Aaron MakiApril 24, 2008

Regeneration in NatureOutstanding Examples

PlanarianCrayfishEmbryos

Inverse RelationshipIncrease complexityDecrease regenerative ability

Regeneration in HumansHigh Moderate Low

Clinical NeedsCardiovascular

Myocardial infarctionStroke

BoneNon-union fracturesTumor resections

NervousSpinal Cord InjuryDegenerative diseases

Stem CellsLong-term self-renewalClonogenicEnvironment-dependent differentiation

Tissue EngineeringRepair/replace damaged tissues

Enhance natural regeneration

Cell SourceEmbryonic stem cells

Adult stem cellsProgenitor cells

SignalsGrowth factors

DrugsMechanical forces

ECMMetals

CeramicsSynthetic polymersNatural polymers

Important VariablesDelivery

Cell SuspensionsTissue-like constructs (scaffolds)

Chemical propertiesGrowth factorsDegradation particlesECM surface

Physical propertiesStructureTopographyRigidityMechanical Loading

Modify Cell BehaviorSurvivalOrganizationMigrationProliferationDifferentiation

Optimize Cellular Response

Stem and Progenitor CellsIsolation/Identification

Signature of cell surface markersSurface adherenceTranscription factors

ClassificationsEmbryonic Stem Cells Adult Stem Cells Induced Pluripotent Stem Cells

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

Potential SolutionsTeratoma Formation

Pre-differentiate cells in culture then insertAnimal pathogens

Feeder-free culture conditions (Matrigel)Immune Response

Somatic cell nuclear transferUniversalize DNA

Ethics

Adult Stem Cells StrengthsEthics, not controversialImmune-privileged

Allogenic, xenogenic transplantation

Many sourcesMost somatic tissues

LimitationsDifferentiation Capacity?Self-renewal?Rarity among somatic cells

Potential SolutionsDifferentiation Capacity

Mimic stem cell nicheLimited Self-renewal

Gene therapyLimited availability

Fluorescence-activated cell sortingAdherence

Heterogenous population works better clinically

Mesenchymal Stem CellsEasy isolation, high expansion, reproducible

Hematopoietic Stem CellsBest-studied, used clinically for 30+ years

Induced Pluripotent Stem Cells

StrengthsPatient DNA matchSimilar to embryonic stem cells? LimitationsSame genetic pre-dispositionsViral gene delivery mechanism

Potential SolutionsSame genetic pre-dispositions

Gene therapy in cultureViral gene delivery mechanism

Polymer, liposome, controlled-releaseUse of known onco-genes

Try other combinations

Soluble Chemical FactorsTransduce signals

Cell type-dependentDifferentiation stage-dependent

Timing is criticalDose-dependence

GrowthSurvivalMotilityDifferentiation

Scaffold purposeTemporary structural support

Maintain shapeCellular microenvironment

High surface area/volumeECM secretionIntegrin expressionFacilitate cell migration

Surface coating

Structural

Ideal Extracellular Matrix3-dimensionalCross-linkedPorousBiodegradableProper surface chemistryMatching mechanical strengthBiocompatiblePromotes natural healingAccessibilityCommercial Feasibility

Modulate PropertiesPhysical, ChemicalCustomize scaffold

Appropriate Trade-offsTissue

Disease condition

“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

Important scaffold variablesSurface chemistryMatrix topography

Cell organization, alignmentFiber alignment -> tissue development

Rigidity5-23 kPa

PorosityLarge interconnectedsmall disconnected

Mechanical ForcesFlow-induced shear stress

Laminar blood flowRhythmic pulses

Uniaxial, Equiaxial stretchMagnitudeFrequency

MechanotransductionConversion of a mechanical stimulus into a biochemical

response

Flow-induced shear stress2D parallel plate flow chamber

Hemodynamic forceLaminar flowPulsatile component

3D matrixInterstitial flowBone: oscillating

Cell-type specific

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

Delivery MethodsInjectable stem cells

Cells or cell-polymer mixLess invasiveAdopt shape of environmentControlled growth factor release

Solid scaffold manufacturingComputer-aided designMatch defect shape

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

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

Cardiovascular Tissue EngineeringCell Source

Embryonic stem cellsMesenchymal stem cells

Endothelial progenitor cellsResident Cardiac SCs

SignalsVEGFTGF-βFGFBMP

PDGFShear stressAxial strain

ECMMatrigelCollagenAlginate

FibrinDecellularized Tissue

PLAPGA

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

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)

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

Future DirectionsStandardization

Central cell processing facilitiesProtocols

Improved antimicrobial methodsAllergies

Synthetic biologyNatural materials made synthetically,

economically

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?

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