STEM CELL SYMPOSIUM ABSTRACTS - Entrepreneur...

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STEM CELL SYMPOSIUM ABSTRACTS

OCTOBER 30, 2018THE HOTEL AT THE UNIVERSITY OF MARYLAND

10TH ANNUAL

ABSTRACT INDEX / POSTER #

# 1 Zimmerlin, L. et al. Human Pluripotent Stem Cells with Improved Functionality and Interspecies Chimera Potential# 2 Zhang, C. et al. Nanofiber-HydrogelComposite-MediatedNeuroprotectionAfterSpinalCordInjury# 3 Shissler, S. et al. ConvertibleNaturalKillerTcellsforCancerImmunotherapy#4 Mertz,J.et al. WholeandPhosphoproteomeAnalysisofEpithelialtoMesenchymalTransitioninHumanStemCellDerived.....# 5 Leng, S. et al. ClinicalEvaluationofLongeveronMesenchymalStemCellsforImprovingVaccineResponseinAgingFrailty#6 Kaushal,S.et al. ClinicalEvaluationofLongeveronMesenchymalStemCellsasAnAdjunctTherapytoSurgicalIntervention.....# 7 Huang, C.Y. et al. ImprovementofTheMaturationStateofHumanInducedPluripotentStemCell-Derived3DCardiac.....#8 Das,D.et al. HumanInducedPluripotentStemCell-DerivedForebrainOrganoidstoAssessNeuronalResponsesto.....#9 Lerman,M.et al. A3D-PrintedPolystyreneScaffoldwithTunableSurfaceChemistrytoEnhanceMesenchymalStemCellGrowth.....# 10 Xian, L. et al. HMGA1isInducedbyProcarcinogenicBacteriawithintheMicrobiometoExpandtheColonStemCellPool.....#11 Machairaki,V.et al. CharacterizationofHumanIPSC-DerivedExtracellularVesiclestoDevelopNovelTherapeuticStrategies# 12 Somers, S. et al. EngineeringHPSC-DerivedSkeletalMuscleGraftsforTheTreatmentofVolumetricMuscleLoss#13 HoTae,L.im,et al. TranscriptionalLandscapeofHumanMyogenesisWithMultipleGeneticReporterEmbryonicStemCellLines#14 Morales,D.et al. C-KIT+CardiacProgenitorCellsinClosedChestSwineModelofMyocardialInfarction#15 Farris,A.et al. TunableOxygen-Releasing,3D-PrintedScaffoldsImproveInVivoOsteogenesis#16 Traficante,M.et al. TreatingTimothySyndromeThroughSpliceModulation#17 Morales,I.et al. InducedPluripotentStemCell-DerivedOligodendrocyteProgenitorCellModeltoStudyNeurodegeneration.....#18 Bian,Q.et al. ASingleCellTranscriptionalAtlasofSynovialJointDevelopment#19 Klarmann,G.et al. DevelopmentofABiphasicMSCDeliverySystemforTheRepairofOsteochondralDefects#20 Hurley-Novatny,A.et al. Multi-LineageCapabilitiesofMesenchymalStemCellsforEngineeringOrthopaedicInterfaces#21 Tian,L.et al. BiliaryAtresiaRelevantHumaniPSCsRecapitulateKeyDiseaseFeaturesinADish# 22 Choi, I.Y. et al. MaintainingHumanPluripotentStemCellswithOpticalStimulationoftheFGFSignalingPathway# 23 Pan, L. et al. CD34+MCSCSRepresentASubsetofSkin-DerivedPrecursorCellsinTheSkin#24 Wang,R.et al. EffectofTraumaonImmunotoleranceAfterMyelinatingProgenitorCellsTransplantationinImmunocompetent.....#25 Sarkar,C.et al. NeuronalDifferentiationofNeuronalStemCellsbyAutophagyInductioninOxidativeEnvironmenttoTreatTBI#26 Hiler,DJ.et al. ModellingPitt-HopkinsSyndrome,AnAutismSpectrumDisorder,withPatientDerivediPSCs#27 Andersen,P.et al. ModelingHeartChamberDevelopmentandDiseaseinCardiacOrganoids#28 Ryu,J.et al. NeuronsfromHumanNeuralProgenitorTransplantsEstablishFunctionalConnectionsinTheAdultCortex.....# 29 Chu, C. et al. UsingStemCellstoRepairRadiation-InducedInjuryAfterTumorEradication–APerspective#30 Zeffiro,T.et al. SafetyandEfficacyofIntravenousAutologousMesenchymalStemCellsforSubacuteIschemicStroke.....#31 Bagnell,A.et al. Generating3DBrainOrganoidsWithEndothelialCellsFromHumaniPSCs.....#32 Kam,TI.et al. RoleofNeurotoxicAstrocyteinNeurodegenerativeDiseases#33 Richard, JP. et al. DevelopmentofImagingBiomarkersforStemCellTransplantationinAmyotrophicLateralSclerosis#34 Durens,M.et al. High-ThroughputMethodsforEvaluatingCellularPhenotypesandNeuralActivityinHumaniPSCs.....# 35 Chun, Y.W. et al. CombinatorialPolymerMatricesEnhanceInVitroMaturationOfHumanInducedPluripotentStemCells.....#36 Nguyen,H.N. InnovativeApplicationsof3DTissueEngineeringTechnology#37 Pavuluri,K.D.et al. IntegratingMotionCorrectionintoCESTMRIpHImaging# 38 Linhong Li, et al. GMP-CompliantNon-ViralCRISPR-MediatedMutationCorrectioninPatientCD34+CellswithSickleCell.....#39 Mishra,R. MechanicalCharacterizationofEngineeredCardiacTissuesforClinicalApplication#40 Kim,S.et al. TransplantedVolarFibroblaststoNonvolarSkinInduceEctopicVolarSkin# 41 Wei, Z. et al. ASelf-HealingHydrogelasAnInjectableInstructiveCarrierforCellularMorphogenesis#42 Gunasekaran,M.et al. ImmunosuppressionIncreasesRegenerativePotentialofAdultRatC-KIT+CardiacProgenitorCells.....# 43 Hu, C. et al. RapidandEfficientGenerationofOligodendrocytePrecursorCells#44 Panicker,N.et al. ActivationoftheNLRP3InflammasomeInHumanDopamineNeuronsAsAConsequenceofParkinDysfunction#45 Xu,J.et al. DivergentStemCellFunctionBetweenSkeletalandSoftTissueCD146+HumanPericytes#46 Thomas,A.et al. Location-AndDose-DependentAttenuationofExperimentalAutoimmuneEncephalomyelitisUsing.....# 47 Southard, S. et al. GenerationandSelectionofPluripotentStemCellsforRobustDifferentiationtoInsulin-SecretingCells.....# 48 Chang, C. et al. Nanofiber-HydrogelCompositeMicroparticlesAsAStemCellDeliveryandPro-RegenerativeMaterialfor.....#49 Srikanth,M.P.et al. ElevatedLevelsofGlucosylsphingosineDeregulateLysosomalCompartmentinNeuronopathicGaucherDisease# 50 He, Y. et al. TheDNAMethylationandRegulatoryElementsDuringChickenGermStemCellDifferentiation# 51 Zhang, C. et al. BuildingACommercialPathforEpiX™technology:ABreakthroughinExpandingandUtilizing.....#52 Suresh,R.et al. NF-KBp50DeficientImmatureMyeloidCells(p50-IMC)AsANovelCancerImmunotherapy# 53 Zhu, W. et al. MonitoringtheDegradationofImplantedHydrogelsUsingCESTMRI#54 Rindone,A.et al. Developmentofa3DFluorescentImagingTechniquetoObserveBloodVesselPhenotypein.....#55 Xu,J.et al. TemporalNextGenerationTranscriptomeScreenofHumanNeuronalPro-SurvivalMoleculesandPathways#56 Elliott,M.et al. FibrinHydrogelSmall-DiameterGraftasAnArterialConduit# 57 Zhu, W. et al. Non-InvasiveImagingofHydrogelScaffoldBiodegradationAndTransplantedCellSurvival#58 Du,J.et al. OptimalElectricalStimulationBoostsNerveRegenerationwithStemCellTherap#59 Macklin,B.et al. InducedPluripotentStemCellDerivedEndothelialCellsDisplayUniquePhenotypeinLowOxygenEnvironments#60 Garza,L. StemCellstoReprogramSkinIdentityandEnhanceProstheticUseinAmputees#61 Guo,W.et al. NF-KAPPABPathwayIsInvolvedinBoneMarrowStromalCell-ProducedPainRelief#62 Cadwell,J.et al. ClinicalScaleProductionandWoundHealingActivityofHumanAdiposeDerivedMesenchymalStemCell.....#63 Hunter,J. AgricultureIsNoLongerBeverlyHillbilliesorGreenAcres#64 Anzmann,A.et al. DevelopmentofaniPSCDerivedCellularModelofBarthSyndrome#65 Park,S.et al. MechanicalCharacterizationofEngineeredCardiacTissuesforClinicalApplication#66 Santoro,M.et al. MechanismsofAngiogenesisWithinBioprintedEndothelial:MesenchymalStemCellCocultures# 67 Sun, C. et al. ADuchenneMuscularDystrophyPatienthiPSC-DerivedMyoblastsBasedDrugScreenUncoversPotential.....#68 Brown,R.et al. AnalysisofTheCordBloodHematopoieticStem/ProgenitorCellPopulationsCulturedInSerum-FreeMedium# 69 Xue, Y. et al. SyntheticmRNAsDriveHighlyEfficientiPSCellDifferentiationtoDopaminergicNeurons#70 Carosso,G.et al. CellularMetabolicDefectsinNeurodevelopmentRevealedbySingleCellAnalysisinPatient-SpecificModels.....#71 Rowley,J. ClosedSystemsEnablingCommercially-ViableStemCellManufacturing#72 Kouser,M.et al. Manganese-enhancedMRIforInterrogatingAstrocyteReplacementinAMouseModelofAmyotrophic.....

FIRST AUTHOR ABSRACT TITLE

Human Pluripotent Stem Cells with Improved Functionality and Interspecies Chimera Potential

Ludovic Zimmerlin, Tea Soon Park, Rebecca Evans, Justin Thomas, Alice He, Leigh Kulpa, Jeffrey Huo, Riya Kanherkar, and Elias T. Zambidis

Johns Hopkins University-SOM, Baltimore, Maryland

Humannaïvepluripotentstemcells(N-hPSC)withimprovedfunctionalitymayhave wide impact in human regenerative medicine due to their potential to con-tributefunctional tissuestoadevelopingembryo.Forexample,stablenaïvehumanembryonicstemcells(N-hESC)maybeemployedfordevelopingtrans-plantablehumanorgansandadultstemcellsindevelopinganimalchimeras,orforgeneratinghumanizedgene-targetedanimalmodelsofdisease.ChemicalinhibitionofGSK3β,ERKandtankyrasesignaling(LIF-3i)wasrecentlydemon-stratedtobesufficientforstablenaïvereversionofabroadrepertoireofcon-ventionalhPSClines.LIF-3inaïvehPSC(N-hPSC)maintainednormalkaryoty-pes,2-4xfolddecreasesinglobal5-methylcytosineCpGmethylationactivities,genome-wideCpGdemethylationatESC-specificgenepromoters,dominantdistalOCT4enhancerusage,phosphorylatedSTAT3signaling,anddecrea-sedERKphosphorylation.LIF-3iincreasedexpressionsofnaïve-specifichu-manpreimplantationepiblastgenes(e.g.,NANOG,KLF2,NR5A2,DNMT3L,HERVH).SurfacemarkeranalysisofN-hPSCvs.theirisogenicconventionalhPSCcounterpartsrevealedsignificantlyincreasedexpressionsofhumannaï-veepiblast-specificmarkersCD325,CD151,andCD44,anddecreasedCD24,CD90,andHLA-ABClevels.Incontrasttoalternativenaïvereversionmethodsthatresultedinlossofimprintedgenomicregions,LIF-3iN-hPSCweredevoidofsystematiclossofimprintedCpGpatternsorlossofDNAmethyltransferaseexpression(e.g.,DNMT1,3A,3B).Methylationarrayanalysisof>1400knownimprinted CpG sites in 12 independent LIF-3i N-hPSC revealed stability ofimprintsalreadyestablishedinisogenicconventionalhPSC.Moreover,LIF-3inaïve reversion resulted in decreased lineage-primed gene expression andimproved functional pluripotency, with concomitant improvement in directeddifferentiationtoallthreegermlayers.Forexample,LIF-3iN-hPSCgeneratedPAX6+SOX1+Nestin+neuralprogenitors,aswellascomplexretinalorganoidsmorerapidlyandefficientlythanconventionalisogenichPSCcounterparts.Torigorouslytestfunctionalpluripotency,N-hPSCexpressingaGFP-puromycinconstructwereinjectedintomurineblastocystsandshowntoefficientlygene-ratemouse-humanchimericembryosatearlystagesofmurinedevelopmentvia human-specific, quantitativemitochondrialDNAandnuclear antigen ex-pression assays. Later-stagemurine embryos showed lower frequencies ofhumancellincorporation(primarilyinmurinefetalliverandnervoussystems)following in vitro culture selection of chimeraswith puromycin.WeproposethattheseLIF-3iN-hPSCwithimprovedfunctionalityandinterspecieschimerapotencywillhavegreatutility ingeneratinghumanizeddiseasemodels,andultimately in generating patient-specific organs and tissues for regenerativetherapies.

Nanofiber-Hydrogel Composite-Mediated Neuroprotection After Spinal Cord Injury

Chi Zhang1-4, Xiaowei Li1-3, Jerry Yan1,3,5, Michelle Seu1,6, Michael Lan1,3,5, Mingyu Yang1-3, Brian Cho1,6, Russ Martin1-3, Yohshiro Nitobe7, Kentaro Yamane7, Aggie Haggerty7, Ines Lasuncion7, Megan Marlow7, Sashank Reddy6, Da-Ping Quan4, Martin Oudega7,8,9, Hai-Quan Mao1-3,5

1Translational Tissue Engineering Center, 2Department of Materials Science & Engineering, 3Institute for NanoBioTechnology, Johns Hopkins University, Baltimore, MD 21205, USA, 4School of Chemistry, Sun Yat-Sen Univeristy, Guangzhou, Guangdong 510275, P. R. China, 5Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD 21205, USA, 6Department of Plastic and Reconstructive Surgery, Johns Hopkins School of Medicine, Baltimore, MD 21287, USA, 7The Miami Project to Cure Paralysis, 8Department of Neurological Surgery, University of Miami, Miami, FL 33136, USA, 9Bruce W. Carter Department of Veterans Affairs Medical Center, Miami, FL 33136, USA.

StatementofPurpose:Contusionistheprevalentmechanismofspinalcordinjury(SCI)inhumans.Theefficacyofavailabletherapiesforspinalcordcon-tusionislimited,withlessthan1%ofthepatientsexperiencingcompleteneu-rologicalrecoverybythetimeofhospitaldischarge.[1]Contusionscauselossofnervoustissueandultimatelytheformationofcysticcavitiesattheinjurysite,whichposesaformidableobstaclefortissuerepairandfunctionalrecovery.[2]Creatingafavorableenvironmentfornervoustissuesparingandregenerationmaysupportimprovedfunctionaloutcomes.Wehavedevelopedaninjectablenanofiber-hydrogelcomposite(NHC)withinterfacialbondingthatenablescellinfiltrationwhileprovidingstructuralintegrityforbridgingcysticcavitiesintheinjuredspinalcord.ThepurposeofthisworkwastoevaluatetheeffectsandpotentialmechanismsofNHConneuroprotectioninanadultratmodelofspi-nal cord contusion. ResultsandDiscussion:GrossanatomicalevaluationofthecontusedspinalcordssuggestedlessdamageovertimeattheinjuryepicenterwiththetreatmentofNHCcomparedwithhydrogelsandPBS.Preliminaryhis-tologicaldataconfirmeddecreasedtissuelossincontusedsegmentsfollowinginjectionofNHC,especiallywhencomparedtoothertreatments.Furthermore,injectionofNHCresultedinthehighestdensitiesofbloodvesselsandaxonswithinthecontusionsite.Thenumberofinflammatorycellsatthecontusionsiteappearedsimilaramongallexperimentalgroups.However,comparedtoother treatmentgroups,NHC-treatedspinalcordpresentedthemostpro-re-generativeM2macrophage phenotypes (Data not shown).Conclusion:Wedeveloped a unique injectable nanofiber-hydrogel composite for supportingrepairofthecontusedspinalcord.Ourresultssuggestthatangiogenesisandimmunomodulationarepossiblemechanismsunderlyingneuroprotectionandaxongrowthinspinalcordlesions.

References:1. National Spinal Cord Injury Statistical Center, Facts and Figures at a Glance. Birmingham, AL: University of Alabama at Birmingham, 2017.2. Hong, A et al. An Injectable Hydrogel Enhances Tissue Repair after Spinal Cord Injury by Promoting Extracellular Matrix Remodeling. Nature Communications, 2017, 8(1): 1-14.

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Poster Abstracts:The 10th Annual

Maryland STEM CELL RESEARCH Symposium

maturedinRPEmediumcontainingB27.RPEmonolayersunderfivecondi-tionswereexaminedinduplicate:1)untreatedmonolayer;2)enzymaticdis-sociationandreplatingfor1hr;3)enzymaticdissociation+12hrreplating;4)treatmentwithTGNF,acombinationoftransforminggrowthfactorbeta(TGFβ)andtumornecrosisfactoralpha(TNFα)for1hr;5)TGNFtreatmentfor12hr.Proteinwas then harvested and prepared for global and phosphoproteomeanalysisby liquidchromatography tandemmassspectrometry (LC-MS/MS).Aftertrypsindigestion,tandemmasstag(TMT)multiplexlabelling,andbasicreverse-phaseLC fractionation,phosphorylatedpeptideswereextractedviaFe3+immobilizedmetalaffinitychromatographyforparallelanalysis.Allfrac-tionswereanalyzedonaQ-ExactiveMS instrumentandpeptidesequencematching, phosphosite assignment, and quantification performed using theMaxQuant software package. Pathway analysis on modified proteins andphosphositeswasperformedusingavarietyofbioinformatictoolsincludingIn-genuityPathwayAnalysisfromQiagen,DAVIDpathwayenrichmenttoolsfromNIH,andtheNetworKinkinasesubstrateanalysissuitefromtheUniversityofCopenhagen.Overall, roughly8000proteinsand9500class Iphosphositeswerequantified fromall tensamples. Interestingly,whileproteinabundanceperturbationsoverlappedextensivelybetweenvariousgroups,amongphos-phorylationchangestheoverlapofsignificantperturbationsafter1hrofeitherdissociationorTGNFwasthemostextensiveofanytwocombinationsofcon-ditions.WhennormalizingforproteinchangesthephosphorylationincreasessharedbytheseconditionsweremostintenseandwidespreadinsubstratesofvariousMAPKs,whilethestrongestdecreaseswereobservedinsubstratesoftheproteinkinaseBandCcomplexes.BothpathwaysareknowntoparticipateinEMT,thoughthespecificphosphorylationeventsobservedheremayleadtonewtherapeutictargetsandpossiblynewmeansofinducingEMTinvitro.

Clinical Evaluation of Longeveron Mesenchymal Stem Cells for Improving Vaccine Response

in Aging Frailty

Leng S1,White O1, Drouillard A2, Pena A2, Peterson K2, Chisolm C2, McClain-Moss LR2, Hare JM2, Oliva AA2.

1Division of Geriatric Medicine & Gerontology, Johns Hopkins University, Baltimore, MD; 2Longeveron LLC, Miami, FL

Agingfrailtyischaracterizedbytheprogressivephysiologicdeclineinmultipleorgansystems,leadingtoincreasedvulnerabilitytodisease,comorbidity,andmortality.Thisincludestheage-relateddeclineoftheimmunesystem,termedimmunosenescence. Immunosenescence results in diminished responsive-nesstoantigenchallengessuchasvaccinations,andincreasesvulnerabilitytoinfectionandassociatedcomplicationssuchasopportunisticinfections,in-creasedhospitalizationrate,anddeath.AgingFrailtyandimmunosenescenceappeardriven,atleastinpart,byachronicpro-inflammatorystate,calledin-flammaging.Therefore,decreasingthispro-inflammatorystatecouldpotentiallyimproveimmunefunctioning.Inthisphase1/2clinicaltrial,weareinvestigating the potential efficacy of Longeveron-produced allogeneicmes-enchymalstemcells(LMSCs)improveimmune-responsetoinfluenzavaccineinAgingFrailtysubjects.LMSCsareaproprietaryformulationofmesenchymalstemcells,whicharemultipotentcellsthathavepowerfulanti-inflammatory

Convertible Natural Killer T-Cells for Cancer Immunotherapy

Susannah C. Shissler, Dominique R. Bollino, Tonya J. WebbDepartment of Microbiology and Immunology, University of Maryland School of Medicine and the Marlene and Stewart Greenebaum Comprehensive Cancer Center, Baltimore, Maryland 21201

Adoptivecellulartransfer(ACT)isanimmunotherapeuticstrategyusedtoen-hanceimmuneresponsesincancerpatients.ACTutilizingengineeredTcellshavethepotentialtoofferlong-termprotection,throughmemory,butaredirec-tedonlytowardscancercellsexpressingthetumorantigenforwhichtheyweredesigned.Thistherapyisthereforelimitedbythepossibilityoftumorescape–a tumor can downregulate the antigen to which the immunotherapy is directed. Analternatecellularstrategyindevelopmentutilizesnaturalkiller(NK)cells,whichrecognizeHLAclassImolecules.NKcellsdirectlylysecancercells,andproducecytokinesandchemokinesthatcanactivateothercomponentsoftheimmunesystem.Here,weproposetoexamineathirdtypeofcellulartherapybasedonnaturalkillerT(NKT)cells.NKTcellsrecognizeantigeninthecontextofCD1d,whichisexpressedineveryone.Moreover,NKTcellscanactivatebothNKcellsandclassicalTcells.Theirdualfunctionisidealforcancertreat-ment, becauseNKT cell-based therapy offers the possibility of inducing aninnatecytotoxictumorresponse,andalsoactivatingtheadaptiveimmunesys-temtoproducetumor-directedcytotoxicTcells(CTL)withlong-livedmemory.WehavedevelopedanovelmethodtogenerateNKTcellsfromhumanadultstemcells.Wehypothesizethatthesestemcell-derived“convertible”NKTcellswillexertpotentantitumorresponses.Thus, thegoalof thisproject is tova-lidateourmethodofgeneratingNKTcells fromadultstemcellsand furthercharacterize theireffector functions.The informationgained in theproposedstudieswillaidinthedevelopmentofanovelimmunotherapy,specificallythegenerationofuniversalNKTcells,thatcanbeusedforthetreatmentofcancer.

Whole and Phosphoproteome Analysis of Epithelial to Mesenchymal Transition in Human Stem Cell

Derived Retinal Pigment Epithelium

Joseph Mertz1,Srinivas Sripathi1, Xu Yang1, Lijun Chen2, Noriko Esumi1, Hui Zhang2, Donald J. Zack1

1Department of Ophthalmology, Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA2Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

Age-related macular degeneration (AMD) is a leading cause of vision im-pairment among the elderly worldwide and there is evidence that epithelial tomesenchymal transition (EMT)maybe involved inAMDpathogenesis invivo.Theadventofembryonicstem(ES)cell-derivedRPEcellsenablesustoinvestigateEMT-relatedpathways inhumanRPEcells ina tightlycontrolledandsystems-basedmanner.WesoughttodiscernthekeyproteinactorsandphosphorylationeventsinRPEEMTtoaidindevelopmentofnoveltherapeuticapproaches to modulating this process. Human ES cells were cultured inmTeSRfollowedbydifferentiation inmediacontaining15%KOserum, then

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properties,andsupportintrinsicrepairandregenerativemechanisms.LMSCsare also immunoprivileged, and thus offer promise as an “off-the-shelf” all-ogeneic therapeutic.This study entails 3 phases. Firstwas aRun-InPhasetodemonstrateprovisionalsafetyand tolerability. Allsubjectsof thisphasehavebeentreatedwithLMSCsandvaccinatedagainstinfluenza.Safetywasdemonstrated,andtheDataMonitoringCommittee(DMC)recommendedtheremainderof thetrialproceedwithoutmodification.Thesecondphase(PilotPhase)wasdesignedtoevaluatetheoptimaltime-intervalbetweenLMSC-tre-atmentandinfluenzavaccination.AllsubjectsofthisPhasehavebeentreatedwithLMSCsandvaccinatedagainst influenza.Safetywasagaindemonstra-ted. Preliminaryefficacydataalsosupport thehypothesis thatLMSCscanimproveimmuneresponseandimmunosenescenceinAgingFrailtysubjects.ThethirdphaseofthisstudyisaPlacebo-Controlled,Double-Blinded,Rando-mizedPhasedesignedtoevaluateefficacy.Thisphaseiscurrentlyenrolling.WeanticipatethattheresultsofthisstudywilldemonstratethatLMSCtherapyis anefficaciousadjuvant therapy that provides significant advantagesoverinfluenzavaccinealone.Wealsoanticipatethattheseresultsshouldbroadlytranslateacrossaspectrumofdiseasesbyrestoringbacktowardsnormalthefunctioningoftheimmunesystem.

Clinical Evaluation of Longeveron Mesenchymal Stem Cells as An Adjunct Therapy to Surgical Intervention for

Hypoplastic Left Heart Syndrome

Kaushal S1, Mehta H1, Hibino N2, Everett AD2, Gentry T3, McClain-Moss LR3, Pena A3, Peterson K3, Oliva AA3, Hare JM3.

1Division of Cardiac Surgery, University of Maryland School of Medicine, 110 S Paca St, 7th Floor, Baltimore, MD; 2Division of Pediatric Cardiac Surgery and Cardiology, Johns Hopkins, Hospital, Zayed Tower Suite 7107, 1800 Orleans St, Baltimore, MD; 3Longeveron LLC, 1951 NW 7th Ave, Suite 520, Miami, FL

Hypoplasticleftheartsyndrome(HLHS)isoneofthemostcomplexformsofcongenitalheartdisease,causingroughly9%ofallcongenitalheartdisease.Whileonceuniformlyfatalshortlyafterbirth,dramaticimprovementsinsurvi-valhadbeenmadethrough3-stagedsurgicalpalliationthatreconstructsthehearttohavetherightventricle(RV)supportsystemiccirculation.Whilethesestrideshavebeenimpressive,themortalityrateneverthelessstillapproaches35%duringthefirstyearoflife,andcontinueshighthereafter.ThisislargelyduetoRVfailure,andimprovingRVfunctioninthesepatientsremainsamajorunmetmedicalneed.WeareconductingaPhase1/2clinicaltrial toevalua-tethesafetyandefficacyofallogeneicLongeveronmesenchymalstemcells(LMSCs)asanadjunctivetherapyfor improvingclinicaloutcomesforHLHSpatients. LMSCs are a proprietary formulation of mesenchymal stem cells(MSCs),whicharemultipotentcellsthatcansupportintrinsicrepairandregenerativemechanisms.MSCscanstimulateendogenousstemcellrecruitment,proliferation, anddifferentiation; inhibit apoptosisand fibrosis; and stimulateneovascularization.MSCsarealsoimmunoprivileged,andthusofferpromiseasan“off-the-shelf”allogeneictherapeutic.Theaimsofthisstudyaretoeva-luatethesafetyandefficacyofintramyocardialinjectionofLMSCsatthestageIIoperationforHLHS.WehypothesizethatLMSCswill improvethefunction

ofthesinglesystemicRVinHLHSpatients,andthusimprovebothshort-andlong-termclinicalmeasuresandoutcomes.Ourapproachisbackedbyprec-linical studies using intracoronary delivery of allogeneicMSCswhich led tosignificant improvementinfunctionaloutcomesinthepressureinducedrightventricle in a swine model—a model which replicates the physiology present HLHSpatients.Phase1isanopen-labelstudyusing10HLHSpatients.Over50%ofthepatientshavebeentreated,withnoadverseevents(AEs)orseriousadverseevents(SAEs)attributabletothecellregenerativetherapy.Prelimina-ryefficacydatasuggeststhetherapeuticpotentialofthistherapytoimproveRVfunction.Phase2bisablinded,randomizedstudyinwhich10subjectswillbetreatedwithLMSCs(n=10),orwillnotreceiveLMSCs(n=10)duringtheStageIIoperation.ThisphasewillcommenceafterallPhase1subjectshavebeentreated,andsafetyevaluated.ThisstudyrepresentsthefirstallogeneicMSCtherapyforHLHS,andweanticipatetheresultscouldleadtoaparadigmshiftintreatmentforthishighlyvulnerablepatientpopulation.

Improvement of The Maturation State of Human Induced Pluri-potent Stem Cell-Derived 3D Cardiac Microtissues by Defined

Chemical Factors

Chen Yu Huang1, Chin Siang Ong2, Rebeca Joca2, Ijala Wilson2, Deborah DiSilvestre2, Gordon Tomaselli2, and Daniel H. Reich1

1Department of Physics and Astronomy, Johns Hopkins University, Baltimore, MD ; 2Division of Cardiology, Johns Hopkins University School of Medicine, Baltimore, MD

Recentadvancesintheunderstandinganduseofpluripotentstemcellshaveproducedmajorchanges in theapproach to thediagnosisand treatmentofhumandisease.Anobstacletotheuseofhumanpluripotentstemcellderivedcardiomyocytes (hiPSC-CMs) for regenerative medicine, disease modelingand drug discovery is their immature state relative to adult myocardium. In this research,3Dcardiacmicrotissues(CMTs)weregeneratedusinghiPSC-CMstorecapitulatethestructural,functionalandmetabolicpropertiesofnormalanddiseasedadultventricularmyocardium.CMTsweretreatedwithontologicallydefinedbiochemical interventions(thyroidhormone,dexamethasoneand in-sulin-like growth factor,TDI) to promote thematurationof hiPSC-CMs.TheeffectsofTDItreatmentonbothstructuralandfunctional(biomechanical,Ca2+handlingandelectrophysiology)propertiesatthetissuelevelwerecharacte-rized. In addition, themolecular correlates ofmaturation of the hiPSC-CMsintheCMTswerestudiedbygeneexpression,proteomicsstudies.OurdatademonstratethatTDItreatmentimprovesbothstructureandfunctionofCMTs.Structurally,thehiPSC-CMsshowimprovedalignmentandlongersarcomerelength, as demonstrated by immunofluorescence and confocal microscopy.Functionally,thecardiacsarcoplasmicreticulumcalciumATPase-2(SERCA2)gene,whichplaysanimportantroleinexcitationcontractioncouplingisincrea-sedaftertreatedwithTDI.TheCMTs’staticanddynamicforcebothincreasefollowingTDItreatment,andopticalmappingshowsthattheCMTsareelectri-callycoupled.This,togetherwiththeenhancedfunctionalpropertiesthatthismaturation approach yields has the potential to yield improved model systems thatcanadvancebothmechanisticstudiesandthedevelopmentofnewthera-piesforthetreatmentofcardiacdiseases.

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1Dept. of Materials Science and Engineering, University of Maryland, College Park, MD; 2Surface and Trace Chemical Analysis Group, Materials Measurement Laboratory, National Institute of Standards and Technology, Gaithersburg, MD; 3Center for Engineering Complex Tissues, College Park, MD; 4Dept. of Bioengineering, Rice University, Houston, TX; 5Fischell Dept. of Bioengineering, University of Maryland, College Park, MD

As cell culture techniques continue to evolve, a balance should be struckbetweenbuildingcomplexstructuresthatbettermimicinvivoconditionsandtechnicaleaseofuse.Whilepolystyrene(PS)hasbeenusedastheprimarysurfaceforculturinghumanmesenchymalstemcells(hMSCs),technologicaladvancementsinmanipulatingthesurfacechemistrycouldimprovetargetedproteinadhesionandhMSCgrowth.Additionally,evolutionof thechemicallycomplexculturesurface froma2D toa3Dplatform isexpected to improveintercellularsignalingthroughbiomimicryofthenativecellenvironment.Impro-vementinthedesignofthe3Dscaffold,throughinvestigationintofloweffectsonhMSCexpansionandosteogenicdifferentiation,couldfurtherthedevelop-mentoffunctionalmodelssuchasthebonemarrownicheforcancerdiagno-sticsanddrugdiscovery.Thisworkseekstoinvestigatetechniquestodirectcell fateonbioreactorscaffoldsandunderstandhowthenativeenvironmentcanbebetterreplicatedinordertobuildafunctionalmodelsystem.Ultimately,weseektobuildabioreactorsystemwherewecanpredictablydirectlong-termhMSCexpansionandosteogenicdifferentiation.PreliminaryresultsshowthatplasmasurfacetreatmentisaneffectivetechniqueforrapidfunctionalizationofPSsurfaces.Aglowdischargeplasmasystemoperatingwith10SCCMoxy-genconverted20.9±1.9at%ofthesurfaceintooxygencontainingspecies,includingcarbonyl(12.2±4.2at%)andcarboxyl(2.6±1.4at%)groups,whichareseenonsurfacesoftissueculturepolystyrene(TCPS).Operatingwith10SCCMammoniaconverted11.9±0.4at%ofthesurfaceintonitrogencontai-ningspecies,primarilyasamines(94.7±7.5at%).Theplasmatreatedsur-faces(oxygen,24.9±2.9°;ammonia36.0±1.4°)havewatercontactangleslowerthaneitherTCPS(50.0±2.0°)orthenativenon-treatedsurfaces(82.9±2.2°).Wefindthaton2Dsurfaces,theammoniatreatedsurfaceshavesig-nificantlyhigherDNAcontent(4.6±0.2xfoldchange)after10daysofgrowthascomparedtotheoxygen(3.6±0.1x)orTCPS(3.3±0.2x).However,theoxygentreatedsurfaceshaveearlierandgreaterexpressionofearlyosteoge-nicmarkers(RUNX2andALP)andgreatercalcification(AlizarinRedStaining)overa3weektimecourse.Thiseffectisboostedwiththeadditionofosteo-genicmedia.Whenthe2Dsurfaceisconvertedinto3Dscaffolds,weseethatthegeneralpropertiesofthefunctionalizedsurfacesareconserved.Scaffoldsaredesignedandplacedinthetreatmentchamberwiththeintentofallowingplasmatopenetratethescaffoldinterior.After10daysofstatic3Dculture,theammonia(3.7±0.3x)treated3DscaffoldshadstatisticallygreaterDNAcon-tentthantheoxygen(3.0±0.2x)treatedscaffolds.Indynamicculture,weseethesetrendscontinueupto7days,wheretheammoniatreatedscaffolds(2.3±0.3x)supportedbettergrowthratherthantheoxygentreatedscaffolds(0.7±0.2x)ataflowrateof3.4mL/min.Ongoingworkincludescharacterizationofosteogenicdifferentiationonthesescaffoldsbothinstaticanddynamicculturetechniques.Additionally,theinfluenceofsurfacechemistryandgeometryoncytokineproductiontosupporthematopoieticstemcellcoculturehasbeenthesubjectofinvestigation.

Human Induced Pluripotent Stem Cell-Derived Forebrain Organoids to Assess Neuronal Responses to

Radiation-Induced DNA Damage

Debamitra Das1,3, Mathew Ladra2, Lawrence Kleinberg2, Sonia Franco2*Vasiliki Machairaki1,3* (*co-last authors)

1Department of Neurology, 2Department of Radiation Oncology and Molecular Radiation Sciences, 3Institute of Cell Engineering,Johns Hopkins University School of Medicine, Baltimore, MD, USA

Stemcellsbyvirtueoftheiruniquepluripotentcapacitycanregeneratevirtuallyanycelltypeandthereforeareappliedinstudiesoforgandevelopment,disea-se modeling, drug screening and cell replacement therapy. Induced pluripotent stemcells(iPSCs)undersuitablecultureconditionsinvitrocanbedifferentia-tedtoformanorganliketissueaka‘Organoid’withmultiplecelltypesthatclo-selyresemblesthetrueorganinvivo.WehypothesizedthathumanforebrainOrganoidsmaybeusedasanexperimentalmodeltostudythemechanismsunderlyingDNArepairinhumanneuronsatdifferentstagesofdevelopment,afterusingradiation-inducedDNAdamage.Tothisend,wehavedesignedaprotocolforforebrainorganoidgenerationthatisreproducibleandtimesaving.These organoids recapitulate key features of human cortical development,includingadefinedSubVentricularZone(SVZ)containingneuralprogenitorcells identifiedbyNestinandSOX2positivecells thatmature topostmitoticcorticalneuronsasevidencedbyTUJ1andCTIP2positivecells.Usingimmu-nofluorescence(IF)todetectDNAdouble-strandbreak(DSB)markersγ-H2AXand53BP1,wehavequantifiedthekineticsofDSBrepairinneuralprogenitorswithintheSVZforupto24hoursafterasingledose(2Gy)ofionizingradiation(IR).EvaluationofcellsurvivalofbothneuralprogenitorsandmatureneuronalcellsafterIR-inducedDNAdamageusinganapoptosismarkerCAS3revealedappearanceofapoptoticcellscorrelatedtemporallypostradiation.Cellcycleanalysisusingproliferationmarkerphosphohistone3(PH3)revealedprolifera-tingcellsneartheventricularsurfaceofearlyOrganoidsthatgraduallydeclinedpostIR.OurdataonDNAdamagerepairinprogenitorversusmatureneuronalcellsindicateasimilartimelinebywhichtheyrepairtheDNAdamageinvolvingahighincidenceofDSBimmediatelypostIRwhichgetsmostlyresolvedby18hourspostradiation.Onthewhole,thisstudysupportstheuseof3DOrganoidculturetechnologyasanovelpreclinicalplatformtostudyDNAdamageresponses in developing and mature neurons.

A 3D-Printed Polystyrene Scaffold with Tunable Surface Chemistry to Enhance Mesenchymal Stem Cell

Growth and Osteogenic Differentiation

Max J. Lerman1,2,3,Shin Muramoto2, Brandon T. Smith3,4, Anushka Gerald3,5, James A. Fookes3,5, Greg Gillen2, Antonios G. Mikos3,4, and John P. Fisher3,5

4

Poster Abstracts:

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The 10th AnnualMaryland STEM CELL RESEARCH Symposium

HMGA1 is Induced by Procarcinogenic Bacteria within the Microbiome to Expand the

Colon Stem Cell Pool and Drive Tumorigenesis

Lingling Xian, Li Luo, Lionel Chia, Shaoguang Wu, Cindy Sears, Linda M. S. Resar

Cancer cells undergo chromatin remodeling and epigenetic reprogramming to co-optstemcellnetworksanddrivetumorprogression,althoughtheunderlyingmechanismsremainpoorlyunderstood.HighMobilityGroupA(HMGA1)chro-matin remodelingproteinsarearchitectural transcription factors that bind toDNAatAT-richsequenceswherethey“open”chromatinandrecruittranscrip-tionalcomplexestomodulategeneexpression.TheHMGA1geneishighlyex-pressedduringembryogenesisandinadultstemcells,butsilencedpostnatallyindifferentiatedtissues.HMGA1isre-expressedinaggressivecancerswherehighlevelsportendadverseclinicaloutcomes.Incolorectalcancer,HMGA1isamongthegenesmosthighlyoverexpressedcomparedtonormalcolonepi-thelium.WepreviouslyreportedthatHMGA1drivestumorprogressioninco-loncancerbyinducinggenesinvolvedinanepithelial-mesenchymaltransition.WealsofoundthatHmga1transgenicmicedevelopaberrantproliferationandpolyposis in the small intestine and colon in addition to lymphoid tumors. In smallintestinalstemcells,Hmga1amplifiesWntsignalstoenhanceself-rene-wal.Surprisingly,Hmga1alsoinducesSox-9todrivePanethcelldifferentiationand“build”astemcellniche. TodeterminehowHMGA1functions incolonepithelialhomeostasisandcarcinogenesis,weexaminedmousemodelswithoverexpressionordeficiencyofHmga1.WediscoveredthatHmga1overex-pression in colon stemcells also leads to expansion in the colon stemcellpool.Further,bothgobletcellsandcolonicmucousareincreased.Indextransodiumsulfatemodelsof inflammatoryboweldisease,Hmga1enhancesre-pairandtissueregeneration,whileHmga1overexpressionpromotesaberrantproliferationandpolyposiswithadvancingageinmice.TobegintodeterminehowHMGA1isinducedduringcarcinogenesis,weexaminedmiceharboringaheterozygousApcloss-of-functionmutation(Min+/-)followingcolonizationwiththe human symbiotic bacteria, enterotoxigenic Bacterioides fragilis (ETBF).ETBFtriggersincreasedWntsignalingbyinducingE-cadherincleavagewithE-cadherinrelease,leadingtoenhanceddistalcolontumorigenesis.Import-antly,ETBFcolonizationinhumansislinkedtobothearlyneoplasiaandcoloncancer. Inpreliminarystudies,wefoundthatETBFinducesHmga1and in-creasesstemcellself-renewal.Inflammatorycytokinegeneswerealsoindu-ced.StudiesareunderwaytoidentifytranscriptionalnetworksandepigeneticalterationsgovernedbyHmga1inthissetting.ThisworknotonlyprovidesnewinsightsintotheroleofHMGA1incolonepithelialhomeostasisbymaintainingboththestemcellpoolandpromotingregenerationfollowinginjury,butalsosuggests thatHMGA1canbeaberrantly inducedbysignals fromthemicro-biome topromote tumorigenesis. Our resultsalsohighlight the interactionsbetweenthemicrobiomeandHMGA1asapotentialtherapeuticopportunityincolon carcinogenesis.

Characterization of Human IPSC-Derived Extracellular Vesicles to Develop Novel Therapeutic Strategies

Vasiliki Machairaki1,3, Senquan Liu2,3 , Kenneth Witwer4, Linzhao Cheng2,3

1Department of Neurology, 2Department of Medicine, 3Institute for Cell Engineering, 4Department of Molecular and Comparative Pathobiology, Johns Hopkins University School of Medicine, Baltimore, MD, USA

HumaninducedPluripotentStemCells(iPSCs)derivedfromsomaticcellsofpatientsprovideanunprecedentedsourceofcellsforresearchintofuturecelltherapies.Unlikemanyadulthumanstemcells,humaniPSCscanbeexpan-dedincultureindefinitelyandefficiently(10-12-foldevery3days),whilemain-taining their developmental potential to generate nearly any cell type. We and othersrecentlydevelopedacompletelydefinedculturesystemtoexpandhu-maniPSCsundereitheradherentorsuspensionconditions.ThissystemusestheE8culturemediumthatischemicallydefinedwiththreerecombinantprote-insplustransferrin,andhumanrecombinantvitronectinasaculturesubstrate.Weanalyzedexosomesand/ormicro-vesicles (collectivelycalledextracellu-larvesiclesorEVs)madebyhumaniPSCs.WecollectediPSC-derivedEVsreleased into the culture conditionedmedium and analyzed them followingstandard methods such as transmission electron microscopy and nanoparticle trackinganalysis(NTA)byNanoSightandZetaViewanalyzers.Weobservedaconcentrationof~109particlespermlofconditionedmedium,withdiametersof100-150nmasdominantpeaks.ThepurifiedEVsbydifferentialultra-centri-fugationcontainRNAs,butnotgenomicDNA.ProteomicandflowcytometricanalysesconfirmedthattheyexpressCD9,CD63andCD81,aswellasmanymembraneandcytoplasmicproteinsofhumaniPSCs.EVsfromhumaniPSCsstimulatedendothelialcellgrowthinculture.WealsoinvestigatedifEVscancarryandtransportaheterologousproteinproducedbyhumaniPSCsthatsta-blyexpress thefirefly luciferase(fluc)geneasamarker.WeconfirmedthatEVsofthegeneticallymodifiediPSCsgeneratedphotonsoncebeingincuba-tedwithacell-permeablesubstrateD-luciferinforfluc,indicatingtheEVsaremembrane-enclosed,containingATP(thereforemetabolicallyactive)aswellastheflucheterologousprotein.WefurtherinvestigatedtheEVuptakeandacti-vitiesinvitro(cellcultures)bymonitoringhorizontaltransferoftheflucactivityintorecipientcells.ThehumaniPSCsthatareculturedwithahighlydefinedmedium(withoutexogenousEVsthatarepresentinFBSandhumanbiologi-cals)provideauniquesystemtoanalyzeuniquephysicalpropertiesaswellastheirbiologicalcomposition.WeenvisionthatinvestigatinghumaniPSC-de-rivedEVswill provide novel approaches of using human iPSCs for cellulartherapeutics without the whole cells.

ThisstudyissupportedinpartbyMSCRFgrants.



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Transcriptional Landscape of Human Myogenesis With Multiple Genetic Reporter Embryonic Stem Cell Lines

HoTae Lim1, In Young Choi1, and Gabsang Lee1, 2, 3, *

Stem cell biologywith including embryonic stem cells (hESCs) is providingunprecedentedopportunityforstudyinghumandevelopment.Generatingske-letalmuscleinvitrowithhESCsopensupnewavenuesfordecipheringessen-tial,butpoorlyunderstoodaspectson transcriptional regulationsonskeletalmuscle genesis. In this study, our multiple human genetic reporter lines and newly devisedmyotube isolationmethodofOCT4::EGFP+embryonic stemcells,MSGN1::EGFP+presomitecells,PAX7::EGFP+putativeskeletalmusc-le stem/precursor cells, MYOG::EGFP+ myoblast cells, and multinucleatedmyotubes,allowustodepictglobalgeneexpressionlandscapeduringhumanskeletalmuscledevelopment.Wecomposedamolecularseparationofhumaninvitromyogenesis,andmappedtranscriptionalchanges,named‘virtualmyo-time’forhumanskeletalmusclegeneration.Additionallywedefinedspeciali-zedsignaturegeneexpressionprofilesfromeachisolatedcellpopulationwithclusteringanalysis.Comprehensive transcriptionalanalysisofhESC-derivedmyogenicspecificationprocesswill provideaunique insight includingnoveltranscriptionfactorsandsignalingpathways.Andstep-wiseRNA-seqprofilingand intensive clusteringanalysiswill be informative for understandinggeneexpressiondatain vitro myogenesis.

C-KIT+ Cardiac Progenitor Cells in Closed Chest Swine Model of Myocardial Infarction

David Morales, Gregory Bittle, Rachana Mishra, Sudhish Sharma, Progyaparamita Saha, Muthukumar Gunasekaran, Mir Arfat, Deqiang Li, Sunjay Kaushal

Afteramyocardial infarction (MI),damaged tissuehasminimalhealing thatoccurs in thehumanheart.The lossof function results in heart failure andincreasespatients’morbidityandmortality.However,cell-based therapyhasshown promising results in myocardial regeneration, especially in myocardial infarction.A recentPhase Iclinical trialofautologous (self-donated)cardiacstemcell therapyreportedthat leftventricular function improvedanaverageof12%intheyearaftertreatment.However,autologoussourcesareexpen-sive, timeconsumingand logisticallycumbersome.Allogeneiccardiacstem/progenitorcells(CPCs)generatedfromneonataltissueofferanattractiveal-ternative.TheKaushallabhasidentifiedanoveltypeofCPCthatexpressthecell-surfacesignalingproteinc-kit.Thesec-kit+CPCsareabletostimulatetheregenerationofcardiacmuscleandrestoreheartfunctioninarodentmodelofmyocardial ischemia.Inaswinemyocardial infarctionmodelwehopetode-monstratec-kit+CPC’scanregeneratemyocardialtissueafterMIinphysiologymoresimilartohumans.Furthermore,withahigh-doseCPC,low-doseCPC,andacontrolgroupthisstudywilldemonstrateproofofconceptanddosingefficacyparametersforc-kit+CPCstemcellbasedregenerativetherapy.

Engineering HPSC-Derived Skeletal Muscle Grafts for The Treatment of Volumetric Muscle Loss

Sarah Somers1*, Jordana Gilbert-Honick¹*, In Young Choi1, HoTae Lim1, Hai-Quan Mao1, Gabsang Lee1, Warren L. Grayson¹

1Johns Hopkins University, Baltimore, MD, US, * Authors contributed equally to this work

Tissueengineeringhasunprecedentedpotentialtocreateaclinicaltreatmentforvolumetricmuscle loss (VML)since thestandardofcare,anautologousmuscleflap,islimitedbytissueavailabilityanddonorsitemorbidity.However,oneof themajor factors limiting the useof a tissueengineered strategy totreatVML is the lackof a translatable stemcell source togenerate robust,non-immunogenic,tissue-engineeredskeletalmusclegrafts.Toovercomethisshortcoming,ourgroupuseshumanembryonicstemcell(hESC)andhumaninducedpluripotentstemcell(hiPSC)derivedmyoblastsincombinationwithanovelelectrospunfibrinfiberscaffoldtoengineerskeletalmuscleconstructs.Thesecellsarejointlyreferredtoashumanpluripotentstemcell(hPSC)-deri-vedmyoblasts.Aspreviouslyreported,1myoblastscanbederivedfromhPSCsthrougha30daydifferentiationprocedurethatincludestheuseofCHIR99021overthefirst4daysofdifferentiationfollowedby8daysofDAPT.ThecellsaremaintainedinserumfreeN2mediafortheremainderofthe30daydiffe-rentiationprocedure.Followingdifferentiation,cellsstainpositive formyosinheavychain(MHC)andmyogenin(MYOG)and30-50%ofthedifferentiatedpopulationexpressdystrophin,Titin,andα-actinin.Currently,ourexperimentsuse thesehPSC-derivedmyoblasts,however, in futurestudieswealsoplantoexploretheeffectsofcell-sortingonmyogenicoutcomesinour3Dsystem,assorting foramyogenicpopulationhasshownpromising results followingdifferentiationina2Dsystem.Specifically,sortingfortheNCAM+/HNK1-popu-lationinthehPSC-derivedmyoblastshasincreasedtheexpressionofMYOD1andMYOG~6foldoverunsortedcellsgrowninmonolayer.1)Tocreatea3DconstructwiththehPSC-derivedmyoblasts,wehavedevelopedelectrospunfibrinscaffoldswithmicroscalealignmentcuesandtunablestiffnessthatinpre-viousstudieshaveenabledrobustregenerationofamurineVMLdefectwhenseededwithC2C12myoblasts. 2)The scaffolds containaporous interior tofacilitatecellinfiltrationandatopographicallyalignedstructurethathascausedcellularalignment inarangeofcell typesincludingC2C12s,primaryhumanmyoblasts,hPSC-derivedmyoblasts,adipose-derivedstemcells,humanum-bilicalveinendothelialcells,andfibroblasts.Thescaffoldsarealsosuturable,biodegradable,andofferscale-upcapabilitiesforlargerdefects.InthecurrentstudywehavecombinedourelectrospunscaffoldswithhPSC-derivedmyo-blaststodevelopatranslatabletissueengineeredskeletalmuscleconstruct.WehaveassessedhPSC-derivedmyoblastgrowth,morphology,andmuscleproteinexpressiononelectrospunfibrinscaffoldscoatedwitharangeofpro-teinsand seededat varyingdensities. hPSC-derivedmyoblastsproliferatedonthescaffoldsupto10daysandexpresseddesminwithmyogenin-positi-venuclei,indicativeoftheirpro-myogenicphenotype.ThecellsalsofusedtoformmultinucleatedmyotubesexpressingMF20,amaturemusclemarker,andalignedwiththescaffold.Wehaveinvestigatedtheeffectofremovingserumfromtheculturemediaatdifferenttimepointsandhavefoundthatlaterserumremovalresultedinincreasedproliferation,moremyotubes,andamoreorga-nizedmyotubemorphologycomparedtosampleswithserumremovedearlier.InfuturestudiesweplantoassessthegrowthofNCAM+/HNK1-hPSC-derivedmyoblastsonthefiberaswellasassestheabilityoftheconstructtohealamouseVMLdefect.

Poster Abstracts:

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Tunable Oxygen-Releasing, 3D-Printed Scaffolds Improve In Vivo Osteogenesis

Ashley L. Farris, Dennis Lambrechts, Nicholas Zhang, S. J. Burris, Alexandra Rindone, Ethan L. Nyberg, Kendall Free, and Warren L. Grayson

Introduction:Theclinicalsuccessoftissueengineeringapproachesiscurrentlylimitedtoacellular,thin,orsmallscaffolds.Onereasonforthis isthelimitedoxygensupplywhencellsareimplanted.Ifatissuehasnovasculature,oxy-genistransportedthroughtissuebydiffusionalone.Toaddressthis,ourgrouppreviouslydemonstratedproof-of-conceptofafacilemethodtodeliveroxygentocellsusing3Dprintedscaffoldscontainingpolymericmicroparticles,calledmicrotanks,thatarehyperbaricallyloadedwithoxygen.However,thetransla-tionalpotentialofthesemicrotankswerelimitedinthattheywerenotcompo-sedofbiodegradablematerials.Inthisstudy,wehavedemonstratedtheabilitytofabricatebiodegradablemicrotanks,tuneoxygenreleasekineticsfrombio-degradable,3D-printedscaffoldsbyalteringthehyperbaric loadingpressureandoxygenreleasekinetics.Furthermore,wehaveshownthatoxygenreleasefromthesematerialscanimproveinvivoectopicboneformationinamurinesubcutaneousdefectmodel.Methods:Toaddressthelimitationsofthecom-merciallyavailablemicrotanks,wehavedevelopedandcharacterizedbiode-gradablemicrotankscreatedusingwater/oil/waterdoubleemulsion.Tocreatetheparticles,anaqueoussolutionofpolyvinylalcohol(PVA)ismixedwithanorganicsolutioncontainingpoly(lactic-co-glycolic)acid(PLGA).Wehaveper-formedcharacterizationofthesemicrotanks,examiningsize,surfacemorpho-logy,andshell thickness.Particlesarethenmixedatvaryingconcentrations(10%,20%,and30%)withpolycaprolactonepowderand3Dprintedusingacustompneumaticprinter.Followingprinting,theprintquality,poresize,strutthickness,surfacemorphology,anddistributionofmicrotanksthroughoutthescaffoldisdetermined.Scaffoldsareloadedwithoxygenusingacustom-madehyperbaricoxygenchamberpressurizedwith100%oxygenatvaryingpressu-resforaperiodof7days.ToassesstheoxygenreleasefromPCLscaffoldswithoutmicrotankandwithvaryingconcentrationsofPLGA/PVAmicrotanks,the oxygen release from hyperbarically-loaded scaffolds and oxygen con-sumptionofadipose-derivedstemcellsweremeasuredusingaSeahorseXFFluxAnalyzerfortemporalmeasurementsandaPresensfluorescent,oxygensensitiveprobe forspatiotemporaloxygenprofiles.Releasecurveswerefit-tedusinganexponentialdecayusingacustomMatlabscriptandcumulativereleasewasdeterminedfromtheexponentialfit.Weassessedtheimpactofoxygendeliveryfromhyperbarically-loadedmicrotankscaffoldsusingamurinesubcutaneousdefectFinally,weimplantedhyperbarically-loadedscaffoldsornon-hyperbarically-loadedscaffoldsintomurinesubcutaneousdefects(n=8)for4weeksandmonitoredectopicosteogenesisusingcomputedtomography.Results:Wehavedemonstratedtunableoxygenreleasekineticsusingourno-velmicrotanksystem.Weareabletotunethecumulativeloadedoxygenbyincreasingmicrotankconcentration,with30%microtankscaffoldscontainingnearlyanorderofmagnitudehigheroxygenthanscaffoldswithoutmicrotanks.Furthermore,wehaveobservedthatincreasingtheloadingpressurecanin-creaseboththetimeconstantofdeliveryandthecumulativeoxygenloadedinthemicrotankscaffolds.Wehavealsocalculatedthat30%microtank-PCLscaffoldscansupporttheoxygenconsumptionadipose-derivedstemcellsat5,000cells/μLforapproximately35hours.

We also examined the biological implant ofmicrotank-scaffolds on adiposederivedstemcellosteogenesisinvivo.Whenhyperbarically-loadedmicrotankscaffoldswereimplantedintomurinesubcutaneousdefectswithadipose-de-rivedstemcells,weobserveda63±0.6%increaseinbonevolumecomparedtoscaffoldsthatwerenothyperbaricallyloaded.Conclusion:Wehavedemon-strated thatmicrotanks can be fabricated using double emulsion.We haveshownthatthesemicrotankscanbe3D-printed,hyperbaricallyloaded,andre-leaseoxygenoveranextendedperiodoftime.Finally,wehavedemonstratedthatoxygenreleasefrommicrotankscanimproveboneformationoutcomes.Theseresultshighlightboththefeasibilityandpromiseofthismethodtodeliveroxygentostemcells.

Treating Timothy Syndrome Through Splice Modulation

Maria Traficante1, Steffi Valdeig1, Josiah Owoyemi1, Moradeke Bamgboye1, Masayuki Yazawa2, and Ivy E. Dick1

1Department of Physiology, University of Maryland; 2Columbia University Stem Cell Initiative

TimothySyndrome(TS) isamultisystemdisorder, featuringneurological im-pairment,autismandcardiacaction-potentialprolongation (longQT)with li-fe-threateningarrhythmias.The lifeexpectancy for thesepatients isoften li-mitedtojustafewyearsofage,duetoalackofadequatetreatmentoptions.However,asmostTSpatientsharbormutationswithinamutuallyexclusiveexon,manipulationofsplicevariationispositionedasapromisingandyetun-exploredtreatmentstrategy.Wethereforeaimtoexpandthetreatmentoptionsforthesepatientsthroughsplicemanipulation.Specifically,wehavedesignedantisenseoligonucleotides (AONs) targeted to thedeleteriousTSexonandevaluated their efficacy in induced pluripotent stem cell derived cardiomyo-cytes(iPSC-CMs).ApplicationofourAONsto iPSC-CMsderivedfromaTSpatientcausedbothadecreaseintheexpressionofthedeleteriousTSexon,aswellasacorrespondingincreaseinthenon-affectedexon.Thus,AONap-plicationsuccessfullydecreasestheburdenofTSchannelsintheheartbyre-placingthemwithfully-functionalalternatively-splicedchannels.Furthermore,AONtreatmentofthecellscorrectedtheabnormallylongactionpotentialsoftheTScells,validatingthefunctionalefficacyofthistreatmentstrategy.Overall,splicemodulatingAONsexhibitagreatpotential for thetreatmentofTSpa-tients.Moreover,theapproachwillserveasamodelfordevelopingnewthera-peuticstrategiesbasedonthemanipulationofproteinsplicing,thusenablingapersonalizedtherapeuticstrategyforselectgeneticdiseases.



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A Single Cell Transcriptional Atlas of Synovial Joint Development

Qin Bian1, Patrick Cahan1

1Biomedical Engineering Department, Johns Hopkins University, Baltimore, MD, 21205, USA

Background: Synovial joints are complex anatomical structures, comprisingdiverse tissue types, including articular cartilage, synovium, fibrous capsuleandligaments,thatplayacrucialroleinskeletalfunction.Synovialjointsde-velopmentbeginswiththeformationofregionswithhighlycondensedflatmes-enchymalcellsatthesitesoftheprospectivejoint,whichiscalledinterzone.Theinterzoneisessentialforsynovialjointdevelopment,iftheregionisremo-vedthroughmicrodissection,thetwoadjacentboneswillfuseandthejointwillnotbeformed.Cellsinthecentralintermediatelaminaareprogressivelylostthroughapoptosis,leadingtotheformationofthejointcavity.However,interzo-necellfate,rolesandmodusoperandihadlongremainedobscure.Alltheab-ovecomponentsarederivedfromanearlysetofGdf5-expressingprogenitorspopulatingtheinterzonedomain.LackofGDF-5inthenaturalmousemutantbrachypodism,causeswidespreadjointdefectsandskeletalgrowthretardati-on.Althoughphysiologicallyandclinicallysignificantthereislimitedinformationonthemechanismsandsignalingmoleculesthatleadtojointmorphogenesis.Despitewiderecognitionthattheinterzoneregionisessentialforjointforma-tion,thereislimitedinformationofthegeneexpressionenvironmentinwhichinterzonecellsemergeanddistinguishthemselvesfromadjacentgrowthplatechondrocytes.Agreaterunderstandingofjointformationcanprovidecriticalin-sightonthepathogenesisofjointdegenerationandtodevelopnovelreparativestrategiestorestorelong-termjointfunctionfollowingtraumaanddisease.Inthisstudy,weapplySingle-cellmRNA-sequencingmethodsenableunbiased,high-throughput,andhigh-resolutiontranscriptomicanalysisofindividualcells.Methods:Here,wegeneratedsinglecellRNAsequencing(scRNAseq)dataofkneejointsfromGdf5-cre::Rosa-EYFPmiceatE12.5,E13.5,E14.5,E15.5andday5.YFP+cellswerefractionatedbyfluorescence-activatedcellsorting(FACS)anddeadcellswereexcludedbyPropidiumIodide.Total3107,4786,1888,1099and4015cellswereprofiledrespectivelybyGemCodeTMSingleCellplatform(10XGenomics).DatawasanalyzedbyLovainclustering,diffe-rentialgeneexpressionandGSEA.WeusedInsituhybridyzationandFAC-ba-sedprospectiveisolationtoverifycelltypesduringsynovialjointdevelopment.Results:Gdf5lineagecellscollectedfromE12.5today5distributeinanlagen,interzone,superficialanddeeperzoneofarticularcartilage,ligament,menisci,synoviumandothersoft tissuesurrounded.E12.5cells fell into fourdistinctclusters.Oneof them isenriched inchondrogenesis relatedgenessuchasCol9a,Col2a1andSox9,whichmightlocalizeinouterinterzone.Thispopula-tionkeptpresentinginlaterstagesandcontributedtodeeperzoneofarticularcartilagewhich isverifiedby ISH.AnotherclusterhighlyexpressingCol1a1,Col1a2,Osr1maydeveloptoperichondriumandsynoviumthatsurroundthejoint.ThethirdclusterwhichissimilartoOsr1enrichedclustermayberelatedtotendondevelopmentsincetwotypicalmarkersoftendonprogenitors:ScxandSix1werehighlyexpressed.TheclusterenrichedinGdf5wasassumedtocontribute to intermediate interzone.Conclusion:Gdf5 lineagecellsdiffe-rentiatetoseveralcell typeswhichcontributetoarticularcartilage, ligament,synoviumandtendonduringsynovialjointdevelopment.Thecellfatemightbeidentifiedbyspecificgeneexpressions.

Induced Pluripotent Stem Cell-Derived Oligodendrocyte Progenitor Cell Model to Study Neurodegeneration

in Progressive Multiple Sclerosis

Itzy Morales1*, Vasiliki Marchairaki1,3, Peter Calabresi1, and Katie Whartenby1,2;

1Neurology, 2Oncology, and 3Institute of Cell Engineering, Johns Hopkins School of Medicine, Baltimore, MD.

Multiplesclerosis(MS)isthemostcommoncauseofprogressiveneurologicaldisability inyoungadults. Ithasbeencharacterizedasanautoimmune res-ponsetomyelin,leadingtodemyelinationanddisruptingtheflowofelectricalimpulses in thecentralnervoussystem.TheetiologyofMS isunknownbutlikely involves a combination of genetic, immunological, and environmentalfactors that influence the pathology, symptomatic presentation, course, andoutcome.MSfallspredominately intotwocategories:relapsing-remittingMS(RRMS),andprogressiveMS(PMS).Advancesindisease-modifyingthera-pieshavesignificantlyimprovedthecourseofRRMS,butPMSremainslargelywithout effective treatments for remyelination and neurodegeneration. BothformsofMSconvergeoncertainpathologicalfeaturesincludingneuronalcelldeathanddemyelinatedaxons,butitremainsunknownwhethertheneuronsortheoligodendrocytes(OLs)arethemainpointofdefect.Recentstudieshaveshownthatoligodendrocyteprogenitorcells(OPCs)arepresentatsitesofin-jury,contradicting thepreviousbelief thatadultswereunable to remyelinateduetolossofOPCsasaconsequenceofaging.Yet,remyelinationremainsinefficient inPMS.MShas been predominately considered an autoimmuneprocess,however,considering the lackof responseto immunosuppressantsandthepresenceofOPCsatsiteofinjury,weproposedthatthecentraldefectofPMSisnotautoimmunebutratheraconsequenceofaninherentinabilityofOPCstodevelopandmatureintoefficientOLs.Theabilitytostudyearlypatho-logicalprocessesinMShasbeenhinderedbytheinaccessibilityoftheaffectedcells,andbyalackofaneffectivemodelsystemtostudyhumanOPCsandOLsthatcloselyrecapitulatesthedisease.Wehavedevelopedamodelsys-tembasedonhumaninducedpluripotentstemcell(iPSC)technology.Inthissystem,peripheralbloodmononuclearcells(PBMCs)obtainedfromacohortofMSpatientsarereprogrammedtoiPSCs,andfurtherdifferentiatedintoOPCs.TheOPCsarethenexposedtoavarietyofcytokinesknowntoplayaroleintheinflammatoryenvironmentcharacteristicofMS,therebyallowingustoidentifyandmonitorearlyresponses,aswellasresponsestosustainedinflammation.Inadditiontofacilitatingstudiesofdiseasepathogenesis,thisnewmodelall-ows invitro testingofpotential therapies thatmightpreventOPCsandOLscelldeath.Furthermore,thissystemaffordsthescreeningofcandidategenesimplicatedinsusceptibilityofOLdifferentiationinMS.Normally,thetargetcellstobeexamined in thisstudy, i.e.OPCswouldbe impracticable toobtainbybiopsyinalivingperson,asthenumberofparticipantpatientswouldbeinsuf-ficientandtheyieldofcellswouldbelow.Insummary,throughadvancesinstemcelltechnologywehavesuccessfullycultivatediPSC-derivedOPCsfromMSdonorbloodsamples.Thisenablesustoworkdirectlywithcellsderivedfromspecificindividualpatients,whichisacriticalfirststepintotheeraofper-sonalizedmedicine.

Poster Abstracts:

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Development of A Biphasic MSC Delivery System for The Repair of Osteochondral Defects

George Klarmann, Avi Gnatt, Saya Vijily, Anita Gugel, Yi Arnold and Luis Alvarez; Theradaptive, Inc, Frederick, Md 21704

Theradaptivehasdevelopedaplatformforatargeteddeliveryofpotentrege-nerativetherapeuticsthatbringabouttissueregenerationinacontrolledman-ner.Thistechnologyisbasedonourdiscoveryofproteinsequencesthatbindtothecrystalfacesofceramicsextremelytightly.Thisallowstherecombinantproductionofceramicbindingvariantsofawidevarietyofproteins.Previously,weappliedthisplatformtodevelopatetherablevariantofBMP2(tBMP2)thatexhibitsextremely tightbindingofβ-TCPceramic.Thus,wecan “paint” thesurfaceoftheTCPwithtBMP2torendertheinertceramicintoapotentosteo-inductivescaffoldmaterial.Suchtargeteddeliverypotentiallyallowssignificantreductionindrugdosagecomparedtowhatisusedincurrentstate-of-theartproductsonthemarket.UndertheMarylandStemCellResearchFoundation,weareadvancing thedevelopment of our implantable osteochondral repairproductcalledConForma®.ConForma®isabiphasicosteochondralcompo-sitewithathincartilage-inducinglayeroverlayingathicksubchondralbone-in-ductingphase.OurputtydesignforbothphasesismadetotheconsistencyofPlay-Doh,containingmostlyβ-TCP.Itiscustomizable,moldable,andcon-formable,allowingthesurgeontointra-operativelymanipulatetheshapeandsizeoftheimplanttorenderitsuitableforuseinawiderangeofosteochondralrepairgeometries. Itdoesnot relyonanypre-operativecustomizationsuchasautologouscellexpansionorcustommachiningofhardwarecomponent,and thesurgeoncanapplyConForma®usingminimally-invasivearthrosco-picsurgical techniques.Thesurgeoncaneasily loadallogeniccellssuchasMSCsduetothehighlyporousstructureandlargesurfacearea.Themostim-portantfeatureofthisimplantisthatnovelbiologicsknownforosteoinductiveandchondro-inductivearetetheredonbothphaseswhichcanpermitcartilageandthesubchondralbonerepairovertimescalesexceedingthreemonths.Thetwoobjectivesofthisworkare1.CompletethedevelopmentoftheConFor-ma® implant system components, and 2. Characterize the performance ofConForma®incell-baseddifferentiationassaysand todevelopmethods toincorporateMSCCD29+intotheimplantwhilemaintaininggoodviabilityandengraftment.SincethestartoftheawardinJulythisyear,wehavebeenfo-cusingontheprocessdevelopmentofthevariantsofseveralgrowthfactorsofinterest,TFG-βandFGFfamiliesinparticular.WehavedevelopedscalablemethodstoexpressourproteininE.coliinclusionbodies.Aftersolubilizationandrefolding,theproteinswerepurifiedusingacombinationofaffinity,ionex-changeandhydrophobicchromatography.Wehavepurifiedupto~120mgofactivetethereddimericBMP2(tBMP2)proteintodate.BiologicalactivityoftheengineeredproteinswasconfirmedwithreporterassaysforBMPandTGF-β3,andproliferationassaysforFGF,andIGF.PositivebindingtoTCPwasde-monstratedforallpurifiedproteins.Wearecontinuingtorefinethepurificationandbindingprocesses,andwillapproachobjective2inthecomingmonths.

Multi-Lineage Capabilities of Mesenchymal Stem Cells for Engineering Orthopaedic Interfaces

Amelia Hurley-Novatny1,2, Navein Arumugasaamy1,2, Megan Kimicata2,3, John Fisher1,2

1Fischell Department of Bioengineering, University of Maryland, College Park, MD 20742; 2Center for Engineering Complex Tissues, University of Maryland, College Park, MD 20742; 3Department of Materials Science and Engineering, University of Maryland, College Park, MD 20742

Background: Interfacesbetweenorthopaedictissues,suchasbone, tendon,cartilageandmuscle,servetotransfermechanicalforcesbetweentissuesofdiffering inherentmechanicalproperties.Boneandtendon,astiff tissueandasoftcomplianttissue,respectively,arejoinedbytheenthesis.Thisenthesishasacomplexstructurecharacterizedbylocalizedandorganizedextracellularmatrixproteinsthatservetotransfertensile,compressive,andshearstressesbetweenthetwotissues.Followinginjuryofthetendon,surgicalinterventiontypically involvescompleteresectionoftheenthesis,resultinginmechanicalinferiority and contributing to poor long-term surgical outcomes. Tissue en-gineering thebone-tendonenthesisprovidesapotentialstrategy to improvehealingandpost-surgical integration.Methods:Bonemorphogenicprotein-2(BMP-2), transforminggrowth factor beta-3 (TGFβ-3), andbonemorphoge-nicprotein-12(BMP-12)wereusedtoinduceosteogenic,chondrogenic,andtenogenic differentiation of mesenchymal stem cells (MSC), respectively.MSCsandpoly(lactic,co-glycolicacid)microparticlescontainingeitherBMP2,TGFβ3,orBMP12,wereencapsulatedinagelatinmethacrylate(GelMA)hyd-rogel.Thesehydrogelswerecastina3Dprinted,biocompatiblemold.Spatialorganizationwasachievedusingamulti-layeringtechnique.Constructswereanalyzedformulti-lineagedifferentiationusingPCRtoassesscelltype-specificgeneexpression(Runx2andbonesapiloprotein1forosteogenesis,Sox9andcollagenXforfibrochondrogenesis,andscleraxisand tenomodulin for teno-genesis)andstainingusingIHCfortissue-specificextracellularmatrix(ECM)components,includingcollagentype(I,II,X,andXII),decorinandaggrecan,andhydroxyapetite.Results:Differentiationofcellsencapsulatedingelswasachievedinmonoculture,asassessedbyPCRforexpressionoftranscriptionfactorsandmatrixproteinsspecific toeach lineage. GelMAconstructscastwith layersofBMP-2,TGFβ-3,andBMP-12 resulted in regionaldifferencesingeneexpressionconsistentwithosteogenesis,chondrogenesis,andteno-genesis, respectively.Additionally,constructshadregionaldifferences inde-positionofcollagentype(IandII),andproteoglycandeposition.EncapsulatedcellsdepositedECMcomponentsspecifictothenativebone-tendonenthesis,includingcollagen(XandXII).Conclusions:TheabilitytodifferentiateMSCsintomultiple lineages inasingleconstructhaspreviouslybeenasignificantchallenge,hinderingthedevelopmentofcomplexandheterotypictissues.Thisstrategycouldpotentiallybeusedtoaidintreatmentoftendoninjuries,whichtypicallyhealpoorlyinadults.TheoverallworkdemonstratesthepotentialuseofMSCsfordevelopmentoforthopedicinterfacetissuessuchasthebone-ten-donenthesistoimproverecoveryoffunctionfollowinginjury.



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Here, we introduce a novel hPSC culture system to employ optical stimulation ofthefibroblastgrowthfactor(FGF)signalingpathway,withoutthedailysup-plementationofrecombinantFGF2protein(akeymoleculefortheirstemness).WithCRISPR/Cas9-basedgeneticincorporationofthelargelight-oxygen-vol-tage (LOV,analgae-/plant-derivedphoto-activableprotein)sensingdomain,weachievedatunablelight-inducedactivationoftheFGFsignalingpathwayinhPSCs.TheopticallymaintainedhPSCsinmultiplepassageshavesimilarcellular andmolecular profiles to those culturedwith FGF2 protein,withoutperturbingdifferentiationcapabilitiesintothreegermlayers.Theseresultsde-monstratethatopticalstimulationoftheFGFsignalingpathwayissufficienttomaintain hPSCs.

CD34+ MCSCS Represent A Subset of Skin-Derived Precursor Cells in The Skin

Li Pan1 *, Sandeep S. Joshi1, Bishal Tandukar1, Emmanual Kalapurakal1 and Thomas J. Hornyak1,2,3

1Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore MD; 2Research & Development Service, VA Maryland Health Care System, Baltimore, MD; 3Department of Dermatology, University of Maryland School of Medicine, Baltimore, MD

Melanocytestemcells (McSCs)whichare located in thehair follicleare re-sponsible forhairpigmentationduringhaircyclesandareakeycomponentofadultstemcellsintheskin.Skin-derivedprecursors(SKPs)havebeeniso-latedanddescribedasamultipotentadultstemcellsfromrodentandhumanskin.TheSKPshave thecapability togive rise toneuralcrest-derivedcellsinvitro.Ourpreviousstudieshaveshown thatCD34+McSCswhich resideintheCD34+bulge/lowerpermanentportion(LPP)regionduringtelogenareneuralcrest-likestemcells.BasedonthesimilaritiesbetweenthepropertiesofCD34+McSCsandSKPs,wehypothesizedthatCD34+McSCsrepresentasubsetofSKPsintheskin.Todeterminethis,CD34+McSCsandSKPswerecultured,differentiatedandanalyzedforsimilarities inneuralcrestderivativemarkerexpression.CD34+McSCs,SKPsandembryonicneuralcreststemcell(eNCSCs)wereisolatedandgrownasspheroidsinneuralcrestmedium(NCCmedium)orSKPmediumfor7days.Subsequently,cellsweredifferent-iatedinneuralcrestorSKPdifferentiationmediumandanalyzedformarkerex-pressionatearly(24hours)andlate(1week)timepoints.Atanearlytimepoint,analysisrevealedthat86%ofCD34+MSCsand76%ofeNCSCsexpressedp75/NGFRwhileonly2%ofSKPsexpressedp75/NGFRinneuralcrestdiffe-rentiationmedium.Thepercentagesofnestin-positiveandfibronectin-positivecellswere similar.At a late timepoint,CD34+McSCsexpressedmore glialmarkersGFAPandCNPasecomparedtoSKPs(44%vs3%and31%vs2%,respectively).However,thepercentagesofβIIItubulin(Tuj1)–positiveneuro-nalcellsandsmoothmuscleactin(α-SMA)-positivecellsweresimilar.Resultswere similar when cells were differentiated in SKP differentiation medium.Takentogether, thedatapresentedheresupport thehypothesisthatCD34+McSCsareneuralcrest-likestemcellsandrepresentasubsetofSKPsintheskin.

Biliary Atresia Relevant Human iPSCs Recapitulate Key Disease Features in A Dish

Lipeng Tian1*, Zhaohui Ye2, Kim Kafka3, Dylan Stewart4, Robert Anders5, Kathleen B. Schwarz3* and Yoon-Young Jang*1,6

1Dept. of Oncology, The Sidney Kimmel Comprehensive Cancer Center; 2Dept. of Medicine; 3Department of Pediatrics; 4Dept. of Surgery; 5Dept. of Pathology; 6Institute for Cell Engineering; Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

Biliaryatresia(BA)isthemostcommoncauseofpediatricend-stageliverdiseaseandthemostrapidlyfibrosingdisease inhuman,but theetiology ispoorlyunderstood.There isnoeffective therapyforBApartlydueto lackofhumanBAmodels.TowardsdevelopinginvitrohumanmodelsofBA,disea-se-specific iPSCs from6BApatientswere generated using non-integratingepisomal plasmids. In addition, to determine the functional significance ofBA-susceptibilitygenesidentifiedbygenomewideassociationstudies(GWAS)inbiliarydevelopment,agenome-editingapproachwasusedtocreateiPSCswith definedmutations in theseGWASBA loci.Using theClusteredRegu-larly InterspacedShortPalindromicRepeats (CRISPR)/Cas9system, isoge-niciPSCsdeficientinBA-associatedgenes(GPC1andADD3)werecreatedfromhealthyiPSCs.BoththeBApatient-iPSCsandtheknockout(KO)iPSCswerestudiedfortheirinvitrobiliarydifferentiationpotential.TheseBA-speci-ficiPSCsdemonstratedsignificantlydecreasedformationofductalstructures,decreasedexpressionofbiliarymarkersincludingCK7,EpCAM,SOX9,CK19,AE2andCFTRandincreasedfibrosismarkerssuchasalphaSMA,Loxl2andCollagen1comparedtocontrols.Boththepatient-andtheKO-iPSCsalsosho-wedincreasedyes-associatedprotein(YAP,amarkerofbileductproliferation/fibrosis).CollagenandYAPwerereducedbytreatmentwiththeanti-fibroge-nicdrugpentoxifylline.Insummary,theseBA-specifichumaniPSCsshoweddeficiency inbiliarydifferentiationalongwith increasedfibrosis, the twokeydiseasefeaturesofBA.TheseBA-relevantiPSCscanprovidenewhumanBAmodelsforunderstandingthemolecularbasisofabnormalbiliarydevelopmentandfibrogenesis,andopportunitiestoidentifydrugsthathavetherapeuticef-fectsonBA.

Maintaining Human Pluripotent Stem Cells with Optical Stimulation of the FGF Signaling Pathway

In Young Choi1*, HoTae Lim1, and Gabsang Lee1, 2, 3, *

1The Institute for Cell Engineering, 2Dept. of Neurology and Neuroscience, 3The Solomon H. Synder Department Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD 21205

Stemcell fate is largelydeterminedbya cell-signalingnetworkandcanbecontrolledbythesupplementationofexogenousrecombinantproteins.Howe-ver,thismaycauseheterogeneousandunsynchronizedsignalingduetotheunevendistributionofrecombinantproteinsandpoorthermostabilty.Suchis-suesarecloselyassociatedwiththespontaneousdifferentiationofhumanplu-ripotentstemcells(hPSCs),whichmayhamperfuturestemcellapplications.

Poster Abstracts:

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Effect of Trauma on Immunotolerance After Myelinating Progenitor Cells Transplantation in Immunocompetent

Myelin Deficient Mice

Rui Wang1,2, A Robert D Stevens 1,2, Piotr Walczak1

1Department of Radiology, Johns Hopkins University School of Medicine, Baltimore, MD, USA; 2Department of Anesthesiology and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA

Celltransplantation-basedtreatmentsforneurologicaldiseasehavebeenpro-mising,yettransplantrejectionremainsamajorbarriertosuccessfulregenera-tivetherapies.Newimmunomodulationmethodsthatinducespecifictolerancehavebeendeveloped fororgan transplantation.Ourgroupandothershaveshownthatlong-lastingtolerancetotransplantedstemcellscanbeachievedviamonoclonalantibodiesblockingco-stimulationsignalinginTcells(co-sti-mulationblockade).Thisapproachopensupopportunityforreplacinglostordysfunctionalcellsinthebrainbyallografting.However,ithasneverbeenstu-diedwhetheradelayedneuroinflammatoryeventsuchasinjury,couldbreakthetoleranceoftheallograftedstemcells?Shouldthatoccuritcouldtriggermassiveand life-threatening rejection.Theaimof this study is toprovideasystematicassessmentof theefficacyandsafetyofco-stimulationblockadeasa tolerance inductionstrategy.Mouseglial restrictedprogenitors (GRPs)werederivedfrommid-gestation(E13)transgenicfLucPLP-GFPmice.mGRPsweretransplantedintoP3immunocompetentshiverermouseneonates(N=10),whilewildtype(C57BL6)neonateswereusedasthecontrolgroup(N=10).Themonoclonalantibodies(anti-mouse-CD154mAbandAbatacept)wereadminis-teredintraperitoneallyondays0,2,4,and6post-grafting.Weusedbiolumi-nescenceimaging(BLI)asanon-invasivemethodtodetectthecellrejection.Theopenfieldtestwasperformedtotestthebehavioralandneurologicalout-come.NeurologicalconsequencesweremonitoredwithMRI.After3monthsthetransplantedmiceweresubjectedtotraumaticbraininjury(TBI).After28daysofTBIthemiceweresacrificedforthehistologicalobservationofgraftedcells,neuroinflammation(Iba1,GFAP,CD45)andstatusofneurons(NeuN).TransplantedcellswereeasilydetectablewithBLIand thesignal intensitiesgraduallyreducedoverthreeweeksaftertransplantationtothebaselinelevelinimmunocompetentmice,bycontrastinshiverermicewithcostimulationblo-ckadethesignalwasmaintainedatarelativelyhighlevel(1.54-2.83foldhighercomparedtobaseline).1weekafterTBItheBLIsignalsignificant increasedinshiverergroup(34-41foldhighercomparedtothatbeforeTBI),throughtheover threeweeksafter injury theBLI signal gradually decreased to plateauat a relatively high level (4.7-6.7 times higher compared to that beforeTBIlevel).Histologicalanalysis28dayspost-TBIshowedmoreGFPpositivecellswithintheTBIinjuredhemispherecomparedwiththecontralateralhemisphe-re(p=0.014).ShiverermicewithmGRPsandcostimulationblockadeshowedareductionincorticalcontusionvolumecomparedwithwild-typeinjuredmice[(18.3%±4.7%)vs(29.7%±2.2%),p<0.01].AfterTBIactivationofmicrogliaandastrocytosiswasasignificantlygreater in theshiverergroupcomparedwiththewildtypemice.Onthecontrary,therewasalowernumberofCD45+infiltra-tingleukocytesinshiverergroupcomparedwithwildtype(p<0.01).Openfieldtestshowedthetrendofincreasedanxiety-likebehaviorafterTBIinshiverer(p>0.05).InMRI28daysafterTBItherewasclearevidenceofbraininjurybutno differenceswere observedbetween thegroupsand that included lesionmorphology,brainedemaorblood-brainbarrieropening.

Theseresultsdemonstratethatco-stimulationblockadeenableslong-termsur-vivalandtolerancetothemGRPsinshiverers.Neuroinflammation(TBI)hasratherpositiveeffecton transplantedcells.28daysafterTBI,shiverermicehadreducedinfiltrationbyCD45+cells,increasedastrocytosisandincreasedactivationofmicrogliacomparedwithwildtypemice.

Neuronal Differentiation of Neuronal Stem Cells by Autophagy Induction in Oxidative Environment to

Treat Traumatic Brain Injury (TBI)

Chinmoy Sarkar1, Robert A. Brown1, Nivedita U. Hegdekar1, Awad Ola2, Ricardo Feldman2 and Marta M. Lipinski1

1Shock, Trauma and Anesthesiology Research (STAR) Center, Department of Anesthesiology, University of Maryland School of Medicine, Baltimore2Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore

Traumaticbraininjury(TBI)isoneofthemajorcausesofdeathamongyoungadultsaccountingforalmost30%ofallinjury-relateddeath.Innon-fatalcasesitleadstolifelongneurologicalimpairmentamongpatientsmainlyduetoacuteandchronicneuronallossintheinjuredbrain.Sincelostneuronscannotbeeffectively replenishedbyanyavailable therapy, implantationofneuralstemcells(NSC)at the injurysiteofbrain isapromisingtherapeuticapproachtotreatTBI.However,highlyoxidativeandinflammatoryenvironmentatthesiteofinjurylimitsthebeneficialeffectofNSCtransplantation,sincesuchconditionisdetrimental forNSCsurvivalanddifferentiation intoneurons. Autophagy,acellulardegradativeprocessthatremovestoxicanddamagedmacromole-cules and organelles, plays important role in neurogenesis. It is also involved inmaintainingintracellularROShomeostasis.However,weobservedimpair-mentofautophagyfluxinthecorticalneuronsaftercontrolledcorticalimpact(CCI)-inducedbraininjuryinmice.Thustheenvironmentattheinjurysiteishighly toxicandnotconducive forNSCsurvivaland theirdifferentiation intoneurons. Wehypothesized that autophagyupregulation inNSCsmaypro-tectcellsagainstoxidativeinsultandwillpromotetheirneuronaldifferentiationintheoxidativeenvironmentat injurysite. Accordingly,wetransducedNSCwithFIP200,acomponentofautophagyinducingUlk-1complex,thenexpo-sedthemtooxidativeenvironmenttoassesstheirsurvivalanddifferentiation.WeobservedhigherlevelofneuronaldifferentiationiniPSC-NSCsexpressingFIP200ascomparedtothecellsexpressingvectorwhenculturedintheoxi-dativecondition.ThisismostlikelyduetotheupregulationofautophagyfluxthroughFIP200andUlk1complex. Thus these resultsclearlydemonstratethatautophagyupregulation isbeneficial inpromotingNSCsurvivalanddif-ferentiationunderhostileoxidativeenvironment.NextwetransplantediPSC-NSCs in the injuredmousebrainatday7after injuryand treated themiceeitherwithautophagyinducingdrugrapamycinorwithvehicle.Weobservedimprovedsurvivalandenhancedneuronaldifferentiationoftransplantedneuralstemcellsinmicetreatedwithrapamycin.TakentogetherourdatashowedthatthetransplantationofNSCswithupregulatedautophagyintheinjuredmousebrainisbeneficialforNSCssurvivalandproperdifferentiationfollowingtrans-plantationinthemousebrainfollowingTBI.



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Neurons from Human Neural Progenitor Transplants Establish Functional Connections in The Adult Cortex:

An Optogenetic Study

Jiwon Ryu, Philippe Vincent, Leyan Xu, Nikolaos Ziogas, Richard Sima, Nicholas Stewart, John Curtin, Shirin Sadeghpour, Sascha du Lac, Elisabeth Glowatzki, Vassilis E. Koliatsos

Johns Hopkins University, School of Medicine, Baltimore, MD, USA

Weproposethatoptogeneticallyengineeredhumanneuralprogenitors(hNPs)isagreat tool in regenerativeneurosciencebecause their integrationwithinthehostnervoussystemandfunctionalityoftheirdifferentiatedneuronalpro-geniescanbeassessedasresponsetolight.Infact,thissameresponsemayeventuallybeleveragedforclinicalpurposesinregenerativeneurology.Here,wetransducedH9embryonicstemcell(ES)-derivedhNPswithalentivirusthatharboredhumanchannelrhodopsin(hChR2)anddifferentiatedthemintoafo-rebrainlineage.WethencharacterizedtheirfateandoptogeneticfunctionalityofhChR2-hNPsinvitrowithelectrophysiologyandimmunocytochemistry.WethentransplantedChR2-hNPsintothemotorcortexofnormalmiceandratsorsubjectsexposed to traumaticbrain injury.Weanalyzed thesurvivalandneuronaldifferentiationofhChR2-hNPsbyimmunohistochemistryfollowedbystereologicalanalysis.Invitro,hChR2-hNPsdemonstratedneuronalphenoty-pesafter70daysofcoculturingwithastrocytes.Progressively,theydisplayedbothinhibitoryandexcitatoryneurotransmittersignatures.ChR2(+)cellsfromtheseculturesgeneratedspontaneousactionpotentials100-120daysintodif-ferentiationandtheirfiringactivitywasclearlyandconsistentlydrivenbyopticalstimulation. Wealsoobserved that,after lightstimulation,neighboringnon-ChR2 neurons generated postsynaptic excitatory and inhibitory responses,evidencethathChR2-hNP-derivedneuronsestablishedfunctionalconnectionswithotherneurons inculture.Threemonthsafter transplantation intomotorcortexofinjuredorshamsubjects,60-70%ofhChR2-hNPsmanifestedneuro-nalcytologiesandphenotypes,whereas60%ofcellsalsoexpressedChR2.Depending on depth of inoculation, transplant-derived neurons extendedaxons through thecorpuscallosumand the internal capsuleandgeneratedterminalfieldseitherinsensorimotorcorticalareaslateraltothetransplant(inthecaseofsuperficialinjectionsthroughlayer3)or,viatheexternalcapsule,in the claustrum, endopiriformarea, and corresponding insular and piriformcortices(inthecaseofdeeperinjectionsinlayer5/6andtheadjacentcorpuscallosum).Atthesesites,theyestablishedsynaptophysin(+)terminals,appro-ximately10%ofwhichexpressedGABAergicmarkers.Therewasnoapparentdifferenceinengraftment,differentiation,orconnectivitypatternbetweeninju-redandshamsubjects.Wearenowanalyzingtheoptogeneticresponsesofterminalfieldsincortexwithc-Fosexpressionandoptogeneticfieldpotentialrecordingsafterlightstimulationofthetransplants.OurfindingsshowthatwecanengineerhNPswithoptogeneticpropertiesthataretransferredtotheirdif-ferentiatedneuronalprogenies.Usingtheseproperties,weshowthatneuronsderivedfromhNPshavethecapacityofestablishingfunctionalsynapseswithpostsynaptic neurons. We conclude that optogenetically endowed hNPs hold greatpromiseastoolstoexplorenewcircuitformationinthebrainand,inthefuture,perhapslaunchanewgenerationofneuromodulatorytherapies.

Modelling Pitt-Hopkins Syndrome, An Autism Spectrum Disorder, with Patient Derived Induced Pluripotent Stem Cells

Hiler, DJ.1, Chen, HY.1, Hamersky, GR.1, Wang Y.1, Rao Sripathy, SR.1, Soudry, OR.1, Warner, MC.1, and Maher, BJ.1,2,3

1The Lieber Institute for Brain Development, Baltimore, MD 21205; 2The Solomon H. Snyder Department of Neuroscience, Johns Hopkins School of Medicine, Baltimore, MD 21205; 3Department of Psychiatry and Behavioral Sciences, Johns Hopkins School of Medicine, Baltimore, MD 21205

Pitt-HopkinsSyndrome (PTHS) isa rareand relativelyunderstudiedautismspectrumdisorderthatiscausedbyanautosomaldominantmutationordele-tioninthegenetranscriptionfactor4(TCF4).TCF4isabasichelix-loop-helix(bHLH) transcription factor thatplaysacritical role inneuronaldevelopmentthroughknowninteractionswithotherproneuralbHLHproteins.TostudytheroleofTCF4 inneurondevelopment,wehavedifferentiatedhumancorticalneuronsfrombothPTHSpatientandcontrolderivedinducedpluripotentstemcells (iPSCs). Usingwhole-cellpatchclamp, immunohistochemistry,qPCR,RNA-seq,and imaginganalysis,wehavecreateda robustandquantifiablepipeline to assess the developmental andmaturation of PTHS derived-hu-mancorticalneurons. Withthispipeline,weanalyzedIPSC-derivedcorticalneuronsfrom6PTHSpatientsand5controlpatientsandhaveidentifiedanexcitabilityphenotypeinPTHSneuronswhencomparedtocontrols.Ouraimistousethisdiscoverytoidentifyanypotentialreceptorsand/orionchannelsthatcontributetothisphenotypeandtoidentifynovelmolecularpathwaysfordrug targeting.

Modeling Heart Chamber Development and Disease in Cardiac Organoids

Peter Andersen, Chlan Kwon

Division of Cardiology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205 USA.

Theheartdevelops from twomolecularlyandmorphologicallydistinctpoolsofcardiacprogenitorcells(CPCs),referredtothefirstandsecondheartfield(FHFandSHF),whichgiverisetodifferentanatomicalstructuresoftheheart.SeveraltypesofinheritedandcongenitalheartdiseasearerestrictedtoregionsoftheheartarisingfromeithertheFHForSHF,thusabnormalCPCdevelop-mentiscloselyassociatedwiththeetiologyofchamber-specificabnormalities.Whilenumerousloss-andgain-of-functionstudieshavedemonstratedtheim-portanceofthevariouspathwaysandsignalsduringearlyheartdevelopmentand formation, their precise roles in specificationandallocationof theFHFandSHFremaintobedetermined.Here,wedevelopedaPSC-basedcardiacorganoid system,wherein heart field specification anddevelopment can bemonitoredbygreenandredfluorescentprotein(GFPandRFP)reporters,andforthefirsttimedemonstratehowtheearlieststepsofheartdevelopmentandformationcanbe recapitulatedandmodeled invitro.Thesefindingsset thestage foramechanisticexplorationofheart field specificationanddevelop-ment,and todevelopaplatform forstudyingchamber-specificdevelopmentand disease in human PSCs.

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Using Stem Cells to Repair Radiation-Induced Injury After Tumor Eradication – A Perspective

Chengyan Chu1, Catherine M. Davis2, Esteban Verlade3, Anna Jablonska1, Miroslaw Janowski1, Jeff W.M. Bulte1, Mihoko Kai3, Piotr Walczak1

1Russell H. Morgan Dept. of Radiology and Radiological Science, 2Dept. of Psychiatry and Behavioral Sciences, 3Dept. of Radiation Oncology, Johns Hopkins University, School of Medicine, Baltimore, MD, USA

Gliomasarethemostcommonanddeadliestprimarybraintumors.Radiothe-rapy is an adjuvant component ofmost treatment strategies.Unfortunately,standardfractionatedradiationhaslimitedefficacywithahighrateofreoccur-renceandresults insevereadverseeffects.Themediansurvivalofpatientssufferingfromglioblastomaisonlyupto14.6months.Clearlythereisadireneedforabreakthroughtreatment.Oligodendrocytesarecharacterizedbyacontinuousturnoverandlikelymostaffectedbyradiation.Recentprogressinregenerativemedicinehasshownthattheycanbereplacedbytransplantationofglialprogenitors,openingup theopportunity for increasing themaximumacceptableradiationdoseifinjurytooligodendrocytescanberepaired.Here,weexplored the feasibility toapplysinglehighdose radiotherapy (40Gy) toeradicate the tumorand rescue the injuredbrainafterward.Wedemonstra-tethathigh-doseradiotherapy isefficient toachievecompleteeradicationof9Lgliomawithin4weeks,asevidencedbyMRIandBLI. LongitudinalMRIdidnotdetectanyabnormalitiesupto10weeksposttreatment;however,be-havioralimpairmentasmeasuredbyarecognitionmemorytestwaspresent.Onhistology,therewasonlymodestevidenceofneuroinflammationwithmildipsilateralastrocytosisandactivationofmicroglia.Inaddition,therewasmilddemyelinationon thesideof irradiationbutnohemorrhage.Thus,ourworkshowsthefeasibilityofescalatingradiationdoseasthereisalong-timewindowbetweendeliveringradiationtherapyandonsetofradiation-induceddamage.Thisprovidesanopportunitytopreventorrepairdamagebasedonmyelinasoneofthekeytargets.

Supported by: R01NS102675, 2017-MSCRFD-3942

Safety and Efficacy of Intravenous Autologous Mesenchymal Stem Cells for Subacute Ischemic Stroke:

A Phase 2 Randomised Controlled Trial

Thomas A. Zeffiro MD, PhD1, Assia Jaillard MD PhD2 , Prof. Marc Hommel MD2, Anaick Moisan PharmD PhD3 , and Prof. Olivier Detante MD PhD4 (for ISIS-HERMES Study Group).

1University of Maryland Medical Center, Department of Radiology, Baltimore, USA; 2Univ. Grenoble Alpes, AGEIS, EA 7407, France; 3Cell Therapy and Engineering Unit, EFS Rhône Alpes Auvergne, Saint Ismier, France; 4Univ. Grenoble Alpes, Grenoble Institut des Neurosciences, GIN, France

Importance:Whilepreclinical studieshavedemonstrated thatmesenchymalstemcells(MSCs)promotefunctionalrecoveryafterstroke,randomizedcon-trolledtrials(RCTs)usingMSCshaveraisedsafetyissues.TheveryfewRCTsthat havebeen carried in subacute stroke reported safety, but low feasibili-tyandnosignificantefficacyofMSC.Objective:Toassesssafety,feasibility,andexploreefficacyofintravenousautologousbonemarrow-derivedMSCsinpatientswithmoderate-severe subacute stroke.Design:The ISIS-HERMEStrialwasasinglecenterPhase2,singleblindRCTwith twoyear follow-up.Setting: Patients were recruited betweenAugust 31, 2010 andAugust 31,2015atGrenoble-AlpesUniversity-Hospital,France.Participants:Weenrol-ledpatientsaged18-70yearswithananteriorischemicstroke,lessthantwoweekspost-onset,withNationalInstitutesofHealthStrokeScale(NIHSS)>7at treatment time. Intervention:Patientswererandomized1:1:1toreceive100millionMSCs,300millionMSCs,ornoMSCsintravenouslyonemonthafterstroke.Real-timedynamicrandomizationwasused,stratifyingbylesionside,age and NIHSS. MainOutcomesandMeasures:Primaryoutcomesassessedsix-month feasibilityandsafety,basedon theproportionofadverseevents.Secondaryefficacyoutcomesassessedglobalandmotorbehavioralrecove-ry.PassivewristmovementfMRIactivityfromprimarymotorcortex(MI)wasassessedasaphysiologicaloutcomemeasure.Aper-protocolanalysiswasperformed tocompare ‘treated’and ‘control’groups.Results:Of31enrolledpatients (median age (IQR) = 53 years (46-59); 22males), 7 received 100millionMSCsand9received300millionMSCs.Feasibilitywas80%.Thetwodosegroupsweremergedforcomparisonwithcontrols.Therewere6adverseeventsintreatedpatientsand9incontrols(p=0.19).WeobservednoeffectsofMSCsonBarthelIndex,NIHSSandmodified-Rankinscore,butsignificantim-provementsinmotor-Fugl-Meyerscore(t=2.43,p=0.019),motor-NIHSSscore(t=2.272,p=0.024)andtask-relatedMIfMRIactivity(t=2.67,p=0.009).Conclu-sionsandRelevance: IntravenousautologousMSCtreatmentwassafeandfeasible.BrainactivitybiomarkerresultssuggestthatMSCtreatmentimprovemotorsystemrecovery.TheobservedbenefitsmightresultfrombrainrepairthroughMSCparacrineeffects.MSCbeneficialeffectsinsubacutestrokeneedtobeconfirmedbyfurtherstudies.TrialRegistration:ClinicalTrials.govIdenti-fierNCT00875654.

Generating 3D Brain Organoids With Endothelial Cells From Human iPSCs

Anna Bagnell, Marie Medynets, Tongguang Wang

Neurodifferentiation Unit, Translational Neuroscience Center, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland

In vitromodelsare important for understanding themechanismsunderlyinghumanneurologicaldiseasepathologiesandthedevelopmentoftherapeutics.Comparedwithtraditionalmonolayerofmixedneuronalcultures,3Dbrainor-ganoidsculturesbasedoninducedpluripotentstemcells(iPSCs)containad-vancedcell-cellinteractionsandorgan-levelstructuresandfunctionscentraltomanydiseaseetiologies.Evidenceindicatethatbloodvesselsandendothelialcellsaspartofhumanbrainplayimportantrolesinhumanbraindevelopmentandneurodegeneration.However,itisstilldifficulttodeveloporganoidscon-tainingbothendothelialandneuralcellsastheyarefromdifferentgermlayers.



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Development of Imaging Biomarkers for Stem Cell Transplantation in Amyotrophic Lateral Sclerosis

Jean-Philippe Richard1, Uzma Hussain1, Sarah Gross1, Arens Taga1, Meh-reen Kouser2, Akshata Almad1, James Campanelli3, Jeff W.M. Bulte2 and Nicholas J. Maragakis1

1Johns Hopkins University School of Medicine, Department of Neurology, Baltimore, MD; 2Johns Hopkins University School of Medicine, Department of Radiology and Institute for Cell Engineering, Baltimore, MD; 3Q-Therapeutics, Inc. Salt Lake City, UT

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative diseasewheremotor neurons progressively degenerate and die.Astrocytes and ot-herglialsubtypesparticipateinALSprogressionandarethereforetargetsfortherapeuticstrategies.Previously,ourlabinvestigatedthepotentialofhumanGlialRestrictedProgenitorCells(QCells) toprovideneuroprotection inALSanimalmodels.ThedevelopmentofQ-CellsasacellulartherapeuticforALShasshownsignificantprogress.AnFDAINDfortheuseofQCellshasbeengranted forourplannedclinical trialdelivering thesecells to thespinalcordofALSpatients.Oneof thechallenges intransplantationtherapeutics intheCNSistheabsenceofdataonmonitoringsurvivalandmigrationofthecellsaftertransplantationinthelivingpatient.UnderstandinghowQCellsbehaveaftertransplantationwillprovidecriticalinformationthatcanhelpusoptimizethenumberofcells for transplantation,understandmigratorypatterns insidethe spinal cord, and monitor cell survival over time. In this study, we will use MRI-basedtechniquestoexaminecellsurvivalandmigrationovertimefollo-wingthetransplantationofCS-1000labeledQ-CellsintorodentmodelsofALS.CS-1000isafluorocarbon-based(19F)emulsionthatallowsin vitrolabelingofQCellsforlaterstudiesofcelltrackingusingMRI.CS-1000canalsoincorpo-ratefluorescenttags(DMRedorDMGreen)toallowforimmunocytochemistry.Important for its clinical use,wenowhave extensive data characterizingQCellsfollowingtheincubationwithCS-1000.WehavebeenabletodeterminethesubcellularlocalizationofthefluorinatedcompoundandhaveperformedseveralexperimentstooptimizethedoseofCS-1000toleratedbyQcellsandthedurationforincubationtoprovidethemaximumloadingeffect.Importantly,wehavealsoshowntheabsenceofanystatisticallysignificantdetrimentalef-fectofCS-1000onthedifferentiationofQCellsintorelevantglialcellsubtypes.Todemonstratethatourlabeledcellswerecapableofsurvival,integrationanddifferentiationintoastrocytesfollowingtransplantationintothespinalcordsofmice,wetransplantedQcellslabeledwithDMGreenCS-1000.Weshowthatthesecellscanbetargetedtotheventralhornandappropriatelydifferentiateintoastrocytes. Importantly,oneweekafter transplantation,wedidnotseeanyuptakeoftheDMGreenCS-1000labelingbyhostmousecells.Conse-quently,thesedatademonstratethattheCS-1000labelingwillgiveusatrueassessmentoftransplantedcellswithoutconcernforfalsepositivelabelingofhost neural cells. In parallel, we conducted 19FNMRexperimentsonCS-1000labeledQSV40cells(animmortalizedQcellline)inordertoquantifytheCS-1000uptakebythelabeledcells.NMRSpectraand19F content analysis shows thatincreasingtheCS-1000concentrationinthelabelingmediaincreasesthe19Fcontentpercell.HavingoptimizedtheinvitroandNMRparadigms,MRIstudiesinrodentsreceivingCS-1000labeledQCellsintotheCNSareunder-way.Takentogether,wearegeneratingpre-clinicaldatanecessaryfordemon-stratingthesafetyandclinicalutilityofCS-1000inpreparationfortranslationaluse in patients.

WehypothesizedthatinducingiPSC-derivedembryoidwithendothelialdiffe-rentiationmediumatearly stage followedbyneural inductionmayhelpge-nerating3Dcerebralorganoidswithmixedneuronalandendothelialcells.Todothat,protocolsweredesignedandoptimizedtopermitthedifferentiationofhuman iPSCembryoidbodies intobothneuronal andendothelial cell typesconcurrently.Geneexpressionanalysisandimmunostainingofspheroidsindi-catedthepresenceofthetwocelltypes.Futurestudieswilllookatstandardi-zingthemodelstomakeitusefulforstudyneurologicaldisorderssuchasALS.

Role of Neurotoxic Astrocyte in Neurodegenerative Diseases

Tae-In Kam1,2,3*, Hyunhee Kim1,2,3*, Jinchong Xu1,2,3, Hyejin Park1,2,3, Aanishaa Jhaldiyal1,2,3, Sung Ung Kang1,2,3, Ted M. Dawson1,2,3,4,5, Valina L. Dawson1,2,3,5,6

1Neuroregeneration and Stem Cell Programs, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA2Department of Neurology, Johns Hopkins University School of Medicine, Bal-timore, MD 21205, USA; 3Adrienne Helis Malvin Medical Research Founda-tion, New Orleans, LA 70130-2685, USA; 4Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; 5Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA6Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA

Activation ofmicroglia by classical inflammatorymediators can convert as-trocytestoaneurotoxicA1phenotypeinmurinecultures.AlthoughactivatedmicrogliaandastrocytesexpressingA1markershavebeen identified in thebrain from patients of neurological disorders includingAlzheimer’s disease(AD)andParkinson’sdisease(PD),theirroleispoorlyunderstood.Herewereportthathumanmicroglial-likecellsandastrocytescanbedifferentiatedfromH1humanembryonicstemcells(hES).ThemRNAtranscriptomeanalysisde-monstratesthattheyexpresshighlyspecificmicroglialandastrocytesmarkers,respectively.ThesehES-derivedmicrogliaandastrocyteshavethephenotype,geneexpressionprofileandfunctionalpropertiesofthesecelltypes.HumanES-derivedmicroglial-likecellsareactivatedandsecreteavarietyofcytoki-nesbya-synucleinpreformedfibrils (PFF)andAβoligomers.The treatmentofactivatedmicrogliaconditionedmedium(MCM)containingTNFa,C1qandIL-1atohumanastrocytesinducesconversionofA2astrocytetoA1astrocy-te.Furthermore,A1astrocyte conditionedmedium (ACM) inducedbya-synPFFandAβoligomerscausesthedeathofhumandopaminergicneuronsandcorticalneurons,respectively.Together,hES-derivedmicroglial-likecellsandastrocytescanbeusedtostudythecontributionofneurotoxicA1astrocytesinneurodegenerativedisorders,providingopportunities for thedevelopmentofnewtreatmentsforneurologicaldisorders.

Poster Abstracts:

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High-Throughput Methods for Evaluating Cellular Phenotypes and Neural Activity in Human Induced Pluripotent Stem Cell

(hIPSC)-Derived 3D-Organoids

Madel Durens, Andre W. Phillips, Jonathan E. Nestor, Jason W. Lunden, Omotayo Oduwole, Michael W. Nestor

The Hussman Institute for Autism, Baltimore, MD 21201, USA

The etiology of neurodevelopmental conditions including autism spectrumcondition(ASC)isoftencomplex.GeneticandenvironmentalheterogeneityinindividualswithASChasnotonlymadeitdifficulttostudyunderlyingbiologybuthaslimitedthetranslatabilityofanimalmodels.Humaninducedpluripotentstemcell(iPSC)technologyprovidesaplatformforexaminingpotentialcellu-larphenotypeswhereintheindividualgeneticbackgroundisconserved.Morerecently,thelimitationsoftraditional2-dimensional(2D)culturesinreflectingtissuecomplexityhaveledtoeffortstodevelopmorecomplex3-dimensional(3D) cultures that canmore faithfullymimic aspects of early neurodevelop-ment.However,issueswithvariability,reproducibility,andsufficientstatisticalpowerhavealsorestrictedtheutilityof3Dmodels.Toaddressthis,highcon-tentscreensthatallowforlargersamplesizeswithgreaterefficiencyhavetobeadaptedto3Dmodels.Inthisstudy,weusedthe3Dserumfreeembryoidbody(SFEB)modeloptimizedbyNestoret.al.,(2013),amodelforearlycor-ticogenesis, and applied several high content assays to probe for phenoty-pes relevant toASC.SFEBsweredifferentiated from iPSCsbyplating ina96-wellV-bottomplate for14days,afterwhich theSFEBswere transferredtocellcultureinsertsandgrownuntil60daysinvitro(DIV).First,weuseda48-wellmulti-electrodearray(MEA)tomeasurespontaneouslocalfieldpoten-tials, as well as responses to various pharmacological agents. We also de-monstratedsynapticplasticityintheSFEBsbychemicallyinducinglong-termpotentiation.TovisualizeneuronalactivityinindividualcellswetransducedtheSFEBswithAAV9-GCamp6,andimagedcalciumactivityafteroneweek.Weimaged and analyzed calcium signaling using theThermoFisherArrayScanXTItoevaluatetheapplicabilityofthismethodtohigh-contentanalysis.Next,weevaluatedneuronalmorphologybycouplingstandardimmunolabelingwithMap2andanalyzingtheimagesusingtheneuritedetectionprotocolontheAr-rayScan.Lastly,becauseexcitatory/inhibitoryimbalancehasemergedasonepossiblehypothesisthatunderliesASCneurobiology,weevaluatedtheratioof γ-aminobutyric acid (GABA)ergic andglutamatergic neuronal populationswithintheSFEBsbyimmunolabelingwithGABAandtheglutamatetranspor-terVGLUT.WeestablishherethatthesecellpopulationscanbeanalyzedinSFEBsusingahighcontentscreen.Altogether,weaimtodevelopaplatformtoestablishneuronalphenotypesin3Dcultures,andusethesemethodologiesforphenotypicdrugscreening.

Combinatorial Polymer Matrices Enhance In Vitro Maturation Of Human Induced Pluripotent

Stem Cell-Derived Cardiomyocytes

Young Wook Chun1 , and Charles C. Hong1,2

Division of Cardiovascular Medicine, University of Maryland School of Medicine, Baltimore, MD

Cardiomyocytes derived from human induced pluripotent stem cells (iPSC-CMs)holdgreatpromiseformodelinghumanheartdiseases.However,iPSC-CMsstudied todate resemble immatureembryonicmyocytesand thereforedonotadequatelyrecapitulatenativeadultcardiomyocytephenotypes.Sinceextracellularmatrixplaysanessentialroleinheartdevelopmentandmatura-tioninvivo,wesoughttodevelopasyntheticculturematrixthatcouldenhancefunctionalmaturationofiPSC-CMsinvitro.Inthisstudy,weemployedalibra-ryofcombinatorialpolymerscomprisingofthreefunctionalsubunits-poly-ε-caprolacton(PCL),polyethyleneglycol(PEG),andcarboxylatedPCL(cPCL)-assyntheticsubstratesforculturinghumaniPSC-CMs.Ofthese,iPSC-CMsculturedon4%PEG-96%PCL(each%indicatesthecorrespondingmolarratio)exhibit thegreatestcontractilityandmitochondrial function.These functionalenhancements are associatedwith increased expression of cardiacmyosinlightchain-2v,cardiactroponinIandintegrinalpha-7.Importantly,iPSC-CMsculturedon4%PEG-96%PCLdemonstratetroponinI(TnI)isoformswitchfromthefetalslowskeletalTnI(ssTnI)tothepostnatalcardiacTnI(cTnI),thefirstre-portofsuchtransitioninvitro.Finally,culturingiPSC-CMson4%PEG-96%PCLalso significantly increased expression of genes encoding intermediate fila-mentsknowntotransduceintegrin-mediatedmechanicalsignalstothemyo-filaments.Insummary,ourstudydemonstratesthatsyntheticculturematricesengineered from combinatorial polymers can be utilized to promote in vitromaturationofhumaniPSC-CMsthroughtheengagementofcriticalmatrix-in-tegrin interactions.

Innovative Applications of 3D Tissue Engineering Technology

Ha Nam Nguyen, PhD ; 3Dnamics, Inc. Baltimore, MD, USA

Thequesttofindnoveltherapeuticsforhumandisordershasbeenhinderedbythelackofaccesstorelevantexperimentalmodels.Conventionalmonolayercellcultureandanimalmodelsoftendonotfullyrecapitulategenuinediseasephenotypesandarepoorpredictorsofhowdrugswillworkinhumans.Thus,majorityofpreclinicaldrugtestingdonottranslatetoclinicalsuccessandhaveahighfailurerate.However,recentprogressin3Dtissueengineeringtechno-logyoffersapromisingplatformthatmaybethekeyinreversingcurrentdrugdevelopmentaltrendandbridgingthegapbetweenconventionalsystemsandhumans.These3D tissues,derived frompluripotentstemcells (PSCs),canmimicarchitecturecomposition,geneticprofile,andphysiologyofthoseorgansfound in vivo.Such3Dtissuesprovideuniquetoolsforstudyingorganogene-sis,generationofclinicallyrelevantcellororganoidtypes,anddevelopmentofnewdiseasemodelsandpharmacotherapy.Here,weshowthatPSC-derived3Dbraintissuescanbeusedtomodelneurologicaldisorders,includingPar-kinson’sdiseaseandAlzheimer’sdisease,andtoperformdrug/toxicityscreen.



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GMP-Compliant Non-Viral CRISPR-Mediated Mutation Correction in Patient CD34+ Cells with Sickle Cell Disease

(SCD) Achieves 80% Wild Type Adult Hemoglobin Expression in Differentiated Erythrocytes

Linhong Li1* , Naoya Uchida2*, Juan Haro Mora2, Selami Demirci2, Lydia Raines2, Cornell Allen1, Suk See De Ravin3, Harry L Malech3, Madhusudan V. Peshwa1, John F Tisdale2

1MaxCyte, Inc., Gaithersburg, MD; 2Sickle Cell Branch, NHLBI, NIH, Bethesda, MD; and 3Laboratory of Clinical Immunology and Microbiology, NIAID, NIH, Bethesda, MD.*Equal contribution

The c.20A>T mutation of β-globin gene causes sickle cell disease (SCD).Allogeneichematopoieticstemcell(HSC)transplantationcancureSCD,butmost lackasuitabledonor.Exvivogene therapystrategies, including lenti-viralmediatedgenetransferorendonucleasemediatedBCL11aknockdownallowingfetalhemoglobin(Hb)induction,arecurrentlyunderevaluation.Cor-rection of the SCDmutation by non-viral gene editing of autologousHSCswould addan alternative strategy andpermit endogenous geneexpressionat itsnatural regulatory locusand thebeneficial reductionof thepathogenicsickleHbproduction.Allogeneictransplantationhasestablishedthatthethera-peutic threshold forclinicalbenefit is≥20%donorchimerism.Wepreviouslyreportedefficientcorrectionofamonogenic “hotspot”mutation in theCYBBgeneinX-linkedchronicgranulomatousdisease(CGD)patientHSCswitharobust, scalable, cGMP, and regulatory compliant process (Sci Transl Med2017)thatwenowapplytoSCD.IninitialstudiesusingaBcellline(B-LCL)createdfromSCDpatientandhealthyvolunteers’CD34+HSCs,wedevelo-pedaSCDmutationspecificguideRNA,andanormalβ-globinspecificguideRNA(converse).TheconverseguidedifferedbyonlyonenucleotidefromtheSCDmutationspecificguide,whereeachguidecouldbeusedtogetherwithasinglestrandedDNAdonortoeffectivelyalterthewildtypetoSCDandtheSCDtowildtype,respectively(ASGCT2017).Atfirst,weoptimizedhomologydirectedrepair(HDR)attheSCDlocusbyintegratingaHindIIIenzymesite.Weobservedefficientsite-specificinsertionoftheHindIII-markerintheB-LCLasevidencedbyHindIIIdigestionofthePCRproducts(~50%),andtargetedsequencing(~35%HDRand~50%Indel).TheoptimizedprocesswasappliedtocorrectSCDCD34+HSCstoachievesimilarbiallelicHDRratesforHindIIIsiteinsertionaswellasgenecorrectionfromtheSCDmutationtothenormalβ-globinsequence(upto~35%correctionand~50%Indel).Interestingly,thiscorrectionwasmaintained during erythroid differentiation in culture.AmongerythrocytesdifferentiatedfromcorrectedSCDCD34+cellsinvitro,wildtypeadultHbprotein levelswereabove60%asassayedbybothreversephaseHPLCandHbelectrophoresis,andsickleHbproductiondecreasedfrom100%to20%aftercorrection.Insummary,basedontheseinvitrocorrectionratesconfirmedbytargetedsequencing,wildtypeadultHbproteinexpression,andsubstantiallydecreasedsickleHbamounts,wearestartingtoevaluateengraft-mentofcorrectedSCDpatientHSCsinimmunodeficientmice.ThehighrateofengraftmentinimmunodeficientmiceofsimilarlycorrectedHSCsobservedinourpublishedCGDstudyputstheseresultsobservedforinvitrocorrectionofSCDwithinthetherapeuticwindowofreversingSCD.

Integrating Motion Correction into CEST MRI pH Imaging

KowsalyaDevi Pavuluri* , Changlin Li*, Michael T. McMahon*#

*Russell H. Morgan Department of Radiology and Radiological Science, Johns Hopkins University School of Medicine; #F. M. Kirby Research Center for Functional Brain Imaging, Kennedy Krieger Research Institute, Baltimore, Maryland 21205, United States

Introduction: Chemical exchange saturation transfer (CEST) imaging is ahighlyversatileMRIcontrastmechanismwellsuitedtoperformingpHimaging.ToproducepHimagessuitable formonitoringstemcell transplants,anMRIacquisitionschemeisusedtocollectaseriesofsaturation transfer imagesoverseveralminutesonexogenousCESTagentsdispersedinhydrogelssup-portingthestemcellgraftsinlivingsubjects.Breathingrelatedimageartifactsarepresent in theresulting imageswhichmustbetaken intoaccount in thepost-processing.Inthisstudy,weidentifyseveralpromisingimageregistrationmethods,integratetheseintoourCESTpost-processingschemesandevalua-tetheirperformanceinmice.Mesenchymalstemcells(MSCs)naturallydiffe-rentiateintoconnectivetissueandarethusattractiveforfacilitatingtherepairofintervertebraldiscsintheback.DetermininghowlongtheengraftedMSCssur-viveisimportanttodetermineiftheproceduresandscaffoldmaterialsshouldbeamended.InourpreviousstudieswehavedevelopedamethodtomonitortransplantedcellsurvivalusingCESTMRIpHimaging.Oneofthechallengesisthattheimageacquisitiontimesarerelativelylong(severalminutes)com-paredtobreathingrates,whichresultsinartifacts,andasaresultstrategiesare requiredwhich takemotion into account during image post-processing.Therespirationratemayfluctuateduringasingle imagingsession,wehaveobserved thisvarying from30 times/min toeven60 times/min formice.Wehavetestedthreeoptionsformotioncorrection:(a)Normalizedmutual infor-mation(NMI)basedrigidregistration;(b)NormalizedCrossCorrelationbasedrigidregistration;andthe(c)Demonsalgorithmbasednon-rigidregistration.Results: Live animal experiments consisted of administering iopamidol at aweightcontrolleddoseof1.5g/kgandacquiredsaturationtransferdataonasingleslicecontainingthecenterofbothkidneys.Weacquirefourimagespriortoadministrationtoallowforagoodreferenceimage,withtheamountofcon-trastinthekidneysbuildingupover30minutesandfairlyuniformacrossbothkidneys,which isnice for testingourmotioncorrections.Aimingtomeasurethesimilaritybetweenthereferenceimageandafter-registration,weusecrosscorrelationas thesimilarity index.Correlationcoefficientdirectly reflects thestructuralsimilaritybetweentwoimagesbutdoesn’ttakeintensityintoaccount,whichisrequiredinthiscasebecausetheintensitycanchangebasedoninflowofcontrastmaterial.TheNMI,NCCanddemonsalgorithmswere testedonimageswith48x48resolutionwithcorrelationcoefficientsof0.9800.982and0.990 respectively.Basedon this,weconcluded that thedemonsalgorithmworkedthebest.Wethencomparedthisalgorithmusingboththeoriginal48x48resolutionandaninterpolationbased256x256resolutionwithcorrelationcoefficientsof0.990and0.997respectively.Visual inspectionalso indicatesobvious improvement in theresultingaligned imageseries.WehavefurtherevaluatedthisbydrawinganROIenclosingasinglekidney,andcomparingthechangeincontrastwithtimeforthisROIbothpre-andpost-registration.Theresultingcurvesdisplayedminimaldifferences incontrastbuildupwith time,furtherindicatingthesuitabilityofthismethod.Insummary,basedonthesefin-dingsthedemonsnon-rigidregistrationalgorithmcanreadilybeintegratedintoCESTpost-processingschemesandissuitableforcorrectingmotionartifacts.Acknowledgement:This study is funded by MD Stem Cell Research Fund.

Poster Abstracts:

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Mechanical Characterization of Engineered Cardiac Tissues for Clinical Application

Rachana Mishra, University of Maryland, Baltimore, MD

Background:Hypoplasticleftheartsyndrome(HLHS)patientshavethehighestmorbidityandmortalityratesofallcongenitalheartdisease(CHD)patientsinspiteofsignificantmedicalandsurgicaladvances.ThehighHLHSmortalityisduetopressure/volumeoverloadofthesinglerightventricle(RV)leadingtoitsdysfunctionandfailureevenaftersurgerypalliation.WehavealreadyshowedthatadministrationofMSCsinneonatalswinemodelofRVpressureoverload.ImprovedtheRVfunctionsbutthemechanismunderlyingthebeneficialeffectsofMSCsonRVfunctionisunclear.Hypothesis:WehypothesizethatinjectedMSCsimprovesRVfunctionbysecretingexosomesenrichedwithgrowthdiffe-rentiationfactor15(GDF15)andmiR-132,whichsynergisticallyactivateano-velGDF15-miR-132-Smad2/3signalingpathwayincardiomyocytesthisleadstopositivecardiacremodelingresponse.Methods:ThisstudywasperformedusingprotocolsthatwerereviewedandapprovedbyourInstitutionalAnimalCareandUseCommitteeandfollowingthe1996GuidefortheCareandUseof LaboratoryAnimals. ImmunosuppressedYorkshire swine (6–9 kg, 14–21daysoflife)underwentpulmonaryartery(PA)bandingfollowedbyinjectionofMSCsorplaceboat30minpostbanding.Echocardiographywasperformedtoassessstructuralandfunctionalchangesatbaseline,postbandingat4wk.StandardRNAinterferencetechnologytoKDGDF15,miR-132andmiR-21ex-pression.ResultsandConclusion:Wehavealreadyshown that fourweeksafterPAbandinginswine,end-systolicandend-diastolicareaswerepreservedintheMSC-injectedgroupcomparedtobaselinewhiletheplacebogroupde-monstratedsignificantpathologicRVdilation(P<0.01),Similarly,RVfunctionwaspreservedintheMSC-treatedgroup(P<0.01).HerewearereportinganovelfindingthatinjectedMSCsleadtopositiveremodelinginpressureover-loadedswinemyocardiumbydecreasingthelevelsofcellularmiR-21andre-leasingexosomesenrichedinGDF15andmiR-132.IdentificationofupstreamregulatorsanddownstreameffectorsoftheGDF15-miR132-Smad2/3pathwaywillbecriticalfortheclinicalsuccessofthistherapy.

Transplanted Volar Fibroblasts to Nonvolar Skin Induce Ectopic Volar Skin

Sooah Kim*, Dongwon Kim*, Seakwoo Lee, Vicky Prizmic, Benjamin Evans, Luis A. Garza

Dept. of Dermatology, Johns Hopkins School of Medicine, Baltimore, MD Amputeeshaveskinproblemsatstumpsitesduetothelong-termuseofpro-sthetics.The conversionof thin skinat stumpsites topalmo-plantar (volar)typeskinwith friction, irritationandpressureresistancecanbeonesolutionfortheseskinproblems.Inpreviousstudy,wedemonstratedthatKERATIN9(KRT9)isthemostuniquelyexpressedgeneinvolarepidermiscomparedtononvolaranditsexpressioncanbeinducedinnonvolarkeratinocytesbyvolarfibroblasts.Asapreclinicalstudy,inthisstudy,wetransplantedmousevolarfibroblastsintonon-volarskinareatoinvestigatetheconversionofskiniden-

titybyevaluatingKrt9expression.DonorvolarfibroblastswereisolatedfrompawofLuciferasepositive/GFPpositive(LUC-eGFP)transgenicmiceandtheninjected intorecipientnonvolarearsof immunodeficientmice(NSG).Lucife-raseactivitiesofdonorinjectedfibroblastswerequantifiedusingIVISLuminaIIimagingsystem.Finally,therecipientearswerecollectedandanalyzedbyqRT-PCRandimmunostainingtoevaluateKrt9expression8weeksaftertrans-plantation.Theluciferasesignalofonemillionvolarfibroblastcellssignificantlydecreasedduringthefirst2weeksaftertransplantationsuchthatonly10%ofinjectedfibroblastsurvivedformoreat2monthspost-injection.Forpreventingthisdecay,weattempted todividedosingof thesame totalamountofcellsseparatedintomultipleinjectionsandinjectsingleinjection.Theluciferaseac-tivitiesoffibroblastdidnotshowasimilardecayinmultipleinjectionandsingleinjection.Finally,wecollected tissues from these treatmentsand found thatKrt9expressionlevelandepidermalthicknessincreased4.8-foldand1.8-fold,respectively,inearsinjectedwithvolarfibroblastscomparetovehicle(n=3andp<0.05).Thesefindingsdemonstratethatmultipleinjectionscouldimprovethesurvival and the regulationof injected site-specific fibroblasts for convertingskinidentity.Therefore,wesuggestthattheconversionofskinidentitywiththeinjectionofvolarfibroblasts to thestumpsiteofamputeesmay improve theprostheticuseandthequalityoflifeforamputees.Furthermore,weexpectourresearchwillcontributetodevelopbothbasicandtranslationalstudytoadvan-cetheknowledgeandtechnologyinthestemcelltherapy.Funding: Maryland Stem Cell Research Fund awarded to Dongwon Kim.

A Self-Healing Hydrogel as An Injectable Instructive Carrier for Cellular Morphogenesis

Zhao Wei, Sharon Gerecht

Department of Chemical and Biomolecular Engineering and Materials Sciences and Engineering, Johns Hopkins University, Baltimore, MD

Transplantationofprogenitorcellscanacceleratetissuehealingandregene-rativeprocesses.Nonetheless,directcelldeliveryfails tosupportsurvivaloftransplantedcellsorlong-termtreatmentofvascularrelateddiseasesduetocompromised vasculature and tissue conditions. Using injectable hydrogelsthatcross-linkinsitu,couldprotectcellsinvivo,buttheirsol-geltransitionistime-dependent and difficult to precisely control. Hydrogels with selfhealingpropertiesareproposedtoaddresstheselimitations,yetcurrentself-healinghydrogelslackbio-functionality,hinderingthemorphogenesisofdeliveredcellsintoa tissuestructure.Hereweestablishagelatin (Gtn)-basedself-healinghydrogelcross-linkedbyoxidizeddextran(Odex)asan injectablecarrier fordeliveryofendothelialprogenitors.Thedynamiciminecross-linksbetweenGtnandOdexconfertheself-healingabilitytotheGtn-l-Odexhydrogelsfollowingsyringeinjection.Theself-healingGtn-l-Odexnotonlyprotectstheprogenitorsfrominjectedshearforcebutitalsoallowscontrollablespatial/temporalplace-mentofthecells.Moreover,owingtothecell-adhesiveandproteolyticsitesofGtn, theGtn-l-Odex hydrogels support complex vascular network formationfromtheendothelialprogenitors,bothinvitroandinvivo.Thisisthefirstreportofinjectable,self-healinghydrogelswithbiologicalpropertiespromotingvascu-larmorphogenesis,whichholdsgreatpromiseforacceleratingthesuccessofregenerative therapies.



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Currently,generationofinducedOPCs(iOPCs)fromhiPSCsandtheirstepwi-sedifferentiation intoOLsare time-consuming (75-120days)and inefficient(20-75%)andgeneratingheterogeneouscells(oligodendrocyteandastrocyte)andtumorigenicpotential(hostgenomemodificationsbyusinglentiviralcons-tructs), and thus have limitations for regenerativemedicine.Here,we haveestablishedandvalidatedarobustandefficientprotocolthatgeneratesexpan-dableiOPCsfromhiPSCswithin10daysusinginnovativetranscriptionfactorscombinedwith theneuralspecificationsignals.Moreover,withsomemodifi-cations,thisprotocolcanbeusedtoconverthumanfibroblastsintoOPC-likecells.Wedemonstratethatcombinationoftranscriptionfactorscocktail(Sox10,Olig2 andASCL1) and smallmolecular cocktail induces 80-90%PDGFR+cellswithin10days.Thesecellsexhibittypicalsmallbi-polarortri-polarOPC-li-kemorphologies,maintaintheirself-renewalcapacityandexpressOPC-spe-cificmarkersNG2,PDGFRα,Sox10andO4.Moreover,theseiOPCsmaintainhighproliferationasshownby60%Ki67+positive.Undermaturedifferentiationcondition,theseiOPCsbecamemultipolarO4+cells,andmyelinbasicproteinMBP+matureoligodendrocyteswithcomplexbranchesforming.Ourprotocoloffersauniqueapproachtocombinetranscriptionfactorcocktailandsmallmo-leculecocktailtogenerateiOPCsfromhiPSCsandhumanfibroblastswithoutgeneticmanipulationinaclinicallyapplicabletimeframe.

Activation of the NLRP3 Inflammasome In Human Dopamine Neurons As A Consequence of Parkin Dysfunction

Nikhil Panicker , Manoj Kumar, Tae-in Kam, Sung Ung Kang, Valina Dawson, Ted M. Dawson

Parkinson’sdisease(PD)isachronic,progressive,neurodegenerativedisor-der that affectsoveramillionpeoplewithin theU.S.Underlying the clinicalsymptomsofPDisthedegenerationofdopamine(DA)neuronslocatedintheparscompactaof thesubstantianigra(SNpc). Inhibitionof theE-3ubiquitinligaseparkiniscentraltofamilialaswellassporadicPDprogression.OneofthemajorcriticismsofPDresearchisthatdespitetheplethoraofstudiesusingrodent or cell culture models, pathologically relevant drug targets have not beenhitherto identifiedbecausetheunderlyingphysiologyof thesesystemsdifferssubstantiallyfromthatofhumanDAneurons.Toaddressthisissue,wegeneratedhumanembryonicstemcell(hES)differentiatedDAneurons(hDAneurons).WealsodevelopedtwoParkinknockouthESlines,allowingustoobtainParkinknockouthDAneurons.PreliminaryresultsfromtheParkinKOhDAneurons revealedupregulationof thecytosolicstress receptorNACHT,LRR,andPYDdomains-containingprotein3(NLRP3),andCaspase-1activa-tion,suggestingthatlossofParkinlicensesNLRP3inflammasomeactivationinhumanDAneurons.FurtherstudiesindicatethatparkininteractswithandpolyubiquitinatesNLRP3,taggingit forproteasomaldegradation.Finally,Im-munohistochemistryanalysisofPDpatientandage-matchedcontrolventralmidbrainbrainsectionsshowsNLRP3upregulationandCaspase-1activationwithin tyrosine hydroxylase (TH) positiveDA neurons under PD conditions.TheseexperimentssuggestthatunconventionalneuronalNLRP3inflammaso-meactivationmayoccurfollowingparkindysfunction.FutureworkwillexploretheconsequencesofneuronalNLRP3inflammasomehyperactivationincon-tributingtoPD-associatedneurodegeneration.

Immunosuppression Increases Regenerative Potential of Adult Rat C-KIT+ Cardiac Progenitor Cells In Allogeneic

Rat Myocardial Infarction Model

Muthukumar Gunasekaran , Rachana Mishra, Sudhish Sharma, Progyaparamita Saha, Mir Yasir Arfat, Chetan Ambastha, David Morales, Gregory J Bittle, Deqiang Li, Sunjay Kaushal

University of Maryland, Baltimore, MD

Rationale:Cardiacprogenitorcells(CPCs,c-kit+)arewell-characterizedstemcelltypeshowntopotentiatecardiacregenerationinmyocardialinfarction(MI)inadultsandpre-clinicalmodels.However,theinnateimmuneresponsedirec-tedtotransplantedadultCPCs(aCPCs)reducescellretentionandMIrecove-ry.WeaimedtoincreasetheregenerativepotentialofaCPCsbyreducingtheimmune responsedirected to the transplantedaCPCsusing cyclosporineA(CSA).Methods:AdultCPCs(aCPCs)wereisolatedfromWisterKyotoratwastransplanted(1X106)toBrownNorwayMIrats.ImmunesuppressantCSAwereadministeredorallytoMIratswithrCPCstransplantationgroup(aCPCs+CSA).CSAaloneandIscove'sModifiedDulbecco'sMedia(IMDM)servedascontrolgroups.EchocardiogramwasperformedtodetermineMIrecoveryonday1,7and28.ThetransplantedrCPCscellretentionwasdetectedusingGFPex-pressingrCPCsonhearttissuebyimmune-histochemistry(IHC).TherateofapoptoticcelldeathoninjuredmyocardiumwasmeasuredusingTUNNELas-say.TheinflammatoryCD-68+cellsweremeasuredbyIHC.Seracollectedonday2,7,14,21and28wereutilizedtomeasuretheinflammatory(IL-2,IL-17,IFN-γ,TGF-β),anti-inflammatory(IL-10)cytokinesandantibodiesagainstcar-diacself-antigens(SAgs)Troponin-TandMyosin.Results:MIratstransplantedwithaCPCs+CSAdemonstratedsignificantincreaseinMIrecoveryandcellre-tentioncomparedtoMIratswithaCPCs,CSAandIMDMcontrols(p<0.05).MIratstransplantedwithaCPCsshowedincreasedinfiltrationofCD-68+cellsandapoptoticcells intheinfarctedmyocardiumcomparedtoaCPCs+CSAgroup(p<0.05).Seracollectedonday2and7ofaCPCsgroupshowedincreasedinflammatorycytokines,antibodiestocardiacSAgsandreducedanti-inflam-matorycytokines.IncontrastMIratstransplantedwithaCPCs+CSAshowedreducedinflammatorycytokinesandincreasedanti-inflammatorycytokineIL-10(p<0.05).Conclusions:Inconclusion,MIratstransplantedwithallogeneicaCPCs+CSAshowedincreasedcellretention,reducedinflammatorycellsandcytokinescompared toaCPCs,CSAand IMDMcontrol.Therefore,CSA re-duces inflammationand increases the retentionof transplantedaCPCs thatresulted in increased myocardial recovery.

Rapid and Efficient Generation of Oligodendrocyte Precursor Cells

Chengchen Hu1,3, Kimberly Wang1,3 , Qingfu Xu1,2,3, Damon Ceylan and Yunqing Li1,3

1Hugo W. Moser Research Institute at Kennedy Krieger, 707 N. Broadway, Baltimore, MD, USA 21205; Department of 3Neurology, The Johns Hopkins University School of Medicine, 600 N. Wolfe Street, Baltimore, MD, USA 21287

Poster Abstracts:

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Divergent Stem Cell Function Between Skeletal and Soft Tissue CD146+ Human Pericytes

Jiajia Xu1, Dongqing Li1, 2, Yiyun Wang1, Leslie Chang1, Carolyn A. Meyers1, Bianca Vezzani2, Mario Gomez Salazar2, Yongxing Gao1, Kristen Broderick1, Carol Morris1, Bruno Peault2,3, Aaron W. James1

1Johns Hopkins University, MD, 2University of Edinburgh, UK, 3University of California, Los Angeles, CA,

INTRODUCTION: Human pericytes demonstrate multilineage differentiationpotential, and their descendants participate in tissue homeostasis and repair. However, increasingevidence fromdevelopmentalbiologysuggests that re-gionalspecificationby tissueoforiginexistsamonghumanpericytes. Inte-restingly,anddespitetheirheterologousorigin,humanadipose-tissuederivedpericyteshavebeenusedwithsuccesstoregeneratebonebyourresearchgroupacrosspre-clinicalmodels. Clearlysomeplasticityexists in therepa-rativephenotypeofhumanpericytes. Insum,understandinghowpericytesregenerate their tissuemicroenvironmentwith such remarkable fidelity is ofcentral importanceforcell-basedeffortsinregenerativemedicine.Here,wesoughttodefineindetailthedifferentiationpotentialandplasticityofCD146+humanpericytesfromskeletalandsofttissuesources.METHODS:Uncultu-redCD146+CD31-CD45-pericyteswerederivedbyfluorescentactivatedcellsorting(FACS)fromhumanperiosteum,adipose,ordermaltissue(N=3patientsamplespertissuetype).Canonicalfeaturesofpericyteswereassayed,inclu-dingcharacteristicpericytemarkerexpression(qRT-PCR,flowcytometry,andimmunocytochemistry),multilineageinvitrodifferentiationpotential(osteoge-nesis,adipogenesis,chondrogenesis),andparacrine-inducedtubulogenesis.Next,quantitativemetricsofinvitroosteogenicandadipogenicdifferentiationwere assessed across human tissues of origin. Intramuscular implantationstudiesassessedectopicboneformationacrosshumanpericytesources.Thetranscriptomeofhumanperiosteum,adipose,ordermalpericyteswasasses-sedbyClariomDmicroarray.RESULTS:PericyteswerefirstvisualizedwithinhumanperiosteumasaCD146expressingperivascularcellpopulation(Fig.1A).UnculturedhumanperiostealpericyteswereisolatedasaCD146+CD31-CD45-cellfraction.FrequencyofCD146+pericyteswithintheperiosteumwas8.17%(7.05%SD),similartothefrequenciesofCD146+pericyteswithinsofttissue.Next,thecomparativeabilityofpericytestodifferentiatedownosteoge-nic and adipogenic lineages was assessed. Periosteal pericytes demonstrated astrikingtendencytoundergoosteoblastogenesis,whilesofttissuepericytesdid not. In contrast, adipose pericytes demonstrated a tendency to under-goadipogenesis. Intramuscular implantsagainrecapitulatedtissueoforigin,withonlyperiostealpericytesdemonstratingneo-osteoblastogenesis.Micro-arrayanalysisbyprincipalcomponentanalysisandunsupervisedhierarchicalclusteringdemonstratedcleardifferencesbyanatomicorigin,andanenrich-mentamongperiostealpericytesinCXCR4signaling.DISCUSSION:Insum,skeletalandsoft tissuepericytesdiffer in their relative lineagedifferentiationpotentialandabilitytoformbone.Plasticityexists,however,andmanipulationofkeysignalingpathwaysmay‘coax’asofttissuepericytetowardanosteo-blastogeniccellfate.OngoingstudiesarefocusingonmanipulationofCXCR4signalinginorderto‘rescue’theosteogenicpotentialofsofttissuepericytes.

Location- And Dose-Dependent Attenuation of Experimental Autoimmune Encephalomyelitis Using

Glial-Restricted Progenitors

Aline M. Thomas1, Shen Li1, Chengyan Chu1, Irina Shats1, Jiadi Xu2, Peter Calabresi3, Peter van Zijl1,2, Piotr Walczak1,2, and Jeff W.M. Bulte1,2

1Department of Radiology and Radiological Science and Cellular Imaging Section, Institute for Cell Engineering, Johns Hopkins University School of Medicine; 2 F.M. Kirby Center for functional brain imaging, Kennedy Krieger Institute Kirby Research Center; 3Department of Neurology, Johns Hopkins University School of Medicine

Introduction:Gliastore lactateat imageable levels tosupplyneurons,whichmaybeapotential target forhaltingMSprogression.Weexplored the regi-ons of the brain benefited by glial transplantation in a primary progressiveexperimentalautoimmuneencephalomyelitis(EAE)animalmodelformultiplesclerosisMSusinglactate-sensitiveMRI.Methods:Luciferase+glial-restrictedprogenitors(GRPs)weretransplantedintonaïveorEAE-inducedmiceatdiffe-rentdosesintothemotorcortexorlateralventricleusingastereotaxicdevice.Lactatewasmonitoredusing1HMRSandon-resonancevariabledelaymul-tiplepulsechemicalexchangesaturationtransfer(onVDMPCEST)MRI.Cellengraftmentandsurvivalwasconfirmedusingbioluminescent imaging(BLI)and histology. Results:onVDMPCESTand1HMRS(lactate)signalschangedspatiotemporallyinEAEmice.GRPsenhancedonVDMPCESTand1HMRS(lactate)signalsincellculturephantoms.onVDMPCESTand1HMRS(lac-tate)signalswereenhancedinnaïvemiceandinEAEmiceuponGRPtrans-plantationatorproximal to the transplantsite.BLIshowedcellengraftmentvariedbytransplantsitelocation.AttenuationofparalysisbyGRPsinthisEAEmodelforMSwasdependentonboththedoseandtransplantsitelocation.

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Generation and Selection of Pluripotent Stem Cells for Robust Differentiation to Insulin-Secreting Cells Capable

of Reversing Diabetes in Rodents

Sheryl Southard, and William Rust ; Seraxis, Inc.

Inducedpluripotentstemcell(iPSC)technologyenablesthecreationandse-lectionofpluripotentcellswithspecificgenetic traits.Wedevelopedapluri-potentcelllinetoformreplacementpancreaticcellsasatherapyforinsulin-de-pendentdiabetes.Beginningwithprimarypancreatictissueacquiredthroughorgandonation,cellswereisolatedandre-programmedusingnon-integratingvectors.EachoftheresultingiPSClineswasthensubjectedtoadifferentiationprotocoltoguidethecellstotheendodermalandpancreaticfate.Thecandi-datelinethatmostconsistentlygeneratedhighlypurepopulationsofendocri-nepancreasprecursorswasselectedforfurtherdevelopment.ThisapproachcreatedaniPSC-variantcellline,SR1423,withageneticprofilecorrelatedwithpreferentialdifferentiationtowardendodermallineageatthelossofmesoder-malpotential.Animproveddifferentiationprotocol,coupledwithSR1423,ge-neratedpopulationsofgreaterthan60%insulin-expressingcellsthatsecreteinsulininresponsetoglucoseandarecapableofreversingdiabetesinrodents.



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ofglucosylsphingosine(GluSph),aneurotoxicmetaboliteofGluCerandkeybiomarkerofGD.Toinvestigatetheimpactofsphingolipidimbalanceinneuro-naldevelopment,wegeneratedneuralprogenitorcells(NPCs)fromnGDandWTiPSCs.nGDNPCshadelevatedlevelsofGluCerandGluSph.Furtheranalysisshowed thatGCasedeficiencycaused lysosomaldepletion innGDNPCsanddifferentiatedneurons.These lysosomal abnormalitieswerepre-ventedbyincubationwithrecombinantGCase.TodirectlytestthehypothesisthatGluSphisresponsibleforthelysosomalabnormalitiesofnGD,weincuba-tedWTNPCswithexogenousGluSph.ImmunofluorescenceanalysisshowedthattreatmentofWT-NPCswithGluSphcausedwidespreadlysosomaldeple-tionasdeterminedbydecreasedexpressionofthelysosomalmarkersLAMP1andLAMP2,aswellaslysotrackerstaining.Thus,GluSphtreatmentpheno-copiedthelysosomaldepletionobservedinnGDNPCs.Lastly,weexaminedtheeffectsofglucosylceramidesynthaseinhibitorsusedinsubstratereductiontherapy,anFDA-approvedtreatmentfortype1GDthatpreventsthebuild-upofGSLs.ThistreatmentrestoredlysosomalhomeostasisinnGDNPCs.Wearecurrentlyworking to identify themolecularsensorsofsphingolipid imba-lancethatareresponsibleforlysosomaldysfunctionandneurodegeneration,andcandidatemoleculeswillbedescribed.Wearealsousingthissystemtoevaluate the therapeuticefficacyofglucosylceramidesynthase inhibitors forreversingneurodegenerationinnGD.

The DNA Methylation and Regulatory Elements During Chicken Germ Stem Cell Differentiation

Yanghua He, Jiuzhou Song

Department of Animal and Avian Sciences, University of Maryland, College Park, Maryland 20742, USA

Theproductionofgermcells invitrowouldopenimportantnewavenuesforhumanmedicine,butthemechanismsofgermcelldifferentiationarenotwellunderstood.Theaimofourstudywastousechickenasamodeltoexploreits regulatory mechanisms. We reported a comprehensive atlas at the geno-me-wideDNAmethylationlandscapeinchickengermstemcellsandtranscrip-tomicdynamicswasalsopresented.ByuncoveringDNAmethylationpatternsonsinglegenes,somegenesaccuratelymodulatedbyDNAmethylationwerefoundtobeassociatedwithcancers,leukemia,andvirusinfectione.g.AKT1,CCND1,MYC,CTNNB1,andPTEN.Chickenuniquemarkerswerefirstdisco-veredforidentifyingmalegermstemcellscomparedtothestudiesofhumanandmouse.Moreover, the integratedepigeneticmechanismswereexploredduringchickenmalegermcelldifferentiation,whichprovidesevidencetoun-derstandtheepigeneticprocessesassociatedwithmalegermcelldifferentia-tioninanimalsandhumans.Collectively,ourcombinedfindingsdemonstratethatgermcelldifferentiation isnotonlyaccompaniedbyanorganizedDNAmethylationchangesbutalsoregulatedbymultifacetedfactorsincludingtran-scriptional regulator complexesandhistonemodifications,whicharesimilaracrossspeciesandhelpusunveilthemechanismsinmodulatinggeneexpres-sionduringgermcelldifferentiation.

Nanofiber-Hydrogel Composite Microparticles As A Stem Cell Delivery and Pro-Regenerative

Material for Soft Tissue Defects

C. Chang1, J. Chua2, R. Martin3, K. Colbert3, HQ. Mao2,3

1Department of Biomedical Engineering 2Department of Materials Science and Engineering, 3LifeSprout, Johns Hopkins University, School of Medicine, Baltimore, MD.

Softtissuedefectsfromtrauma,cancer,developmentaldefects,andagingaf-fectsmillionsofpatientsayear,leadingtodisability,chronicpain,andphysicaldefects.Therefore,thereisaneedtoimprovenaturalsofttissueregenerationandtoimprovetheintegrationofnewlygeneratedtissuewiththehostenviron-ment.Currently,fatgraftingandfreetissuetransferaretheprimaryapproachestorepairingsofttissuedefects.However,duetotheinvasiveness,costs,andcomplicationsarising fromsurgicalprocedures, there isaneed foranalter-nativeapproachthatmimicsthenaturalhealingprocessoffat,butalsoiscon-venientandaccessibleforclinicians.Thenanofiber-hydrogelcomposite(NHC)materialusingacombinationofhyaluronicacidandpolycaprolactonenanofi-bershasbeenformulatetomatchthestiffnessofnativesofttissue,whileall-owingforadequateporosityforhosttissueinfiltration.PreviousanimalstudiesusingNHCasabulkgelhasyieldedimprovedhosttissueregenerationandangiogenesisintoinjectedsite.However,asvolumeoftheinjectionincreased,theextentofregeneratedtissuebecamelimited.Therefore,anewformulationofNHC,throughparticulatingthematerialtomicroparticles(~250m)waspro-duced.ThemicroparticleNHCformulationwasabletobeloadedwithhMSCs,while proliferating cellswere able to penetrate the core of the particles.ByincreasingthesurfaceareaofhMSCstointeractwiththemicroparticleNHCs,thehMSC-NHCcombinationcanbeusedasaninjectablematerialtoimprovedrateofsoft-tissueremodeling.

Elevated Levels of Glucosylsphingosine Deregulate Lysosomal Compartment in Neuronopathic Gaucher Disease

M.P. Srikanth1, O. Awad1, R.A. Feldman1

1Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD 21201, USA

Downregulation of β-glucocerebrosidase (GCase) is a frequent event in in-herited and idiopathic neurodegenerative diseases. GCase is encoded byGBA1andcatalyzesthehydrolysisofglucosylceramide(GluCer),thefirststepin the biosynthesis of glycosphingolipids (GSLs). Mutations in enzymes ofsphingolipidmetabolismare the causeof >70 lysosomal storagedisorders.Mostsphingolipidosescauseneurodegeneration, indicating thatsphingolipidbalanceisessentialforneuronalsurvival.Severebi-allelicmutationsinGBA1causefataltypes2and3neuronopathicGaucherdisease(nGD),whilemo-no-allelicGBA1mutationsare thehighest known risk factor forParkinson'sdisease(PD).BrainsfrompatientswithnGDhave1000-foldelevatedlevels

Poster Abstracts:

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Building A Commercial Path for EpiX™ technology: A Breakthrough in Expanding and Utilizing

Tissue-Resident Stem Cells

Chengkang (CK) Zhang1, Anura Shrivastava1, Hermann Bihler2, Jan Harrington2, Isaac Musisi2, Martin Mense2, Scott H. Randell3, Matthew J. Bruehl4, Marianne S. Muhlebach4, Travis J. McQuiston1, Sherry S. Challberg1 and Brian A. Pollok1

1Propagenix Inc., 9605 Medical Center Drive, Suite 325, Rockville, MD 208752Cystic Fibrosis Foundation, 44 Hartwell Avenue, Lexington, MA 024213Marsico Lung Institute, UNC School of Medicine, Chapel Hill, NC 275994Department of Pediatrics, UNC School of Medicine, Chapel Hill, NC 27599

WiththesupportofMSCRFcommercializationgrant,wesuccessfullybuiltthecommercialpathfortheEpiX™technology–abreakthroughinculturingtis-sue-residentepithelialstemandprogenitorcellsfromdiversetissuesforstudy-ingepithelialcellbiologyinvitroandunleashingtheirpotentialforregenerativecelltherapy.Inthisproject,ourworkaimedat1)illustratingtheunparalleledcompetitiveadvantageofEpiX technologyoverconventionalmethodsusinghumanairwayepithelial cells asanexample; 2) demonstrating theutility ofEpiX technology forpersonalizedmedicineapplicationsbyexpandingnasalepithelialcellsoutofminimal-invasivenasalbrushingsamplesobtainedfromcysticfibrosispatients;and3)scalingupEpiXmediumproductionfrombenchscale to lotsizeof100 liters, toprovidesupport formovingforwardwith fullcommercialization.Summary:InAIM1,weverifiedthattheEpiXmediumsup-portedtheexpansionofprimaryairwayepithelialcellsfromtwodifferentdo-norsforover50populationdoublings,whileretainingconsistentfunctionalityasassessedbyvariousmolecularandcellularcharacterizationassaysbytheendof1Q2018.Thisisasignificantimprovementovercurrentpractice,andallowsresearcherstogeneratebillion-foldmoreairwayepithelialcellsfortheirresearch study thanusing the conventionmethods. InAIM2,we testedapersonalizedmedicineapproachforcysticfibrosispatientsbyusingpatient-de-rivednasalepithelialcellsforfunctionalinvitrodrugresponseevaluationas-say.Wesuccessfullygenerated50–100millionsofnasalepithelialcellsusingbrushingsamplesfrom7adolescentCFpatients(4malesand3females,7/7,100%success),andtestedtheirresponsestoIvacaftorandLumacaftorusingtheUssingchamberassay,a“goldstandard”drugefficacyevaluationassayforCFTR-directedtherapeutics.TheresultsrevealeddistinctresponsestowardsthesamedrugdespitethatthesepatientssharethesameCFTRmutation.InAIM3,wehavesuccessfullyestablishedtheproductionSOPsandQCspeci-ficationsfortheEpiXmedium,includinganalyticalandfunctionalqualitytestsand specifications suchas standardpH, sterility, osmolality, endotoxin levelandsupportofcellgrowthanddifferentiation.Sofar,wehavemanufactured4lotsofEpiXmediumatascaleof100litersanddeliveredhigh-quality,reprodu-cibleproductsformanyresearchersworkingwithairwayepithelialcells,inclu-dinginternationalcustomers.LicenseofEpiXtechnology:STEMCELLTechno-logies,aleaderofresearchtoolcompanydedicatedinsupportingthestemcellresearchcommunity,hassignedanexclusivelicenseagreementwithPropa-genixtocommercializeEpiXtechnologyandbringthisrevolutionaryproducttotheresearchcommunitythroughitsdistributionnetwork.Grant-relatedpubli-cations:Oneposterpresentedat2018ATSconference,andonemanuscriptsubmittedtoCellReports(inrevision)Patents:ThreeUSpatents(9,790,471,9,963,680and10,066,201)ontheEpiX™technologyhavebeengranted.

NF-KB p50 Deficient Immature Myeloid Cells (p50-IMC) As A Novel Cancer Immunotherapy

Rahul Suresh, Theresa Barberi, David J. Barakat, Kenneth J. Pienta, and Alan D. Friedman

Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, MD, USA

Multiple solid tumors, including melanoma, fibrosarcoma, colon, prostate,andpancreaticcarcinoma,glioblastoma,andneuroblastomaexhibitimpairedgrowthinp50-/-micelackingtheinhibitoryNF-kBp50subunit,associatedwithactivated tumor-associatedmacrophagesandTcells. Wehypothesize thattheseeffectsresult fromintrinsicactivationof tumormyeloidcells,withcon-sequenttumor-infiltratingTcellactivation,andenvisionacell-basedtherapywhereinpatient-derivedmyeloidprogenitorsareexpandedwhiletargetingtheirp50allelesandtheninfusedbackintopatients.Tovalidatethistherapeuticap-proach,micewereinoculatedwithHi-Mycprostatecancer(PCa),GL261glio-blastoma (GBM),orK-Ras(G12D)pancreaticductal carcinoma (PDC)cells,thelattertwolinesexpressingexogenousluciferase.Oncetumorgrowthwasevident,micereceivedadoseof5-fluorouracil(5FU)followedfivedayslaterbymyeloidcelltherapy.5FUwasprovidedtoreducebonemarrowproductionofcompetingmyeloidcells,aswellastoreducetumormyeloidcellnumbersandtoreleasetumorneoantigenstofavorTcellactivation.Togenerateimmaturemyeloid cells (IMC) for infusion,marrowcells fromp50-/- orwild-type (WT)micewereexpandedinmediacontainingSCF/TPO/FLandtransferredbrieflytoM-CSF.TheIMCwerethenadministeredintravenouslyevery3-4days(1E7cells/doseforthreedoses).Immatureratherthanmaturecellswereinfusedastheseareexpectedtomoreeffectivelyby-passlungandlivertoreachtumorandlymphnodes.MicebearingPCashowedsignificantlyslowedtumorgrowthafterreceiving5FUfollowedbyp50-IMCcomparedwithmicereceivingWT-IMCor5FUalone (p=0.001),with5-fold reduced tumor volumeonday35.FormiceimplantedwithGBM,3of5manifestedverysmalltumorsonday21,incontrasttothecontrolgroups,and4of10PDCtumorsshrank>10-foldinresponseto5FUfollowedbyp50-IMC.ProgenyofCD45.2+IMCweretrackedinCD45.1+tumor-bearinghosts.p50-IMC-derivedCD11b+myeloidcellswereevident in prostate tumor, draining lymph nodes, spleen, and marrow, with tu-morandnodalF4/80+macrophagesdisplayinganactivatedMHCII+CD11c+phenotype.Inaddition,tumorCD8Tcellnumberswere5-foldincreasedafterp50-IMCcomparedwithWT-IMC,andthesemanifestedanactivatedphenoty-pe,withincreasedinterferonexpressioninresponsetoPMA/ionomycin.Adop-tivecelltransferofp50-IMC,followingadoseof5FU,activatestumormyeloidandTcellstoslowmurinetumorgrowthandpredictsthetherapeuticutilityofhumanp50-IMCagainstmultiplesolidtumors.



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showninbandphotonquantification(n=4).800CWand680LTcorrespondtolabeled gelatin (green) andHA (red), respectively. (d)Correlation of in vivoCESTsignalwithNIRfluorescenceintensityvaluesshowsanexcellentcorre-lationforgelatin(R2=0.94),butnotforHA(R2=0.45,datanotshown).Conclusion:Hydrogeldegradationcanbevisualizedinanon-invasiveandla-bel-freemanner.GelatinwasfoundtobethemajorcontributorofCESTcon-trastandalsothemaindegradationcomponentinthehydrogel.Thisapproachmaybeusedfurthertodevelophydrogelswithoptimalbiodegradationproper-tiesforscaffoldedstemcells.References:[1]MadlCM,etal.NatureMaterials.2017;16:1233.[2]LiangY.etal.Biomaterials2015;42:144.

Development of a 3D Fluorescent Imaging Technique to Observe Blood Vessel Phenotype in

Tissue-Engineered Bone Grafts

Alexandra N. Rindone1,2 , and Warren L. Grayson1,2,3,4

1Dept. of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD; 2Translational Tissue Engineering Center, Johns Hopkins University School of Medicine, Baltimore, MD; 3Dept. of Materials Science and Engineering, Johns Hopkins University, Baltimore, MD.4Institute for NanoBioTechnology, Johns Hopkins University, Baltimore, MD.

Tissueengineeredbonegrafts(TEBGs)havedemonstratedtocompletelyhealcritical-sizeddefectsinsmallanimalmodels,buttheirclinicalsuccessislimitedduetoa lackofunderstandingintothemechanismsofrobustbonehealing.In a physiological environment, specific blood vessel phenotypes – charac-terizedbyCD31/endomucinco-expressionandmorphology–are intimatelyassociatedwithosteoprogenitors,andarenecessaryforproperbonegrowthandremodeling.However, there isminimalknowledgeonhowbloodvesselphenotypecontributestoboneformationinTEBGs.Theobjectiveofthisstudyistodevelopa3Dimagingplatformforevaluatingbloodvesselphenotypeandvessel-osteoprogenitorrelationshipsinTEBGs.Asamplepreparationmethodwasoptimized toobservevesselsandosteoprogenitors in1)nativemurinecalvariaand2)TEBGscontainingpolycaprolactone(PCL)-fibrinscaffoldsandhumanadipose-derivedstemcellsimplantedintocritical-sizedmurinecalva-rialdefects.Briefly,harvestedsampleswerefixed in4%PFA,decalcified in10%EDTA,andtreatedwithchloroformtodissolvethePCLscaffolds.Then,sampleswerestainedwithantibodiesandopticallyclearedwiththiodiethanoltoallowforwhole-mountimaging.Highresolution3Dimagesweregeneratedbytaking2x2or3x3tilescansonalightsheetmicroscope(10xmagnification,~1.3 x1.5 x0.8mm3per tile, 20%x-y tile overlap), andprocessedusingImageJ.Ourresultsdemonstratethatthistechniqueenabledvisualizationofdistinct blood vessel phenotypes – characterized by CD31 and endomucinexpression– inbothnative calvariaand in implantedTEBGs.Furthermore,wewereable tovisualize thedistributionofRunx2-stainedosteoprogenitorsin native calvaria.Futureworkwill focusondevelopingquantitativemetricsforvesselphenotype,andelucidatingthespatiotemporalrelationshipbetweenbloodvesselphenotypesandosteoprogenitorsinTEBGs.Thisworkwillprovi-deabetterunderstandingofthebiologicalmechanismsgoverningrobustboneregeneration,andinformstrategiesfordesigningclinically-successfulTEBGs.

Monitoring the Degradation of Implanted Hydrogels Using CEST MRI

Wei Zhu1,2, Chengyan Chu1,2, Shreyas Kuddannaya1,2, Yue Yuan1,2,Piotr Walczak1,2, Anirudha Singh3,4, Xiaolei Song1,2, Jeff W.M. Bulte1,2,4

1Russell H. Morgan Dept. of Radiology and Radiological Science, Division of MR Research, The Johns Hopkins University School of Medicine, Baltimore, MD, USA; 2Cellular Imaging Section, Institute for Cell Engineering, The Johns Hopkins University School of Medicine, Baltimore, MD, USA; 3Dept. of Urology, The James Buchanan Brady Urological Institute, The Johns Hopkins School of Medicine, Baltimore, MD, USA; 4Dept. of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD, USA.

Introduction:Stemcelltherapyhasgarneredmuchattentionfortreatmentofneurodegeneration.However,theharshmicroenvironmentatthesiteofdama-gedtissueoftenimpairstheretentionandsurvivaloftransplantedstemcells.Scaffoldingcellswithhydrogelsisapromisingstrategytoovercomeinitialcelllossandmanipulatecellfunctionpost-transplantation.Whilescaffoldmaterialsareessential for improvingsurvivalofcellulargrafts,matrixdegradation isamajorrequirementforfunctionalgraftintegrationfromimplantedbiomaterials.[1].Therefore,monitoringthedegradationofhydrogelsisessentialfordevelo-pingsuccessfulscaffoldedstemcelltherapies.WeinvestigatedwhetherCESTMRIcanvisualizeimplantedhydrogelscaffolddegradationinmousebrain.Methods:Acovalentlycross-linkedhydrogelcomposingof20mg/mLthiol-mo-difiedgelatin, 20mg/mL thiol-modifiedhyaluronic acid (HA), and20mg/mLpolyethylene(glycol)diacrylate(PEGDA)(crosslinker)wasprepared.Thera-tioofgelatintoHAwasvariedinanefforttoachievetheoptimalformulationforCESTcontrast.TheamountofPEGDAequals25%ofthetotalvolumeofgelatinandHA.InvitroCESTMRIwascarriedoutat37°C.Forinvivovisu-alization,3μLhydrogelwasinjectedintomousestriatum.CESTMRIwasper-formedusingahorizontalbore11.7TBrukerscannertill42days.TovalidatetheInvivoCESTMRIfindingsandidentifythemaindecomposingcomponentinthehydrogel,gelatinandHAwerelabeledgreenandrednear-infrared(NIR)dyes,respectively.ThestainedhydrogelwasthenvisualizedovertimeusingaLI-CORopticalinvivoimagingsystem.ResultsandDiscussion:InvitroCESTMRIdemonstratedapeakat3.6ppmforsaturationfieldstrengthsfrom1.2to7.2μT.Avalueof3.6μTexhibitedacomparableCESTsignalwithhigherB1values,butalessbroadspectrum,andwasthereforeusedforallfurtherexpe-riments.Whenweexaminedthethreeindividualcomponentsofthehydrogel,wefoundthatgelatinisthemajorcontributortotheCESTsignalat3.6ppm,inagreementwithearlierstudies[2].Wethenincreasedtheproportionofgelatinin thehydrogel.The increaseofgelatinenhancedCESTcontrast (Fig. 1a).Whenthehydrogelwasinjectedintothebrainofmice,itwasclearlydistingu-ishedfromthesurroundingnativetissue(Fig.1b).ThisattenuationofCESTcontrast indicates the feasibilityofCESTMRI tomonitor thedegradationofhydrogelovertime.TheNIRsignalofgelatindecreasegraduallyover42days,while theHA signal remained relatively stable (Fig. 3c). This suggests thatgelatin,themainsourceofCESTsignalat3.6ppm,isthemaindecomposingcomponent in thehydrogel.Anexcellentcorrelationwas foundbetween thedecayofCESTsignalandgelatinNIRfluorescencesignal(R2=0.94,Fig.3d).Figure1.(a)CESTmapofdifferenthydrogelsinjectedinthestriatumobtainedat3.6ppmforB1=3.6μT.Ratiosrepresentgelatin:HA.(b)T2-wCESTover-layimagesof4:1hydrogelmeasuredat3.6ppmfromday1today42.Arrowindicateshydrogelinjectionsite.(c)SerialNIRopticalimagesofthesamemice

Poster Abstracts:

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Temporal Next Generation Transcriptome Screen of Human Neuronal Pro-Survival Molecules and Pathways

Xu, J.-C.*, Kang, S.-U.*, Zhu, J., Fan, J., Frazee, A., Antonescu, C., Zhou, W., Chen, L., Chi, Z., Yang, S.-Z., Ji, H., Salzberg, S., Leek, J., Dawson, T.M., Dawson, V.L.

Neuroregeneration and Stem Cell Programs, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Department of Neurology, Jinling Hospital, Nanjing University School of Medicine, 305 East Zhongshan Road, Nanjing, Jiangsu 210002, China; Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

Alzheimer’s disease and Related Dementias (AD/ADRD) and Stroke arethemajordevastatingneurologicaldisorders.Stroke resulting frombrain in-farcts,oftenco-existsandpotentiallyinteractswithAD.Intriguing,inbothADandstroke,significantdysregulationofN-MethylD-Asparticacid receptors -(NMDAR,theprimetargetforhumancognition),aswellasreducedneuronalavailabilityofoxygen&glucose(associatewithhumanbrainevolution)occureitherascauseorinteractivepathologies12-15.Thus,theidentificationofma-jor regulatory factorsgoverning these ‘antiquated’pathwaysassociatedwithNMDARandoxygen&glucoseisinterestingandimperative.Themanipulationof these pathwaysmay then provide potential targets for interventions. Ex-tensive researches had been studied on these pathways.However, humanbraintissuehasinaccessiblenature.ThesnapshotofADandstrokethroughpostmortemtissuehashinderedtheidentificationofdynamicchanges,whichmayleadtoneurondegenerationorsurvival.Modelsusedtoidentifydynamicchangesinhumanneuronsneedtobeestablished.Usinghumanembryonicstem(ES)cellsandinducedpluripotentstem(iPS)cells,wehavedevelopedahigh-efficiencymethodofgeneratingauthentichumancortical neurons16,positioningus to investigate the longitudinalsignalingactivated.Weprofiledthetime-seriestranscriptomefollowingactivationofthei)NMDAR-ii)oxygen&glucosepathway- regulatedsurvivalpathways inhumancorticalneurons.TranscriptomicanalysisrevealsthathumanneuronsrespondverysimilarlytoeitherNMDARoroxygen&glucoseactivation, indicatingconvergeddowns-treamsignaling.Functionalclusteringanalysisof thedifferentiallyexpressedgenes shows theenrichment of genesmediating neuroplasticity, learning&memoryandneuronsurvival.FurtherupstreamanalysisofregulatorynetworksofthesedifferentiallyexpressedgenesidentifiedPrdm3(MECOM/EVI1)astheoneofthemajorregulatoryfactors.Intriguingly,thetemporallifespanexpres-sionpatternofPrdm3andseveralotherpotentialmasterregulatorsinhumanprefrontalcortex18areverysimilar to thehighercognitive trajectory19,20 inhumanbrain.ThisindicatesPrdm3anditsmono-methyltransferaseactivityonhistone3lysine9(H3K9me1)mayregulatebraincognitionandagingofbrainthroughinductionofgeneexpression

Fibrin Hydrogel Small-Diameter Graft as An Arterial Conduit

Morgan Elliott1,2, Brian Ginn2,3,4, Takuma Fukunishi5, Djahida Bedja6, Abhilash Suresh1,2, Theresa Chen2,7, Harry C. Dietz6,8,9, Lakshmi Santhanam10, Hai-Quan Mao1,2,3,4, Narutoshi Hibino2,5, Sharon Gerecht2,3, 7,11

1Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD, USA; 2Institute for NanoBioTechnology, Johns Hopkins University, Baltimore, MD, USA; 3Department of Materials Science and Engineering, Johns Hopkins University Whiting School of Engineering, Baltimore, MD, USA; 4Translational Tissue Engineering Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA; 5Department of Surgery, Johns Hopkins University School of Medicine, Baltimore, MD, USA; 6Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA; 7Department of Chemical and Bio-molecular Engineering, Johns Hopkins University Whiting School of Enginee-ring, Baltimore, MD, USA; 8Department of Pediatrics and Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA; 9Howard Hughes Medical Institute, Chevy Chase, MD, USA; 10Department of Anesthesiology and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA; 11Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, MD, USA

Autologousandsyntheticpolymersmall-diametergraftsusedforarterialby-passsurgeryareinsufficientandoftenleadtomorbidityandmortality.Here,wereportasmalldiametertissue-engineeredvasculargraft(sTEVG)preparedfromanaturalpolymerforarterialbypasssurgery.Usingahollowfibrinmicro-fibertubewithcontrolledsurfacetopography,weenableimmediate,controlledperfusionandformationofanaligned,confluentendotheliumbothinvitroandinvivo. Implantationofeitheracellularorendothelializedgrafts (1.09±0.17mmindiameter)inmouseabdominalaortasupportednormalbloodflowandvesselpatency for24weeks, indicatingnochange invascular functionduetosTEVGimplantation.Acellulargraftsdevelopedclotson the luminalwallsbyweekone in1of5of cases,butnoevidenceof theseclotswasvisiblein either graft type at later time points.Endothelialized grafts evidenced in-creased overall cell concentration, including macrophages, smooth muscle cells(SMCs),andendothelialcellsatweek1comparedtoacellularsTEVGs.Forbothacellularandendothelializedgrafts,SMCpenetrationwithcollagenandelastindepositionwasapparentbyweek4andincreasedovertime.Themediallayerwassignificantlythickeratweek8andcalcificationwasreducedinweek8and24endothelializedgraftsrelativetoacellulargrafts, indicatingenhancedremodeling.Thetunicamedia,composedofcollagen,elastin,andSMCs,becamemoreorganizedintolayers,similartothebandingseeninthenativeaortabyweek24.Afterimplantation,theultimatecircumferentialstressandstrainandtheelasticityoftheacellularandendothelializedgraftsclose-lyapproximatedthenativeabdominalaorta.Additionally, theelasticityof theweek-24sTEVGscloselyapproximatedthenativecontrol.Overall,weshowthatoursTEVGcomposedofanaturalpolymermatchesthenativevesselsizeand mechanical properties, incorporates into the native vascular tissue, has lowthrombogenicity,andexhibitsaclinicallyrelevantshelflife;thus,itwarrantsfurtherinvestigationtowardsarterialbypasssurgeryclinicaltrials.



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alsothemaindegradationcomponentinthehydrogel.ScaffoldingofGRPsinbiocompatibleandmechanicallystablehydrogelscanconferhighergraftsta-bilitywithminimalcellloss,leadingtoprolongedcellsurvival.Ourdual-modeimagingapproachmaybeusedtodevelophydrogelswithoptimalbiodegra-dationpropertiesforscaffoldedstemcells.REFERENCES:[1]MadlCM,etal.NatureMaterials.2017;16:1233.[2]LiangY.etal.Biomaterials2015;42:144.ACKNOWLEDGEMENTS:ThisstudywassupportedbyMSCRFD-3899.

Optimal Electrical Stimulation Boosts Nerve Regeneration with Stem Cell Therapy

Jian Du1, Gehua Zhen2, Huanwen Chen1, Shuming Zhang3,4, Liming Qing1, Xiuli Yang1, Gabsang Lee5,6, Hai-Quan Mao3,4, Xiaofeng Jia1,3,7,8,9*

Department of 1Neurosurgery, 7Orthopedics, 8Anatomy and Neurobiology, University of Maryland School of Medicine, Baltimore, MD 21201, Dept. of 2Orthopaedics, 3Biomedical Engineering, 4Materials Science and Engineering, 4Neurology, 5Neuroscience, 9Anesthesiology and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205

Peripheralnerveinjuriesoftenleadtoincompleterecoveryandcontributetosignificantdisability toapproximately360,000people in theUSAeachyear.Stemcelltherapyholdssignificantpromiseforperipheralnerveregeneration,butmaintenanceofstemcellviabilityanddifferentiationpotential invivoarestillmajorobstaclesfortranslation.Hereweinvestigatedtheeffectsofdifferentelectricalstimulatingparametersonhumanneuralcreststemcells(NCSCs)differentiation in vitro, and then evaluated the effect of optimized electricalstimulation(ES)onhumanneuralcreststemcell(NCSC)transplantationforperipheralnerveregeneration.15mmcritical-sizedsciaticnerveinjuriesweregeneratedwithsubsequentsurgicalrepairinsixtyathymicnuderats.Injuredanimalswere randomly assigned into five groups (N=12per group): blankcontrol,ES,NCSC,NCSC+ES,andautologousnervegraft.TheoptimizedESfromtheinvitrostudieswasappliedimmediatelyaftersurgicalrepairfor1hinESandNCSC+ESgroups.Recoverywasassessedbybehavioral(CatWalkgaitanalysis),wetmuscle-mass,histomorphometric,andimmunohistochemi-calanalysesateither6or12weeksaftersurgery(N=6pergroup).Gastrocne-miusmusclewetmassmeasurementsinES+NCSCgroupwerecomparabletoautologousnervetransplantationandsignificantlyhigherthanothergroups(p<0.05).Quantitative histomorphometric analysis and catwalk gait analysisshowedsimilarimprovementsbyESonNCSCs(p<0.05).AhighernumberofviableNCSCswasshownviaimmunochemicalanalysis,withhigherSchwanncells(SC)differentiationintheNCSC+ESgroupcomparedtotheNCSCgroup(p<0.05).Overall,ESonNCSCtransplantationsignificantlyenhancednerveregenerationafter injury and repair, andwas comparable toautograft treat-ment.Thus,EScanbeapotentalternativetobiochemicalandphysicalcuesformodulatingstemcellsurvivalanddifferentiation.Thisnovelcell-basedinter-ventionpresentsaneffectiveandsafeapproachforbetteroutcomesafterpe-ripheral nerve repair.

AcknowledgmentsSupported by Maryland Stem Cell Research Fund

Non-Invasive Imaging of Hydrogel Scaffold BiodegradationAnd Transplanted Cell Survival

Wei Zhu, Kang, Shreyas Kuddannaya, Chengyan Chu, Yue Yuan,Piotr Walczak, Anirudha Singh, Xiaolei Song, Jeff Bulte

Russell H. Morgan Dept. of Radiology and Radiological Science and Cellular Imaging Section, Institute for Cell Engineering, The Johns Hopkins University School of Medicine, Baltimore, MD, USA

INTRODUCTION:Stemcelltherapyhasgarneredmuchattentionfortreatmentofneurodegeneration.However,theharshmicroenvironmentatthesiteofda-maged tissue often impairs the retention and survival of transplanted cells.Scaffoldingcellswithhydrogelsisapromisingstrategytoovercomeinitialcelllossandmanipulatecellfunctionpost-transplantation.Whilescaffoldmaterialsareessential for improvingsurvivalofcellulargrafts,matrixdegradation isamajorrequirementforfunctionalgraftintegrationfromimplantedbiomaterials[1].Therefore,monitoringthedegradationofhydrogelsisessentialfordevelo-pingsuccessfulscaffoldedstemcelltherapies.Itisalsoessentialtostudythesurvivaloftransplantedstemcellstoeventuallypromoteintegrationanddiffe-rentiationinthehosttissue.WeinvestigatedwhetherCESTMRIcanvisualizeimplantedhydrogelscaffolddegradationinmousebrain,andusedbiolumine-scenceimaging(BLI)toprobetransplantedcellsurvival.METHODS:Acova-lentlycross-linkedhydrogelcomposingof20mg/mLthiol-modifiedgelatin,20mg/mLthiol-modifiedhyaluronicacid(HA),and20mg/mLpolyethylene(glycol)diacrylate(PEGDA)(crosslinker)wasprepared.InvitroCESTMRIwascarriedoutat37°C.Forinvivovisualization,3μLhydrogelwasinjectedintomousestriatum.CESTMRIwasperformedover42daysusingahorizontalbore11.7TBrukerscanner.TovalidatetheinvivoCESTMRIfindingsandidentifythemaindecomposingcomponent inthehydrogel,gelatinandHAwerelabeledwithgreenandrednear-infrared(NIR)dyes,respectively.Thelabeledhydro-gelwasthenvisualizedovertimeusingaLI-CORopticalinvivoimagingsys-tem.Additionally,mouseglialrestrictedprogenitorcells(mGRPs)expressinggreenfluorescentprotein(GFP)andtransducedwithalentiviralvectorcarryingfirefly luciferase(pLenti4-CMV-Luc)wereencapsulatedwithin thehydrogels.Thesurvivalofthetransplantedcellswith/withoutscaffolding(hydrogel)wasmonitoredwithaPerkinElmerIVISSpectrum/CTinstrument.RESULTSANDDISCUSSION:InvitroCESTMRIdemonstratedapeakat3.6ppmforsatura-tionfieldstrengthsfrom1.2to7.2μT.Avalueof3.6μTexhibitedacomparableCESTsignalwithhigherB1values,butalessbroadspectrum,andwasthere-foreusedforallfurtherexperiments.Whenweexaminedthethreeindividualcomponentsofthehydrogel,wefoundthatgelatinisthemajorcontributortotheCESTsignalat3.6ppm,inagreementwithearlierstudies[2].Whenthehydrogelwasinjectedintothebrainofmice,itcouldbeclearlydistinguishedfromthesurroundingnativetissue.ThisattenuationofCESTcontrastindica-tes the feasibilityofCESTMRI tomonitor thedegradationofhydrogelovertime.TheNIRsignalofgelatindecreasegraduallyover42days,whiletheHAsignalremainedrelativelystable.Thissuggeststhatgelatin,themainsourceofCESTsignalat3.6ppm,isthemaindecomposingcomponentinthehydro-gel.An excellent correlationwas found between the decay ofCEST signalandgelatinNIRfluorescencesignal(R2=0.94).WhenmGRPswereinjectedintobrain,scaffoldedmGRPsshowedprolongedinvivosurvivalandrestrictedlocalizationattheinjectionsiterelativetounscaffoldedmGRPs.CONCLUSI-ONS:Hydrogeldegradationcanbevisualizedinanon-invasiveandlabel-freemanner.GelatinwasfoundtobethemajorcontributorofCESTcontrastand

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Induced Pluripotent Stem Cell Derived Endothelial Cells Display Unique Phenotype in Low Oxygen Environments

Bria Macklin1,Sharon Gerecht1,2

1Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD 21208, 2Department of Materials Science and Engineering, Johns Hopkins University, Baltimore, MD 21218, United States

Objective:Hypoxia,orlowoxygentension(<5%),isaconditionthathasbeenshown togovernvasculogenesisas theembryodevelopsand theneed foroxygenincreasesinnewlyformedtissues.Here,weaimtostudyhowhypo-xiaregulatesvascularnetworkformationofhumaninducedpluripotentstemcell (hiPSCs) derived endothelial cells (ECs).Methods:Using our bi-potentdifferentiationprotocol,wegenerateearlyvascularcells (EVCs) thatconsistof PDGFRβ+/SM22α+ early pericytes and VECad+/CD31+ early ECs fromhiPSCs,whichcanbefurthermaturedintofunctionalvascularcells.VECad+/CD31ECsorHUVECsareencapsulatedincollagenhydrogels,stimulatedwithVEGFandallowedtoformvascularnetworksover3days.“Hypoxichydrogels”aregeneratedbybalancingO2diffusionwithO2cell consumption.Oxygensaturation in hydrogels ismeasured using non-invasive fluorescence quen-chingO2sensors.Results:HUVECsencapsulatedinthe“hypoxichydrogels”reachedO2saturation~1%andgeneratedvascularnetworkssimilartothoseinthenonhypoxichydrogels.Incontrast,hiPSC-ECsencapsulatedin“hypoxichydrogels” formedlargerandthickertubeswithoverall increasednumberoftubesascomparedtononhypoxichydrogels,butdidnotreachbelow10%O2saturation.TreatmentwithaROSinhibitor,diphenyleneiodonium(DPI),ledtosimilarnetworkinhibitioninbothmatureandhiPSC-ECs.Examiningtheoxy-genuptakeratewefoundanadaptationofthehiPSC-ECtohypoxiabutnotinHUVECs.Currentstudiesfocusondelineatingtheunderlyingmechanism.

Stem Cells to Reprogram Skin Identity and Enhance Prosthetic Use in Amputees

Luis Garza, MD-PhD, Johns Hopkins University School of Medicine

Background:Amputation rates in recent conflicts have been high.Althoughmajoradvancementshavebeenmadeinprosthetics,theiruseisstill limitedbytheskinatthestumpsite.Mostnotablydiscomfortandpain,butalsoskinbreakdownresultbecause theskinflapat thestumpsiteswasnotadaptedtobearweight.Weproposetoreprogramtheskinatthestumpsitetobethethickskinatthepalmsandsoles(volarskin)tomakeitmoreweight-,friction-and irritant-resistant.Methods:Under grants fromMSCRFweare currentlyperformingacellular therapyclinical trialonhumansubjects tocreateecto-picvolarskin.Weareinjectingautologousvolarfibroblaststonon-volarareasandmeasuring for changes towards volar skin.Wearealso testing in vitrowhatfactorscontrolreprogramingofnon-volarepithelialcellstovolarepithelialcells.Results:Wehavefullregulatoryapprovalandhaveenrolledmorethan15 subjects testing a variety of variables.Using genome-wide analysis,weshowthatKERATIN9(KRT9)isthemostuniquelyenrichedtranscriptinvolarkeratinocytes.KRT9expressioninkeratinocytesisupregulatedbyvolarcom-paredtonon-volarfibroblastinbothmousemodelsandinvitroco-cultures.In

ourPhase1clinicaltrial,KRT9proteinexpressionisselectivelyupregulatedbyvolarcomparedtononvolarfibroblasts.Additionally,wefindthatcytoplasmicsize—notablyhigherinnativevolarkeratinocytes--alsoincreasesinnon-volarkeratinocytesexposedtovolarfibroblasts.Theseresultssuggestthiscellulartherapyismodifyingskinidentitybothingeneexpressionandinoverallarchi-tecture.Conclusions:Cellulartherapyisapromisingavenuetoreprogramskinidentity inamputeesatthestumpsitetobecomethickpalmo-plantarskin inanefforttoenhanceprostheticuse.Planningforaphase2trialinamputeesiscurrently in progress.

NF-KAPPAB Pathway Is Involved in Bone Marrow Stromal Cell-Produced Pain Relief

Wei Guo1,Satoshi Imai1,2, Jia-Le Yang1, Shiping Zou1, Huijuan Li1,3, Huakun Xu4, Kamal D. Moudgil5, Ronald Dubner1, Feng Wei1, Ke Ren1*

1Dept. of Neural & Pain Sciences, School of Dentistry, & Program in Neuroscience, Univ. of Maryland, Baltimore, MD; 2Present Affiliation: Dept. of Clinical Pharmacology & Therapeutics, Kyoto Univ. Hospital, Kyoto, Japan; 3Dept. of Neurology, The 3rd Affiliated Hospital of Sun Yat-sen Univ., Guangdong Province, China; 4Dept. of Advanced Oral Sciences and Therapeutics, Univ. of Maryland School of Dentistry, Baltimore, MD ; 5Dept. of Microbiology & Immunology, Univ. of Maryland, Baltimore, MD

Bonemarrowstromalcells(BMSCs)producelong-lastingattenuationofpainhypersensitivity.ThiseffectinvolvesBMSC’sabilitytointeractwiththeimmunesystemandactivationoftheendogenousopioidreceptorsinthepainmodula-torycircuitry.ThenuclearfactorkappaB(NF-ϰB)proteincomplexisakeytran-scriptionfactorthatregulatesgeneexpressioninvolvedinimmunity.Wetestedthehypothesis that theNF-ϰBsignalingplaysa role inBMSC-inducedpainrelief.Wefocusedontherostralventromedialmedulla(RVM),akeystructureinthedescendingpainmodulatorypathway,thathasbeenshowntoplayanimportant role inBMSC-producedantihyperalgesia. InSpragueDawley ratswitha ligation injuryof themassetermuscletendon(TL),BMSCs(1.5M/rat)fromdonorratswereinfusedi.v.at1wpost-TL.P65exhibitedpredominantneuronallocalizationintheRVMwithscattereddistributioninglialcells.At1w,butnot8wafterBMSCinfusion,westernblotandimmunostainingshowedthatp65ofNF-ϰBwassignificantlyincreasedintheRVM.GiventhatchemokinesignalingiscriticaltoBMSCs’pain-relievingeffect,wefurtherevaluatedaroleofchemokinesignaling inp65upregulation.Prior to infusionofBMSCs,wetransducedBMSCswithCcl4shRNA, incubatedBMSCswithRS102895,aCCR2bantagonist,ormaraviroc,aCCR5antagonist.Theantagonismofche-mokinessignificantlyreducedBMSC-inducedupregulationofp65,suggestingthatupregulationofp65wasrelatedtoBMSCs’pain-relievingeffect.Wethentested theeffectofaselectiveNF-ϰBactivation inhibitor,BAY11-7082.ThemechanicalhyperalgesiaoftheratwasassessedwiththevonFreymethod.Inthepre-treatmentexperiment,BAY11-7082(2.5and25pmol)wasinjectedintotheRVMat2hpriortoBMSCinfusion.PretreatmentwithBAY11-7082attenuatedBMSCs’ antihyperalgesia, but post-treatment at 5w post-BMSCwasnoteffective.Onthecontrary, inTLratsreceivingBAY11-7082withoutBMSCs,TL-inducedhyperalgesiawasattenuated,consistentwithdualrolesofNF-κBinpainhypersensitivityandBMSC-producedpainrelief.TheseresultsindicatethattheNF-ϰBsignalingpathwayinthedescendingcircuitryisinvol-ved in initiationofBMSC-producedbehaviouralantihyperalgesia.Supportedby theMSCRFandNIH;HL is supportedbyTheTeam-BuildingProject forStemCellResearch,SYSU.



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ITworkforcedevelopmentandwebelievewecanstartthatprocesswiththeOpenDataSTEAMSummerCampprogram.WemustadaptourITworkforcetocontinuetodelivervalue,primarilybyrecognizinginformationasastrategicassetandbycreatingan information-focused ITorganization. Severalnewkindsofinformationprofessionalswillstaffthisorganization(e.g.datascien-tists, informationmanagers, information architect, information leader, digitalarchivists,dataandinformationvisualizationdesigners,data/informationste-wards).Roleswillalsobeneededtogovern,manage,andanalyzeopenandbigdata.Theskillsrequiredtoperformtheserolesgenerallydonotexistatinabundancetoday,whichwillresultinashortfallforthesepositions.Weneedtoprepareforthisevolutionandcultureshifttoday.TheOpenDataSTEAMSum-merCampprogramwillenableorganizationsto:1)linkworkforcerequirementsdirectly tomissionsandaid in data collection; 2) developa comprehensivepictureof thedata throughaprocessofcatalogingandanalysis;3) identifyandimplementdataqualitystrategies,especiallyforprioritydatasets;and4)identifyandovercomeinternalandexternalbarrierstoaccomplishingstrategicdataidentificationgoals.VulcanEnterprises,LLC.proposesaroadmapfortheOpenDataSTEAMSummerCamp thatwouldestablishacontinuouscyclebetween your organization and an academic institution, industry or govern-ment.Thislifecyclewouldbecomprisedofsevenbasicsteps:#1.Proposalpre-sentationandagreement;#2.CommunicationPlandevelopmentanddelivery;#3.Applicationdevelopmentanddelivery;#4.Applicantacceptance;#5.Campimplementation;#6.EvaluateandMeasure(Endofsummer);#7.LessonsLe-arned/AdjustPlanasNecessaryfornextyear.

Development of an iPSC Derived Cellular Model of Barth Syndrome

Arianna Franca Anzmann1, Steven M. Claypool2, Hilary J. Vernon1,3

1McKusick-Nathans Institute of Genetic Medicine, 2Dept. of Physiology, 3Dept. of Pediatrics, Johns Hopkins University School of Medicine, Baltimore MD.

BarthSyndrome(BTHS,3-methylglutaconicaciduriatypeII,MIM#302060)isanX-linkedinbornerrorofmitochondrialphospholipidmetabolism,causedbypathogenicvariantsinthegenetafazzin(TAZ).TAZencodesforatransacylaseinvolvedinthefinalremodelingstepofcardiolipin(CL),aphospholipidlocali-zedtotheinnermitochondrialmembranewithkeyrolesincristaeformation,or-ganizationofthemitochondrialrespiratorychain,andintheapoptoticcascade.DeficiencyofTAZresultsinabnormalCLcontent,includinganaccumulationofmonolysocardiolipin(MLCL)andareductionofremodeledCL.Asistypicalofmostprimarymitochondrialdiseases,BTHSisamultisystemdisorderandischaracterizedbycardiomyopathy,skeletalmyopathy,andneutropenia,amongother features.However, in sharp contrast tomanymitochondrial diseases,BTHShasminimalneurologicalburden.It isnotknownwhyTAZ,anubiqui-touslyexpressedprotein, leadsto theuniquetissuedistributionofBTHS. Inordertoexplorenovelareasofcellulardysfunction,weappliedamulti-omicsdiscoveryapproach toaCRISPR-editedTAZ-deficientHEK293cell line thatwasdevelopedandphenotypically validated inour laboratory.With thisap-proach,wediscovereddifferentialexpressionofproteinsinvolvedindynamicresponses tomitochondrialstress(i.e.machineryoffission, fusion,andmit-ophagy)andbioenergeticresponsestomitochondrialstress(i.e.downregula-tionofROSproduction).

Clinical Scale Production and Wound Healing Activity of Human Adipose Derived Mesenchymal Stem Cell Extracellular Vesicles

from a Hollow Fiber Bioreactor

John J.S. Cadwell, FiberCell Systems Inc. and John Ludlow, Zen Bio

Introduction:Productionofextracellularvesicles(EV)suchasexosomesatthescalerequiredforclinicalapplicationsremainsachallenge.Currentmethodscanutilizelargenumbersofflasksandserumstarvationinabatchmodepro-cess.HollowfiberbioreactorsareperhapsidealforproducinglargequantitiesofEVat100Xhigherconcentrations thanconventionalprotocols.Hollowfi-berbioreactorssupportthecultureoflargenumbersofcellsathighdensities,1-2X10e8cells/ml.Cellsareboundtoaporoussupportwitha20kDamolecularweightcutoff(MWCO)socellpassagingisnotrequiredandEVcannotcrossthefiberineitherdirection.Methods:1X109adiposederivedadultMSCwereculturedinaFiberCellhollowfiberbioreactorfor8weeksinDMEM+10%FBSinthecirculatingmediumonly.Cellsdidnotexpand(monitoredbyglucoseup-takerate)nordidtheydifferentiate(bymultipleimmunocytochemistryassays)overthistime.40mlofconditionedmediumfromtheextra-capillaryspacewasharvestedweekly.Ascratchclosuretestandratskinwoundhealingassaywasused to measure wound healing activity. Results:8.6X1011EVparticlesinavolumeof120mlswereharvestedfromtheadultadiposederivedMSCculture.18litersofDMEMwereconsumedover8weeks.Additionally,EV/proteinwas10-foldhigherinharvestsfromthebioreactorsuggestinghigherpurityaswell.Woundhealingassaysdemonstratedsignificantaccelerationofwoundhea-ling. Summary/conclusion:Hollowfiberbioreactorshavedemonstratedpoten-tialforthemanufacturingscaleproductionofEVusingcGMPcompliantmate-rialsandmethods.TheEVisolatedfromadultadiposederivedMSCculturedinanHFBRshowactivitythatcanpromotewoundhealingbothininvitroandinvivoassays.Hollowfiberbioreactorsprovideanumberofsignificantadvan-tagescomparedtoflaskbasedprotocolsincludinghigherconcentrations,largecapacity,timeandspaceefficiency,andperhapsEVquality.CurrentavailabletechnologypermitstheproductionofgramquantitiesofEV,withpotentialuseforclinicalapplications.

Agriculture Is No Longer Beverly Hillbillies or Green Acres

Joyce Hunter, Vulcan Enterprises, LLC.

Triggeringstudents’interestinpursuingmoretechnicalfieldsbeginsinschools.TherearemanyeffectivestrategiesforengagingstudentsandimprovingtheirperformanceinSTEAMsubjectswhichmakesthebusinesscasefortheOpenDataScienceTechnologyEngineeringAgricultureandMath(STEAM)SummerCampProgramcompelling.Onevitalbenefitofopendata,makinginformationresourceseasytofind,accessible,andusablecanfuelentrepreneurship,inno-vation,andscientificdiscoverythatimprovesAmericans'livesandcontributessignificantlytojobcreation.OpenDatahasbecomebothatacticalandstrate-gicroleinorganization’soperationsandpoliciesandwillrequirepersonneltofulfillemergingskill-setssuchasdataanalysts,qualityassurancespecialistsanddataofficers.OrganizationsthathaveOpenDataaspartoftheirstrategicworkforceplanningareabletoanticipatefutureneedsandutilizeleadtimetofoster the development of new skills internally before they becomemissioncritical.Theyneedtobuildthecapacitytothinkasasingleenterpriseaboutits

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Withthesefindingsinhand,wenextsetouttodeterminewhetherthedynamicandbioenergeticresponsestomitochondrialstressaremoredysregulatedincelltypesthatareclinicallyaffectedinBTHS.Inordertoaccomplishthis,wedevelopedanovelcellularmodelofTAZdeficiencyiniPSCs.CRISPR/Cas9-gRNAwasusedtoedittheiPSCscellswithinexon2ofTAZ.Aresultant50bpdeletionwasidentifiedin4individuallyisolatedclones.TheframeshiftdeletionresultsincompletelossofTAZ.Furtherstudiesinthisnovelmodel,withdiffe-rentiation to cardiomyocytes and neurons, has the potential to provide insight into the roleofCLcomposition inmitochondrial function,aswellas tooffernoveltreatmenttargetsforBTHS.

Mechanical Characterization of Engineered Cardiac Tissues for Clinical Application

Seungman Park1, Cecillia Lui2, Wei-Hung Jung1, Debonil Maity1, Chin Siang Ong2, Joshua Bush3, Venkat Maruthamuthu3, Narutoshi Hibino2,5, Yun Chen1,4,5*

1Department of Mechanical Engineering, Johns Hopkins University, MD, USA2Division of Cardiac Surgery, Johns Hopkins University, MD, USA; 3Department of Mechanical and Aerospace Engineering, Old Dominion University, VA, USA; 4Center for Cell Dynamics, Johns Hopkins University, MD, USA; 5Institute for NanoBio Technology, Johns Hopkins University, MD, USA

Heart failures (HFs) is the leadingcauseofdeath in theU.S.Currently, themosteffectivewaytotreatHFisorgantransplantation.However,thismethodhaslimitationsduetothelackoforgandonors,immuneresponsetothetrans-plantedorgan,andhighcosts.Recently, tissueengineeringapproachesarebeingwidelyexploredtoaddressthechallenges.Amongthose,cardiacpatchisapromisingreplacementforthedamagedtissues.Acellularpatchesconsis-tingofECMproteinswereshowntoeffectivelyresuscitateinjuredmyocardiuminanimalmodels. Inaddition,3Dbioprinting,wherecellsandECMproteinsareorganizedinspecificgeometryanddimensionstoyieldfunctionalcardiacpatchwarrantsapotentialsolutiontotreatHFs.Nonetheless,amajorobstacledescribedinthefollowingremainstobesurmountedbeforeengineeredtissu-escanbeusedasageneraltreatmentoption.Directmappingofmechanicalforceandstressover3Dcardiactissuesisyettobedeveloped,asopposedtoits2Dcounterparts,despitethat3Dcardiactissuesaremorephysiologicallyrepresentativeandcanbereadilyappliedclinically.Inthisstudy,asaproofofconcept,3Dbioprintedcardiactissuesderivedfromhumaninducedpluripotentstemcells(hiPSCs)aretested.First,theengineeredcardiactissueslabeledwithparticlesarerecordedandtrackedtodeterminespatiallyandtemporallyvariable contraction forces.Viscoelasticpropertiesaremeasuredusingma-gnetic tweezers.The results are used to compute 3D force and stress dis-tributionovertheengineeredtissuebyfiniteelementmethod.Insummary,aframework isdeveloped toassessclinical-gradeengineeredcardiac tissuesanddeterminetheappropriatevaluerangessuitableforimplantation.There-sultsrelatingcontractility,intrinsicmechanicalpropertiesandstressdistributionintheengineeredtissue,canalsoinformabetterdesignforfuturefabricationofengineeredtissues.

Mechanisms of Angiogenesis Within Bioprinted Endothelial: Mesenchymal Stem Cell Cocultures

Marco Santoro, Tolulope Awosika, Kirstie L. Coombs, John P. Fisher

Fischell Department of Bioengineering, University of Maryland College Park, College Park, Maryland; Center for Engineering Complex Tissues, University of Maryland College Park, College Park, Maryland

Introduction:Bioprintingholdsgreatpromiseinthefabricationofvascularizedscaffolds,acentralchallengeintissueengineering.Acommonstrategytofa-bricatesuchvasculaturereliesonthedepositionofendothelialcells(ECs)andmesenchymalstemcells(MSCs).However,studieshavereportedcontrastingresultsontheroleofMSCsinarteriogenesisandangiogenesis.Therefore,inthisstudyweinvestigatedhowthecombinationofEC:MSCdrivestheforma-tionofnewvasculaturewithinbioprintedscaffoldsbyregulatingkeysignalingpathways. MaterialsandMethods:Humanumbilicalveinendothelialcells(HU-VECs)andratorhumanMSCs(rMSCsorhMSCs,respectively)wereencap-sulatedingelatinmethacrylate(GelMA)indifferentcocultureratios(1:0,3:1,1:1,1:3,and0:1)andculturedfor2weeks.Cell-ladenGelMAwasextrudedasfibers(400μmdiameter)andevaluatedforcellviability(Live/Dead)andexpres-sionofangiogenicmarkers(CD34,VE-cadherin,α-SMA)viafluorescentmicro-scopy.Geneexpressionwasanalyzedviart-PCRagainstVEGF,PDGF-BB,TIE-2andTGF-β1-relatedsignalingpathways.ExpressionofVEGF,TGF-β1,PDGF-BB,andAng-1ligandswasmeasuredviaELISA.Alldatawereanalyzedusingaone-wayANOVAtest(p<0.05),followedbyaposthocTukey’sHSDtest. Results andDiscussion:Cell-ladenGelMA scaffoldswere successfullybioprintedwithnodetrimentaleffectsoncellviability.GeneexpressionshowedupregulationofangiogenicmarkersintheEC:MSC3:1groupcomparedtoho-motypiccultures.Interestingly,weobservedaphysiologicallyrelevantpatternwithinitialupregulationofangiogenesisinECs(VEGF,PDGF)followedbyen-hancedarteriogenesisinMSCs(PDGF,TGF-β1)withinhybridHUVEC/rMSCcoculturesatlatertimepoints.ThesefindingswerecorroboratedinfullyhumanHUVEC/hMSCcocultures,wherehMSCexertanantiangiogeniceffectearlyinthecultureandaproangiogeniceffectafter7daysincoculture.Conclusions:InbioprintedEC:MSCcocultures,earlyangiogenesisisdrivenbyECs,whilela-terarteriogenesisismainlyregulatedbyMSCs.Duringthisprocess,EC:MSCcrosstalksynergisticallypromotesup-regulationofangiogenicandarteriogenicpathwaysinbothcells.Infuturestudies,thisbasicknowledgewillserveasthedesigncriteriatofabricatevascularizedscaffoldsofaclinicallyrelevantsize.

A Duchenne Muscular Dystrophy Patient hiPSC-Derived Myoblasts Based Drug Screen Uncovers Potential

Compounds for DMD Treatment

Congshan Sun, Inyong Choi, Gabsang Lee, Kathryn Wagner

Duchennemusculardystrophy(DMD),themostcommonmusculardystrophy,ischaracterizedbyprogressivemuscledegenerationandweaknessresultinginthelossofambulationduringadolescenceandultimatelydeathinearlyadult-hood.CurrentlythereareonlytwoFDAapproveddrugstargetingDMD,andthereisagreatneedofmoretreatmentoptionsforthisdevastatingdisease.



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dium.The3D-ECMstructuresledtothemaintenanceofamoreprimitivesub-populationofhematopoieticstemandprogenitorcellswhenculturedinQBSF60Medium.TheseresultssuggestthatitisfeasibletocultureandexpandcordbloodCD34+cellsunderclinicallyrelevantconditions.

Synthetic mRNAs Drive Highly Efficient iPS Cell Differentiation to Dopaminergic Neurons

Yingchao Xue1, Xiping Zhan8, Senthilkumar S. Karuppagounder2,4,9, Shuli Xia1,2, Valina L. Dawson2,3,4,6,9, Ted M. Dawson2,3,4,7,9, John Laterra1,2,3,5 and Mingyao Ying1,2

1Hugo W. Moser Research Institute at Kennedy Krieger, Baltimore, Maryland, USA ; 2Department of Neurology; 3Department of Neuroscience; 4Neuroregeneration and Stem Cell Programs, Institute for Cell Engineering5Department of Oncology; 6Department of Physiology Department of Pharmacology and Molecular Sciences; 7Johns Hopkins University School of Medicine, Baltimore, Maryland, USA; 8Department of Physiology and Biophysics, Howard University, Washington, DC, USA; 9Adrienne Helis Malvin Medical Research Foundation, New Orleans, Louisiana, USA

Proneuraltranscriptionfactors(TFs)drivehighlyefficientdifferentiationofplu-ripotent stem cells to lineage-specific neurons. However, current strategiesmainlyrelyongenome-integratingviruses.Here,weusedsyntheticmRNAscodingtwoproneuralTFs(Atoh1andNgn2)todifferentiateiPScells(iPSCs)intomidbraindopaminergic(mDA)neurons.mRNAscodingAtoh1andNgn2withdefinedphosphositemodifications led tohigherandmorestableprote-in expression, and inducedmore efficient neuron conversion, as comparedtomRNAscodingwild-typeproteins.Using thesetwomodifiedmRNAswithmorphogens,weestablisheda5-dayprotocolthatcanrapidlygeneratemDAneuronswith>90%purityfromnormalandParkinson'sdiseaseiPSCs.Afterin-vitromaturation, thesemRNA-inducedmDA (miDA) neurons recapitulatekey biochemical and electrophysiological features of primarymDA neuronsandcanprovidehigh-contentneuroncultures fordrugdiscovery.ProteomicanalysisofAtoh1-bindingproteinsidentifiedthenon-muscleMyosinII(NM-II)complexasanewbindingpartnerofnuclearAtoh1.TheNM-IIcomplex,com-monlyknownasanATP-dependentmolecularmotor,bindsmorestronglytophosphosite-modifiedAtoh1thanthewildtype.Blebbistatin,anNM-IIcomplexantagonist,andbradykinin,anNM-II complexagonist, inhibitedandpromo-ted,respectively,thetranscriptionalactivityofAtoh1andtheefficiencyofmiDAneurongeneration.ThesefindingsestablishedthefirstmRNA-drivenstrategyforefficientiPSCdifferentiationtomDAneurons.WefurtheridentifiedtheNM-IIcomplexasapositivemodulatorofAtoh1-drivenneurondifferentiation.Themethodologydescribedherewill facilitate thedevelopment ofmRNA-drivendifferentiationstrategiesforgeneratingiPSC-derivedprogenieswidelyapplica-bletodiseasemodelingandcellreplacementtherapy.

However,duetoa lackofefficientDMDdiseasemodel,drugdiscoveryanddevelopmenthavebeenslow.Human inducedpluripotentstemcell (hiPSC)whichovercomestheinterspeciesdifferenceofanimalmodelsandlimitedex-pandabilityofprimarycelllinesmakesthelarge-scaledrugdiscoverypossible.Our laboratory has developeda novel serum-free hiPSC-to-skeletalmusclelineage differentiation protocol. We also characterized one of DMD patienthiPSC-derivedmyoblastrelatedphenotypesasfusiondefectcomparingmul-tiplelinesofDMDpatienthumaninducedpluripotentstemcell(hiPSC)-derivedmyoblasts,whichwasusedasaphenotype toscreencompounds forDMDtreatment. We performed a tiered medium-scale high-throughput chemicalcompound screen, composedof 1524 compounds fromJohnsHopkinsCli-nicalCompoundLibrary,onmyoblastsderivedfromoneDMDpatientspecifichiPSCline.Myotubeformationandassociatedproteinexpressionwereusedas parameters to select hit compounds. We selected primary hit compounds fromtheprimaryscreeningfollowedbysecondaryscreeningtovalidateandoptimizetheconcentrationofpositivehitcompounds.Twofinalhitcompoundswere selected from the in vitro screening, followedby testing inMDXmicemodel.Currentlywearealsoworkingontheactionmechanismsoftheselec-teddrugusingunbiased transcriptionalanalysis.OurworksetsanexampletoapplyhiPSC-basedcompoundscreening toDMDfield,whichovercomesthelimitationofotherDMDmodelsandprovideaplatformforfuturemusculardystrophy drug discovery.

Analysis of The Cord Blood Hematopoietic Stem/Progenitor Cell Populations Cultured In Serum-Free Medium

Under Various Conditions

Ronald L. Brown, Ph.D.1, Grace Almeida-Porada, M.D., Ph. D.2, Christopher D. Porada Ph.D.2, and Valerie Fremont, Ph.D1.

1Quality Biological, Inc., Department of Research and Development, Gaithersburg, MD, USA, 20879; and 2Wake Forest Institute for Regenerative Medicine, Winston-Salem, North Carolina, USA, 27157

Theuseofcordbloodforadulttransplantshasbeenhamperedbytherelati-velylownumberofhematopoieticstemcells.Usingaclinicalgradeserum-freemedium,QBSF60,weevaluatedseveralcombinationsofearlyactingcytoki-nesfortheirabilitytosupporttheexvivo2-dimensionalcultureofcordbloodCD34+cellswhilemaintainingtheir long-termengraftingability.ThecultureswerefirstevaluatedforthephenotypicexpressionoftheCD34cellmarkerandthenfunctionallyfortheirshort-termandlong-termengraftingabilityusingthesheep-humanxenograftmodel.After14daysofculture,allthecytokinecombi-nationssupportedanincreaseintotalnumberofCD34+cellsfromtheprimitiveprogenitor cells as well as the more mature clonogenic progenitor cells. For invivoengraftingstudies,weselectedacytokinecombinationforitsabilitytoyieldsignificantnumbersoftheprimitiveprogenitorswithoutexcessivediffe-rentiation. We demonstrated that the selected culture conditions support the maintenanceand/ortheexpansion(23-fold)ofthelong-termengraftingHSCovertwoweeksofculture.Furthermore,QBSF60supportsthe3-dimensionalcultureofCD34+cellsandallowsforexvivoexpansionanddifferentiationofCD34+stemcells.Bioengineeredliverconstructsmadefrom3Dliver-extra-cellular-matrix(3D-ECM)seededwitheitherhepatoblasts, fetal-liver-derived,orbone-marrowderivedstromalcells,yieldedoptimalgrowthinQBSF60Me-

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Cellular Metabolic Defects in Neurodevelopment Revealed by Single Cell Analysis in Patient-Specific

Models of Kabuki Syndrome

Giovanni Carosso, Jonathan Augustin, Leandros Boukas, Ha Nam Nguyen, Gabrielle Cannon, Briana Winer, Johanna Robertson, Kasper Hansen, Loyal Goff & Hans Bjornsson

Mutations in thechromatin-modifyinggeneKMT2Darecausative in70%ofKabukisyndrome(KS)cases,inwhichreducedintellectualcapacitybeginninginearlychildhoodisthoughttoarisefromdampenedcontroloftheepigenome.Thislossofgeneexpressioncontrolcouldmanifestatanynumberoftargetsgenome-wideinavarietyofcelltypes,andhasbeenshowntonegativelyim-pactadultneurogenesisinamousemodel,butKS-specificchangesincellularnetworksduringneurodevelopmenthaveyettobediscovered.WegeneticallyreprogrammedKSpatientskincellsintoinducedpluripotentstemcells(iPSC),enablinggenerationofthefirsthumanneuralprogenitorcell(NPC)modelofKS.Comparedtohealthycontrols,thesecellsshowrestrictedproliferationandalteredcellcycleprogression,aswellashigherratesofapoptoticcelldeath.BecausedevelopingNPCsexistalongaheterogeneousspectrumofdifferen-tiationstages,weleveragedthepowerfulresolutionofsinglecellexpressionprofilingtoidentifytheprecisestatesofmaturationandcellcycleoccupancyofindividualcellsfollowingneuralinductionfromiPSCs.Surprisingly,wefoundthat KSNPCs exhibit transcriptional hallmarks of precocious differentiation,witha~20-foldincreaseddensityofcellsatthemostmatureNPCstages,andthistrendwasconfirmedattheproteinlevelbyflowcytometricanalyses.ThemosttranscriptionallybluntedgenenetworksinKSNPCsgovernthemetabo-licprocessesofglycolysis,proteinqualitycontrol,andoxygensensing,whileproteinsynthesis,mRNAdegradation,andapoptoticexecutionpathwaysweremarkedlyincreased.ByseparatingNPCsintosubsetsalongadifferentiationtrajectory,wespecifyenergy-relatedmetabolicdysregulationasaprincipaldri-verofKSgeneexpressioninearlyNPCs,whilematureNPCdifferencesarebiasedtowardmRNAdegradationandribosomalpathways.ExtendingthesefindingswithCRISPR/Cas9geneeditinginanisogenichippocampalneuronKSmodel,we identifyconcordantmetabolicexpressionabnormalities,morepreciselydefineacellcyclearrestduringmitoticdivision,andobserveaccu-mulationofmisfoldedproteinaggregates.Identificationofcellcontext-specific,genome-wideKMT2DbindingsitesrefinedalistofKStranscriptionaltargetsheavilybiasedtowardcellularoxygensensingdefects,andfunctionalexperi-mentsdemonstrategenotype-dependenteffectsofhypoxiaoncelldeath in-duction in KS cells. Finally, we test our findings in vivo in a Kmt2dmousemodel,measuringgeneexpressioninproliferatinghippocampalprecursorstorevealconcordantKS-associatedgeneexpressionprofiles,similarmitoticdi-visionarrest,anddepletionofthehippocampaladultstemcellpool.Together,ourfindingssupportamodelinwhichalteredexpressionofKMT2D-controlledcellularmetabolic gene targets underlies an acceleratedmaturation of, andsubsequentdepletion in,poolsofneuralprecursorcells inKS.Morebroad-ly,wedemonstrate the investigativepowerofsinglecell resolution inhighlyheterogeneouscellcontextstowardidentificationofdisease-drivingmolecularmechanismsfortherapeuticdevelopmentinneurodevelopmentaldisorders.

Closed Systems Enabling Commercially-Viable Stem Cell Manufacturing

Jon Rowley, PhD, RoosterBio, Inc., Frederick, MD 21703, USA

The need for affordable production of cells, including humanMesenchymalStemCells(hMSC),inclosedscalablesystemshasbeenidentifiedasa“Cur-rentChallenge”intheTechnologyRoadmapestablishedbytheNIST-fundedCellManufacturingConsortium.Additionally,hMSCshavebeenidentifiedasakeycriticalrawmaterialformanyregenerativemedicineproductsasreportedinover800clinicaltrialswithanestimated60Billioncellsnecessaryforma-nufactureperclinicaltrial(Olsen,etal2018).Therefore,acriticalbottleneckimpedingthegrowthofRegenerativeMedicineistheavailabilityofcGMP-com-plianthMSCforclinicaldevelopmentpathways.Hencethereisanimmedia-teneedtodevelopscalableplatformsystemsthatsimplify theproductionofcGMP-compliantstemcellsandradicallyreduceclinicaltranslationtimelines.Theobjectiveof thiswork specifically focusesononecritical componentofa complete scalable closed system: the development and qualification of afunctionallyclosedversionofbonemarrowderivedhMSCworkingcellbanksforuseinscalablestemcellmanufacturing.ToachievethisgoalinAIM1,wedevelopedthedocumentsandproductionchainofclosedbagsystemscon-taininga100McellhMSCworkingcellbank.Thisincludedtheevaluationandvalidationofmultiplecommerciallyavailablecryobagsaswellassourcingandcustomizationofafillingmanifoldsystemforproductproduction.Anoptimizedcontrolledratefreezingprotocolwasevaluatedforfinalproductmanufacturing,basedonthepostthawviability,viablecellconcentration,recoveredtotalvolu-me,and5dayhMSCexpansionprofiles.Followingproductdevelopmentop-timization,AIMIIofthisstudywastocompleteanddocumentacomparabilitystudybetweentheclosedsystembaggedhMSCswithstandardopensystemcryovials, in2half-scaleand1 full-scaleproduction runs.Thecomparabilityincludedassaysfocusingoncellviability,hMSCidentity,cellrecovery,aswellas on characterization of hMSC differentiation potential, cytokine secretionprofiles,andimmunomodulatorypotential.CompletionofbothAIMIandIIwillresult inafinalProduct/ProcessDevelopmentReportwith full comparabilitypackagetosupportdownstreamproductdocumentation.Within6-12monthsofcompletingthisproposal,RoosterBioexpectstobringtomarketthenovelclosedsystemhMSCworkingbanks,labeled‘ForFurtherManufacturing’.ThisprojectsupportedbytheMSCRF/TEDCOresultedincontinuedjobgrowthwit-hin thestateofMarylandaswellasadirect impact forRoosterBio throughin-stateexpenditurestosupportthiswork.Thefinalproductwillshaveseve-ralyearsandmillionsofdollarsoffthetypicalRegenerativeMedicineproductdevelopment timelines along with having a direct downstream impact through theapplicationofthedevelopedprocessesandknowledgetodevelopfurthernewandinnovativeproducts.InpartduetothesupportofMSCRFandTED-CORoosterBioiswellpoisedtobealeadingmanufacturerforRegenerativeMedicineforyearstocome.



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Manganese-enhanced MRI for Interrogating Astrocyte Replacement in A Mouse Model of Amyotrophic

Lateral Sclerosis

Mehreen Kouser1 , Jean-Philippe Richard2, Michael T. McMahon3, Nicholas J. Maragakis2, and Jeff W.M. Bulte1,3

1Johns Hopkins University School of Medicine, The Russell H. Morgan Department of Radiology and Radiological Science, Baltimore, MD; 2Johns Hopkins University School of Medicine, Department of Neurology and Neurosurgery, Baltimore, MD; 3Johns Hopkins University School of Medicine, Kennedy Krieger Institute, F.M. Kirby Center for Functional Brain Imaging, Baltimore, MD

AmyotrophicLateralSclerosis(ALS)isadevastatingdiseasewithnear100%mortality.Animportantrolehasbeenassignedto(over)activatedastrocytes.Given the fewavailable treatmentoptions,stemcell therapy isnowactivelybeingpursuedasanewtreatmentparadigm,aimedateitherimmunomodulati-on(usingmesenchymalstemcells-MSCs),astrocytereplacement(usingglialprogenitors),ormotorneuronrestoration,withseveralclinicaltrialscurrentlyinprogress.Ourgoalistobetterunderstandthemechanismsunderlyingastrocy-teactivationthatcreatesatoxicenvironmentformotorneuronscausingtheirdeath.Weaimtoreplacehostglial-restrictedprogenitors(GRPs)topreservethe neuronal environment using intraspinal transplantation and to develop no-velimagingbiomarkersthatcanreportnotonlyonthefateoftransplantedcellsbutalsoonchangesintheALShostenvironment.Tothisend,weinvestigatedtheactivationstateofhostcellsinvivousingmanganese(MnCl2)-enhancedmagneticresonanceimagingMRI(MEMRI).WeusedtransgenicSOD1G93AmalemicefromJAXasthemodelforALSandcomparedthemtotheageandsexmatchedwildtypecontrols.BothSOD1G93AandWTmicewererandomlyassignedtoeitherthesalineorMnCl2groupresultinginfourgroupstotalwhichwere, WT-Saline, WT-MnCl2, SODG93A-Saline, and SODG93A-MnCl2. Allmicewereimagedatpre-symptomatic(60day),onset(90day),andpost-sym-ptomatic(120day)stagesofALS.Imagesofthelumbarregionofthespinalcordwereobtainedbeforeand24hoursafteri.p.injectionofafreshsolutionof30mMMnCl2at0.4mmol/kgbodyweightinsalineattheabovestatedages.OurdatashowthatbothWTandSOD1G93AmiceexhibitanincreaseinT1wimagecontrast24hafterMnCl2injection.Moreover,incontrasttoourhypo-thesis, there isnoobservableT1wimagecontrastdifferenceasthediseaseprogressesingreyorwhitematterofthespinalcord.Immunohistologicalana-lysisofneuroinflammation,motorneurondegeneration,andastrocytepatholo-gyiscurrentlyunderway.MeanwhilewearealsoexploringotherbrainregionswherewemaybeabletoobserveactivatedhostastrocytesusingMEMRI.

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