aaron maki april 24, 2008. regeneration in nature outstanding examples planarian crayfish embryos...
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Aaron MakiApril 24, 2008
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Regeneration in NatureOutstanding Examples
PlanarianCrayfishEmbryos
Inverse RelationshipIncrease complexityDecrease regenerative ability
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Regeneration in HumansHigh Moderate Low
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Clinical NeedsCardiovascular
Myocardial infarctionStroke
BoneNon-union fracturesTumor resections
NervousSpinal Cord InjuryDegenerative diseases
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Stem CellsLong-term self-renewalClonogenicEnvironment-dependent differentiation
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Tissue EngineeringRepair/replace damaged tissues
Enhance natural regeneration
Cell SourceEmbryonic stem cells
Adult stem cellsProgenitor cells
SignalsGrowth factors
DrugsMechanical forces
ECMMetals
CeramicsSynthetic polymersNatural polymers
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Important VariablesDelivery
Cell SuspensionsTissue-like constructs (scaffolds)
Chemical propertiesGrowth factorsDegradation particlesECM surface
Physical propertiesStructureTopographyRigidityMechanical Loading
Modify Cell BehaviorSurvivalOrganizationMigrationProliferationDifferentiation
Optimize Cellular Response
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Stem and Progenitor CellsIsolation/Identification
Signature of cell surface markersSurface adherenceTranscription factors
ClassificationsEmbryonic Stem Cells Adult Stem Cells Induced Pluripotent Stem Cells
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Embryonic Stem CellsHighest level of pluripotency
All somatic cell typesUnlimited self-renewal
Enhanced telomerase activityMarkers
Oct-4, Nanog, SSEA-3/4
LimitationsTeratoma FormationAnimal pathogensImmune ResponseEthics
Strengths
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Potential SolutionsTeratoma Formation
Pre-differentiate cells in culture then insertAnimal pathogens
Feeder-free culture conditions (Matrigel)Immune Response
Somatic cell nuclear transferUniversalize DNA
Ethics
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Adult Stem Cells StrengthsEthics, not controversialImmune-privileged
Allogenic, xenogenic transplantation
Many sourcesMost somatic tissues
LimitationsDifferentiation Capacity?Self-renewal?Rarity among somatic cells
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Potential SolutionsDifferentiation Capacity
Mimic stem cell nicheLimited Self-renewal
Gene therapyLimited availability
Fluorescence-activated cell sortingAdherence
Heterogenous population works better clinically
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Mesenchymal Stem CellsEasy isolation, high expansion, reproducible
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Hematopoietic Stem CellsBest-studied, used clinically for 30+ years
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Induced Pluripotent Stem Cells
StrengthsPatient DNA matchSimilar to embryonic stem cells? LimitationsSame genetic pre-dispositionsViral gene delivery mechanism
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Potential SolutionsSame genetic pre-dispositions
Gene therapy in cultureViral gene delivery mechanism
Polymer, liposome, controlled-releaseUse of known onco-genes
Try other combinations
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Soluble Chemical FactorsTransduce signals
Cell type-dependentDifferentiation stage-dependent
Timing is criticalDose-dependence
GrowthSurvivalMotilityDifferentiation
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Scaffold purposeTemporary structural support
Maintain shapeCellular microenvironment
High surface area/volumeECM secretionIntegrin expressionFacilitate cell migration
Surface coating
Structural
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Ideal Extracellular Matrix3-dimensionalCross-linkedPorousBiodegradableProper surface chemistryMatching mechanical strengthBiocompatiblePromotes natural healingAccessibilityCommercial Feasibility
Modulate PropertiesPhysical, ChemicalCustomize scaffold
Appropriate Trade-offsTissue
Disease condition
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“Natural” MaterialsPolymers
CollagenLamininFibrinMatrigelDecellularized matrix
CeramicsHydroxyapatiteCalcium phosphateBioglass
Perfusion-decellularized matrix: using nature's platform to engineer a bioartificial heart.
Ott, et al.Nat Med. 2008 Feb;14(2):213
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Important scaffold variablesSurface chemistryMatrix topography
Cell organization, alignmentFiber alignment -> tissue development
Rigidity5-23 kPa
PorosityLarge interconnectedsmall disconnected
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Mechanical ForcesFlow-induced shear stress
Laminar blood flowRhythmic pulses
Uniaxial, Equiaxial stretchMagnitudeFrequency
MechanotransductionConversion of a mechanical stimulus into a biochemical
response
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Flow-induced shear stress2D parallel plate flow chamber
Hemodynamic forceLaminar flowPulsatile component
3D matrixInterstitial flowBone: oscillating
Cell-type specific
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Models for Tissue EngineeringIn vitro differentiation
Construct tissues outside body before transplantation
Ultimate goal Most economical Least waiting time
In situ methodologyHost remodeling of environment
Ex vivo approachExcision and remodeling in culture
Combine physical and chemical factors
Optimize stem cell differentiation and organization
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Delivery MethodsInjectable stem cells
Cells or cell-polymer mixLess invasiveAdopt shape of environmentControlled growth factor release
Solid scaffold manufacturingComputer-aided designMatch defect shape
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Cardiovascular Tissue EngineeringHeals poorly after damage (non-functional
scar tissue)Myocardial infarction
60% survival rate after 2 years>40% tissue death requires transplantation
More patients than organ donors
Heart attack and strokesFirst and third leading causes of deathPatient often otherwise healthy
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Current interventionsBalloon angioplasty
Expanded at plaque site, contents collectedVascular stent
Deploy to maintain openingSaphenous vein graft
Gold StandardForm new conduit, bypass blockage
All interventions ultimately fail10 years maximum lifetime
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Cardiovascular Tissue EngineeringCell Source
Embryonic stem cellsMesenchymal stem cells
Endothelial progenitor cellsResident Cardiac SCs
SignalsVEGFTGF-βFGFBMP
PDGFShear stressAxial strain
ECMMatrigelCollagenAlginate
FibrinDecellularized Tissue
PLAPGA
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Clinical QuestionsWhat cell source do you use?How should cells be delivered?What cells within that pool are beneficial?How many cells do you need?When should you deliver the cells?What type of scaffold should be used?
These answers all depend on each other
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Very sensitive to methodology!2 nearly identical clinical trials, opposite results
Autologous Stem cell Transplantation in Acute Myocardial Infarction (ASTAMI)
Reinfusion of Enriched Progenitor cells And Infarct Remodeling in Acute Myocardial Infarction (REPAIR-AMI)
Same inclusion criteriaSame cell source (Bone marrow aspirates)Same delivery mechanism (intracoronary infusion)Same timing of deliverySIMILAR cell preparation methods
Seeger et al. European Heart Journal 28:766-772 (2007)
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Cell preparation comparisonBone marrow aspirates
diluted with 0.9% NaCl (1:5)
Mononuclear cells isolated on Lymphoprep™ gradient 800rcf 20 min
Washed 3 x 45 mL saline + 1% autologous plasma (250rcf)
Stored overnight 4°C saline + 20 autologous plasma
Bone marrow aspirates diluted with 0.9% NaCl (1:5)
Mononuclear cells isolated on Ficoll™ gradient 800rcf 20 min
Washed 3 x 45mL PBS (800rcf)
Stored overnight room temperature in 10 + 20% autologous serum
Courtesy of Dr. Tor Jensen
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Future DirectionsStandardization
Central cell processing facilitiesProtocols
Improved antimicrobial methodsAllergies
Synthetic biologyNatural materials made synthetically,
economically
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Long-term: “clinical-grade” cell linesAnimal-substance free conditions
Human feeder cells, chemically-defined mediaFeeder-free culture
No immune rejection, no immunosuppressive drugsSomatic cell nuclear transferGenetic engineering, reprogramming
Goals: understand normal/disease development, then repair/replace diseased organs and vice versaTissue engineering approach
ex vivo, in situ for now In vitro for the future?
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SummaryRight combination of cell, scaffold, and
factors depends on clinical problemExtensive physician/scientist/engineering
collaboration is vital to successTissue engineering is leveraging our
knowledge of cell biology and materials science to promote tissue regeneration where the natural process is not enoughStem cells are an excellent tool for this task