Os-MSC-BMIFN-120574TNF-120572
1 05 43 plusmn 21b 11 plusmn 03 33 plusmn 03 17 plusmn 02
1 025 45 plusmn 14 10 plusmn 03 31 plusmn 01 18 plusmn 05
1 012 53 plusmn 19 11 plusmn 04 32 plusmn 10 23 plusmn 12
1 006 54 plusmn 24 14 plusmn 05 26 plusmn 11 39 plusmn 31
1 003 43 plusmn 17 14 plusmn 05 22 plusmn 02 21 plusmn 12
aPBMC from healthy individuals were used as responder cells towards BM-derived MSCs pretreated with IFN-120574 and TNF-120572 (MSC-BM IFN-120574TNF-120572) or not (MSC-BM) as stimulating cells at various responder stimulator(R S) ratios Similar experiments were performed with BM-derived MSCscommitted to osteodifferentiation process and pretreated with IFN-120574 andTNF-120572 (Os-MSC-BM IFN-120574TNF-120572) or not (Os-MSC-BM) as stimulatingcellsbData are mean plusmn sem of alloproliferation percentage obtained with 5and 3 distinct healthy donors for MSC-BM and Os-MSC-BM experimentsrespectively This percentage is calculated considering PBMC stimulatedwith LCLlowast as 100 alloproliferation
agreement with recent findings we observed enhancedHLA-Gexpression byMSCs following osteodifferentiation (Figures3(a) 3(b) and 4(b)) [13]
Table 2 MSC from AT after osteodifferentiation and licensing byIFN-120574 and TNF-120572 do not induce PBMC proliferation in allogenicconditions
R Saratio MSC-AT MSC-AT
IFN-120574TNF-120572 Os-MSC-AT Os-MSC-ATIFN-120574TNF-120572
1 05 56 plusmn 10b 72 plusmn 31 11 plusmn 02 09 plusmn 06
1 025 55 plusmn 15 52 plusmn 26 13 plusmn 03 08 plusmn 05
1 012 53 plusmn 15 47 plusmn 29 10 plusmn 03 11 plusmn 08
1 006 53 plusmn 20 55 plusmn 30 10 plusmn 02 13 plusmn 10
1 003 46 plusmn 11 39 plusmn 20 14 plusmn 03 18 plusmn 13
aPBMC from healthy individuals were used as responder cells towards AT-derived MSCs pretreated with IFN-120574 and TNF-120572 (MSC-AT IFN-120574TNF-120572) or not (MSC-AT) as stimulating cells at various responder stimulator(R S) ratios Similar experiments were performed with AT-derived MSCscommitted to osteodifferentiation process and pretreated with IFN-120574 andTNF-120572 (Os-MSC-AT IFN-120574TNF-120572) or not (Os-MSC-AT) as stimulatingcellsbData are mean plusmn sem of alloproliferation percentage obtained with 4and 3 distinct healthy donors for MSC-AT and Os-MSC-AT experimentsrespectively This percentage is calculated considering PBMC stimulatedwith LCLlowast as 100 alloproliferation
Then we investigated the immunomodulatory propertiesof MSCs as third-party cells in MLR Results showed thatbothBM-derived andAT-derivedMSCsdisplay immunosup-pressive properties in a dose-responsemanner (Figure 6) It is
8 Journal of Immunology Research
MSC-BM 940 911 864938 896 929
Allo
prol
ifera
tion
inhi
bitio
n (
)A
llopr
olife
ratio
n in
hibi
tion
()
Allo
prol
ifera
tion
inhi
bitio
n (
)A
llopr
olife
ratio
n in
hibi
tion
()
461 628 750379 676 796
0
50
100
850 803 893600 725 895
0
50
100
0
50
100
919 879 879909 867 887
0
50
100
Bone marrow-derived mesenchymal stem cells
Adipose tissue-derived mesenchymal stem cells
No MBCP
Bone marrow-derived mesenchymal stem cells
Adipose tissue-derived mesenchymal stem cells
MBCP
MSC-BM IFN120574TNF120572
MSC-BMMSC-BM IFN120574TNF120572
MSC-ATMSC-AT IFN120574TNF120572
MSC-ATMSC-AT IFN120574TNF120572
1 05 04 1 05 02 1 05 01 1 05 04 1 05 02 1 05 01
1 05 04 1 05 02 1 05 011 05 04 1 05 02 1 05 01
Responder stimulator MSC ratio Responder stimulator MSC ratio
Responder stimulator MSC ratio Responder stimulator MSC ratio
Figure 7 Both BM- andAT-derived MSCs when combined to MBCP biomaterial keep their immunosuppressive properties PBMC fromhealthy individuals were used as responder cells towards irradiated LCLlowast used as stimulating cells in presence of either BM-derived MSCs(MSC-BM) or AT-derived MSCs (MSC-AT) that were pretreated with IFN120574 and TNF120572 (IFN120574TNF120572) or not and used as third-party cellsat various responder stimulator MSC ratios MSCs were combined to MBCP biomaterial (MBCP) or not (No MBCP) Results are given asmean percentage of alloproliferation inhibition plusmn sem when compared to PBMC+LCLlowast using PBMC from 3 distinct healthy donors
Table 3 Immunogenicity of MSC from bone marrow and adiposetissue combinedwithMBCPbiomaterial and licensing by IFN-120574 andTNF-120572
MSC-BM MSC-BMIFN-120574TNF-120572 MSC-AT MSC-AT
IFN-120574TNF-120572No MBCPa 75 plusmn 22b 98 plusmn 40 139 plusmn 39 230 plusmn 52
MBCP 519 plusmn 141 411 plusmn 116 81 plusmn 08 110 plusmn 08
aPBMC from healthy individuals were used as responder cells towards BM-or AT-derivedMSCs pretreated with IFN-120574 and TNF-120572 or not as stimulatingcells at 1 02 responder stimulator ratiobData are mean plusmn sem of alloproliferation percentage obtained with 3distinct healthy donors This percentage is calculated considering PBMCstimulated with LCLlowast as 100 alloproliferation
of note that (i) AT-derivedMSCs aremore potent at low dosescompared to BM-derived MSCs (119875 lt 005) (Figures 6(a) and6(b)) and (ii) licensing with cytokines reduces significantlythe immunosuppressive properties of AT-derived MSCs(119875 lt 005) (Figure 6(b)) When combined to biomaterial(MBCP) both BM-derived and AT-derived MSCs still exertimmunosuppressive properties as they greatly inhibit T cell
alloproliferation with or without being seeded with MBCP(Figure 7) Although not statistically significant addition ofMBCP reduces the immunomodulatory properties of BM-derived MSCs at high responder stimulator MSC ratiosThis could be due to steric hindrance when high numbersof cells are used Indeed such MBCP effect is no longerobserved at low ratios (Figure 7) Also both BM-derivedand AT-derived MSCs when committed to preosteoblasticMSCs inhibit T cell alloproliferation and remain thus able toinduce a tolerogenicmicroenvironment (Figure 8) Licensingwith IFN-120574 and TNF-120572 did not modify such MSC-derivedimmunosuppression (Figures 7 and 8) It is of note thatonce osteodifferentiated AT-derived MSCs are more potentat low doses compared to BM-derived MSCs as they displayhigher immunosuppressive effects (119875 lt 005) (Figure 8)Such higher immunomodulatory capacity of adipose tissue-derived multipotent stromal cells compared to their bonemarrow-derived counterparts has been previously reported[19]
Our present results are in agreement with previousreports showing that differentiation of stem cells does not
Journal of Immunology Research 9
894 889 942 649 528956 925 976 678 367
0
50
100
Allo
prol
ifera
tion
inhi
bitio
n (
)
Osteodifferentiated bone marrow-derived MSC
MSC-BMOs-MSC-BM IFN120574TNF120572
1 05 05 1 05 025 1 05 0125 1 05 006 1 05 003
Responder stimulator MSC ratio
(a)
888 926 917 895 854936 935 913 859 780
0
50
100
Allo
prol
ifera
tion
inhi
bitio
n (
)Osteodifferentiated adipose tissue-derived MSC
Responder stimulator MSC ratio
MSC-ATMSC-AT IFN120574TNF120572
1 05 05 1 05 025 1 05 0125 1 05 006 1 05 003
(b)
Figure 8 Both BM- andAT-derived MSCs when committed to osteodifferentiation process keep their immunosuppressive propertiesPBMC from healthy individuals were used as responder cells towards irradiated LCLlowast used as stimulating cells in presence of eitherosteodifferentiated BM-derived MSCs (Os-MSC-BM) or AT-derived MSCs (Os-MSC-AT) that were pretreated with IFN120574 and TNF120572(IFN120574TNF120572) or not and used as third-party cells at various responder stimulator MSC ratios Results are given as mean percentage ofalloproliferation inhibition plusmn sem when compared to PBMC+LCLlowast using PBMC from 3 and 5 distinct healthy donors in BM- and AT-derived MSCs experiments respectively
alter their low immunogenicity and immunomodulatoryproperties For instance human amniotic epithelial cellswhich have stem cell-like properties retain their immuno-suppressive functions after differentiation into hepatocyte-like cells [20] Also human Whartonrsquos jelly-derived MSCsmaintain the expression of immunomodulatory moleculessuch as HLA-G when subjected to osteogenic differentiationin vitro [21]
In conclusion MSCs from BM or AT display tolerogenicproperties which are maintained following osteodifferenti-ation process or addition of biomaterial and may thus beconsidered as allogenic sources for regenerating bone defectsin orthopaedic and maxillofacial surgery [22]
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This project has received funding from the European UnionrsquosSeventh Programme for research technological develop-ment and demonstration underGrant agreement no 241879
References
[1] R Langer and J P Vacanti ldquoTissue engineeringrdquo Science vol260 no 5110 pp 920ndash926 1993
[2] C Laurencin Y Khan and S F El-Amin ldquoBone graft substi-tutesrdquo Expert Review of Medical Devices vol 3 no 1 pp 49ndash572006
[3] H Petite V ViateauW Bensaıd et al ldquoTissue-engineered boneregenerationrdquo Nature Biotechnology vol 18 no 9 pp 959ndash9632000
[4] M Di Trapani G Bassi M Ricciardi et al ldquoComparative studyof immune regulatory properties of stem cells derived fromdifferent tissuesrdquo StemCells andDevelopment vol 22 pp 2990ndash3002 2013
[5] AUccelli V Pistoia andLMoretta ldquoMesenchymal stemcells anew strategy for immunosuppressionrdquo Trends in Immunologyvol 28 no 5 pp 219ndash226 2007
[6] M Najar G Raicevic H Fayyad-Kazan et al ldquoImmune-relatedantigens surface molecules and regulatory factors in human-derived mesenchymal stromal cells the expression and impactof inflammatory primingrdquo Stem Cell Reviews vol 8 pp 1188ndash1198 2012
[7] R Rizzo D Campioni M Stignani et al ldquoA functional role forsoluble HLA-G antigens in immune modulation mediated bymesenchymal stromal cellsrdquoCytotherapy vol 10 no 4 pp 364ndash375 2008
10 Journal of Immunology Research
[8] F Morandi L Raffaghello G Bianchi et al ldquoImmunogenicityof human mesenchymal stem cells in HLA-class I-restricted T-cell responses against viral or tumor-associated antigensrdquo StemCells vol 26 no 5 pp 1275ndash1287 2008
[9] A Naji N Rouas-Freiss A Durrbach E D Carosella LSensebe and F Deschaseaux ldquoConcise review combininghuman leukocyte antigen G and mesenchymal stem cells forimmunosuppressant biotherapyrdquo Stem Cells vol 31 pp 2296ndash2303 2013
[10] G La Rocca and R Anzalone ldquoPerinatal stem cells revisiteddirections and indications at the crossroads between tissueregeneration and repairrdquoCurrent Stem Cell Research ampTherapyvol 8 pp 2ndash5 2013
[11] Z Selmani ANaji EGaiffe et al ldquoHLA-G is a crucial immuno-suppressive molecule secreted by adult human mesenchymalstem cellsrdquo Transplantation vol 87 no 9 supplement pp S62ndashS66 2009
[12] Z Selmani A Naji I Zidi et al ldquoHuman leukocyte antigen-G5 secretion by human mesenchymal stem cells is required tosuppress T lymphocyte andnatural killer function and to induceCD4+ CD25ℎ119894119892ℎFOXP3+ regulatory T cellsrdquo Stem Cells vol 26no 1 pp 212ndash222 2008
[13] F Deschaseaux J Gaillard A Langonne et al ldquoRegulationand function of immunosuppressivemolecule human leukocyteantigen G5 in human bone tissuerdquo The FASEB Journal vol 27pp 2977ndash2987 2013
[14] M Krampera ldquoMesenchymal stromal cell licensing a multistepprocessrdquo Leukemia vol 25 no 9 pp 1408ndash1414 2011
[15] P Moreau F Adrian-Cabestre C Menier et al ldquoIL-10 selec-tively induces HLA-G expression in human trophoblasts andmonocytesrdquo International Immunology vol 11 no 5 pp 803ndash811 1999
[16] O Brugiere GThabutM Pretolani et al ldquoImmunohistochem-ical study of HLA-G expression in lung transplant recipientsrdquoAmerican Journal of Transplantation vol 9 no 6 pp 1427ndash14382009
[17] S Lefebvre S Berrih-Aknin F Adrian et al ldquoA specificinterferon (IFN)-stimulated response element of the distalHLA-G promoter binds IFN-regulatory factor 1 and mediatesenhancement of this nonclassical class I gene by IFN-betardquoTheJournal of Biological Chemistry vol 276 no 9 pp 6133ndash61392001
[18] N Rouas-Freiss S Bruel C Menier C Marcou P Moreauand E D Carosella ldquoSwitch of HLA-G alternative splicing ina melanoma cell line causes loss of HLA-G1 expression andsensitivity to NK lysisrdquo International Journal of Cancer vol 117no 1 pp 114ndash122 2005
[19] S M Melief J J Zwaginga W E Fibbe and H RoelofsldquoAdipose tissue-derived multipotent stromal cells have a higherimmunomodulatory capacity than their bone marrow-derivedcounterpartsrdquo StemCells TranslationalMedicine vol 2 pp 455ndash463 2013
[20] J Y Tee V Vaghjiani Y H Liu P Murthi J Chan and UManuelpillai ldquoImmunogenicity and immunomodulatory prop-erties of hepatocyte-like cells derived from human amnioticepithelial cellsrdquo Current Stem Cell Research amp Therapy vol 8pp 91ndash99 2013
[21] G La Rocca M Lo Iacono T Corsello S Corrao F Farina andR Anzalone ldquoHuman Whartonrsquos jelly mesenchymal stem cellsmaintain the expression of key immunomodulatory moleculeswhen subjected to osteogenic adipogenic and chondrogenic
differentiation in vitro new perspectives for cellular therapyrdquoCurrent Stem Cell Research ampTherapy vol 8 pp 100ndash113 2013
[22] C Menard and K Tarte ldquoImmunoregulatory properties of clin-ical grade mesenchymal stromal cells evidence uncertaintiesand clinical applicationrdquo Stem Cell Research amp Therapy vol 4article 64 2013
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
8 Journal of Immunology Research
MSC-BM 940 911 864938 896 929
Allo
prol
ifera
tion
inhi
bitio
n (
)A
llopr
olife
ratio
n in
hibi
tion
()
Allo
prol
ifera
tion
inhi
bitio
n (
)A
llopr
olife
ratio
n in
hibi
tion
()
461 628 750379 676 796
0
50
100
850 803 893600 725 895
0
50
100
0
50
100
919 879 879909 867 887
0
50
100
Bone marrow-derived mesenchymal stem cells
Adipose tissue-derived mesenchymal stem cells
No MBCP
Bone marrow-derived mesenchymal stem cells
Adipose tissue-derived mesenchymal stem cells
MBCP
MSC-BM IFN120574TNF120572
MSC-BMMSC-BM IFN120574TNF120572
MSC-ATMSC-AT IFN120574TNF120572
MSC-ATMSC-AT IFN120574TNF120572
1 05 04 1 05 02 1 05 01 1 05 04 1 05 02 1 05 01
1 05 04 1 05 02 1 05 011 05 04 1 05 02 1 05 01
Responder stimulator MSC ratio Responder stimulator MSC ratio
Responder stimulator MSC ratio Responder stimulator MSC ratio
Figure 7 Both BM- andAT-derived MSCs when combined to MBCP biomaterial keep their immunosuppressive properties PBMC fromhealthy individuals were used as responder cells towards irradiated LCLlowast used as stimulating cells in presence of either BM-derived MSCs(MSC-BM) or AT-derived MSCs (MSC-AT) that were pretreated with IFN120574 and TNF120572 (IFN120574TNF120572) or not and used as third-party cellsat various responder stimulator MSC ratios MSCs were combined to MBCP biomaterial (MBCP) or not (No MBCP) Results are given asmean percentage of alloproliferation inhibition plusmn sem when compared to PBMC+LCLlowast using PBMC from 3 distinct healthy donors
Table 3 Immunogenicity of MSC from bone marrow and adiposetissue combinedwithMBCPbiomaterial and licensing by IFN-120574 andTNF-120572
MSC-BM MSC-BMIFN-120574TNF-120572 MSC-AT MSC-AT
IFN-120574TNF-120572No MBCPa 75 plusmn 22b 98 plusmn 40 139 plusmn 39 230 plusmn 52
MBCP 519 plusmn 141 411 plusmn 116 81 plusmn 08 110 plusmn 08
aPBMC from healthy individuals were used as responder cells towards BM-or AT-derivedMSCs pretreated with IFN-120574 and TNF-120572 or not as stimulatingcells at 1 02 responder stimulator ratiobData are mean plusmn sem of alloproliferation percentage obtained with 3distinct healthy donors This percentage is calculated considering PBMCstimulated with LCLlowast as 100 alloproliferation
of note that (i) AT-derivedMSCs aremore potent at low dosescompared to BM-derived MSCs (119875 lt 005) (Figures 6(a) and6(b)) and (ii) licensing with cytokines reduces significantlythe immunosuppressive properties of AT-derived MSCs(119875 lt 005) (Figure 6(b)) When combined to biomaterial(MBCP) both BM-derived and AT-derived MSCs still exertimmunosuppressive properties as they greatly inhibit T cell
alloproliferation with or without being seeded with MBCP(Figure 7) Although not statistically significant addition ofMBCP reduces the immunomodulatory properties of BM-derived MSCs at high responder stimulator MSC ratiosThis could be due to steric hindrance when high numbersof cells are used Indeed such MBCP effect is no longerobserved at low ratios (Figure 7) Also both BM-derivedand AT-derived MSCs when committed to preosteoblasticMSCs inhibit T cell alloproliferation and remain thus able toinduce a tolerogenicmicroenvironment (Figure 8) Licensingwith IFN-120574 and TNF-120572 did not modify such MSC-derivedimmunosuppression (Figures 7 and 8) It is of note thatonce osteodifferentiated AT-derived MSCs are more potentat low doses compared to BM-derived MSCs as they displayhigher immunosuppressive effects (119875 lt 005) (Figure 8)Such higher immunomodulatory capacity of adipose tissue-derived multipotent stromal cells compared to their bonemarrow-derived counterparts has been previously reported[19]
Our present results are in agreement with previousreports showing that differentiation of stem cells does not
Journal of Immunology Research 9
894 889 942 649 528956 925 976 678 367
0
50
100
Allo
prol
ifera
tion
inhi
bitio
n (
)
Osteodifferentiated bone marrow-derived MSC
MSC-BMOs-MSC-BM IFN120574TNF120572
1 05 05 1 05 025 1 05 0125 1 05 006 1 05 003
Responder stimulator MSC ratio
(a)
888 926 917 895 854936 935 913 859 780
0
50
100
Allo
prol
ifera
tion
inhi
bitio
n (
)Osteodifferentiated adipose tissue-derived MSC
Responder stimulator MSC ratio
MSC-ATMSC-AT IFN120574TNF120572
1 05 05 1 05 025 1 05 0125 1 05 006 1 05 003
(b)
Figure 8 Both BM- andAT-derived MSCs when committed to osteodifferentiation process keep their immunosuppressive propertiesPBMC from healthy individuals were used as responder cells towards irradiated LCLlowast used as stimulating cells in presence of eitherosteodifferentiated BM-derived MSCs (Os-MSC-BM) or AT-derived MSCs (Os-MSC-AT) that were pretreated with IFN120574 and TNF120572(IFN120574TNF120572) or not and used as third-party cells at various responder stimulator MSC ratios Results are given as mean percentage ofalloproliferation inhibition plusmn sem when compared to PBMC+LCLlowast using PBMC from 3 and 5 distinct healthy donors in BM- and AT-derived MSCs experiments respectively
alter their low immunogenicity and immunomodulatoryproperties For instance human amniotic epithelial cellswhich have stem cell-like properties retain their immuno-suppressive functions after differentiation into hepatocyte-like cells [20] Also human Whartonrsquos jelly-derived MSCsmaintain the expression of immunomodulatory moleculessuch as HLA-G when subjected to osteogenic differentiationin vitro [21]
In conclusion MSCs from BM or AT display tolerogenicproperties which are maintained following osteodifferenti-ation process or addition of biomaterial and may thus beconsidered as allogenic sources for regenerating bone defectsin orthopaedic and maxillofacial surgery [22]
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This project has received funding from the European UnionrsquosSeventh Programme for research technological develop-ment and demonstration underGrant agreement no 241879
References
[1] R Langer and J P Vacanti ldquoTissue engineeringrdquo Science vol260 no 5110 pp 920ndash926 1993
[2] C Laurencin Y Khan and S F El-Amin ldquoBone graft substi-tutesrdquo Expert Review of Medical Devices vol 3 no 1 pp 49ndash572006
[3] H Petite V ViateauW Bensaıd et al ldquoTissue-engineered boneregenerationrdquo Nature Biotechnology vol 18 no 9 pp 959ndash9632000
[4] M Di Trapani G Bassi M Ricciardi et al ldquoComparative studyof immune regulatory properties of stem cells derived fromdifferent tissuesrdquo StemCells andDevelopment vol 22 pp 2990ndash3002 2013
[5] AUccelli V Pistoia andLMoretta ldquoMesenchymal stemcells anew strategy for immunosuppressionrdquo Trends in Immunologyvol 28 no 5 pp 219ndash226 2007
[6] M Najar G Raicevic H Fayyad-Kazan et al ldquoImmune-relatedantigens surface molecules and regulatory factors in human-derived mesenchymal stromal cells the expression and impactof inflammatory primingrdquo Stem Cell Reviews vol 8 pp 1188ndash1198 2012
[7] R Rizzo D Campioni M Stignani et al ldquoA functional role forsoluble HLA-G antigens in immune modulation mediated bymesenchymal stromal cellsrdquoCytotherapy vol 10 no 4 pp 364ndash375 2008
10 Journal of Immunology Research
[8] F Morandi L Raffaghello G Bianchi et al ldquoImmunogenicityof human mesenchymal stem cells in HLA-class I-restricted T-cell responses against viral or tumor-associated antigensrdquo StemCells vol 26 no 5 pp 1275ndash1287 2008
[9] A Naji N Rouas-Freiss A Durrbach E D Carosella LSensebe and F Deschaseaux ldquoConcise review combininghuman leukocyte antigen G and mesenchymal stem cells forimmunosuppressant biotherapyrdquo Stem Cells vol 31 pp 2296ndash2303 2013
[10] G La Rocca and R Anzalone ldquoPerinatal stem cells revisiteddirections and indications at the crossroads between tissueregeneration and repairrdquoCurrent Stem Cell Research ampTherapyvol 8 pp 2ndash5 2013
[11] Z Selmani ANaji EGaiffe et al ldquoHLA-G is a crucial immuno-suppressive molecule secreted by adult human mesenchymalstem cellsrdquo Transplantation vol 87 no 9 supplement pp S62ndashS66 2009
[12] Z Selmani A Naji I Zidi et al ldquoHuman leukocyte antigen-G5 secretion by human mesenchymal stem cells is required tosuppress T lymphocyte andnatural killer function and to induceCD4+ CD25ℎ119894119892ℎFOXP3+ regulatory T cellsrdquo Stem Cells vol 26no 1 pp 212ndash222 2008
[13] F Deschaseaux J Gaillard A Langonne et al ldquoRegulationand function of immunosuppressivemolecule human leukocyteantigen G5 in human bone tissuerdquo The FASEB Journal vol 27pp 2977ndash2987 2013
[14] M Krampera ldquoMesenchymal stromal cell licensing a multistepprocessrdquo Leukemia vol 25 no 9 pp 1408ndash1414 2011
[15] P Moreau F Adrian-Cabestre C Menier et al ldquoIL-10 selec-tively induces HLA-G expression in human trophoblasts andmonocytesrdquo International Immunology vol 11 no 5 pp 803ndash811 1999
[16] O Brugiere GThabutM Pretolani et al ldquoImmunohistochem-ical study of HLA-G expression in lung transplant recipientsrdquoAmerican Journal of Transplantation vol 9 no 6 pp 1427ndash14382009
[17] S Lefebvre S Berrih-Aknin F Adrian et al ldquoA specificinterferon (IFN)-stimulated response element of the distalHLA-G promoter binds IFN-regulatory factor 1 and mediatesenhancement of this nonclassical class I gene by IFN-betardquoTheJournal of Biological Chemistry vol 276 no 9 pp 6133ndash61392001
[18] N Rouas-Freiss S Bruel C Menier C Marcou P Moreauand E D Carosella ldquoSwitch of HLA-G alternative splicing ina melanoma cell line causes loss of HLA-G1 expression andsensitivity to NK lysisrdquo International Journal of Cancer vol 117no 1 pp 114ndash122 2005
[19] S M Melief J J Zwaginga W E Fibbe and H RoelofsldquoAdipose tissue-derived multipotent stromal cells have a higherimmunomodulatory capacity than their bone marrow-derivedcounterpartsrdquo StemCells TranslationalMedicine vol 2 pp 455ndash463 2013
[20] J Y Tee V Vaghjiani Y H Liu P Murthi J Chan and UManuelpillai ldquoImmunogenicity and immunomodulatory prop-erties of hepatocyte-like cells derived from human amnioticepithelial cellsrdquo Current Stem Cell Research amp Therapy vol 8pp 91ndash99 2013
[21] G La Rocca M Lo Iacono T Corsello S Corrao F Farina andR Anzalone ldquoHuman Whartonrsquos jelly mesenchymal stem cellsmaintain the expression of key immunomodulatory moleculeswhen subjected to osteogenic adipogenic and chondrogenic
differentiation in vitro new perspectives for cellular therapyrdquoCurrent Stem Cell Research ampTherapy vol 8 pp 100ndash113 2013
[22] C Menard and K Tarte ldquoImmunoregulatory properties of clin-ical grade mesenchymal stromal cells evidence uncertaintiesand clinical applicationrdquo Stem Cell Research amp Therapy vol 4article 64 2013
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Journal of Immunology Research 9
894 889 942 649 528956 925 976 678 367
0
50
100
Allo
prol
ifera
tion
inhi
bitio
n (
)
Osteodifferentiated bone marrow-derived MSC
MSC-BMOs-MSC-BM IFN120574TNF120572
1 05 05 1 05 025 1 05 0125 1 05 006 1 05 003
Responder stimulator MSC ratio
(a)
888 926 917 895 854936 935 913 859 780
0
50
100
Allo
prol
ifera
tion
inhi
bitio
n (
)Osteodifferentiated adipose tissue-derived MSC
Responder stimulator MSC ratio
MSC-ATMSC-AT IFN120574TNF120572
1 05 05 1 05 025 1 05 0125 1 05 006 1 05 003
(b)
Figure 8 Both BM- andAT-derived MSCs when committed to osteodifferentiation process keep their immunosuppressive propertiesPBMC from healthy individuals were used as responder cells towards irradiated LCLlowast used as stimulating cells in presence of eitherosteodifferentiated BM-derived MSCs (Os-MSC-BM) or AT-derived MSCs (Os-MSC-AT) that were pretreated with IFN120574 and TNF120572(IFN120574TNF120572) or not and used as third-party cells at various responder stimulator MSC ratios Results are given as mean percentage ofalloproliferation inhibition plusmn sem when compared to PBMC+LCLlowast using PBMC from 3 and 5 distinct healthy donors in BM- and AT-derived MSCs experiments respectively
alter their low immunogenicity and immunomodulatoryproperties For instance human amniotic epithelial cellswhich have stem cell-like properties retain their immuno-suppressive functions after differentiation into hepatocyte-like cells [20] Also human Whartonrsquos jelly-derived MSCsmaintain the expression of immunomodulatory moleculessuch as HLA-G when subjected to osteogenic differentiationin vitro [21]
In conclusion MSCs from BM or AT display tolerogenicproperties which are maintained following osteodifferenti-ation process or addition of biomaterial and may thus beconsidered as allogenic sources for regenerating bone defectsin orthopaedic and maxillofacial surgery [22]
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This project has received funding from the European UnionrsquosSeventh Programme for research technological develop-ment and demonstration underGrant agreement no 241879
References
[1] R Langer and J P Vacanti ldquoTissue engineeringrdquo Science vol260 no 5110 pp 920ndash926 1993
[2] C Laurencin Y Khan and S F El-Amin ldquoBone graft substi-tutesrdquo Expert Review of Medical Devices vol 3 no 1 pp 49ndash572006
[3] H Petite V ViateauW Bensaıd et al ldquoTissue-engineered boneregenerationrdquo Nature Biotechnology vol 18 no 9 pp 959ndash9632000
[4] M Di Trapani G Bassi M Ricciardi et al ldquoComparative studyof immune regulatory properties of stem cells derived fromdifferent tissuesrdquo StemCells andDevelopment vol 22 pp 2990ndash3002 2013
[5] AUccelli V Pistoia andLMoretta ldquoMesenchymal stemcells anew strategy for immunosuppressionrdquo Trends in Immunologyvol 28 no 5 pp 219ndash226 2007
[6] M Najar G Raicevic H Fayyad-Kazan et al ldquoImmune-relatedantigens surface molecules and regulatory factors in human-derived mesenchymal stromal cells the expression and impactof inflammatory primingrdquo Stem Cell Reviews vol 8 pp 1188ndash1198 2012
[7] R Rizzo D Campioni M Stignani et al ldquoA functional role forsoluble HLA-G antigens in immune modulation mediated bymesenchymal stromal cellsrdquoCytotherapy vol 10 no 4 pp 364ndash375 2008
10 Journal of Immunology Research
[8] F Morandi L Raffaghello G Bianchi et al ldquoImmunogenicityof human mesenchymal stem cells in HLA-class I-restricted T-cell responses against viral or tumor-associated antigensrdquo StemCells vol 26 no 5 pp 1275ndash1287 2008
[9] A Naji N Rouas-Freiss A Durrbach E D Carosella LSensebe and F Deschaseaux ldquoConcise review combininghuman leukocyte antigen G and mesenchymal stem cells forimmunosuppressant biotherapyrdquo Stem Cells vol 31 pp 2296ndash2303 2013
[10] G La Rocca and R Anzalone ldquoPerinatal stem cells revisiteddirections and indications at the crossroads between tissueregeneration and repairrdquoCurrent Stem Cell Research ampTherapyvol 8 pp 2ndash5 2013
[11] Z Selmani ANaji EGaiffe et al ldquoHLA-G is a crucial immuno-suppressive molecule secreted by adult human mesenchymalstem cellsrdquo Transplantation vol 87 no 9 supplement pp S62ndashS66 2009
[12] Z Selmani A Naji I Zidi et al ldquoHuman leukocyte antigen-G5 secretion by human mesenchymal stem cells is required tosuppress T lymphocyte andnatural killer function and to induceCD4+ CD25ℎ119894119892ℎFOXP3+ regulatory T cellsrdquo Stem Cells vol 26no 1 pp 212ndash222 2008
[13] F Deschaseaux J Gaillard A Langonne et al ldquoRegulationand function of immunosuppressivemolecule human leukocyteantigen G5 in human bone tissuerdquo The FASEB Journal vol 27pp 2977ndash2987 2013
[14] M Krampera ldquoMesenchymal stromal cell licensing a multistepprocessrdquo Leukemia vol 25 no 9 pp 1408ndash1414 2011
[15] P Moreau F Adrian-Cabestre C Menier et al ldquoIL-10 selec-tively induces HLA-G expression in human trophoblasts andmonocytesrdquo International Immunology vol 11 no 5 pp 803ndash811 1999
[16] O Brugiere GThabutM Pretolani et al ldquoImmunohistochem-ical study of HLA-G expression in lung transplant recipientsrdquoAmerican Journal of Transplantation vol 9 no 6 pp 1427ndash14382009
[17] S Lefebvre S Berrih-Aknin F Adrian et al ldquoA specificinterferon (IFN)-stimulated response element of the distalHLA-G promoter binds IFN-regulatory factor 1 and mediatesenhancement of this nonclassical class I gene by IFN-betardquoTheJournal of Biological Chemistry vol 276 no 9 pp 6133ndash61392001
[18] N Rouas-Freiss S Bruel C Menier C Marcou P Moreauand E D Carosella ldquoSwitch of HLA-G alternative splicing ina melanoma cell line causes loss of HLA-G1 expression andsensitivity to NK lysisrdquo International Journal of Cancer vol 117no 1 pp 114ndash122 2005
[19] S M Melief J J Zwaginga W E Fibbe and H RoelofsldquoAdipose tissue-derived multipotent stromal cells have a higherimmunomodulatory capacity than their bone marrow-derivedcounterpartsrdquo StemCells TranslationalMedicine vol 2 pp 455ndash463 2013
[20] J Y Tee V Vaghjiani Y H Liu P Murthi J Chan and UManuelpillai ldquoImmunogenicity and immunomodulatory prop-erties of hepatocyte-like cells derived from human amnioticepithelial cellsrdquo Current Stem Cell Research amp Therapy vol 8pp 91ndash99 2013
[21] G La Rocca M Lo Iacono T Corsello S Corrao F Farina andR Anzalone ldquoHuman Whartonrsquos jelly mesenchymal stem cellsmaintain the expression of key immunomodulatory moleculeswhen subjected to osteogenic adipogenic and chondrogenic
differentiation in vitro new perspectives for cellular therapyrdquoCurrent Stem Cell Research ampTherapy vol 8 pp 100ndash113 2013
[22] C Menard and K Tarte ldquoImmunoregulatory properties of clin-ical grade mesenchymal stromal cells evidence uncertaintiesand clinical applicationrdquo Stem Cell Research amp Therapy vol 4article 64 2013
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
10 Journal of Immunology Research
[8] F Morandi L Raffaghello G Bianchi et al ldquoImmunogenicityof human mesenchymal stem cells in HLA-class I-restricted T-cell responses against viral or tumor-associated antigensrdquo StemCells vol 26 no 5 pp 1275ndash1287 2008
[9] A Naji N Rouas-Freiss A Durrbach E D Carosella LSensebe and F Deschaseaux ldquoConcise review combininghuman leukocyte antigen G and mesenchymal stem cells forimmunosuppressant biotherapyrdquo Stem Cells vol 31 pp 2296ndash2303 2013
[10] G La Rocca and R Anzalone ldquoPerinatal stem cells revisiteddirections and indications at the crossroads between tissueregeneration and repairrdquoCurrent Stem Cell Research ampTherapyvol 8 pp 2ndash5 2013
[11] Z Selmani ANaji EGaiffe et al ldquoHLA-G is a crucial immuno-suppressive molecule secreted by adult human mesenchymalstem cellsrdquo Transplantation vol 87 no 9 supplement pp S62ndashS66 2009
[12] Z Selmani A Naji I Zidi et al ldquoHuman leukocyte antigen-G5 secretion by human mesenchymal stem cells is required tosuppress T lymphocyte andnatural killer function and to induceCD4+ CD25ℎ119894119892ℎFOXP3+ regulatory T cellsrdquo Stem Cells vol 26no 1 pp 212ndash222 2008
[13] F Deschaseaux J Gaillard A Langonne et al ldquoRegulationand function of immunosuppressivemolecule human leukocyteantigen G5 in human bone tissuerdquo The FASEB Journal vol 27pp 2977ndash2987 2013
[14] M Krampera ldquoMesenchymal stromal cell licensing a multistepprocessrdquo Leukemia vol 25 no 9 pp 1408ndash1414 2011
[15] P Moreau F Adrian-Cabestre C Menier et al ldquoIL-10 selec-tively induces HLA-G expression in human trophoblasts andmonocytesrdquo International Immunology vol 11 no 5 pp 803ndash811 1999
[16] O Brugiere GThabutM Pretolani et al ldquoImmunohistochem-ical study of HLA-G expression in lung transplant recipientsrdquoAmerican Journal of Transplantation vol 9 no 6 pp 1427ndash14382009
[17] S Lefebvre S Berrih-Aknin F Adrian et al ldquoA specificinterferon (IFN)-stimulated response element of the distalHLA-G promoter binds IFN-regulatory factor 1 and mediatesenhancement of this nonclassical class I gene by IFN-betardquoTheJournal of Biological Chemistry vol 276 no 9 pp 6133ndash61392001
[18] N Rouas-Freiss S Bruel C Menier C Marcou P Moreauand E D Carosella ldquoSwitch of HLA-G alternative splicing ina melanoma cell line causes loss of HLA-G1 expression andsensitivity to NK lysisrdquo International Journal of Cancer vol 117no 1 pp 114ndash122 2005
[19] S M Melief J J Zwaginga W E Fibbe and H RoelofsldquoAdipose tissue-derived multipotent stromal cells have a higherimmunomodulatory capacity than their bone marrow-derivedcounterpartsrdquo StemCells TranslationalMedicine vol 2 pp 455ndash463 2013
[20] J Y Tee V Vaghjiani Y H Liu P Murthi J Chan and UManuelpillai ldquoImmunogenicity and immunomodulatory prop-erties of hepatocyte-like cells derived from human amnioticepithelial cellsrdquo Current Stem Cell Research amp Therapy vol 8pp 91ndash99 2013
[21] G La Rocca M Lo Iacono T Corsello S Corrao F Farina andR Anzalone ldquoHuman Whartonrsquos jelly mesenchymal stem cellsmaintain the expression of key immunomodulatory moleculeswhen subjected to osteogenic adipogenic and chondrogenic
differentiation in vitro new perspectives for cellular therapyrdquoCurrent Stem Cell Research ampTherapy vol 8 pp 100ndash113 2013
[22] C Menard and K Tarte ldquoImmunoregulatory properties of clin-ical grade mesenchymal stromal cells evidence uncertaintiesand clinical applicationrdquo Stem Cell Research amp Therapy vol 4article 64 2013
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom