Deng and Jianping Fu Department of Biomedical Engineering and Department of Mechanical Engineering University of Michigan Ann Arbor Michigan ABSTRACT Dissociationinduced apoptosis of human embryonic stem cells hESCs hampers their largescale culture ID: 77718
Download Pdf The PPT/PDF document "Biophysical Letter Improving Survival of..." is the property of its rightful owner. Permission is granted to download and print the materials on this web site for personal, non-commercial use only, and to display it on your personal computer provided you do not modify the materials and that you retain all copyright notices contained in the materials. By downloading content from our website, you accept the terms of this agreement.
BiophysicalLetterImprovingSurvivalofDisassociatedHumanEmbryonicStemCellsbyMechanicalStimulationUsingAcousticTweezingCytometryDiChen,YubingSun,CheriX.Deng,andJianpingFuDepartmentofBiomedicalEngineeringandDepartmentofMechanicalEngineering,UniversityofMichigan,AnnArbor,MichiganABSTRACTDissociation-inducedapoptosisofhumanembryonicstemcells(hESCs)hamperstheirlarge-scaleculture.Here-inweleveragedthemechanosensitivityofhESCsandemployed,toourknowledge,anoveltechnique,acoustictweezingcy- BiophysicalJournalVolume108March2015131513171315 plates.Asaresult,67.02.0%(5)ofsuchcellssur-vivedwithin24hafterdisassociation(MB;Fig.1MovieS2).hESCsthathavealreadyspreadbeforeATCtreatmentsalsosurvivedforatleast24hregardlessofthepresenceofMBsorUStreatment(Fig.1),suggestingthatcellspreadingandadhesionmightbecriticalforpro-motinghESCsurvival.Interestingly,ultrasoundapplicationalone(MB)orthepresenceofRGD-MBsaloneMB)hadmarginalbutsigniÞcanteffectinenhancinghESCsurvival,resultinginasurvivalrateof28.62.2%(5)forhESCswithoutMBsbuttreatedwithultrasound(MB)(Fig.1),and35.75)forhESCswithRGD-MBsbutwithoutultrasoundtreatment(Fig.1).TheseresultssuggestthatbindingofRGD-MBsmaytriggeradhesionsignalingandpromotehESCsurvival.Tofurtherinvestigatetheroleofintegrin-mediatedadhe-sioninhESCsurvival,weconductedexperimentstoapplyATCtreatmentstohESCsconjugatedwithMBscoatedwithacetylatedlow-densitylipoprotein(AcLDL;AcLDL-MBs),aligandfortransmembranemetabolicreceptorsthatdoesnotbindintegrins.UnderthesameATCtreat-ments,thesurvivalrateofhESCswithAcLDL-MBswasunchangedcomparedtocellswithoutMBs(MB;Fig.1),eventhoughAcLDL-MBsex-hibitedsigniÞcantlygreaterdisplacementsthanRGD-MBs(datanotshown).Thesedatasuggestedthatintegrin-medi-atedadhesionwasrequiredforimprovedhESCsurvivalbyacousticactuationofintegrin-anchoredRGD-MBs.Toascertaindetailsofacousticactuationofintegrin-anchoredRGD-MBsonhESCs,wedividedoriginallyroundedsinglehESCsintodifferentsubgroupsbasedontheinitialnumberofRGD-MBsattachedtoeachcellFig.2).WeexaminedhESCsurvivalrateandthecorre-spondingMBdisplacementsforeachsubgroup.hESCswithtwoormoreRGD-MBspercellexhibitedasigniÞ-cantlygreatersurvivalratethancellswithnooronlyoneRGD-MBunderATCtreatments(Fig.2).Thetotalaccu-mulativeRGD-MBdisplacementpercellforhESCswithtwotofourRGD-MBswasalsosigniÞcantlygreaterthanthatforhESCswithonlyoneRGD-MBpercell(Fig.2ItshouldbenotedthattheheightenedtotalaccumulativedisplacementofRGD-MBswasnotonlyduetothegreaternumberofRGD-MBspercell,butalsofromthesecondaryacousticradiationforcesbetweenMBs(),whichcouldefÞ-cientlydisplaceMBstowardeachother(MovieS3).For FIGURE1()ATCstimulationbyacousticexcitationofMBsattachedtocells.()Bright-ÞeldimageshowinghESCsattachedwithMBs.Scalebar,20m.()Illustrationofultrasoundproto-col.()LivecellimagingshowinghESCsexposedtoATC.RoundedhESCs(redcircles)withspreadhESCs(bluecirclesScalebar,10m.()SurvivalrateofhESCsexposedtoATCtreatmentswithRGD-MBs,AcLDL-MBs,andwithoutMB5,numberofcellsineachexperiment200).Errorbars,SE;**0.01,*0.05,StudentÕs-test.ToseethisÞgureincolor,goonline. FIGURE2()DistributionofMBspercell(Þveindependentex-200cellsforeach).()SurvivalrateofhESCswithdifferentinitialnumbersofRGD-MBs/cell;200cellsforeachexperiment,20cellsforeachsubgroupwithdifferentnumberofMBspercell.Errorbars,meanSE;**0.01,*-test.()AccumulativeMBdisplacementperATCtreatment.Errorbars,meanSE;**0.01,*0.05,StudentÕs-test.()Changeofcellareafordifferentsubgroups.BiophysicalJournal108(6)1315Ð1317BiophysicalLetters hESCswiththreeRGD-MBspercell,thetotalaccumulativeMBdisplacementwassimilartotwo-bubblecases(Fig.2becauseoneofthethreeMBsoftenexhibitedaminimalmovement(MovieS4)withbalanceofforcesinthepres-enceofmultipleMBsandmutualinteractionsamongthem.Four-MBcasescouldbeconsideredasacombinationofone-,two-,and/orthree-MBcaseswithvariationsinthepatternsofindividualMBmovementsdependingontheirrelativelocationswitheachotheronthecells(Fig.2MovieS5).Inaddition,initiallyroundedhESCsboundwithRGD-MBsandtreatedwithATCstimulations(MB)initiatedtheirspreading~5haftercellseedingandbecamefullyspreadby12h(Fig.2),signiÞcantlyslowerthanconventionalcell-spreadingprocesses().AreasofhESCswithoutMBs(MB)orwithMBsbutwithoutultrasoundtreatments(MB)remainedunchangedduringtheÞrst24hoursaftercellseeding(Fig.2),corre-spondingtolowsurvivalratesforthesegroups.Toevaluatepossiblelong-termeffectsofATCtreatment,weconductedanalkalinephosphatase(ALP)assaytodeter-minehESCcloningefÞciency.Consistentwithimprovedinitialsurvivalrate,cloningefÞciencyofATC-treatedhESCsincreasedapproximatelythreefoldcomparedtoun-treatedMBcontrols(Fig.3),suggestingthattheeffectofATCwasmainlytheimprovementofinitialsurvivalofdisassociatedhESCsratherthancellprolifera-tionormigration().ATCtreatmentsdidnotadverselyimpacthESCstemnessmarkerexpression,asshownbypos-itiveimmunostainingofpluripotencymarkersOct4,Sox2,andE-cadherin7daysafterATCtreatments(Fig.3Collectively,ourdataindicatethatATCstimulationsimprovetheclonogenicityofdisassociatedhESCsbyincreasingtheirinitialsurvivalrate.WhilethemolecularmechanismunderlyingsuchmechanosensitivebehaviorofhESCsrequiresfurtherstudy,ourresultssuggestthatinteg-rin-mediatedadhesionformationandstrengtheningbyATCstimulationsmayfacilitatespreadingofdisassociatedhESCs,whichinturnrescuesthecellsfromhyperactivatedactomyosinactivitiestriggeringdownstreamcaspase-medi-atedapoptoticsignalingpathways.Comparedtoestablishedmethodsforapplyingsubcellularforcesusingsolidmicrobeads(i.e.,opticalandmagnetictweezers),ATCutilizesMBsthatdonotexhibitcellularinter-nalizationandcanbeeasilyremovedfromhESCswithoutleavingbehindexogenousmaterials,providingapromisingbiocompatibleplatformforlarge-scalehESCculture.SUPPORTINGMATERIALSupportingMethods,SupportingDiscussion,andÞvemoviesareavailableathttp://www.biophysj.org/biophysj/supplemental/S0006-3495(15)00128-9ACKNOWLEDGMENTSThisworkissupportedinpartbytheNationalScienceFoundation(grantsNo.CMMI1129611andNo.CBET1149401),theNationalInstitutesofHealth(grantsNo.R21HL114011andNo.R21EB017078),andtheAmer-icanHeartAssociation(grantNo.12SDG12180025).REFERENCES1.Watanabe,K.,M.Ueno,,Y.Sasai.2007.AROCKinhibitorpermitssurvivalofdissociatedhumanembryonicstemcells.Nat.Biotechnol.25:681Ð686.2.Chen,G.,Z.Hou,,J.A.Thomson.2010.Actin-myosincontractilityisresponsibleforthereducedviabilityofdissociatedhumanembryonicstemcells.CellStemCell.3.Ohgushi,M.,M.Matsumura,,Y.Sasai.2010.Molecularpathwayandcellstateresponsiblefordissociation-inducedapoptosisinhumanpluripotentstemcells.CellStemCell.4.Sun,Y.,L.G.Villa-Diaz,,J.Fu.2012.Mechanicsregulatesfatede-cisionsofhumanembryonicstemcells.PLoSONE.5.Keung,A.J.,P.Asuri,,D.V.Schaffer.2012.Softmicroenviron-mentspromotetheearlyneurogenicdifferentiationbutnotself-renewalofhumanpluripotentstemcells.Integr.Biol.(Camb).6.Sun,Y.,K.M.Yong,,J.Fu.2014.Hippo/YAP-mediatedrigidity-dependentmotorneurondifferentiationofhumanpluripotentstemNat.Mater.13:599Ð604.7.Fan,Z.,Y.Sun,,J.Fu.2013.Acoustictweezingcytometryforlive-cellsubcellularmodulationofintracellularcytoskeletoncontractility.Sci.Rep.8.Heureaux,J.,D.Chen,,A.P.Liu.2014.Activationofabacterialme-chanosensitivechannelinmammaliancellsbycytoskeletalstress.Mol.Bioeng.9.Dayton,P.A.,K.E.Morgan,,K.W.Ferrara.1997.Apreliminaryevaluationoftheeffectsofprimaryandsecondaryradiationforcesonacousticcontrastagents.IEEETrans.44:1264Ð1277.10.Bardsley,W.G.,andJ.D.Aplin.1983.Kineticanalysisofcellspreading.I.Theoryandmodelingofcurves.J.CellSci.61:365Ð373.11.Barbaric,I.,V.Biga,,P.W.Andrews.2014.Time-lapseanalysisofhumanembryonicstemcellsrevealsmultiplebottlenecksrestrictingcolonyformationandtheirreliefuponcultureadaptation.StemCell12.Li,L.,B.H.Wang,,L.Wang.2010.Individualcellmovement,asymmetriccolonyexpansion,rho-associatedkinase,andE-cadherinimpacttheclonogenicityofhumanembryonicstemcells.Biophys.J.98:2442Ð2451. FIGURE3()ALPassayfordissociatedhESCswithorwithoutATCtreatmentsasindicated.Scalebar,1mm.()RelativeratioofALPcoloniestothenumberofhESCsinitiallyseeded.)Immunostainingwithanti-Oct4,anti-Sox2,andanti-E-cad-herinantibodiesforhESCcolonies7daysafterATCtreatments.Scalebar,100m.ToseethisÞgureincolor,goonline.BiophysicalJournal108(6)1315Ð1317BiophysicalLetters1317