Mesenchymal Stem Cells Isolation

O R I G I N A L A R T I C L EOriginal ArticleMesenchymal Stem Cells:Isolation,Characterisation and In Vivo Fluorescent Dye TrackingChristopher Weir,PhD a ,b ,d ,Marie-Christine Morel-Kopp,PhD b ,d ,Anthony Gill,FRCPA c ,d ,Kellie Tinworth,MSc a ,d ,Leigh Ladd,PhD a ,d ,Stephen N.Hunyor,MD a ,d ,∗and Christopher Ward,PhD b ,daCardiac Technology Centre &Kolling Institute,Sydney,AustraliabNorthern Blood Research Centre and Department of Haematology,Sydney,Australiac Anatomical Pathology Department,Sydney,Australiad University of Sydney at Royal North Shore Hospital,Sydney,AustraliaCell therapies have been used to regenerate the heart by direct myocardial delivery,by coronary infusion and by surface attached scaffolds.Multipotent mesenchymal stem cells (MSC)with capacity to differentiate into cardiomyocytes and other cell lines have been predominantly trialled in rodents.However,large animal models are increasingly needed to translate basic research into new,safe regenerative therapies.Understanding the mode of action of cell therapies in the mammalian heart has been limited by cell tracking capability.This study examined the ability to track the fate of allogeneic MSC in sheep using various fluorescent dyes.MSC isolated from sheep bone marrow were grown in culture following extraction and flow cytometric characterisation.After labelling with fluorescent tracking dyes (e.g.CFSE and DiI)cells were tested for in vitro and in vivo signal up to six belling effect on cell division and differentiation was studied.Several dyes lost fluorescence and slowed cell division.However,the thiol reactive dye CM-DiI showed detectable in vivo fluorescence in labelled MSC six weeks after injection into sheep skeletal muscle and two weeks after implantation of an MSC coated biomaterial scaffold.CM-DiI labelled MSC differentiated in vitro showed label retention over four weeks.The fluorescent membrane dye CM-DiI tracks implanted sheep MSC and provides an alternative to traditional cell markers such as gene modified GFP .(Heart,Lung and Circulation 2008;17:395–403)©2008Australasian Society of Cardiac and Thoracic Surgeons and the Cardiac Society of Australia andNew Zealand.Published by Elsevier Inc.All rights reserved.Keywords.Mesenchymal stem cells (MSC);CM-DiI;Cell division and differentiation;Sheep;Biomaterials;ScaffoldIntroductionCoronary artery disease leading to myocardial ischaemia and infarction is characterised by loss of cardiomyocytes,with frequent progression to heart failure whose incidence has increased markedly due to coronary angioplasty/stenting,bypass surgery and pharmacotherapy.Stem cell therapy has been proposed as a repair mechanism,preferably before hearts enter a phase of rapid decline in pump function.Received 13December 2007;received in revised form 15January 2008;accepted 20January 2008;available online 8April 2008∗Corresponding author at:Cardiac Technology Centre,Depart-ment of Cardiology,Royal North Shore Hospital,St Leonards,Sydney,NSW 2065,Australia.Tel.:+61299268679;fax:+61299014097.E-mail address:stephenh@.au (S.N.Hunyor).The potential for mesenchymal stem cells (MSC)to dif-ferentiate into specialised cell lineages,such as osteocytes,adipocytes and chondrocytes has been documented,1as has differentiation into cardiomyocytes in vitro .2However,there is evidence that MSC exert their beneficial effect pre-dominantly via paracrine mechanisms resulting from cell death.3Short-term in vivo rat studies show that MSC injected into damaged myocardium adopt characteristics of car-diomyocytes and occupy a major part of the damaged area.4In another study Orlic et al.5mobilised bone marrow (BM)cells in mice with myocardial infarction by inject-ing granulocyte colony stimulating factor (G-CSF),with cells localised in the infarcted zone generating myocardial cells.Despite such promise,doubts persist concerning long-term cell survival,and reliable MSC markers and evidence of differentiation into cardiomyocytes is lacking,especially©2008Australasian Society of Cardiac and Thoracic Surgeons and the Cardiac Society of Australia and New Zealand.Published by Elsevier Inc.All rights reserved.1443-9506/04/$30.00doi:10.1016/j.hlc.2008.01.006ORIGINAL ARTICLE 396Weir et al.Heart,Lung and Circulation Fluorescent stem cell tracking2008;17:395–403in large animal models.6Sheep appear particularly suit-able for refining stem cell techniques for heart repair.Current cellular therapies involve taking either uns-elected autologous BM cells7or homogenous antibodyselected‘specific’sub-populations,8which aim pre-dominantly for vasculogenesis in ischaemic or necroticmyocardium.In our sheep we used MSC adherenceselection and growth under standardised conditions toprovide a cell population capable of differentiation intocardiomyocytes.7,9Work on human MSC has utilised spe-cific antibody labelling(e.g.STRO-1,V-CAM)to isolateand define particular cells10but use of specific selectedcells for cardiac repair in animals is limited.3Because avail-able specific ovine monoclonal antibodies do not reactwith sheep MSC(except anti-ovine CD44),we examinedlabelling and tracking of MSC withfluorescent ingdyes such as CFSE or CM-DiI(Molecular Probes,USA)we optimised ovine MSC labelling in vitro,noted its effecton cell division and studied dye retention in differenti-ating MSC.Having defined optimal in vitro staining,weassessed in vivo dye stability following MSC implantationdirectly into muscle,or subcutaneously when grown on abiomaterial scaffold.While recent work suggests that adult skin cells can begenetically modified to recreate pluripotent cells resem-bling hESCs,11their clinical application appears remote.Exploring the full potential of non-immunogenic,read-ily available MSC for heart repair is therefore warranted.This study focused on the potential of CM-DiI as an MSCtracking dye for use in heart regeneration.Materials and MethodsMerino-cross sheep(42.5–44.5kg)received humane carein compliance with the Guide for the Care and Use of Labora-tory Animals(US National Institutes of Health,publication85–23,revised1996)in a protocol approved by the Institu-tional Animal Care and Ethics Committee.Surgical PreparationSterile surgery was performed under alfaxalone(1–1.5mg/kg)induced general anaesthesia maintainedwith 1.5–2%isoflurane in40%O2.Following intuba-tion expired CO2was maintained at30–35mmHg.Nopost-operative drug therapy was given.Bone Marrow(BM)ExtractionBM was collected aseptically into Na2EDTA from thesheep’s iliac crest under general anaesthetic.The buffycoat was isolated by centrifugation(2500×g,10min)andresuspended into1.5ml PBS.A500␮l sample served forflow cytometric analysis and residual for culture(A).Thebuffy coat(1ml)wasfiltered through a meshfilter(Bec-ton Dickinson,USA)and residualfiltrate washed off withsterile PBS;half that cell suspension was cultured(B).Thefiltered buffy coat left(500␮l)was layered onto10mlFicoll(Amersham Bioscience,Uppsala,Sweden)and cen-trifuged(1200×g,20min).Cells at the interface wereremoved,washed twice in sterile PBS and split into twofractions(500␮l).One fraction was cultured directly(C)and the other selected for CD106(V-CAM)(D)usingindirect immuno-magnetic beads(MACs TM microbeads,Miltenyti Biotech,Bergish Gladbach,Germany)accordingto manufacturer’s protocol.All cell groups were culturedin DMEM with10%foetal calf serum(FCS),1×antibiotics(penicillin,streptomycin)and5mM L-glutamine.Bone Marrow AnalysisA small BM sample was analysed for anti-ovine markersCD44,CD45,CD34,CD11b,CD31(Serotec,Oxford,UK)and the anti-human marker CD106(BD Pharmingen TM,San Diego,USA).Briefly,30␮l of complete marrow wasadded to the antibodies and incubated at room tempera-ture for15min in the dark.Cells werefixed(0.5ml10%formaldehyde,0.6ml10×HBSS and0.9ml dH2O)for10min,400␮l dH2O added and red blood cells allowed tolyse for10min at4◦C,followed byflow cytometric analysis.Cultured MSC AnalysisAfter culturing the cells in DMEM with10%FCS and peni-cillin and streptomycin as antibiotics(Trace Biosciences)for1–2weeks until each fraction had reached confluencein a75cm2flask,MSC from each sample were analysedbyflow cytometry using anti-ovine markers CD44,CD45and CD11b and anti-human CD106.Carboxyfluorescein Diacetate,Succinimidyl Ester(CFSE)LabellingCFSE dye stock was made up to a concentration of100␮M/ml in DMSO as recommended by manufacturer(Molecular Probes,Oregon,USA).MSC were detachedusing trypsin and resuspended at a concentration of1×107cells/ml in PBS and then labelled at1␮M,2␮Mand4␮M concentrations(6×106cells)for10min at37◦Cbefore washing in PBS and putting back into culture formonitoring offluorescent staining retention over a periodof two weeks using unlabelled control MSC.Chloromethylbenzamido-1,1 -Dioctadecyl-3,3,3 3 -Tetramethylindocarbocyanine Perchlorate(CM-DiI;Lipophilic Carbocyanine Dye)LabellingCM-DiI stock was made up at a1mg/ml concentrationin ethanol as recommended by manufacturer(MolecularProbes,USA)and MSC staining at4␮M,6␮M and8␮Mconcentrations was performed for15or30min at37◦Cor room temperature,andfinally for15min at4◦C to opti-mise staining levels.All staining was performed on75cm2flasks with a confluent layer of3–4×belledcells were cultured,maintained at sub-confluence andmonitored forfluorescence levels overfive weeks usingflow belled and unlabelled control MSCwere monitored for cell growth with cell numbers countedusing a haemocytometer.CM-DiI Labelling Effect on Cell Growth and ViabilityT o assess labelling effect on cell growth aflask of MSCs waslabelled with4␮M CM-DiI for15min at37◦C and4◦C.A plate of unlabelled cells served as control.Cells fromO R I G I N A L A R T I C L EHeart,Lung and Circulation Weir et al.3972008;17:395–403Fluorescent stem cell trackingTable 1.Staining Characteristics and Follow-up of MSCs ±Biomaterial Scaffold Implanted Directly into Muscle or Subcutaneously in SheepImplantation SiteT wo WeeksSix Weeks(i)MSC directly into muscle2×106MSC in 0.2ml serum-free DMEMLateral gastrocnemiusPeak staining group (stained 2days pre-injection)7.5×107CM-DiI labelled MSCs in serum-free DMEM (high dose).Cranial tibial CM-DiI stained group (stained on the day) 2.5×106unlabelled MSC in 0.4%trypan blue (low dose)Dorsal gastrocnemius Control unlabelled MSC1×107CM-DiI labelled MSC in 0.4%trypan blue (low dose)Central cranial tibial 7.5×106MSC in serum-free DMEM (high dose)(ii)MSC on biomaterialCranial dorsal region Control unlabelled MSC2×106unlabelled MSCMiddle dorsal regionCM-DiI labelled MSC (stained 2days pre-injection)0.5×106CM-DiI labelled MSC spiked on biomaterial (LD)Caudal dorsal regionCM-DiI labelled MSC +additional 2×106cells1.5×107CM-DiI labelled MSC (HD)both flasks were washed in PBS and detached using 0.25%trypsin-EDTA.Exactly 100cells were sorted by flow cytom-etry based on FL-2v SSC both for CM-DiI labelled and unlabelled cells in triplicate groups for each day (i.e.days 4,7,14)and placed on six-well plates.On sampling days media was removed and cells washed in sterile PBS before treatment,spun down (6000rpm/1min)and counted using a haemocytometer.In Vivo Two-and Six-Week CM-Dil Label Retention Trials (T able 1)Flasks of MSC were grown to confluence and stained with 4␮M CM-DiI.A sample of cells showed pre-injection staining level.MSC were also cultured for two weeks in DMEM and 10%FCS with Penicillin-Streptomycin on a prosthetic silicone biomaterial scaffold that has random sized and random shaped cavities in its matrix structure (Seare Matrix ®,Seare Biomatrix Systems,Inc,Salt Lake City,USA).The biomaterial had been sterilised and then cut into circular sections to cover the whole well of a six-well plate.The cells were stained on the biomaterial two days prior to its implantation.This served to establish proof of principle for MSC growth in this matrix material and also to provide a substrate for in vivo MSC tracking studies.Extra labelled cells were placed on one bioma-terial sample prior to its insertion back into the sheep.A negative control consisted of biomaterial plus unla-belled MSC.The biomaterial was inserted subcutaneously in sheep that also had direct MSC injection.Six-week in vivo CM-DiI labelling persistence served to mimic the timeframe of ovine myocardial infarct repair.Differentiation of MSCMultipotent capacity of ovine MSC was proven after in vitro culturing with specific supplements by induc-ing differentiation into chondrogenic and cardiomyocyte phenotypes.Samples were compared with control cells from the same cell lineage cultured without differentiating supplement.Chondrogenic supplement comprised 1%ITS-premix (BD Biosciences,Australia),100␮M ascorbate-2phosphate,10−7M dexamethasone and 10ng/ml TGF-␤1.12Cardiomyocyte supplement was 3␮M/l 5-azacytidine (Sigma,Australia).2Differentiation of CM-DiI MSCAfter CM-DiI dye labelled cells were induced to differ-entiate,chondrogenic and cardiomyocyte differentiating groups were monitored for four weeks by flow cytome-try (FL-2)and fluorescence microscopy (Olympus BX51epi-fluorescent microscope with a 520–550nm filter block).CM-DiI labelled undifferentiated MSC and unlabelled MSC served as controls.Chondrogenic induced cells were filtered (mesh:BD,USA)to produce a single cell popula-tion.ImmunohistochemistryMorphological analysis and immunohistochemistry was performed on cell block preparations.Briefly,cells grow-ing in culture medium were suspended in Hanks solution and centrifuged to form a pellet of cellular material which was fixed in 10%buffered formalin,processed routinely and paraffin embedded.This tissue was sec-tioned onto positively charged glass slides (Superfrost plus,Menzel-Glaser,Germany).Sequential slides were stained with haematoxylin and eosin (H&E)and used for desmin immunohistochemistry and correlation with mor-phology.Desmin immunohistochemistry used a BondmaX TM autostainer (Vision Biosystems,Mt Waverley,Victoria,Australia)with manufacturer’s settings.Antigen retrieval was performed in a pH 6buffered medium (Bond TM Epitope Retrieval solution 1,AR9961,Vision Biosys-tems)for 30min at 97◦C.Slides were incubated with mouse monoclonal anti-human desmin antibody (clone D33,M0760,Dako Carpinteria CA,USA)for 30min at 25◦C.Washing and antigen detection were performed using Bond Define biotin-free detection system (DS9713,Vision Biosystems)according to manufacturer’s instruc-tion.ORIGINAL ARTICLE 398Weir et al.Heart,Lung and Circulation Fluorescent stem cell tracking2008;17:395–403Figure1.Flow cytometry marrow screening using anti-ovine CD44/45/34/11b and anti-human CD106.( )Isotype control.H&E stained and desmin labelled slides were exam-ined together by a single pathologist not blinded to otherdata.T wo blinded pathologists independently assessedslides for morphological and histochemical evidence ofcartilaginous differentiation(H&E and alcian blue stainedsections).ResultsMSC Flow CharacterisationInitial screening of sheep BM demonstrated cell pop-ulations with hemopoetic(CD34+,CD45+)andmesenchymal(CD44+and CD106+)stem cellpopu-Figure2.CD106selected and adherent MSC populations using anti-ovine CD11b/44/45and anti-human CD106after3weeks culture.(—)CD 106,(....)adherent.O R I G I N A L A R T I C L EHeart,Lung and Circulation Weir et al.3992008;17:395–403Fluorescent stem celltrackingFigure bel retention (A and B)and cell division (C and D).(A)CFSE,(B)CM-DiI;cell division (C)DiI,(D)CM-DiI.lations (Fig.1),indicating similarity to human mark-ers.Adherence or anti-human CD 106was used to select ovine MSC characterised by anti-ovine CD 11b,CD 44,CD 45and anti-human CD 1063weeks post-culture (Fig.2).Similarly,residual marrow and filtrate showed no significant difference in population characteristics.All MSC populations extracted were CD 44+,CD 106+,CD 11b −,CD 34−and CD 45−,indicating they were non-haemopoeitic.All MSC groups lost CD 106sig-nal after two more passages.Because of equivalence of isolation methods for cell markers,adherent selec-tion was chosen for further experiments because of rapid cell production rate and reproducible cell num-bers.CFSE and CM-DiI Cell LabellingInitial CFSE labelling of ovine MSC showed rapid signal loss over eight days at all staining concentrations (FL-1;flow cytometry—Fig.3A;and fluorescent microscopy).This rendered CFSE unsuitable for cell tracking.Initial studies of cells stained with 4␮M,6␮M or 8␮M CM-DiI concentrations optimised dye labelling at 37◦C and showed a fluorescent spike at day 2,with highest signal at 8␮M (Fig.3B).By day 7,cell fluores-cence dropped significantly and levelled out but remained readily detectable by flow cytometry and fluorescence microscopy,and significantly above control for five weeks at all concentrations (Fig.3C).While stain retention did not differ between 4␮M and 8␮M CM-DiI incubation of MSC,concentrations of ≥6␮Mimpaired cell division (Fig.3D).Therefore,4␮M staining was chosen.Staining at 37◦C compared to room temperature or 4◦C produced a stronger signal at 4␮M and temperature sensitivity was also evident with 6–8␮M.Staining time (15–30min)at 37◦C (4␮M)showed no effect.Optimised labelling was therefore standardised at 4␮M CM-DiI con-centration for 15min at 37◦C.In Vivo CM-DiI Labelling ExperimentsInitial injection of CM-DiI labelled MSC into skeletal mus-cle showed dye retention for six weeks (Fig.4B and D).Similarly CM-DiI stained cells on biomaterial show dis-tinct staining two weeks after implantation (Fig.4F and H).Tissue sections typically show moderate red auto-fluorescence (Fig.4E and G).This pattern of stain retention supports CM-DiI as a suitable labelling system for in vivo cell tracking.MSC DifferentiationLineage-specific differentiation of ovine MSC was carried out after a period of four weeks.T wo blinded surgical pathologists both identified cartilaginous differentiation by morphology and by presence of alcian blue positive extra-cellular matrix in treated MSC and not in other cell lines or controls (Fig.5C,F,G,H).Immunohistochemistry for desmin in the chondrogenic cell lines was focally positive.No alcian blue positive extra-cellular matrix was present in the control or cardiomyocyte cells.MSC induced with 5-azacytidine to cardiomy-ocyte formation did not show characteristic cell ‘beating’ORIGINAL ARTICLE 400Weir et al.Heart,Lung and Circulation Fluorescent stem cell tracking2008;17:395–403Figure4.MSC±biomaterial CM-DiI retention:6weeks post MSCmuscle implantation.Unlabelled:(A)×20;(C)×100.DiI labelled:(B)×20;(D)×100.Two weeks post biomaterial+MSC implantation:(E,F)control and CM-DiI labelled,×40;(G,H)×100.Cells cultured onglass slides(I)control;(J)CM-DiI label,×40.reported in approximately30%of murine MSC,2,13rais-ing the possibility of species differences.Genetic andimmuno-histochemical markers of myocyte differentia-tion have however,been reported in porcine studies.14Theovine MSC when induced with5-azacytidine did demon-strate different growth and adherence patterns and alsostained desmin positive indicating primitive differentia-tion towards muscle cells.CM-DiI Labelled Cell DifferentiationMSC labelled with CM-DiI were split into four groups toassess whether CM-DiI staining affected differentiationand whether staining was retained.All CM-DiI labelledMSC differentiation groups showed identicalhistologicalFigure5.Differentiated MSC morphology:(A)control,(B and C)cardiomyocyte,chrondrogenic induced.No morphological features ofcardiomyocyte differentiation.Desmin immunohistochemistry:(D)negative control,(E)cardiomyocytes,(F)chondrogenic cells.Chondrogenic differentiation:(G)morphologic,(H)alcian bluestaining.(All H&E stain except(H).)and immunohistochemical features to unlabelled differ-entiated MSC.Monitoring of CM-DiI label retention by differentiatedMSC usingflow cytometry demonstrated that all groupsexcept chondrocyte maintainedfluorescence levels higherthan control(Fig.6E).The cardiomyocyte induced groupshowed highestfluorescence signal after four weeks.When examined byfluorescence microscopy however allgroups showedfluorescence similar to control.DiscussionThe lineage-specific differentiation potential of MSC hassuggested a therapeutic role in cardiac repair,15but stud-ies have mostly been in small animals with limited abilityto refine strategies and techniques for human use.Theovine model currently lacks‘tools’such as monoclonalantibodies and GFP to track,identify and analyse MSC.Therefore the key objective of this study was to optimiseisolation of ovine MSC and to test cell tracking dyes foreffectiveness,stability and cell damage.O R I G I N A L A R T I C L EHeart,Lung and Circulation Weir et al.4012008;17:395–403Fluorescent stem celltrackingFigure 6.CM-DiI fluorescence of differentiated MSC groups after 3weeks in culture (×40).(A and B)Negative/positivecontrol—unlabelled/labelled undifferentiated MSC,(C and D)cardiomyocyte/chondrogenic induced MSC,(E)flow cytometric mean fluorescence intensity of CM-DiI labelled MSC after 4weeks in vitro differentiation (n =3,mean ±S.D.).Initial work focused on isolation and characterisation of ovine MSC which,unlike murine MSC,have not been well characterised or isolated according to surface mark-ers such as c-kit and SCA-1.8Initial selection involved flask adherence,well documented for MSC isolation from hemopoietic stem cells.7Subsequently,anti-human CD 106antibody with magnetic bead separation isolated a small population of cells which formed adherent colonies after several days,indicating that V-CAM antigen is species conserved.However,several weeks in culture changed surface markers with loss of CD 106.Adherent and CD 106selected populations showed no significant difference in surface markers after several weeks culture.Carboxyfluorescein diacetate,succinimidyl ester (CFSE)labelling,initially trialled as tracker for ovine MSC had been used to track cell division,e.g.lymphocytes,16but MSC so stained and grown in culture rapidly lost staining.In contrast,MSC staining intensity with chloromethylbenzamido-DiI (CM-DiI)labelling peaked at day 2and levelled out by day 7.It was maintained for four to five weeks in vitro in sub-confluence maintained cultures.In experiments labelling MSC with CM-DiI before 100cells were sorted by flow cytometry and then cultured,CM-DiI signal was lost after five divisions.While expected in rapidly dividing cultures,MSC were expected not to divide rapidly in vivo .Therefore CM-DiI (4␮M concentration)labelling which did not affect cell division was chosen.The use of a non-specific DiI version for in vivo track-ing over 24hours of injected endothelial progenitor cells into rat heart has been documented.17Our initial two-week sheep study showed retention of CM-DiI staining in MSC injected into skeletal muscle or placed subcuta-neously on biomaterial.Thicker sections served to clearly visualise cells by fluorescence microscopy at six weeks.Additionally,biomaterial proved effective in culturing and applying MSC in different applications.The reservoir of cells in such scaffolds could replace cells,compensating for loss during direct injection,shown to approach 80–90%in beating pig and rat heart.18DiI,without the CM derivative,has been used to stain rat MSC in vivo to six months,19and similar stain retention after eight weeks with nerve growth 20indicates that cells with low division rates retain DiI stain for long periods.CM-DiI has improved solubility giving better dye reten-tion and stability through fixation and permeabilisation procedures 21without requiring cytotoxic DMSO.Our ovine MSC were induced to chondrogenic differentiation 12and after four weeks of culture were examined by two surgical pathologists blinded to con-ditions.The cells showed early morphological and histochemical features of cartilaginous differentiation not present in other cell lines.These cells also showed pos-itive labelling for the muscle marker desmin.Desmin expression (“myochondroblastic phenotype”)is likely to be a feature of regenerating,dividing and differentiat-ing cartilage cells 22akin to myofibroblastic cells present in regenerating fibrous tissue.Differentiation of ovine MSC into cardiomyocytes did not demonstrate the characteristic ‘beating’and myotube formation described by Makino et al.2whether using large numbers of 5-azacytidine induced cells or cloned MSCs.However,the cardiomyocyte induced MSC did show desmin staining although morphological identifica-tion provided no evidence of myocyte differentiation.Only chondrogenic and cardiomyocyte lines showed desmin positivity as opposed to desmin negative controls.Unlabelled and CM-DiI labelled MSC when induced to differentiate,produced the same histological pat-terns,demonstrating that 4␮M CM-DiI does not affect cell division/differentiation.Flow cytometry showed cardiomyocyte induced MSC retained the highest flu-orescence for four weeks.Chondrogenic induced cells showed a rapid seven-day drop in fluorescence,per-haps due to CM-DiI dye incorporation into different cell areas or excretion during ing fluores-cent microscopy each differentiated group had a differentORIGINAL ARTICLE 402Weir et al.Heart,Lung and Circulation Fluorescent stem cell tracking2008;17:395–403staining pattern indicating CM-DiI’s incorporation intodifferent cell components.Chondrogenic CM-DiI stainingis quite distinctive compared to cardiomyocyte inducedcells,possibly relative to level of differentiation.Others have shown that DiI dye is retained in MSC wheninjected into an infarcted heart region in rats,where theyalso begin to show differential changes.19Left longer in theinfarct zone(up to six months)cells showed more musclemarkers(␣-actinin,myosin heavy chain)but no decreasein DiI signal.19This corresponds with our results in sheepshowing CM-DiI retained even with MSC differentiatingand expressing different cell markers.Similar to our invitro differentiation studies Dai et al.reported that whileMSC expressed cardiac cell markers,differentiation wasincomplete.19The key advantage of CM-DiI and derivatives is pro-vision of a stable marker should MSC surface markers(CD44)change during differentiation.It also obviatesneed for genetic manipulation of cells used to induceGFP.ConclusionIn conclusion,our study demonstrated that characterisa-tion of ovine MSC reveals similar markers to other species(i.e.CD44+,and CD45−and CD11b−).However,lack ofdistinct ovine specific MSC markers(CD105,CD106,CD271,SCA-1and c-kit)make in vitro characterisation andstudy difficult.In our studies in vitro differentiation andidentification by blinded pathologists confirmed them asMSC.CM-DiI dye studies demonstrated it to be a simple alter-native for tracking cells that had been prepared in a fewhours,both in vitro and in vivo.In contrast to GFP there isno genetic manipulation.We demonstrated that CM-DiIlabelling provides a stablefluorescent tracking system notinfluenced by changes in surface markers or 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