research article osteodifferentiated mesenchymal stem...

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

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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


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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


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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


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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


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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




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






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


MSC-BM 940 911 864938 896 929

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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


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



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


[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


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


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


(a)

Cou

nts

Cou

nts

100 101 102 103 104 100 101 102 103 104

R11 R12

MSC-BM

R13 R14

IgG1HLA-G5tubulin


FL 2 FL 2

(b)

020406080

100120140160 Controls

020406080

100120140160

MSC-BM

LCLlowast

MSC

-BM

(CPM

times10

minus3)

(CPM

times10

minus3)


PBM

C15

MSC

-BM

IFN120574

TN

F120572

PBM

C15

LCL

lowast


05

1 025

1 012

1 006

1 003


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


100 101 102 103 104

FL 2

100 101 102 103 104

FL 2

Cou

nts

Cou

nts

R11 R12

IgG1HLA-DR

R13 R14


(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


IFN120574TNF120572

(b)

0102030405060708090

100Controls

0102030405060708090

100

(CPM

times10

minus3)

(CPM

times10

minus3)


LCLlowast

MSC

-AT

PBM

C59-

MSC-AT

lowastPB

MC

59

- LC

L

MSC

-AT

IFN120574

TN

F120572


05

1 025

1 012

1 006

1 003


3H

-thym

idin

e inc

orpo

ratio

n

3H

-thym

idin

e inc

orpo

ratio

n

(c)




2PO4 and






IgG1ALPtubulin

100 101 102 103 104

FL 2

100 101 102 103 104

FL 2

R11 R12

Cou

nts

Cou

nts

R13 R14


(a)

IgG1ALPtubulin

100 101 102 103 104

FL 2

100 101 102 103 104

FL 2

R11 R12

Cou

nts

Cou

nts

R13 R14


(b)

0102030405060708090

100 Controls

0102030405060708090

100

Os-MSC-BM


(CPM

times10

minus3)

(CPM

times10

minus3)

LCLlowast

Os-

MSC

-BM

PBM

C00

Os-

MSC

-BM

IFN120574

TN

F120572 lowastPB

MC

00

LCL


05

1 025

1 012

1 006

1 003


3H

-thym

idin

e inc

orpo

ratio

n

3H

-thym

idin

e inc

orpo

ratio

n

(c)









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


(a)

100 101 102 103 104

FL 2

100 101 102 103 104

FL 2

R12R11 R13 R14

Os-MSC-AT


Cou

nts

Cou

nts


(b)

0102030405060708090

Controls


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


05

1 025

1 012

1 006

1 003


3H

-thym

idin

e inc

orpo

ratio

n

3H

-thym

idin

e inc

orpo

ratio

n

(c)









0

10

20

30

40

50


PMtimes10

minus3)

LCLlowast

MSC

-BM

0

10

20

30

40

50


(CPM

times10

minus3)

LCLlowast

MSC

-AT

0

10

20

30

40


(CPM

times10

minus3)

+

0

10

20

30

40


(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






MSC-BM 954 885 667 339 165888 812 647 310 153

0

50

100

Allo

prol

ifera

tion

inhi

bitio

n (

)


1 05 05 1 05 025 1 05 0125 1 05 006 1 05 003


(a)

MSC-AT 878 824 879 859 823883 831 741 662 572

0

50

100

Allo

prol

ifera

tion

inhi

bitio

n (




1 05 05 1 05 025 1 05 0125 1 05 006 1 05 003

(b)























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



No MBCP



MBCP





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













894 889 942 649 528956 925 976 678 367

0

50

100

Allo

prol

ifera

tion

inhi

bitio

n (

)



1 05 05 1 05 025 1 05 0125 1 05 006 1 05 003


(a)

888 926 917 895 854936 935 913 859 780

0

50

100

Allo

prol

ifera

tion

inhi

bitio

n (




1 05 05 1 05 025 1 05 0125 1 05 006 1 05 003

(b)






Acknowledgments


References






























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















100 101 102 103 104

FL 2

100 101 102 103 104

FL 2

Cou

nts

Cou

nts

R11 R12

IgG1HLA-DR

R13 R14


(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


IFN120574TNF120572

(b)

0102030405060708090

100Controls

0102030405060708090

100

(CPM

times10

minus3)

(CPM

times10

minus3)


LCLlowast

MSC

-AT

PBM

C59-

MSC-AT

lowastPB

MC

59

- LC

L

MSC

-AT

IFN120574

TN

F120572


05

1 025

1 012

1 006

1 003


3H

-thym

idin

e inc

orpo

ratio

n

3H

-thym

idin

e inc

orpo

ratio

n

(c)




2PO4 and






IgG1ALPtubulin

100 101 102 103 104

FL 2

100 101 102 103 104

FL 2

R11 R12

Cou

nts

Cou

nts

R13 R14


(a)

IgG1ALPtubulin

100 101 102 103 104

FL 2

100 101 102 103 104

FL 2

R11 R12

Cou

nts

Cou

nts

R13 R14


(b)

0102030405060708090

100 Controls

0102030405060708090

100

Os-MSC-BM


(CPM

times10

minus3)

(CPM

times10

minus3)

LCLlowast

Os-

MSC

-BM

PBM

C00

Os-

MSC

-BM

IFN120574

TN

F120572 lowastPB

MC

00

LCL


05

1 025

1 012

1 006

1 003


3H

-thym

idin

e inc

orpo

ratio

n

3H

-thym

idin

e inc

orpo

ratio

n

(c)









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


(a)

100 101 102 103 104

FL 2

100 101 102 103 104

FL 2

R12R11 R13 R14

Os-MSC-AT


Cou

nts

Cou

nts


(b)

0102030405060708090

Controls


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


05

1 025

1 012

1 006

1 003


3H

-thym

idin

e inc

orpo

ratio

n

3H

-thym

idin

e inc

orpo

ratio

n

(c)









0

10

20

30

40

50


PMtimes10

minus3)

LCLlowast

MSC

-BM

0

10

20

30

40

50


(CPM

times10

minus3)

LCLlowast

MSC

-AT

0

10

20

30

40


(CPM

times10

minus3)

+

0

10

20

30

40


(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






MSC-BM 954 885 667 339 165888 812 647 310 153

0

50

100

Allo

prol

ifera

tion

inhi

bitio

n (

)


1 05 05 1 05 025 1 05 0125 1 05 006 1 05 003


(a)

MSC-AT 878 824 879 859 823883 831 741 662 572

0

50

100

Allo

prol

ifera

tion

inhi

bitio

n (




1 05 05 1 05 025 1 05 0125 1 05 006 1 05 003

(b)























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



No MBCP



MBCP





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













894 889 942 649 528956 925 976 678 367

0

50

100

Allo

prol

ifera

tion

inhi

bitio

n (

)



1 05 05 1 05 025 1 05 0125 1 05 006 1 05 003


(a)

888 926 917 895 854936 935 913 859 780

0

50

100

Allo

prol

ifera

tion

inhi

bitio

n (




1 05 05 1 05 025 1 05 0125 1 05 006 1 05 003

(b)






Acknowledgments


References






























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















IgG1ALPtubulin

100 101 102 103 104

FL 2

100 101 102 103 104

FL 2

R11 R12

Cou

nts

Cou

nts

R13 R14


(a)

IgG1ALPtubulin

100 101 102 103 104

FL 2

100 101 102 103 104

FL 2

R11 R12

Cou

nts

Cou

nts

R13 R14


(b)

0102030405060708090

100 Controls

0102030405060708090

100

Os-MSC-BM


(CPM

times10

minus3)

(CPM

times10

minus3)

LCLlowast

Os-

MSC

-BM

PBM

C00

Os-

MSC

-BM

IFN120574

TN

F120572 lowastPB

MC

00

LCL


05

1 025

1 012

1 006

1 003


3H

-thym

idin

e inc

orpo

ratio

n

3H

-thym

idin

e inc

orpo

ratio

n

(c)









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


(a)

100 101 102 103 104

FL 2

100 101 102 103 104

FL 2

R12R11 R13 R14

Os-MSC-AT


Cou

nts

Cou

nts


(b)

0102030405060708090

Controls


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


05

1 025

1 012

1 006

1 003


3H

-thym

idin

e inc

orpo

ratio

n

3H

-thym

idin

e inc

orpo

ratio

n

(c)









0

10

20

30

40

50


PMtimes10

minus3)

LCLlowast

MSC

-BM

0

10

20

30

40

50


(CPM

times10

minus3)

LCLlowast

MSC

-AT

0

10

20

30

40


(CPM

times10

minus3)

+

0

10

20

30

40


(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






MSC-BM 954 885 667 339 165888 812 647 310 153

0

50

100

Allo

prol

ifera

tion

inhi

bitio

n (

)


1 05 05 1 05 025 1 05 0125 1 05 006 1 05 003


(a)

MSC-AT 878 824 879 859 823883 831 741 662 572

0

50

100

Allo

prol

ifera

tion

inhi

bitio

n (




1 05 05 1 05 025 1 05 0125 1 05 006 1 05 003

(b)























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



No MBCP



MBCP





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













894 889 942 649 528956 925 976 678 367

0

50

100

Allo

prol

ifera

tion

inhi

bitio

n (

)



1 05 05 1 05 025 1 05 0125 1 05 006 1 05 003


(a)

888 926 917 895 854936 935 913 859 780

0

50

100

Allo

prol

ifera

tion

inhi

bitio

n (




1 05 05 1 05 025 1 05 0125 1 05 006 1 05 003

(b)






Acknowledgments


References






























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















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


(a)

100 101 102 103 104

FL 2

100 101 102 103 104

FL 2

R12R11 R13 R14

Os-MSC-AT


Cou

nts

Cou

nts


(b)

0102030405060708090

Controls


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


05

1 025

1 012

1 006

1 003


3H

-thym

idin

e inc

orpo

ratio

n

3H

-thym

idin

e inc

orpo

ratio

n

(c)









0

10

20

30

40

50


PMtimes10

minus3)

LCLlowast

MSC

-BM

0

10

20

30

40

50


(CPM

times10

minus3)

LCLlowast

MSC

-AT

0

10

20

30

40


(CPM

times10

minus3)

+

0

10

20

30

40


(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






MSC-BM 954 885 667 339 165888 812 647 310 153

0

50

100

Allo

prol

ifera

tion

inhi

bitio

n (

)


1 05 05 1 05 025 1 05 0125 1 05 006 1 05 003


(a)

MSC-AT 878 824 879 859 823883 831 741 662 572

0

50

100

Allo

prol

ifera

tion

inhi

bitio

n (




1 05 05 1 05 025 1 05 0125 1 05 006 1 05 003

(b)























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



No MBCP



MBCP





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













894 889 942 649 528956 925 976 678 367

0

50

100

Allo

prol

ifera

tion

inhi

bitio

n (

)



1 05 05 1 05 025 1 05 0125 1 05 006 1 05 003


(a)

888 926 917 895 854936 935 913 859 780

0

50

100

Allo

prol

ifera

tion

inhi

bitio

n (




1 05 05 1 05 025 1 05 0125 1 05 006 1 05 003

(b)






Acknowledgments


References






























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

10

20

30

40

50


PMtimes10

minus3)

LCLlowast

MSC

-BM

0

10

20

30

40

50


(CPM

times10

minus3)

LCLlowast

MSC

-AT

0

10

20

30

40


(CPM

times10

minus3)

+

0

10

20

30

40


(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






MSC-BM 954 885 667 339 165888 812 647 310 153

0

50

100

Allo

prol

ifera

tion

inhi

bitio

n (

)


1 05 05 1 05 025 1 05 0125 1 05 006 1 05 003


(a)

MSC-AT 878 824 879 859 823883 831 741 662 572

0

50

100

Allo

prol

ifera

tion

inhi

bitio

n (




1 05 05 1 05 025 1 05 0125 1 05 006 1 05 003

(b)























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



No MBCP



MBCP





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













894 889 942 649 528956 925 976 678 367

0

50

100

Allo

prol

ifera

tion

inhi

bitio

n (

)



1 05 05 1 05 025 1 05 0125 1 05 006 1 05 003


(a)

888 926 917 895 854936 935 913 859 780

0

50

100

Allo

prol

ifera

tion

inhi

bitio

n (




1 05 05 1 05 025 1 05 0125 1 05 006 1 05 003

(b)






Acknowledgments


References






























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















MSC-BM 954 885 667 339 165888 812 647 310 153

0

50

100

Allo

prol

ifera

tion

inhi

bitio

n (

)


1 05 05 1 05 025 1 05 0125 1 05 006 1 05 003


(a)

MSC-AT 878 824 879 859 823883 831 741 662 572

0

50

100

Allo

prol

ifera

tion

inhi

bitio

n (




1 05 05 1 05 025 1 05 0125 1 05 006 1 05 003

(b)























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



No MBCP



MBCP





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













894 889 942 649 528956 925 976 678 367

0

50

100

Allo

prol

ifera

tion

inhi

bitio

n (

)



1 05 05 1 05 025 1 05 0125 1 05 006 1 05 003


(a)

888 926 917 895 854936 935 913 859 780

0

50

100

Allo

prol

ifera

tion

inhi

bitio

n (




1 05 05 1 05 025 1 05 0125 1 05 006 1 05 003

(b)






Acknowledgments


References






























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















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



No MBCP



MBCP





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













894 889 942 649 528956 925 976 678 367

0

50

100

Allo

prol

ifera

tion

inhi

bitio

n (

)



1 05 05 1 05 025 1 05 0125 1 05 006 1 05 003


(a)

888 926 917 895 854936 935 913 859 780

0

50

100

Allo

prol

ifera

tion

inhi

bitio

n (




1 05 05 1 05 025 1 05 0125 1 05 006 1 05 003

(b)






Acknowledgments


References






























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















894 889 942 649 528956 925 976 678 367

0

50

100

Allo

prol

ifera

tion

inhi

bitio

n (

)



1 05 05 1 05 025 1 05 0125 1 05 006 1 05 003


(a)

888 926 917 895 854936 935 913 859 780

0

50

100

Allo

prol

ifera

tion

inhi

bitio

n (




1 05 05 1 05 025 1 05 0125 1 05 006 1 05 003

(b)






Acknowledgments


References






























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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