Effect of DiD Carbocyanine Dye Labeling on Immunoregulatory ...

Research ArticleEffect of DiD Carbocyanine Dye Labeling on ImmunoregulatoryFunction and Differentiation of Mice Mesenchymal Stem Cells

Maryam Sadat Mohtasebi12 Fatemeh Nasri12 and Eskandar Kamali Sarvestani13

1Department of Immunology Shiraz University of Medical Sciences Shiraz 7134845794 Iran2Student Research Committee Shiraz University of Medical Sciences Shiraz 7134845794 Iran3Autoimmune Diseases Research Center Shiraz University of Medical Sciences Shiraz 7134845794 Iran

Correspondence should be addressed to Eskandar Kamali Sarvestani immunol2sumsacir

Received 24 August 2014 Accepted 18 November 2014 Published 11 December 2014

Academic Editor Armand Keating

Copyright copy 2014 Maryam Sadat Mohtasebi et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Mesenchymal stem cells (MSCs) have been used to treat a variety of degenerative disorders Labeling of MSCs with an appropriatetracer is vital to demonstrate the in vivo engraftment and differentiation of transplanted MSCs DiD is a lipophilic fluorescent dyewith near infrared emission spectra that makes it suitable for in vivo tracing Therefore in the present study the consequences ofDiD labeling on induction of oxidative stress and apoptosis as well as inhibition of biological functions of mesenchymal stem cells(MSCs) were investigated DiD labeling did not provoke the production of ROS induction of apoptosis or inhibition of productionof immunosuppressive factors (PGE2 and IL-10) by MSCs In addition there were no statistical differences between DiD-labeledand unlabeled MSCs in suppression of proliferation and cytokine production (IFN-120574 and IL-17) by in vitro stimulated splenocytesor improvement of clinical score in EAE after in vivo administration In addition DiD labeling did not alter the differentiationcapacity of MSCs Taken together DiD can be considered as a safe dye for in vivo tracking of MSCs

1 Introduction

Among stem cells with the ability to differentiate into variouscell types mesenchymal stem cells (MSCs) with immuno-suppressive functions are promising tools for therapeuticapplications in autoimmune diseases even in allogeneic set-tings The efficacy of MSCs in the treatment of autoimmunediseases is principally dependent on suppression of ongoinginflammatory responses by production of anti-inflammatorycytokines as well as their ability in differentiation intofunctional cells [1ndash3] To investigate the migration of MSCsto damaged tissue or their in vivo differentiation into injuredcells availability of an appropriate tracing system is necessaryDifferent imaging methods have been used to track thelocation of injected MSCs without sacrificing the animalHowever noninvasive imaging techniques based on biolu-minescence single-photon emission computed tomographyusing enzymatic conversionretention and positron emissiontomography need stable integration of the transgene [4 5]The requirement for genetic manipulation of stem cell and

intravenous injection of potentially immunogenic substratefor each imaging session are among disadvantages of thesemethods Nuclear imaging has also several disadvantagesincluding exposure to radiation high costs and short half-life of almost all of nuclear tracers [6]The technique of choiceto trace the cell fates after injection is fluorescence-based invivo optical imaging that is a safe and noninvasive methodwith capability of in vivo chasing of labeled cells over timeBeside biocompatibility and resolution concerns the lack ofinfluence of selected tracer on desired biological functionsof MSCs is also quite important Therefore any tracer thatis used for in vivo MSCs-tracking should affect neitherimmunomodulatory functions nor differentiation capacity ofMSCs

Fluorescent lipophilic carbocyanine dyes are insolublein water but their fluorescence is readily detected whenincorporated into membranes They are classified as oneof the most appropriate families of dyes in labeling andtracking Lipophilic carbocyanine dyes incorporate into cell

Hindawi Publishing CorporationStem Cells InternationalVolume 2014 Article ID 457614 10 pageshttpdxdoiorg1011552014457614

2 Stem Cells International

membranes and diffuse laterally within the cellular plasmamembranes resulting in staining of the entire cell [7 8]Simple staining procedure structural similarity with cellmembrane phospholipids and prolonged dye retention incells are among the advantages of these dyes for using inlive organisms [9 10] Amongst the members of this familyDiD with long wavelength excitation and emission spectrais proper for in vivo imaging due to the fact that NIRfluorescence avoids the interference of the target tissue withthe background fluorescence and creates a high contrastwhen the dye is tracked by in vivo imaging system [10 11]Interestingly DiD has been used for labeling of MSCs withno interference with MSCs differentiation into chondrocytes[11] However the effects of DiD labeling on immunosup-pressive functions and differentiation of MSCs towards anadipogenic or osteogenic cell fate remained to be investigatedAccordingly the present study aimed to shed light on possibleeffects of DiD labeling on in vitro differentiation of DiD-labeled MSCs into adipogenic osteogenic and neural pro-genitor cells as well as in vitro and in vivo immunosuppressivefunction of DiD-labeled MSCs

2 Materials and Methods

21 Animals Female C57BL6 and male BalbC mice werepurchased from Pasteur Institute of Iran and maintainedunder the 12 h lightdark condition at 25∘C BalbC micebetween 4 and 5 weeks of age were used for isolationof the MSCs Experimental autoimmune encephalomyelitis(EAE) was induced in C57BL6 mice All the animals weremaintained underNational Institute of HealthGuide for Careand Use of Laboratory Animals (NIH publication 1985) inanimal house of Shiraz University of Medical Sciences

22 MSCs Isolation Culture Characterization and DiDLabeling MSCswere isolated from femur and tibia accordingto the previously reported method by Da Silva Meirellesand Nardi [12] Briefly bone marrow cells from femurand tibia of BalbC mice were suspended in proper mediaand maintained in 5 CO

2atmosphere at 37∘C with 95

humidity After 24 h the suspended cells were removed bychanging the medium After reaching 70ndash90 confluencethe adherent cells were trypsinized and moved to a newflask with suitable density In order to separate the pureMSCs population trypsinization of MSCs was repeated forat least 4 times when the cells reached suitable confluenceMSCs in passage 5 were trypsinized and analyzed with flowcytometry (BDFACSCalibur USA) for surfacemarkers usingphycoerythrin- (PE-) conjugated anti-CD45 anti-CD44 andanti-Sca-1 as well as fluorescein isothiocyanate- (FITC-)conjugated anti-CD34 (eBioscience UK) and anti-MHCII(BD Pharmingen USA) antibodies Proper isotype controlswere used in all experiments The data were collected andanalyzed by Cell Quest and Flow Jo software (version 76)respectivelyMSCswere labeled with different concentrationsof DiD to determine the highest nontoxic concentration ofthe dyewhich could be traced aftermultiple division ofMSCsAccordingly staining of MSCs was performed with 5120583M of

DiD Briefly 1 times 106 MSCs were suspended in 1mL DMEMcontaining 5 120583M DiD (AAT Bioquest USA) for 20min at37∘C 95 humidity and 5 CO

2 The labeled cells were

washed three times with PBS to remove excess DiD and usedin further experiments

23 Proliferation Capacity of DiD-Labeled MSCs Prolifera-tive capacity of the MSCs was assessed after labeling with5 120583M DiD A total of 2 times 103 labeled and control cellswere counted and plated in a 96-well plate After a cultureperiod of 30 h the MSCs were pulsed with 05 120583Ci of [3H]-methylthymidine (MP Biomedical USA) for 18 hours todetermine the fold of expansion

24 Reactive Oxygen Species (ROS) Production of MSCsProduction of ROS in the DiD-labeled MSCs was measuredusing dichlorofluorescein diacetate (DCFDA InvitrogenUSA) staining [13] DiD-labeled and unlabeled MSCs (2 times105) were incubated in a 6-well plate with 2mL DMEM forone day Then DCFDA (final concentration 10mM) wasadded to the culture for 30min in CO

2incubator After that

cells were washed with PBS for three times and trypsinisedand the dye intensity was quantified by flow cytometer

25 Mitochondrial Membrane Potential (MMP) of MSCsDecrease of MMP is an early indicator of cell apopto-sis According to the procedure reported by Chang etal [14] 2 times 105 DiD-labeled or unlabeled MSCs wereseeded in a 6-well plate and incubated with 40 nM of 33-dihexyloxacarbocyanineDiOC6(3) (Enzo life Sciences USA)at 37∘C for 20min After incubation MSCs were washedand resuspended in PBS and their fluorescent intensity wasdetermined by flow cytometry

26 Differentiation of DiD-Labeled MSCs MSCs cells wereincubated with 5120583M DiD for 20min and kept in the culturefor a day before starting the differentiation protocols Controlwells were treated with the same except for labeling with DiDFor differentiation towards adipocytes sim70 confluent cellswere cultured for 10 days in DMEM supplemented with 1120583Mdexamethasone 05 120583M ascorbic phosphate and 200120583Mindomethacin [15]Themedium was changed every 2-3 daysIntracellular accumulation of lipid vacuoles was also shownby staining with 05 Oil Red O for 10 minutes Osteogenicdifferentiation was done with a medium supplementedwith 1 120583M dexamethasone 10mM 120573-glycerophosphate and05 120583M ascorbic phosphate for 10 days [16] Cells were fedby changing media approximately once every three daysCalcium deposition was revealed by 1 Alizarin Red stainingfor 10 minutes For neural precursor (NP) differentiation 1 times106mLMSCs frompassages 7ndash10were cultured in neurobasaldifferentiation media on agarose gel coated plates whichwere used for bolster neurosphere induction as describedby Shiri et al [17] Neurobasal media were supplemented by1 B27 1 insulin-transferring-selenite 2mM L-glutamine1 penicillinstreptomycin 10 FBS 1 ngmL b-FGF and1 120583MDHEA All materials for differentiation were purchasedfrom Sigma USA After 7 days neurosphere formation was

Stem Cells International 3

identified by microscopic examination and the cells werecollected to investigate the expression of neuronal stem cellmarker (nestin) by real time-PCR Total cellular RNA wasisolated from neurosphere by RNAX plus kit (CinnagenCompany Iran) In addition cDNA was synthesized from2 120583g total RNA using random hexamer primer and M-MuLV reverse transcriptase (Fermentas USA) for 60min at42∘C Nestin primer sequences (forward reverse) were asfollows 51015840-TACAGAGTCAGATCGCTCAGATCC-31015840 51015840-CAGCAGAGTCCTGTATGTAGCCAC-31015840 Both primershad an intron spanning to avoid genomic DNA contam-ination Moreover PCR reactions were normalized usingselective forward 51015840-AGCTTCTTTGCAGCTCCTTCG-31015840and reverse 51015840-CATCCATGGCGAACTGGTG-31015840 primersfor 120573-actin as internal control The cDNA was amplified asfollows incubation at 95∘C for 10min followed by 40 cycles of10 s at 95∘C 1min at 60∘CThemelting curves of the real-timePCR products were observed for specificity of the productsafter the end of the reaction

27 Mixed Leukocyte Reaction (MLR) MLR in the presenceof MSCs was done to compare the immunomodulatoryfunction of unlabeled and DiD-labeled MSCs 3 times 104 cellsfrom passages 4ndash6 were seeded in 96-well plates in DMEMcontaining 10 FBS 100UmL penicillin and 100 120583gmLstreptomycin After 8 hours the MSCs were washed twicewith PBS and 3times 105 CFSE-labeled splenocytes (15times 105 fromC57BL6 and 15 times 105 from BALBc mice) were added to theMSCs Splenocytes were labeled with 10 120583M CFSE in 150 120583Lof RPMI for 15minutes 1120583gmlConcanavalinAwas added toeach well except unstimulated splenocytes (negative control)After five days the splenocytes were harvested and the rateof proliferation was evaluated based on diminution of CFSEfluorescent intensity in cells using flow cytometry

28 Treatment of EAE by DiD-Labeled MSCs MOG35ndash55

peptide (MEVGWYRSPFSRVVHLYRNGK Peptron Korea)was used to induce chronic EAE inC57BL6mice [18] Briefly200120583g of MOG

35ndash55 diluted in 200120583L PBS and emulsifiedwith 200120583L incomplete Freundrsquos adjuvant (Gibco Germany)and 1mgmL heat inactivated mycobacterium tuberculosis(Difco USA) in PBS was injected subcutaneously AfterMOG

35minus55administration 200 ng pertussis toxin (List Bio-

logical Laboratory USA) was injected intraperitoneally onthe day of immunization and two days later Clinical scores ofthe EAEwere recorded at least 3 times perweek EAE signwasscored as follows 0 = no disease 1 = limp tail 2 = hind limbparalysis 3 = paralysis of all four limbs 4 = moribund con-dition and 5 = death [19] The mice were divided into threegroups each containing 5 mice with the mean clinical scoreof 22 OnemillionDiD-labeled or unlabeledMSCs in 02mLof PBS were injected on days 22 29 and 36 into the peritoneaof the treated groups while the control EAE mice received02mL of diluents on the same days The mice were followedup until day 45 To examine the effect of DiD-labeled MSCson antigen-specific immunosuppression 24 days after initialadministration ofMSCs splenocytes fromEAEmice (treatedand untreated) were stimulated with MOG

35ndash55 peptide andproliferation was assessed by 3H-thymidine incorporation

Briefly 2 times 105 splenocytes from treated- and untreated-EAEmice were stimulated by 20120583gwell of MOG

35minus55 After a cul-

ture period of 72 h splenocytes were pulsed with 05 120583Ciwellof [3H]-methylthymidine (MP Biomedical USA) for 18 h todetermine the fold of expansion Radionuclide uptake wasmeasured by 120573-scintillation counter (Wallac Germany)

29 Cytokine Detection by ELISA Supernatants of 1 times 106DiD-labeled and unlabeled MSCs in 2mLs DMEM werecollected after 24 h of culture and kept frozen at minus70∘c untildetermination of cytokine levels Supernatants of MLR werealso collected and kept in the same condition for cytokineanalysis The levels of PGE2 and IL-10 in the supernatantsof MSCs as well as levels of PGE2 IL-10 IFN-120574 and IL-17in the supernatants of MLR were quantitatively analyzed byenzyme-linked immunosorbent assay (ELISA) according tothe manufacturerrsquos instructions (Mabtech UK) Sera fromtreated- (with labeled and unlabeled MSCs) and untreated-EAE mice were also evaluated for the levels of inflammatory(IFN-120574 and IL-17) and anti-inflammatory (IL-10) cytokinesby ELISA

210 Data Analysis The data are presented as mean plusmn SDKruskal-Wallis test was used to check the overall differencebetween groups Also Mann-Whitney 119880 was used to analyzethe difference in clinical scores and cytokine levels betweentreated- and untreated-EAE groups SPSS 15 software wasused for statistical analysis 119875 lt 005 was considered asstatistically significant

3 Results

Both labeled and unlabeled MSCs grow in a homogenousfibroblast shaped pattern In addition flow cytometric anal-ysis of MSCs for expression of CD44 Sca-1 MHC II andhematopoietic stem cell markers (CD45 and CD34) showedmore than 95 purity in the fifth passage (data not shown)

31 Proliferation Capacity of DiD-Labeled MSCs To assesspossible effect of DiD labeling on proliferation of MSCs thecells were stained with DiD and after 48 hours of culturewere pulsed with [3H]-methylthymidine for 18 hours Nosignificant differences were observed between DiD-labeledand intact MSCs (27014plusmn2001CPM and 26747plusmn3540CPMresp 119875 = 079 Figure 1(a))

32 Multilineage Differentiation Capacity of DiD-LabeledMSCs Following 10 days of culture under differentiationconditions cells were stained by Alizarin Red and Oil RedO to confirm calcium deposition and accumulation of lipidvacuoles in cytoplasm of theMSCs respectively Microscopicexaminations revealed no remarkable differences betweenosteogenic differentiation capacities of unlabeled MSCs(Figure 2(a)) and DiD-labeled MSCs (Figure 2(b)) LikewiseDiD-labeled MSCs (Figure 2(d)) showed the sameadipogenic differentiation capacity as the intact MSCs(Figure 2(c)) In addition differentiation of unlabeled


0

10000

20000

30000

40000

NS

cpm

DiD-MSC MSC

MSCDiD-MSC

(a)

0

2000

4000

6000

8000

NS

NS

PG

E2 (p

gm

L)

24h 48h

MSCDiD-MSC

(b)

0

20

40

NS

NS

IL-1

0 (p

gm

L)

24h 48h

MSCDiD-MSC

(c)

Figure 1 Evaluation of IL-10 and PGE2 production and proliferation assay in intact and DiD-labeled MSCs No significant difference wasobserved between unlabeled and DiD-labeled MSCs in proliferation capacity by [3H]-thymidine incorporation (a) levels of PGE2 (b) andIL-10 (c) production after 24 h and 48 h in culture These data were expressed as mean plusmn SD of three independent experiments in triplicate

and DiD-labeled MSCs into NPs was also assessed afterseven days of culture in proper differentiation mediaMicroscopic examination demonstrated the differentiationof untaggedMSCs (Figure 2(e)) as well as DiD-labeledMSCs(Figure 2(f)) into NPs according to neurosphere formationIn addition while mRNA expression level of nestin wasincreased almost 2-fold in NPs compared to MSCs nosignificant difference was observed in nestin mRNAexpression between NPs derived from unlabeled MSCs andDiD-labeled MSCs (23 plusmn 04 and 216 plusmn 02 resp 119875 = 07)

33 Mitochondrial Membrane Potential and ROS Productionin DiD-Labeled MSCs In the context of toxicity of DiDlabeling mitochondrial membrane potential and ROS pro-duction by DiD-labeled MSCs were assessed by DiOC6(3)and DCFDA labeling respectively The result showed thatthere were no significant differences in mean fluorescentintensity (MFI) of DiOC6(3) between DiD-labeled and unla-beled MSCs (259 plusmn 142 and 2396 plusmn 153 resp 119875 = 035Figure 3(a)) To further consolidate safety of DiD labeling onMSCs function the possible occurrence of apoptosis was alsoinvestigated by DCFDA staining No significant differencesin MFI of DCFDA were observed between DiD-labeled andunlabeled MSCs (157 plusmn 1568 and 149 plusmn 209 resp 119875 = 052Figure 3(b)) Therefore the results confirmed DiD labelingdid not affect the viability of MSCs

34 PGE2 and IL-10 Production by DiD-Labeled MSCs Theresults of the present study showed that unlabeled and DiD-labeled MSCs are able to produce detectable and statisticallysimilar levels of PGE2 (5316 plusmn 328 and 5338 plusmn 109 pgmLresp 119875 = 091 Figure 1(b)) and IL-10 (210 plusmn 23 and1853 plusmn 55 pgmL resp 119875 = 05 Figure 1(c)) after 24 hours

of cultureTherewere also no significant statistical differencesin PGE2 production (6058plusmn424 and 5828plusmn229 pgmL resp119875 = 045) or IL-10 synthesis (235 plusmn 23 and 27 plusmn 57 pgmLresp 119875 = 02) between unlabeled and DiD-labeled MSCssubsequent to 48 hours of culture

35 Inhibition of Splenocytersquos Proliferation and Cytokine Pro-duction by DiD-Labeled MSCs To test the antiprolifera-tive function of MSCs on splenocytes MLR reaction wasconducted in the presence and absence of MSCs Evidentproliferation was detected in two-way MLR in the absenceof MSCs (7115 plusmn 725 Figure 4(a)) while the proliferativeresponse was significantly decreased in 1 10 cell ratio ofMSCsplenocyte whether MSCs were unlabeled (2142 plusmn766 119875 = 00001 Figure 4(b)) or labeled with DiD (2278 plusmn590 119875 = 00001 Figure 4(c)) This observation couldbe a result of PGE2 overproduction since analysis of MLRsupernatants revealed upregulation of PGE2 in the presenceof unlabeled and DiD-labeled MSCs (5832 plusmn 362 pgmL and6181 plusmn 147 pgmL resp) in comparison to supernatant ofMLR reaction in the absence of MSCs (1089 plusmn 938 pgmL119875 = 005 for both comparison Figure 4(d)) Interestingly asshown in Figure 4(d) in comparison to MLR in the absenceof MSCs the presence of unlabeled and DiD-labeled MSCssignificantly decreased the levels of IL-10 (3260 plusmn 400 pgmL500 plusmn 96 pgmL and 618 plusmn 174 pgmL resp 119875 = 005for both) IFN-120574 (3903 plusmn 697 pgmL 963 plusmn 168 pgmL and873 plusmn 147 pgmL resp 119875 = 005 for both) and IL-17 (223 plusmn526 pgmL 515 plusmn 104 pgmL and 676 plusmn 73 pgmL resp119875 = 005 for both) in the culture supernatant while thelevels of the abovementioned cytokines were not significantlydifferent between supernatants collected from MLR in thepresence of unlabeled and labeled-MSCs


(a) (b)

(c) (d)

(e) (f)Figure 2 Lineage differentiation of untagged (a c and e) and DiD-labeled MSCs (b d and f) into adipocytes osteocytes and NPsCulturing of unlabeled and DiD-labeled MSCs in differentiation media followed by staining with Oil Red O for lipid (a and b resp originalmagnifications 400x) or Alizarin Red for calcium deposition (c and d resp original magnifications 400x) and microscopic examinationsfor NPs production (e and f resp) were used to confirm their similar differentiation capacity (original magnifications 100x)

36 Improvement of Clinical Score of EAE Three timesinjection of allogeneic MSCs with or without DiD labelingwere similarly reduced clinical score of EAE (165 plusmn 024 and167 plusmn 021 resp) compared to those of control untreated-EAE mice (256 plusmn 017 119875 = 0001 for both comparisonsFigure 5(a)) In agreement with clinical scores sera levels ofproinflammatory cytokines (IFN-120574 and IL-17)were decreasedsignificantly in the sera of mice treated with labeled (520 plusmn371 pgmL and 770 plusmn 311 pgmL resp 119875 = 0008 and119875 = 001 resp Figure 5) or unlabeled MSCs (670 plusmn322 pgmL and 430 plusmn 299 pgmL resp 119875 = 001 and119875 = 0007 resp Figure 5) compared to those in untreatedcontrol mice (1437 plusmn 1170 pgmL and 1850 plusmn 916 pgmLresp) Of interest the levels of IL-10 were significantly higherin untreated control mice (210 plusmn 38 pgmL) compared tothose in mice treated with labeled MSCs (390 plusmn 159 119875 =

001 Figure 5(b)) or unlabeled MSCs (420 plusmn 121 119875 =0008 Figure 5(b)) Similar to proinflammatory cytokinesno significant difference in the levels of IL-10 was observedbetween mice treated with unlabeled MSCs and DiD-labeledMSCs (424 plusmn 121 pgmL and 390 plusmn 159 pgmL resp 119875 =07 Figure 5(b)) Also in vitro stimulation of splenocytesfrom EAE-treated mice with MOG

35minus55peptide showed

lesser proliferation than control untreated mice though thisdifference was not significant (119875 = 013 for unlabeled and119875 = 02 for DiD-labeled MSCs group resp Figure 5(c))

4 Discussion

Recent studies have shed light on the MSCs as potentcandidates for treatment of inflammatory and autoimmune-mediated diseases Privileged advantages of MSCs include


400

300

200

100

0

100 101 102 103 104

Cou

nt

DiOC6(3)

(a)

100 101 102 103 104

Cou

nt

250

200

150

100

50

0

DCFDA

(b)

Figure 3 Flow cytometric analysis of ROS production and mitochondrial membrane potential of MSCs (a) DiD-labeled MSCs stainedwith DiOC6(3) (dash line) emitted quite similar fluorescent intensity compared to unlabeled MSCs (solid line) at 520 nm (b) No significantdifferences in fluorescent intensity of DCFDA were also observed between DiD-labeled (dash line) and unlabeled MSCs (solid line) Thesedata were expressed as mean plusmn SD of three independent experiments in triplicate

their ability in differentiation to various cell types [1 220] and suppressive impact on immune responses [3 21]However lack of distinctive and authenticated markers onMSCs for in vivo tracing has made the researchers employvarious nonspecific trackingmethods Tracking ofMSCswithoptical imaging using biocompatible fluorescent dyes such asDiI has been assessed in several studies [22 23] Howeverusage of ldquoDiDrdquo with long wavelength emission spectra ispreferred due to the lower interference with backgroundfluorescence [10 11] Accordingly DiD can be considered asa suitable substitute for DiI Due to the lack of publishedinvestigations onpossible effects ofDiD labeling on biologicalfunctions of MSCs in this study the consequences of DiDlabeling on viability proliferation immunosuppressive activ-ity and differentiation potential of MSCs were investigatedThe results of the present study have shown that labeling with5 120583M DiD did not induce the production of ROS in MSCsFurthermore as a marker of early apoptosis mitochondrialmembrane potential in the DiD-labeled MSCs remainedunchanged Furthermore no significant differences wereobserved between the DiD-labeled and controlMSCs regard-ing their proliferative potency As a result 5120583M DiD haveneither apoptotic nor cytostaticcytotoxic effects on MSCsThis result is compatible with previous reports indicatinglabeling with 4120583M DiI has no cytostaticcytotoxic effects onMSCs [22 23]

The undesirable effect of labeling on the differentiationcapacity of MSCs has always been another apprehension ofresearchers Current study showed that DiD labeling does notaffect differentiation capability of MSCs to different progen-itors including adipocytes and osteocytes and differentiation

towards neural precursors at the morphological and molecu-lar levels (expression of nestin mRNA) Sutton et al showedthat DiD-labeled human MSCs maintained their capabilityto differentiate into chondrocytes lineage [10] Although theglycosaminoglycan level of the labeled MSCs was reducedupon differentiation labeling had no evident effects on cellmorphology [10] Weir et al [22] and Dai et al [24] have alsoshown that labeling of MSCs with DiI did not interfere withtheir capacity to differentiate into cardiomyocytes Overallin contrast to some reports that have revealed the negativeeffects of the CdSeZnS quantum dot labels or Feridex tracerson the MSCsrsquo differentiation capacity [25 26] DiD had noeffects on the mice MSCs differentiation

It has been shown that MSCs can inhibit immune cellsfunctions by either physical contact or secretion of solublefactors [27ndash30] Therefore we investigated whether labelingofMSCs with 5 120583MDiDmight affect the immunosuppressiveability ofMSCS in prevention of splenocytes proliferation andcytokine production in MLR assay The results of the presentstudy showed that unlabeled and DiD-labeled MSCs havethe same ability in production of two well-known inhibitorymediators (PGE2 and IL-10 Figure 3) that inhibit lympho-cytes activation [31 32] In accordance with these resultsDiD-labeled MSCs were able to decrease the proliferation ofsplenocytes in two-way MLR as efficiently as the unlabeledMSCs (119875 = 0001 Figure 4) In addition the productionof inflammatory (IFN-120574 and IL-17) and anti-inflammatory(IL-10) cytokines in the culture supernatant of MLR wassignificantly reduced in the presence of both DiD-labeledand unlabeled MSCs (Figure 4(d)) It can be hypothesizedthat production of high levels of PGE2 by both DiD-labeled


800

600

400

200

0

100 101 102 103 104

763237

Cou

nt

CFSEMLR without MSCs

(a)

3000

2000

1000

0

100 101 102 103 104

190810

CFSEMSCs splenocytes 1 10

(b)

2500

2000

1500

1000

500

0

100 101 102 103 104

188812

CFSEDiD-MSCs splenocytes 1 10

(c)

2000

1500

1000

500

0

100 101 102 103 104

703930

CFSEUnstimulated splenocytes

(d)

0

2000

4000

6000

8000

MLR (2 unrelated splenocytes)

NS

NSNS

NS

Con

cent

ratio

n (p

gm

l)

PGE2 IL-10 IL-17

lowastlowast

lowastlowast

lowast

MSCs splenocytes 1 10DiD-MSCs splenocytes 1 10

IFN-120574

(e)

Figure 4 MSCs inhibit proliferation and cytokine production by activated splenocytes Unlabeled and DiD-labeled BALBc-derived MSCswere added into 2-way MLR reaction of splenocytes (at ratio 1 10) and the proliferation of splenocytes (based on CFSE reduction) waschecked after 5 days Histogram of cell proliferation in MLR reaction in the absence of MSCs (a) and in the presence of unlabeled (b) orDiD-labeled MSCs (c) demonstrated suppression of splenocytes proliferation by MSCs whether labeled with DiD or not Unstimulatedsplenocytes showed the lowest level of CFSE dilution (d) Three days after initiation of MLR reaction downregulation of IL-17 IFN-120574 andIL-10 and upregulation of PGE2 were observed in the culture supernatants of MLR reactions in the presence of unlabeled or DiD-labeledMSCs (e) NS not significant lowast119875 lt 005 lowastlowast119875 lt 001 These data were expressed as mean plusmn SD of three independent experiments in duplicate

and unlabeled MSCs (Figure 3(b)) might lead to preventionof lymphocyte activation and cytokine expression In thisrespect inhibitory effect of PGE2 on IFN-120574 production [33]or inhibition of lymphocyte proliferation by MSCs throughPGE2 production [34] has been reported previously Theeffects of PGE2on IL-10 production are controversial [35ndash38]However given the fact that PGE2 is involved in suppressionof IL-10 production in certain conditions [37 38] in ourresults reduced production of IL-10 in the presence of DiD-labeled or unlabeled MSCs might be explained accordingto the presence of high levels of PGE2 in the coculture ofMSCs and splenocyte [37 38] Overall both intact and DiD-labeled MSCs are equally competent in production of PGE2and employment of other mechanisms that eventually reducecytokines production by splenocytes in MLR assay

The present study indicated that similar to unlabeledMSCs DiD-labeled MSCs significantly improve the clinicalscores of the EAE compared to the untreated controls

(167 plusmn 021 and 165 plusmn 024 versus 256 plusmn 017 resp119875 = 00001) In addition in comparison to untreated con-trols DiD-labeled and unlabeled MSCs-treated mice showedsignificant reduction in the serum levels of IFN-120574 (193 plusmn117 pgmL versus 52plusmn371 pgmL and 67plusmn322 pgmL resp119875 = 001) and IL-17 (185 plusmn 91 pgmL versus 77 plusmn 311 pgmLand 43 plusmn 299 pgmL resp 119875 = 001) and increased levels ofIL-10 (210 plusmn 38 pgmL versus 390 plusmn 159 pgmL and 42 plusmn121 pgmL resp 119875 = 005 and 119875 = 001 resp)These resultscan be explained by improved peripheral tolerance resultingfrom DiD-labeled or unlabeled MSCs administration anddeviation of cytokine profile to Th2Treg pattern As a resultour study revealed no significant differences between the invivo effects of unlabeled and DiD-labeledMSCs in the reduc-tion of the disease symptoms However the specificity of DiDlabeling in tracing of MSCs needs to be investigated in moredetail since the possibility of DiI dissociation from labeledcells and donor-to-host transfer of dye [39] has been reported


0005101520253035

Control groupNonlabeled MSCDiD-labeled MSC

Time (days)Cl

inic

al sc

ore

0 5 10 15 20 25 30 35 40 45

lowastlowastlowast

Mice MSCs injection 3 times

(a)

0

100

200

300

EAE nontreated groupNonlabeled MSC groupDiD-labeled MSC group

Con

cent

ratio

n (p

gm

l)

lowastlowast

lowastlowast

lowastlowast

lowast

lowastlowast

lowastlowast

IL-10 IL-17IFN-120574

(b)

0

5000

10000

15000

20000

cpm

Basal MOG


(c)

Figure 5 Therapeutic effects of allogenic MSCs in EAE mice The mean plusmn SEM of clinical score of EAE in mice treated with threeintraperitoneal injections of 1 times 106 allogeneicMSCs with or without DiD as well as EAE controls is depicted in (a)MSCs with or without DiDlabeling decrease the levels of IFN-120574 and IL-17 and increase IL-10 levels in sera of treated mice compared to those in control EAE group (b)In vitro stimulation of splenocytes by MOG

35ndash55 peptide revealed no significant reduction in splenocytes proliferation in treatment groupscompared to those of control group (c) lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 These data were collected as mean plusmn SD from five mice ineach group

5 Conclusion

The results of the present study indicated that DiD mightbe an appropriate candidate dye for MSCs labeling and invivo tracking since DiD labeling did not show any effect ondifferent biological behaviors and functions of themiceMSCsin both in vitro and in vivo conditions

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This study was financially supported by the Shiraz Universityof Medical Sciences Shiraz Iran (Grant nos 6152 6022 and5147) Hereby the authors would like to thankDr SamieeMrMalekmakan Ms Taki Mr Hashemi and Ms Dehghan for

their technical support This paper was extracted fromMSctheses of M S Mohtasebirsquos and F Nasrirsquos theses for fulfillingtheir MSc degrees

References

[1] S Ghannam C Bouffi F Djouad C Jorgensen and D NoelldquoImmunosuppression by mesenchymal stem cells mechanismsand clinical applicationsrdquo Stem Cell Research amp Therapy vol 1no 1 article 2 2010

[2] D Karussis C Karageorgiou A Vaknin-Dembinsky et alldquoSafety and immunological effects of mesenchymal stem celltransplantation in patients with multiple sclerosis and amy-otrophic lateral sclerosisrdquo Archives of Neurology vol 67 no 10pp 1187ndash1194 2010

[3] S Aggarwal and M F Pittenger ldquoHuman mesenchymal stemcells modulate allogeneic immune cell responsesrdquo Blood vol105 no 4 pp 1815ndash1822 2005

[4] Y A Cao A J Wagers A Beilhack et al ldquoShifting foci ofhematopoiesis during reconstitution from single stem cellsrdquo


Proceedings of the National Academy of Sciences of the UnitedStates of America vol 101 no 1 pp 221ndash226 2004

[5] S S Gambhir H R Herschman S R Cherry et al ldquoImagingtransgene expression with radionuclide imaging technologiesrdquoNeoplasia vol 2 no 1-2 pp 118ndash138 2000

[6] K Stojanov E F J de Vries D Hoekstra A van WaardeR A J O Dierckx and I S Zuhorn ldquo[18F]FDG labelingof neural stem cells for in vivo cell tracking with positronemission tomography inhibition of tracer release by phloretinrdquoMolecular Imaging vol 11 no 1 pp 1ndash12 2012

[7] M Studeny F C Marini J L Dembinski et al ldquoMesenchymalstem cells potential precursors for tumor stroma and targeted-delivery vehicles for anticancer agentsrdquo Journal of the NationalCancer Institute vol 96 no 21 pp 1593ndash1603 2004

[8] C S von Bartheld D E Cunningham and E W RubelldquoNeuronal tracingwithDiI decalcification cryosectioning andphotoconversion for light and electron microscopic analysisrdquoJournal of Histochemistry amp Cytochemistry vol 38 no 5 pp725ndash733 1990

[9] K X Hu M H Wang C Fan L Wang M Guo and H SAi ldquoCM-DiI labeled mesenchymal stem cells homed to thymusinducing immune recovery of mice after haploidentical bonemarrow transplantationrdquo International Immunopharmacologyvol 11 no 9 pp 1265ndash1270 2011

[10] E J Sutton S E Boddington A J Nedopil et al ldquoAn opticalimaging method to monitor stem cell migration in a model ofimmune-mediated arthritisrdquo Optics Express vol 17 no 26 pp24403ndash24413 2009

[11] S E Boddington E J Sutton T D Henning et al ldquoLabel-ing human mesenchymal stem cells with fluorescent contrastagents the biological impactrdquo Molecular Imaging and Biologyvol 13 no 1 pp 3ndash9 2011

[12] L Da Silva Meirelles and N B Nardi ldquoMurine marrow-derived mesenchymal stem cell isolation in vitro expansionand characterizationrdquo British Journal of Haematology vol 123no 4 pp 702ndash711 2003

[13] E Pawelczyk A S Arbab S Pandit E Hu and J AFrank ldquoExpression of transferrin receptor and ferritin followingferumoxides-protamine sulfate labeling of cells implications forcellularmagnetic resonance imagingrdquoNMR inBiomedicine vol19 no 5 pp 581ndash592 2006

[14] Y Chang T-L Chen J-R Sheu and R-M Chen ldquoSuppressiveeffects of ketamine on macrophage functionsrdquo Toxicology andApplied Pharmacology vol 204 no 1 pp 27ndash35 2005

[15] S Nadri M Soleimani R H Hosseni M Massumi A Atashiand R Izadpanah ldquoAn efficient method for isolation of murinebone marrow mesenchymal stem cellsrdquo International Journal ofDevelopmental Biology vol 51 no 8 pp 723ndash729 2007

[16] C M Digirolamo D Stokes D Colter D G Phinney R Classand D J Prockop ldquoPropagation and senescence of humanmarrow stromal cells in culture a simple colony-forming assayidentifies samples with the greatest potential to propagate anddifferentiaterdquo British Journal of Haematology vol 107 no 2 pp275ndash281 1999

[17] E H Shiri N Z Mehrjardi M Tavallaei S K Ash-tiani and H Baharvand ldquoNeurogenic and mitotic effects ofdehydroepiandrosterone on neuronal-competent marrow mes-enchymal stem cellsrdquo International Journal of DevelopmentalBiology vol 53 no 4 pp 579ndash584 2009

[18] G Constantin C Laudanna S Brocke and E C ButcherldquoInhibition of experimental autoimmune encephalomyelitis by

a tyrosine kinase inhibitorrdquoThe Journal of Immunology vol 162no 2 pp 1144ndash1149 1999

[19] S Pluchino A Quattrini E Brambilla et al ldquoInjection of adultneurospheres induces recovery in a chronic model of multiplesclerosisrdquo Nature vol 422 no 6933 pp 688ndash694 2003

[20] M E J Reinders W E Fibbe and T J Rabelink ldquoMultipotentmesenchymal stromal cell therapy in renal disease and kidneytransplantationrdquo Nephrology Dialysis Transplantation vol 25no 1 pp 17ndash24 2010

[21] M Krampera S Glennie J Dyson et al ldquoBone marrow mes-enchymal stem cells inhibit the response of naive and memoryantigen-specific T cells to their cognate peptiderdquo Blood vol 101no 9 pp 3722ndash3729 2003

[22] C Weir M-C Morel-Kopp A Gill et al ldquoMesenchymal stemcells isolation characterisation and in vivo fluorescent dyetrackingrdquoHeart Lung andCirculation vol 17 no 5 pp 395ndash4032008

[23] M Rutten M A Janes B Laraway C Gregory and K GregoryldquoComparison of quantumdots andCM-DiI for labeling porcineautologous bone marrow mononuclear progenitor cellsrdquo TheOpen Stem Cell Journal vol 2 pp 25ndash36 2010

[24] W Dai S L Hale B J Martin et al ldquoAllogeneic mesenchymalstem cell transplantation in postinfarcted rat myocardiumshort- and long-term effectsrdquoCirculation vol 112 no 2 pp 214ndash223 2005

[25] S-C Hsieh F-F Wang C-S Lin Y-J Chen S-C Hung andY-J Wang ldquoThe inhibition of osteogenesis with human bonemarrow mesenchymal stem cells by CdSeZnS quantum dotlabelsrdquo Biomaterials vol 27 no 8 pp 1656ndash1664 2006

[26] L Kostura D L Kraitchman A M Mackay M F Pittengerand J MW Bulte ldquoFeridex labeling of mesenchymal stem cellsinhibits chondrogenesis but not adipogenesis or osteogenesisrdquoNMR in Biomedicine vol 17 no 7 pp 513ndash517 2004

[27] A Uccelli L Moretta and V Pistoia ldquoImmunoregulatoryfunction of mesenchymal stem cellsrdquo European Journal ofImmunology vol 36 no 10 pp 2566ndash2573 2006

[28] W T Tse J D Pendleton W M Beyer M C Egalka and EC Guinan ldquoSuppression of allogeneic T-cell proliferation byhuman marrow stromal cells implications in transplantationrdquoTransplantation vol 75 no 3 pp 389ndash397 2003

[29] I Rasmusson ldquoImmune modulation by mesenchymal stemcellsrdquo Experimental Cell Research vol 312 no 12 pp 2169ndash21792006

[30] M di Nicola C Carlo-Stella M Magni et al ldquoHumanbonemarrow stromal cells suppress T-lymphocyte proliferationinduced by cellular or nonspecificmitogenic stimulirdquoBlood vol99 no 10 pp 3838ndash3843 2002

[31] A J Cutler V Limbani J Girdlestone and C V NavarreteldquoUmbilical cord-derived mesenchymal stromal cells modulatemonocyte function to suppress T cell proliferationrdquoThe Journalof Immunology vol 185 no 11 pp 6617ndash6623 2010

[32] S H Yang M-J Park S-Y Kim et al ldquoSoluble mediatorsfrom mesenchymal stem cells suppress T cell proliferation byinducing IL-10rdquo Experimental ampMolecularMedicine vol 41 no5 pp 315ndash324 2009

[33] S G Harris J Padilla L Koumas D Ray and R PPhipps ldquoProstaglandins as modulators of immunityrdquo Trends inImmunology vol 23 no 3 pp 144ndash150 2002

[34] M Najar G Raicevic H I Boufker et al ldquoMesenchymalstromal cells use PGE2 tomodulate activation and proliferationof lymphocyte subsets combined comparison of adipose tissue


Whartonrsquos Jelly and bone marrow sourcesrdquo Cellular Immunol-ogy vol 264 no 2 pp 171ndash179 2010

[35] K F MacKenzie K Clark S Naqvi et al ldquoPGE2 inducesmacrophage IL-10 production and a regulatory-like pheno-type via a protein kinase A-SIK-CRTC3 pathwayrdquo Journal ofImmunology vol 190 no 2 pp 565ndash577 2013

[36] G Napolitani E V Acosta-Rodriguez A Lanzavecchia andF Sallusto ldquoProstaglandin E2 enhances Th17 responses viamodulation of IL-17 and IFN-120574 production bymemory CD4+ Tcellsrdquo European Journal of Immunology vol 39 no 5 pp 1301ndash1312 2009

[37] K Boniface K S Bak-Jensen Y Li et al ldquoProstaglandin E2regulates Th17 cell differentiation and function through cyclicAMP and EP2EP4 receptor signalingrdquo Journal of ExperimentalMedicine vol 206 no 3 pp 535ndash548 2009

[38] C H Chu S H Chen Q Wang et al ldquoPGE2 inhibits IL-10production via EP2-mediated 120573-arrestin signaling in neuroin-flammatory conditionrdquoMolecular Neurobiology 2014

[39] W Schormann F J Hammersen M Brulport et al ldquoTrackingof human cells in micerdquo Histochemistry and Cell Biology vol130 no 2 pp 329ndash338 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

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Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


membranes and diffuse laterally within the cellular plasmamembranes resulting in staining of the entire cell [7 8]Simple staining procedure structural similarity with cellmembrane phospholipids and prolonged dye retention incells are among the advantages of these dyes for using inlive organisms [9 10] Amongst the members of this familyDiD with long wavelength excitation and emission spectrais proper for in vivo imaging due to the fact that NIRfluorescence avoids the interference of the target tissue withthe background fluorescence and creates a high contrastwhen the dye is tracked by in vivo imaging system [10 11]Interestingly DiD has been used for labeling of MSCs withno interference with MSCs differentiation into chondrocytes[11] However the effects of DiD labeling on immunosup-pressive functions and differentiation of MSCs towards anadipogenic or osteogenic cell fate remained to be investigatedAccordingly the present study aimed to shed light on possibleeffects of DiD labeling on in vitro differentiation of DiD-labeled MSCs into adipogenic osteogenic and neural pro-genitor cells as well as in vitro and in vivo immunosuppressivefunction of DiD-labeled MSCs

2 Materials and Methods

21 Animals Female C57BL6 and male BalbC mice werepurchased from Pasteur Institute of Iran and maintainedunder the 12 h lightdark condition at 25∘C BalbC micebetween 4 and 5 weeks of age were used for isolationof the MSCs Experimental autoimmune encephalomyelitis(EAE) was induced in C57BL6 mice All the animals weremaintained underNational Institute of HealthGuide for Careand Use of Laboratory Animals (NIH publication 1985) inanimal house of Shiraz University of Medical Sciences

22 MSCs Isolation Culture Characterization and DiDLabeling MSCswere isolated from femur and tibia accordingto the previously reported method by Da Silva Meirellesand Nardi [12] Briefly bone marrow cells from femurand tibia of BalbC mice were suspended in proper mediaand maintained in 5 CO

2atmosphere at 37∘C with 95

humidity After 24 h the suspended cells were removed bychanging the medium After reaching 70ndash90 confluencethe adherent cells were trypsinized and moved to a newflask with suitable density In order to separate the pureMSCs population trypsinization of MSCs was repeated forat least 4 times when the cells reached suitable confluenceMSCs in passage 5 were trypsinized and analyzed with flowcytometry (BDFACSCalibur USA) for surfacemarkers usingphycoerythrin- (PE-) conjugated anti-CD45 anti-CD44 andanti-Sca-1 as well as fluorescein isothiocyanate- (FITC-)conjugated anti-CD34 (eBioscience UK) and anti-MHCII(BD Pharmingen USA) antibodies Proper isotype controlswere used in all experiments The data were collected andanalyzed by Cell Quest and Flow Jo software (version 76)respectivelyMSCswere labeled with different concentrationsof DiD to determine the highest nontoxic concentration ofthe dyewhich could be traced aftermultiple division ofMSCsAccordingly staining of MSCs was performed with 5120583M of

DiD Briefly 1 times 106 MSCs were suspended in 1mL DMEMcontaining 5 120583M DiD (AAT Bioquest USA) for 20min at37∘C 95 humidity and 5 CO

2 The labeled cells were

washed three times with PBS to remove excess DiD and usedin further experiments

23 Proliferation Capacity of DiD-Labeled MSCs Prolifera-tive capacity of the MSCs was assessed after labeling with5 120583M DiD A total of 2 times 103 labeled and control cellswere counted and plated in a 96-well plate After a cultureperiod of 30 h the MSCs were pulsed with 05 120583Ci of [3H]-methylthymidine (MP Biomedical USA) for 18 hours todetermine the fold of expansion

24 Reactive Oxygen Species (ROS) Production of MSCsProduction of ROS in the DiD-labeled MSCs was measuredusing dichlorofluorescein diacetate (DCFDA InvitrogenUSA) staining [13] DiD-labeled and unlabeled MSCs (2 times105) were incubated in a 6-well plate with 2mL DMEM forone day Then DCFDA (final concentration 10mM) wasadded to the culture for 30min in CO

2incubator After that

cells were washed with PBS for three times and trypsinisedand the dye intensity was quantified by flow cytometer

25 Mitochondrial Membrane Potential (MMP) of MSCsDecrease of MMP is an early indicator of cell apopto-sis According to the procedure reported by Chang etal [14] 2 times 105 DiD-labeled or unlabeled MSCs wereseeded in a 6-well plate and incubated with 40 nM of 33-dihexyloxacarbocyanineDiOC6(3) (Enzo life Sciences USA)at 37∘C for 20min After incubation MSCs were washedand resuspended in PBS and their fluorescent intensity wasdetermined by flow cytometry

26 Differentiation of DiD-Labeled MSCs MSCs cells wereincubated with 5120583M DiD for 20min and kept in the culturefor a day before starting the differentiation protocols Controlwells were treated with the same except for labeling with DiDFor differentiation towards adipocytes sim70 confluent cellswere cultured for 10 days in DMEM supplemented with 1120583Mdexamethasone 05 120583M ascorbic phosphate and 200120583Mindomethacin [15]Themedium was changed every 2-3 daysIntracellular accumulation of lipid vacuoles was also shownby staining with 05 Oil Red O for 10 minutes Osteogenicdifferentiation was done with a medium supplementedwith 1 120583M dexamethasone 10mM 120573-glycerophosphate and05 120583M ascorbic phosphate for 10 days [16] Cells were fedby changing media approximately once every three daysCalcium deposition was revealed by 1 Alizarin Red stainingfor 10 minutes For neural precursor (NP) differentiation 1 times106mLMSCs frompassages 7ndash10were cultured in neurobasaldifferentiation media on agarose gel coated plates whichwere used for bolster neurosphere induction as describedby Shiri et al [17] Neurobasal media were supplemented by1 B27 1 insulin-transferring-selenite 2mM L-glutamine1 penicillinstreptomycin 10 FBS 1 ngmL b-FGF and1 120583MDHEA All materials for differentiation were purchasedfrom Sigma USA After 7 days neurosphere formation was















3 Results





0

10000

20000

30000

40000

NS

cpm

DiD-MSC MSC

MSCDiD-MSC

(a)

0

2000

4000

6000

8000

NS

NS

PG

E2 (p

gm

L)

24h 48h

MSCDiD-MSC

(b)

0

20

40

NS

NS

IL-1

0 (p

gm

L)

24h 48h

MSCDiD-MSC

(c)








(a) (b)

(c) (d)






4 Discussion



400

300

200

100

0

100 101 102 103 104

Cou

nt

DiOC6(3)

(a)

100 101 102 103 104

Cou

nt

250

200

150

100

50

0

DCFDA

(b)







800

600

400

200

0

100 101 102 103 104

763237

Cou

nt


(a)

3000

2000

1000

0

100 101 102 103 104

190810


(b)

2500

2000

1500

1000

500

0

100 101 102 103 104

188812


(c)

2000

1500

1000

500

0

100 101 102 103 104

703930


(d)

0

2000

4000

6000

8000


NS

NSNS

NS

Con

cent

ratio

n (p

gm

l)

PGE2 IL-10 IL-17

lowastlowast

lowastlowast

lowast


IFN-120574

(e)






0005101520253035


Time (days)Cl

inic

al sc

ore

0 5 10 15 20 25 30 35 40 45

lowastlowastlowast


(a)

0

100

200

300


Con

cent

ratio

n (p

gm

l)

lowastlowast

lowastlowast

lowastlowast

lowast

lowastlowast

lowastlowast

IL-10 IL-17IFN-120574

(b)

0

5000

10000

15000

20000

cpm

Basal MOG


(c)



5 Conclusion




Acknowledgments



References




















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology















3 Results





0

10000

20000

30000

40000

NS

cpm

DiD-MSC MSC

MSCDiD-MSC

(a)

0

2000

4000

6000

8000

NS

NS

PG

E2 (p

gm

L)

24h 48h

MSCDiD-MSC

(b)

0

20

40

NS

NS

IL-1

0 (p

gm

L)

24h 48h

MSCDiD-MSC

(c)








(a) (b)

(c) (d)






4 Discussion



400

300

200

100

0

100 101 102 103 104

Cou

nt

DiOC6(3)

(a)

100 101 102 103 104

Cou

nt

250

200

150

100

50

0

DCFDA

(b)







800

600

400

200

0

100 101 102 103 104

763237

Cou

nt


(a)

3000

2000

1000

0

100 101 102 103 104

190810


(b)

2500

2000

1500

1000

500

0

100 101 102 103 104

188812


(c)

2000

1500

1000

500

0

100 101 102 103 104

703930


(d)

0

2000

4000

6000

8000


NS

NSNS

NS

Con

cent

ratio

n (p

gm

l)

PGE2 IL-10 IL-17

lowastlowast

lowastlowast

lowast


IFN-120574

(e)






0005101520253035


Time (days)Cl

inic

al sc

ore

0 5 10 15 20 25 30 35 40 45

lowastlowastlowast


(a)

0

100

200

300


Con

cent

ratio

n (p

gm

l)

lowastlowast

lowastlowast

lowastlowast

lowast

lowastlowast

lowastlowast

IL-10 IL-17IFN-120574

(b)

0

5000

10000

15000

20000

cpm

Basal MOG


(c)



5 Conclusion




Acknowledgments



References




















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

10000

20000

30000

40000

NS

cpm

DiD-MSC MSC

MSCDiD-MSC

(a)

0

2000

4000

6000

8000

NS

NS

PG

E2 (p

gm

L)

24h 48h

MSCDiD-MSC

(b)

0

20

40

NS

NS

IL-1

0 (p

gm

L)

24h 48h

MSCDiD-MSC

(c)








(a) (b)

(c) (d)






4 Discussion



400

300

200

100

0

100 101 102 103 104

Cou

nt

DiOC6(3)

(a)

100 101 102 103 104

Cou

nt

250

200

150

100

50

0

DCFDA

(b)







800

600

400

200

0

100 101 102 103 104

763237

Cou

nt


(a)

3000

2000

1000

0

100 101 102 103 104

190810


(b)

2500

2000

1500

1000

500

0

100 101 102 103 104

188812


(c)

2000

1500

1000

500

0

100 101 102 103 104

703930


(d)

0

2000

4000

6000

8000


NS

NSNS

NS

Con

cent

ratio

n (p

gm

l)

PGE2 IL-10 IL-17

lowastlowast

lowastlowast

lowast


IFN-120574

(e)






0005101520253035


Time (days)Cl

inic

al sc

ore

0 5 10 15 20 25 30 35 40 45

lowastlowastlowast


(a)

0

100

200

300


Con

cent

ratio

n (p

gm

l)

lowastlowast

lowastlowast

lowastlowast

lowast

lowastlowast

lowastlowast

IL-10 IL-17IFN-120574

(b)

0

5000

10000

15000

20000

cpm

Basal MOG


(c)



5 Conclusion




Acknowledgments



References




















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a) (b)

(c) (d)






4 Discussion



400

300

200

100

0

100 101 102 103 104

Cou

nt

DiOC6(3)

(a)

100 101 102 103 104

Cou

nt

250

200

150

100

50

0

DCFDA

(b)







800

600

400

200

0

100 101 102 103 104

763237

Cou

nt


(a)

3000

2000

1000

0

100 101 102 103 104

190810


(b)

2500

2000

1500

1000

500

0

100 101 102 103 104

188812


(c)

2000

1500

1000

500

0

100 101 102 103 104

703930


(d)

0

2000

4000

6000

8000


NS

NSNS

NS

Con

cent

ratio

n (p

gm

l)

PGE2 IL-10 IL-17

lowastlowast

lowastlowast

lowast


IFN-120574

(e)






0005101520253035


Time (days)Cl

inic

al sc

ore

0 5 10 15 20 25 30 35 40 45

lowastlowastlowast


(a)

0

100

200

300


Con

cent

ratio

n (p

gm

l)

lowastlowast

lowastlowast

lowastlowast

lowast

lowastlowast

lowastlowast

IL-10 IL-17IFN-120574

(b)

0

5000

10000

15000

20000

cpm

Basal MOG


(c)



5 Conclusion




Acknowledgments



References




















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


400

300

200

100

0

100 101 102 103 104

Cou

nt

DiOC6(3)

(a)

100 101 102 103 104

Cou

nt

250

200

150

100

50

0

DCFDA

(b)







800

600

400

200

0

100 101 102 103 104

763237

Cou

nt


(a)

3000

2000

1000

0

100 101 102 103 104

190810


(b)

2500

2000

1500

1000

500

0

100 101 102 103 104

188812


(c)

2000

1500

1000

500

0

100 101 102 103 104

703930


(d)

0

2000

4000

6000

8000


NS

NSNS

NS

Con

cent

ratio

n (p

gm

l)

PGE2 IL-10 IL-17

lowastlowast

lowastlowast

lowast


IFN-120574

(e)






0005101520253035


Time (days)Cl

inic

al sc

ore

0 5 10 15 20 25 30 35 40 45

lowastlowastlowast


(a)

0

100

200

300


Con

cent

ratio

n (p

gm

l)

lowastlowast

lowastlowast

lowastlowast

lowast

lowastlowast

lowastlowast

IL-10 IL-17IFN-120574

(b)

0

5000

10000

15000

20000

cpm

Basal MOG


(c)



5 Conclusion




Acknowledgments



References




















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


800

600

400

200

0

100 101 102 103 104

763237

Cou

nt


(a)

3000

2000

1000

0

100 101 102 103 104

190810


(b)

2500

2000

1500

1000

500

0

100 101 102 103 104

188812


(c)

2000

1500

1000

500

0

100 101 102 103 104

703930


(d)

0

2000

4000

6000

8000


NS

NSNS

NS

Con

cent

ratio

n (p

gm

l)

PGE2 IL-10 IL-17

lowastlowast

lowastlowast

lowast


IFN-120574

(e)






0005101520253035


Time (days)Cl

inic

al sc

ore

0 5 10 15 20 25 30 35 40 45

lowastlowastlowast


(a)

0

100

200

300


Con

cent

ratio

n (p

gm

l)

lowastlowast

lowastlowast

lowastlowast

lowast

lowastlowast

lowastlowast

IL-10 IL-17IFN-120574

(b)

0

5000

10000

15000

20000

cpm

Basal MOG


(c)



5 Conclusion




Acknowledgments



References




















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0005101520253035


Time (days)Cl

inic

al sc

ore

0 5 10 15 20 25 30 35 40 45

lowastlowastlowast


(a)

0

100

200

300


Con

cent

ratio

n (p

gm

l)

lowastlowast

lowastlowast

lowastlowast

lowast

lowastlowast

lowastlowast

IL-10 IL-17IFN-120574

(b)

0

5000

10000

15000

20000

cpm

Basal MOG


(c)



5 Conclusion




Acknowledgments



References




















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Effect of DiD Carbocyanine Dye Labeling on Immunoregulatory ...

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Transcript of Effect of DiD Carbocyanine Dye Labeling on Immunoregulatory ...