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Hematology, Transfusion and Cell Therapy

Original article

Standardization and quality assessment for clinical grade mesenchymal stem cells from human adipose tissue

Tanya Debnath

a

, Lakshmi Kiran Chelluri

b,∗

aGlobalMedicalEducation&ResearchFoundation,Hyderabad,India

bTransplantImmunology&StemCellUnit,GleneaglesGlobalHospitals,Hyderabad,India

a r t i c l e i n f o

Articlehistory:

Received9December2017 Accepted2May2018 Availableonline19June2018

Keywords:

Differentiation Geneticstability

Humanadiposederivedstemcell Mesenchymalstemcells Proliferation

a bs t r a c t

Background:Mesenchymalstemcellshaveimmensepotentialinstemcell-basedtherapies, however there is a pre-requisite to develop a curative cell dose. Adipose tissue- derivedmesenchymalstemcellsarepromisingmainlyduetotheirpotentialabundance, immunomodulatoryeffectandremarkabledifferentiationpotential.Nevertheless,senes- cencemaydevelopduringtheirinvitroexpansionduetotheincidenceofgeneticinstability.

Hence,itisimportanttoattainanidealbalancebetweenmesenchymalstemcellgrowth, qualityandgeneticintegritybeforetheirclinicaluse.

Methods:Stromalvascularfractionwasobtainedfromomentumtissueofpatientsunder- goingliposuctionproceduresformorbidobesity.Thisstudystandardizedaclosedsystem protocolwhichcanbeutilizedforclinicalgradestemcellderivation.Stagesofcellgrowth andcharacterizationofhumanadiposetissue-derivedmesenchymalstemcellswerealso assessedalongwiththechromosomalstabilityintheseinvitrocultures.

Results:Humanadiposetissue-derivedmesenchymalstemcellsmaintainedtheirspindle- shapedmorphologyandwereabletoproliferate andrenew,confirmingtheirsuitability forinvitrocultivationandgenerateclinicalgrademesenchymalstemcells.Immunophe- notypingindicatesthatthecellsexpressedclusterofdifferentiation(CD)73/CD90/CD105, mesenchymal stem-cell markers, while lackedCD34/CD45/Human Leukocyteantigen- antigen Drelated (HLA-DR)expression(hematopoietic cellmarkers). Acell cyclestudy demonstrated growth kinetics underinvitro culture conditions.Human adiposetissue derived mesenchymal stem cells expressednormal cell karyotypeby chromosomalG- banding indicating their genetic stability at Passage 5. Mesenchymal stem cells also demonstratedtrilineagedifferentiation.

Conclusions: Availabilityofadiposetissueinabundanceisa majoradvantageforclinical applications.Furthermore,detailedcharacterizationofhumanadiposetissue-derivedmes- enchymalstemcells,theirgenomicstabilityanddifferentiationpotentialfromstromalvas- cularfractionofhumanadiposetissuewouldhelpassistintissueregenerationandrepair.

©2018Associac¸ ˜aoBrasileiradeHematologia,HemoterapiaeTerapiaCelular.Published byElsevierEditoraLtda.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Correspondingauthorat:TransplantImmunology&StemCellUnit,GleneaglesGlobalHospitals,Lakdi-ka-Pool,Hyderabad,India.

E-mailaddress:lkiran@globalhospitalsindia.com(L.K.Chelluri).

https://doi.org/10.1016/j.htct.2018.05.001

2531-1379/©2018Associac¸ ˜aoBrasileiradeHematologia,HemoterapiaeTerapiaCelular.PublishedbyElsevierEditoraLtda.Thisisan openaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

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Introduction

Stemcellsarecharacterizedbytheirself-renewalproperties andbytheircapacitytogeneratedifferentiatedcelllineages providingprospectsforcell-basedtissueregeneration.Stem cellsaremaintainedduringthelifeoftheorganismtohelp inlineage-specificdifferentiation.1,2 Althoughstemcellsare maintainedintheiradulttissues,someofthecellsundergo divisionwithoutdifferentiation,suchthatoneoftheprogeny remainsundifferentiated,whiletheotherproliferatesanddif- ferentiatestogeneratenewtissuemass.3

Mesenchymalstemcells(MSCs)arepresentintissuesor organsfromwhichtheyexitintothecirculationtohomeinto sitesofinjuryor toothertissues. TheMSCs are wellchar- acterizedbasedontheirsurfacemarkersandalsohavethe abilitytoregenerateintovariouscellsandtissues.4,5 Human MSCs(MSCs)areauniqueprogenitorcellthatcanberecovered frommostvascularizedtissues.Themostsalientfeaturesof MSCsalsoincludetheirimmunomodulationpropertiesand trophiccapabilities.MSCsactasimmune-conductorsoftis- suerepairandregenerationbasedontheirabilitytosecrete trophicfactorsthatstimulateneighboringparenchymalcells tostartrepairingdamagedtissues.6

TheclinicaluseofMSCshasbeengrowingenormouslyin bothhumanandanimalresearch.MSCsexhibitaremarkable feature of transdifferentiation into ectodermal, neuroecto- dermal and endodermal cells, phenomena referred to as

‘stem cell plasticity’. This knowledge opened the possibil- ity of clinical applications of MSCs in the regeneration of other tissues such as cornealreconstruction, treatment of acutelunginjury,oralmucosalregeneration,homingofMSCs forregenerationatsitesofinjury,etc.Thoughevidencehas accrueddemonstratingthisphenomenon,thereisstillagapin understandingthemolecularmechanismoftransitionsthat willbeimportanttocontroltheprocess efficiently.Studies alsodemonstrated the utilizationofdiverse organ-resident perivascularMSC-likecellsand bonemarrow-derivedMSCs intissueregeneration.7,8Thesecellscanbeculturedtocreate homogenouspopulationsundercontrolledcultureconditions.

Moreover, MSCs also possess immunosuppressive effects, implying their possible clinical applications in regenera- tivemedicineandtherapiesfortreatment-resistantimmune disorders.

MSCsaredescribedasimmaturecellswithinthebonemar- row,peripheralblood,menstrualblood,andnearlyalladult tissuesand solidorgans.9,10 MSCshavealsobeenshownto possessimmunogenicpropertiesandapowerfulimmunosup- pressivepotential,whichmakethemattractiveforallogeneic celltherapy.11

Adiposetissuehasevolvedasanotherpotentialalterna- tivesourceofstemcells.Adiposetissue-derivedpopulations ofcellsfromthestromalvascularfraction(SVF)areharvested bydigestingthefatandconcentratingtheremainingcells.Ear- lier,Youngetal.12reportedthatthesestemcellsfromtheSVF offathadsimilarmorphologytoMSCs.Subcutaneousadipose depositsareaccessible,abundant,andreplenishable,thereby providingapotentialadultstemcellreservoirforeveryindi- vidual.

Humanadiposetissue-derived stemcells(ADSCs),when comparedwithbonemarrow-derivedstemcells(BMSCs),can beeasilyculturedandproliferaterapidlywithahighcellu- lar activity.13 Izadpanah et al.reportedthat humanADSCs can maintain their chromosomal stability for more pas- sages(30)comparedtohumanBMSCs(20)beforebecoming senescent.14 All thesequalitiesmakeadipose tissueause- ful source of MSCs. Jurgens et al. showed that the yield, proliferation and differentiation of MSCs also depend on the tissue harvesting site.15 Thus, it is also important to monitortheprocessestoexpandMSCsinarobustandrepro- ducibleway,whileassuringtheirpurityandqualityforclinical applications.

Althoughthe currentwork isnotincremental,it isvery importanttoreproduceclinicalgradeMSCsforclinicalpur- posesanddemonstratethesamephenotypicandgenotypic characteristics.Hence,thepresentstudydealswiththeiso- lationandpropagationofhumanMSCsderivedfromtheSVF ofadiposetissue.Thegrowthpattern,long-termchromoso- malstabilityandtrilineagedifferentiationpotentialofhuman ADSCs was alsostudiedin order toemploythesecells for chronicailments.

Methods

Low-glucose Dulbecco’s modified eagle medium (L-DMEM;

4.5mmol/Lglucose),typeIcollagenaseandfetalbovineserum (FBS)were purchasedfromGibco(BRL,USA).All antibodies were purchasedfromBDBioscience(SanJose,USA).Allthe finechemicalswerepurchasedfromSigma(St.Louis,USA).

Prior written informed consent was obtained from donors aged from 26 to 57 years undergoing liposuction procedures for morbid obesity. The study was approved by the Global Hospitals, Institution Ethics Committee (IEC – Ref no. GMERF/BS/SAC/IEC/ICSCR2014/01). The adipose tissue for the study was collected in sterile Dulbecco’s modified eagle medium (DMEM) supple- mented with antibiotics in accordance with the code of ethics of the World Medical Association (Declaration of Helsinki).

Stromalvascularfractionisolationfromomentumtissue

Cellswereisolatedfrom2–3gofadiposetissueusingamod- ified procedure of Zuk et al.16 The collected tissues were washedtwoorthreetimeswithphosphatebuffersaline(PBS).

The tissuewas minced into pieces of1–3mmin diameter andtreatedwithpre-warmedcollagenasetypeI(2mg/mL)at 37Cwithintermittentvortexingfor1–2h.Thedigestedtis- suewasfilteredusinga40-␮mcellstrainer,thefiltratewas centrifuged at600gfor10min.Thesupernatantcontaining thematureadipocyteswasdiscarded.Thepelletwasidenti- fiedasthehumanadiposetissue-derivedMSCsconsistinga heterogeneousmixtureofcells.

Thecellswereseededatadensityof4000cells/cm2incom- pleteMSCculturemediafor3–5daysforinitialattachment.

TheSVFwascharacterizedforvarioushematopoietic,mes- enchymalandsidepopulationcellsurfacemarkersusingflow

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cytometry.Theculturedcellswerewashedandcountedusing Trypanblue todistinguish viableand deadcells,and then frozeninfreezingmediumconsistedofDMEMwith90%FBS and10%dimethylsulfoxide(DMSO).

Assessmentofcellviability

The cell viability of cryopreserved human ADSCs (n=5) after revival was determined by incubatingwith 7-amino- actinomycinD(7-AAD,BDPharmingen,USA)for20mininthe dark.7-AADviabilityassayswerecarriedoutbyflowcytome- tryusingCellQuestsoftware(BectonDickinson,USA)ateach passagelevel.Approximately,1×106cells/mLwereincubated with20␮Lofthedyefor10minbeforeanalysis.Thisreagent isusedasaviabilityprobeformethodstoexcludedeadcells basedonlightscatteranduptakeof7-AADasdetectedinthe fluorescence3(FL3)channel.

Characterizationofhumanadiposetissue-derivedstem cells

Morphologicalcharacterization-staining

Human ADSCs were maintained in growth media till they reached 60% confluency. Giemsa staining was done to assess the basic morphology of human ADSCs. Approxi- mately1×103cells/cm2 were cultured and later fixed with 3.7% paraformaldehyde for 20min at room temperature.

Cellswere washedand stainedwithGiemsastainfollowed by hematoxylin & eosin (H&E) staining for morphological evaluation.

Markerexpressionbyconfocalmicroscopy

HumanADSCs were furthercharacterizedbyimmunocyto- chemistry to detect the presenceor absence of the CD90, CD73andHLA-DR,CD45,andCD34markers.HumanADSCs (1×103cells/cm2) were grown on cover slips in a humidi- fiedatmosphere. After18h,the coverslipswere fixedwith 3.7%paraformaldehyde (Sigma–Aldrich,St.Louis,MO,USA) for20minatroomtemperature.Thecover slipswere incu- batedat4Covernightwithprimaryantibodies againstthe followingsurfacemarkers:CD90(1:100),CD73(1:100),HLA-DR (1:50),CD34(1:100),andCD45(1:100).Thecoverslipsweresub- sequentlyincubatedwithAlexaFluor488(Molecularprobes, Lifetechnologies,USA)andlabeledwithsecondaryantibod- iesatroomtemperaturefor1h.Further,theyweremounted on slides with mounting medium (Vectashield) containing 4,6-diamidino-2-phenylindole(Abcam,Cambridge,UK) and incubatedfor2–3minatroomtemperatureinthedark.The imagingwas performedusingaconfocalmicroscope(Leica TCS,CLSM)andtheimageswereprocessedusingLeicasoft- ware.

Immunophenotypingbyflowcytometry

Thecellsweretrypsinizedandthepelletwasre-suspended inPBS.Acellsuspensionwasmadewith1×106cells/mLand incubated inthe dark for30min at4C with the antibod- ies. Cell surface antigen expression ofhuman ADSCs was detected at each passage using flow cytometry. Epitopes, such as CD90 (FITC), CD34 (PE), CD73 (APC), CD45 (FITC),

and HLA-DR (PE),were evaluated forpositive versus nega- tiveexpressionbyflowcytometryusingCellQuestsoftware (Becton Dickinson). All these anti-human antibodies were raised in mouse and were obtainedfrom BD Pharmingen, USA.

Proliferationkineticsofhumanadiposetissue-derived stemcells

ADSCswereseededatadensityof1×104cells/cm2tostudy thegrowthkineticsateachpassage(n=5).Periodically,after 24h,48h,72h,96h,120hand144hthecellsweretrypsinized, harvestedandcounted.Agraphwasplottedofthenumberof cellsagainsttimepointindicatingthegrowthphasesofstem cells.

Cellcycleanalysis

Cellcycledemonstratesthestagesofcellgrowth.Proliferat- inghumanADSCs(n=5)wereharvestedandfixedincold70%

ethanol.Ethanolwasaddeddropwisetothepelletwhilevor- texing,ensuringfixationofallthecells.Thepelletwasallowed tofixfor30minat4C.Thecellpelletwasstainedwith200␮L propidiumiodidefrom50␮g/mLstocksolution.

Karyotypingofhumanadiposetissue-derivedstemcells

The cytogenetic analysis of adipose tissue-derived MSCs helpsinassessingtheirstabilityandtumorigenicity.Human ADSCs(n=5)were seededatadensityof8.0×103cells/cm2 in complete basal media. The actively dividing cells were arrested at the mitotic phase after 48h. The cells were treatedwithcolchicine(0.1␮g/mL) (Sigma–Aldrich,USA)for 3–4hpriortoterminatingcultureandharvestedusing0.25%

trypsin-EDTA. Thecells were washed and re-suspended in pre-warmed hypotonic solution (0.075M KOH) for 20min at 37C. The pellet was fixed and incubated in chilled methanol andglacialacetic acid (3:1)solution overnightat 4C.Thefixedcellsweredroppedontoapre-warmedslide byholdingtheslideata60 angle.Imagesof5metaphase spreadsforeachsamplewerekaryotypedusingspectralimag- ing software at a resolution of 550 bands per haploid set resolution.

Trilineagedifferentiationpotential

Adipogenicdifferentiation

ExponentiallygrowinghumanADSCs(n=5)wereculturedasa monolayerfor21daysinthepresenceofhigh-glucoseDMEM, 10%FBS,andadipogenicsupplements(1␮Mdexamethasone, 1␮g/mL insulin and 0.5mM 3-isobutyl-1-methylxanthine) (Sigma–Aldrich, USA). To visualize adipocytes, cultures were fixed in 10% neutral buffered formalin solution (Sigma–Aldrich, USA) for30min,then washed and stained withOilRedO(3mg/mLinH2O)for10min.

Osteogenicdifferentiation

Thehuman ADSCswere differentiated into osteogenic lin- eagesbygrowingtheminosteogenicinductionmedia.Human ADSCs(n=5)wereseededintheculturemediaataseeding

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Adipose tissue

PBS wash

0.075% collagenase type l @ 37°C for

30 min

Inactivate digestion by

supplementing serum Centrifuge @ 1500 rpm

Filter using 40µm cell strainer

SVF pellet Plate SVF pellet in complete culture media

Figure1–Isolationofmesenchymalstemcellsfromadiposetissue.Schematicrepresentationofenzymaticdigestionof adiposetissueusingcollagenasetypeI.Isolationandexpansionofthemesenchymalstemcellspopulationfromthe stromalvascularfraction.

Figure2–Humanadiposetissue-derivedstemcellsculture,viabilityandcharacterization.(A)Humanmesenchymalstem cellswereisolatedfromadiposetissueinlow-glucoseDulbecco’smodifiedeaglemedium.Thecellsformedsmallcolonies withinthreedaysofcultureasshownbythearrows.Theplateshowedadherent,spindle-shapedfibroblast-likecells comingoutofthecolonies.Humanmesenchymalstemcellsisolatedinlow-glucoseDulbecco’smodifiedeaglemedium reached80–85%confluencybyDay10afterseeding(magnification10×).(B)Viabilityofadiposetissue-derivedstemcells afterfreeze/thawcycles(n=5).Forwardscatterversuslogfluorescence3(FL3)channel(7-AADfluorescence)dotplotshows themeanpercentageofviableanddeadcells.(C)Theadheredmesenchymalstemcellsshowedspindle-shaped

morphologybyhematoxylin&eosinstaining.Giemsastainingalsoconfirmedthespindle-shapedmorphologyofadipose tissue-derivedstemcells.(D)CD34/45andHLA-DRexpressionswerenegative,whileCD90/CD73/CD105expressionwas observedonthecellsurface(showningreen).Thenucleuswasstainedwith4,6-diamidino-2-phenylindole(blue).

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Figure3–Characterizationofthecellsurfacemarkersforhumanadiposetissue-derivedstemcellsbyflowcytometry.

CD34-PE,CD45-FITC,HLA-DR-PE,CD90-FITC,CD105-PerCPandCD73-APC.Theshadedhistogramsrepresentunstained negativecontrolcells.Passage1didnotexpressCD34/45markers(0.2%and2.5%,respectively),whilelowlevelsofHLA-DR expression(2.2%)wereobserved.Humanadiposetissue-derivedstemcellswerepositiveforCD90(98%)andCD73(99%) expression,indicatingastemcellimmunophenotype(n=5).

densityof200cells/cm2 inculturedishes. Whenthe plates reached 90% confluency, human ADSC culture media was replacedwithosteogenicinductionmediacontaining100nM dexamethasone, 10mM ␤-glycerophosphate and 0.1mg/mL ascorbate-2-phosphate in the presence of 10ng/mL bone morphogenetic protein 2 (BMP-2). After three weeks, the cultureswerefixedin10%neutralbufferedformalinsolution (Sigma–Aldrich,USA)andwereevaluatedforthepresenceof calciumdepositsbyvonKossastaining.

Chondrocytedifferentiation

TheconfluenthumanADSCs(n=5)were removedfromthe cultureflaskand acellpelletwasobtainedbycentrifugeat 100gfor5–10min.Thecellsweresuspendedinchondrogenic induction media containing high-glucose DMEM supple- mentedwith10%FBS,50␮g/mLascorbicacid,1%ITS-premix, and0.1␮Mdexamethasoneinthepresenceof10ng/mLtrans- forminggrowthfactorbeta(TGF-␤–Sigma–Aldrich,USA).The cellswereculturedinapelletsystemfor21days.Thediffer- entiatedcellswerestainedwithAlcianblue.

Statisticalanalysis

All quantitative data are represented as means±standard error(SE).

Results

Isolationofhumanadiposetissue-derivedstemcellsfrom stromalvascularfraction

HumanMSCsweresuccessfullyisolatedfromadiposetissue (n=5).Figure1depictstheisolationprocessofSVFfromadi- posetissue.TheSVFwasplatedincultureflasks.Anaverage yieldof1×106cells/mLwasobtainedfrom2–3gadiposetis- sue.MSCswereseentoattachtoculturedishessparselyand mostcellsdisplayedaspindle-likeshape.Thesecellsbegan toproliferateataboutDay4andgraduallygrewtoformsmall colonies.

Characterizationofhumanadiposetissue-derivedstem cells

Cultureofhumanadiposetissue-derivedstemcells

In large colonies, cells were more densely distributed and showedaspindleshape.Thecoloniescontinuedtogrow,grad- uallyexpandinginsizeandreachedconfluencybyDay10.The platereached80–85%confluencyreadyforpassaging14days afterseeding(Figure2A).

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Figure4–Growthprofileofhumanadiposetissue-derivedstemcells.(A)Growthcurveobtainedforthehumanadipose tissue-derivedstemcells(n=5)showsanexponentialgrowthphase.ThedeclinephasestartedafterDay5andcellnumber decreasedto1.5×103cells/mL.(B)Thegraphrepresentsmeanpercentageofhumanadiposetissue-derivedstemcellsat differentstagesofthecellcycle.ThepercentageofcellsintheSphasedecreasedsignificantlyafter72h.(CandD)Allthe stagesofcellcycleofhumanadiposetissue-derivedstemcellsweretestedafter24hand96husingflowcytometry.hADSC:

humanadiposetissue-derivedstemcells.

Viabilityofhumanadiposetissue-derivedstemcells

Thecellsweretrypsinizedandpassagedfurther.Theisolated adherentpopulationshowedthin,longspindleshapedfibrob- lastlikecells.Thefreeze-thawedcellsalsoshowedmorethan 87%viabilityby7-AADstaining(Figure2B).

Morphologicalevaluation

Morphologically,humanADSCsshowedtheirspindleshape andfibroblast-likeappearanceonDay14ofcultureusingH&E and Giemsastaining. Cells exhibitedhigh nucleus tocyto- plasmicratioonGiemsastaining.TheadheredMSCsshowed spindleshapedmorphologyonH&Estaining(Figure2C).

Immunocytochemistryofhumanadiposetissue-derived stemcells

Further,tocharacterizehumanADSCs,animmunocytochem- ical procedure was carried out using several cell surface markers.Theywererecognizedfortheirimmunoreactivityto MSC-specificcelltypemarkers.Asshown,MSCsweremarked byCD90,CD105andCD73,butwerenotimmunopositivefor CD45,CD34andHLADR(Figure2D).

Immunophenotypingofhumanadiposetissue-derivedstem cells

TheisolatedSVFshowedpositiveexpressionofCD34/45indi- catingtheheterogeneoushematopoieticcellpopulation.Upon culture,ateachpassagehumanADSCsrepresentedonly1–3%

expressionofCD34/45andHLADR.Thepercentageexpression ofCD90/CD73/CD105increasedupto90–99%ateverypassage (P1–P5).Morethan90%oftheSVFexpressedsurfacemarkers forstemcells(n=5).Thedatasuggestedthatthethirdpas- sagehumanADSCsexhibitaMSCorigin,andthattheyare notinterminglingwithhematopoieticcells(Figure3).

Proliferationofhumanadiposetissue-derivedstemcells Thegrowthrateofadiposetissue-derivedMSCswasdefinedby plottingagrowthcurve.Onanaverage,humanADSCsexhib- itedashortlagtimeintheirgrowthcurveateachpassage.

ThegrowthcurveforthesehumanADSCs(n=5)includeda lagphasebutcellnumbersincreasedaftertwodays.Thecells wereatlogphasefor48handlaterthecellnumberdecreased indicatingtheirdeathphase,representingasigmoidgrowth curveofhumanADSCs(Figure4A).

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Passage 5 Passage 2

A

Giemsa

B

Giemsa

Karyogram Karyogram

1 2 3

6 7 8 9 10 11 12

17 18 16

22 21 19 20

13 14 15

X Y

6 7 8 9 10 11 12

17 18 16 14 15

13

19 20 21 21 X Y

4 5

1 2 3 4 5

Figure5–Karyotypicanalysisofarepresentativestemcellisolatedfromhumanadiposetissue.(A)Karyotypicanalysisof Passage2humanadiposetissue-derivedstemcellsfromafemaledonorwithanormalfemaleKaryotype(46,XX).(B)At Passage5,humanadiposetissue-derivedstemcellsfromamaledonorexpressednormalamalekaryotype(46,XY).

DNAcontentofculturedhumanadiposetissue-derivedstem cells

Thephasesofcellcycleandtheexponentiallygrowinghuman ADSCs were also studied(Figure 4B).The cell cycle analy- sisemphasizestheneedforearliersubculturingtooptimize growthrateandnon-differentiation.Datashowthatafter72h, thepercentageinSphaseisreduced,asevidentfromthemean channelshift(MFI)valuesfromtheflowcytometrybasedCell Questanalysisreportandthus,revealingadecreaseinDNA replicationandindicatingcellsshouldbesubculturedaccord- ingtorecommendedcriteria.Cellswerealsoanalyzedat96h withoutsubculturing.Asignificantdecreaseinthepercent- ageofcellsinSphasewasobservedasthecells remainin cultureandbecomemoreconfluentovertime(Figure4Cand D).Thus,subculturingpriorto80%confluencyisrequiredfor anoptimizedgrowthratewithoutdifferentiation.

Genomicstabilityofhumanadiposetissue-derivedstemcells Theprimaryscreeningofthechromosomalspreadswasdone byGiemsastaining.ThekaryogramofculturedhumanADSCs (n=5)fromafemaledonorrevealedanormalfemale(46,XX) atPassage2(n=5)asshowninFigure5A.Whereas,human ADSCsisolatedandculturedfromtheSVFfractionofamale donoralsoconservedthenormalmalekaryotype(46,XY)at Passage5(n=5)asshowninFigure5B.Thechromosomenum- bersandstructureremainedstableafterexpansionbyserial passaging.Chromosomeswerepairedbasedonbandingpat- ternsandpositionoftheirrespectivecentromere.

Trilineagedifferentiationpotential

Differentiationintoadipogeniclineage

ThehumanADSCs(n=5)weredifferentiatedintoadipocytes when growninadipogenic inductionmedia. Oncethe adi- pogenicinductionmediawasadded,cellproliferationstopped and the cells appearedflattenedand increased insize.Oil droplets started accumulating in the differentiating cells, whichincreasedinnumberandsizeasthedifferentiationpro- gressed(Figure6A.a).OilRedOstainingshowedoildroplets stainedred,confirmingthedifferentiationofhumanADSCs intoadipocytes.Althoughtherewaschangeiscellmorphol- ogyintheuninducedcultures,therewasnovisiblestainingof lipiddroplets.

Differentiationintoosteogeniclineage

WhenhumanADSCs(n=5)weregrowninosteogenicinduc- tion media, theydifferentiated into osteogeniclineage and showedmineralization.Rapidcellproliferationwasobserved in the early phase of differentiation, which was gradually reduced atthe onset ofmineralization;vonKossastaining for calcium demonstrated the secreted calcium containing mineraldeposits(Figure6B.b)therebyconfirmingosteoblast differentiation on Day 21 of differentiation. Plates with L- DMEMmediadidnotshowanymineralizationevenafter21 daysofculture.

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Figure6–Trilineagedifferentiationpotentialofhumanadiposetissue-derivedstemcells.(A)Phasecontrastmicrographs showingthedifferentstagesofdifferentiationofhumanadiposetissue-derivedstemcellstoadipocytes.Adipocyteswith oildropletsarevisibleonDay14ofadipogenicinduction;thesizeandnumberincreasedasthedifferentiationprogressed.

OilRedOstainedthelipiddropletsredonDay21(magnification20×).(B)Phasecontrastmicrographsshowingdifferent stagesofthedifferentiationofhumanadiposetissue-derivedstemcellstoosteoblasts.Day14ofosteogenicinduction showedvisiblemineraldeposits.TherewasasignificantincreaseinthemineralizationonDay21ofosteogenicinduction (magnification20×).(C)Pelletcultureofhumanadiposetissue-derivedstemcellsonDay21.Hematoxylin&eosinstaining showedhomogenouscelldistribution.Alcianbluestainingofacidshowedmucopolysaccharideaggregatesinthe

differentiatedchondrocytes.

Differentiationintochondrogeniclineage

Pellet culture was used for chondrogenic differentiation.

The human ADSCs (n=5) cell pellet was supplemented with chondrogenic differentiation media. A spherical cell pellet was observed after seven days of culture. There was a progressive increase in the size of the cell pel- let mass after 21 days (Figure 6C.a). The paraffin fixed pellets were sectioned and stained with H&E and Alcian

blue stains.Thedifferentiatedcells inchondrogenicmedia were observed to have large nuclei that became progres- sively bigger and morerounded over 21 days ofculturing.

Clearpericellularlacunaewerealsoobservedintheinduced chondrogenic culture. The deposits of acid mucopolysac- charides were confirmedby Alcian bluestainingas shown in Figure 6C.c.An uninduced hADSC pellet was usedas a control.

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Discussion

Thestromalcomponentofmesenchymaltissuesisthoughtto harborstemcellsthatdisplayextensiveproliferativecapacity andmultilineagepotential.Stromalcellsisolatedfromvarious mesodermaltissuesshare keycharacteristics,includingthe abilitytoadheretoplastictoformfibroblasticlikecolonies (calledCFU-F),extensiveproliferativecapacityandabilityto differentiateintoseveralmesodermallineages.Considering thesecharacteristics,stromalcellsmaypotentiallybeuseful inthefieldofregenerativemedicine.

AstudyhasfoundthattheSVFfromadiposetissuepro- vides 500 times more stem cells than bone marrow (only 0.01–0.001%).17Adiposetissueisconsideredthemainenergy sourceoftheorganism.Adiposetissuecontainsalargenum- ber of stromal stem cells.18 In 2003, van Harmelen et al.

showedthatthe ratioofadipocytestoMSCsisconstant in humans,independent ofbody mass index and age.19 This allowsagreaterharvestofMSCsfrompatientsorsubjectswith ahigherBMI.

Thisworkfocusedonthestandardizationoftheisolation procedureofhumanstemcellsfromtheSVFofadiposetissue.

Thetypeofculturemediausedforisolationandpropagation greatlyinfluencetheviability,growthcharacteristicsandthe differentiationpotentialofhumanMSCs.20,21Primarycultures maintainedfor5–10 daysinL-DMEM mediasupplemented with10%FBS,l-glutamineandantibioticsshowedenriched, adherentcellshavingfibroblast-likemorphology.Inthisstudy, thesurfacemarkerexpressionofthesehumanADSCsfrom Passages1to5wasobserved.Ideally,Passage3expresseda higherpercentageofstemcell-relatedsurfacemarkers.The adiposetissue-derivedSVF cellsisolatedandpropagated in L-DMEMhad positive MSC surface markersand were neg- ativeforthehematopoieticmarkers.However,theendoglin (CD105)expressionwasalteredatlaterpassagesowingtothe inclinationofhumanADSCs towardosteogenicdifferentia- tion.Mitchelletal.alsoobservedsimilarexpressionpatterns ofCD105 andCD166 markers.22 Theisolated cellsalsohad CD90,CD73proteinexpressionsontheirsurfaceasobserved byconfocalimaging.

The phenotype of human ADSCs is quite similar to BMSCs.23Thestagesofthecellcycleinproliferatinghuman ADSCswerestudiedatdifferenttimepoints.Thegrowthkinet- ics of cultured human ADSCs consistently represented a sigmoidalgrowthcurveinallthe5passagesdemonstrating anexponentialpatternofgrowth.Thecellcyclepatternalso revealedthatafter72hthecelldivisionissloweddowninthe cultures.Myungetal.reportedthattheintegrityofthegenome andgeneticstabilityisprotectedinthe Sphaseofthecell cycle.24

InvitroexpandedhumanADSCswerechromosomallysta- blewithoutanyclonalalterations.Theirgeneticintegritywas maintainedwithoutanychromosomalabnormalitiesorstruc- turalaberrationsuntilPassage5.Theseresultsarebeneficial todeterminethequalityoftheADSCsfortherapeutictrans- lation. Unaltered karyotypes can be considered one ofthe importantqualityindicatorsofMSCsinclinicalimplications.

SimilartoBorgonovoetal.,thedataofthisstudyalsosuggest thatconventionalkaryotypingcanbeusefulasapreliminary

tooltodeterminethe chromosomalstability duringinvitro expansionofMSCs.25

This study also showed that MSCs are capable of forming osteoblasts, chondrocytes and adipocytes in vitro.

Furthermore,ourpreviousstudiesdemonstratedsimilartri- lineagedifferentiationpotentialofhumanADSCsonvarious biopolymers.26Thus,theSVFfromadiposetissueholdsgreat promiseintissueengineeringandregenerationalongwithits effectiveutilizationincellbasedtherapeutics.

Conclusion

In conclusion,advancesincell biologyhave ledtoinnova- tive and newtherapeutic potentials ofusing fat tissuefor regenerativemedicine.TheSVFfromadiposetissuehasasuf- ficiently largepopulationofMSCs.Particularly,in vitroMSC cultureconditionsmustbevalidatedproperlytoavoidchro- mosomal abnormalities.Thewell characterizedautologous humanADSCscouldhaveclinicalapplicabilityforcell-based therapies.

Conflicts of interest

Theauthorsdeclarenoconflictsofinterest.

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