Therapeutic and Research Potential of Human Stem Cells...
Transcript of Therapeutic and Research Potential of Human Stem Cells...
Therapeutic and Research Potential of Human Stem Cells –
Prospects and Challenges
Dr Stephen L MingerDr Stephen L MingerStem Cell Biology LaboratoryStem Cell Biology Laboratory
Wolfson Centre for AgeWolfson Centre for Age--Related DiseasesRelated DiseasesKingKing’’s College Londons College London
Focal Point, Biomedicine in ChinaFocal Point, Biomedicine in China
Stem Cell Expert Stem Cell Expert –– UK Gene Therapy UK Gene Therapy Advisory Committee (GTAC)Advisory Committee (GTAC)
Manipulation of Stem Cells for Manipulation of Stem Cells for Research &/or Therapeutic UseResearch &/or Therapeutic Use
Expansion & Differentiation of Pluripotent Expansion & Differentiation of Pluripotent Cells for TransplantationCells for TransplantationStimulation of Endogenous TissueStimulation of Endogenous Tissue--Specific Specific Stem/Progenitor Cells for Tissue RepairStem/Progenitor Cells for Tissue RepairExpansion & Differentiation of Pluripotent Expansion & Differentiation of Pluripotent Cells for Drug Discovery/ToxicologyCells for Drug Discovery/ToxicologyGeneration of DiseaseGeneration of Disease--Specific Pluripotent Specific Pluripotent Cell Line for Cell Line for ““Disease in a dishDisease in a dish”” ModellingModelling
ParkinsonParkinson’’s Disease and the Brains Disease and the Brain
Progressive and sustainedimprovement in transplantfunction over ten years inhuman graft recipient
Piccini et al, 1999, NatureNeurosci, 2
Stem Cells – Types, Flavours and Sources
Pluripotent Multipotent
Multipotent
Multipotent
Cell transplantationCell transplantation and stem cells: and stem cells: minimal requirements for successminimal requirements for success
Stem/progenitor cells must proliferate for extended Stem/progenitor cells must proliferate for extended periods and generate 100,000,000,000+s of cellsperiods and generate 100,000,000,000+s of cellsStem/progenitor cell phenotype must be stable over time Stem/progenitor cell phenotype must be stable over time with no loss of cellular potencywith no loss of cellular potencyStem/progenitor cells must be capable of generating Stem/progenitor cells must be capable of generating required cell types upon differentiationrequired cell types upon differentiationStem/progenitor cells or their differentiated progeny Stem/progenitor cells or their differentiated progeny must survive implantation, functionally integrate into must survive implantation, functionally integrate into host adult tissue, evade immune rejection, and provide host adult tissue, evade immune rejection, and provide longlong--term therapeutic benefit. term therapeutic benefit.
Austin Smith, 2003
With Good Karma, Embryos, andCulture Conditions, Human ESCells Can Be Established At Frequency of ~10%
A, B, Cystic Fibrosis Line (CF-1)C HES Cell Line (WT-4)
A B
CC
Oct-4
β-Tubulin(ectoderm)
Smooth Muscle Actin (mesoderm) Albumin (endoderm)
ES cells
Pluripotent MultipotentTissue-specificstem cells
Neural Stem Cells
Pancreatic Stem Cells
Bi or Tri- potentialProgenitor cells
Other Stem Cells
Neurones
Oligos
Astrocytes
Tissue-specific cell types
Insulin Glucagon
Generation of multipotent somatic stem cells from pluripotent embryonic stem cells
Generation of Differentiated Somatic Cell Generation of Differentiated Somatic Cell Populations from Human ES CellsPopulations from Human ES Cells
Embryoid Body (EB) FormationEmbryoid Body (EB) FormationEfficient process Efficient process –– gives rise to many diff cell typesgives rise to many diff cell typesVery mixed populations (Very mixed populations (endoendo--, , mesomeso-- ectoderm)ectoderm)
EB Formation with Defined EB Formation with Defined MitogenicMitogenic FactorsFactorsUncertainty as to which factors support cell types of interestUncertainty as to which factors support cell types of interest
Direct Selection by Direct Selection by MitogenicMitogenic SelectionSelectionOnly cells capable of responding to individual factors should prOnly cells capable of responding to individual factors should proliferateoliferate
CoCo--Culture of Culture of EBsEBs/ES cells with Somatic Cell Lines/ES cells with Somatic Cell LinesOften requires physical contact/close proximity to target cellsOften requires physical contact/close proximity to target cellsCells often of animal origin and require serum for growthCells often of animal origin and require serum for growth
Lineage Specification and SelectionLineage Specification and SelectionRequires cell surface antigen unique to cell population of interRequires cell surface antigen unique to cell population of interestestRequires knowledge of transcription factors expressed early in dRequires knowledge of transcription factors expressed early in development of evelopment of target tissue target tissue –– genetic modification and selectiongenetic modification and selectionRequires Selection of Target Population Requires Selection of Target Population --> Gene Transfer, > Gene Transfer, FACsFACs, , ImmunoselectionImmunoselection
Human Embryoid Bodies – 6 days post-differentiation
In vitro expression of germ layer markers during differentiation.
0 5 10 15 20
4002501001007550250
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WT3-
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-NCAM-1AFPVEGFR2
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Differentiation time (Days)
All data are expressed relative to an undifferentiated hES cell and normalised for the GAPDH house-keeping gene. n=5. Bars represent S.E.M.
Human Neural Stem/Progenitor Cells Derived from Human Embryonic Stem Cells
Taylor and Minger, unpublished
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PSA-NCAM Nucleostemin Sox-2 Musashi-1
Neural stem cell marker
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Generation of Differentiated Somatic Cell Generation of Differentiated Somatic Cell Populations from Human ES CellsPopulations from Human ES Cells
Embryoid Body (EB) FormationEmbryoid Body (EB) FormationEfficient process Efficient process –– gives rise to many diff cell typesgives rise to many diff cell typesVery mixed populations (Very mixed populations (endoendo--, , mesomeso-- ectoderm)ectoderm)
EB Formation with Defined EB Formation with Defined MitogenicMitogenic FactorsFactorsUncertainty as to which factors support cell types of interestUncertainty as to which factors support cell types of interest
Direct Selection by Direct Selection by MitogenicMitogenic SelectionSelectionOnly cells capable of responding to individual factors should prOnly cells capable of responding to individual factors should proliferate oliferate
CoCo--Culture of Culture of EBsEBs/ES cells with Somatic Cell Lines/ES cells with Somatic Cell LinesOften requires physical contact/close proximity to target cellsOften requires physical contact/close proximity to target cellsCells often of animal origin and require serum for growthCells often of animal origin and require serum for growth
Lineage Specification and SelectionLineage Specification and SelectionRequires cell surface antigen unique to cell population of interRequires cell surface antigen unique to cell population of interestestRequires knowledge of transcription factors expressed early in Requires knowledge of transcription factors expressed early in development of target tissue development of target tissue –– genetic modification and selectiongenetic modification and selectionRequires Selection of Target Population Requires Selection of Target Population --> Gene Transfer, > Gene Transfer, FACsFACs, , ImmunoselectionImmunoselection
Neuroepithelial stem cell
Dopaminergic Progenitor Cell
Post-mitotic differentiating DA neuron
Differentiated neuron
Lmx1aMsx1
Aldh1, Lmx1bNgn2
Nurr-1En1, En2, Wnt1, Pax2, Pax5
Ptx3 TH, RET
Nurr-1, Ptx3, Lmx1b, En1, En2, Aldh1, TH
Gestational week 4 6.55
Proposed induction and development of mesencephalic dopaminergic neurons in the human midbrain.
Onset Of DA Neuron Markers During Embryonic Onset Of DA Neuron Markers During Embryonic Development Of Human MidbrainDevelopment Of Human Midbrain
Taylor and Minger, 2005
Sox1
Timeline for Clinical Translation of PD Cell Therapy
Human LineageHuman Lineage--Specific Specific Transcription FactorsTranscription Factors
Cloned Cloned Sox 1Sox 1 neuroectodermneuroectodermOligOlig 22 oligodendroglialoligodendroglialPaxPax 66 retinal & S cordretinal & S cordNkxNkx 2.5 2.5 cardiaccardiacGATAGATA--44 cardiac cardiac Tbx5Tbx5 cardiaccardiacPdxPdx--11 pancreaticpancreaticLmx1bLmx1b dopaminergicdopaminergicLmx1aLmx1a dopaminergicdopaminergicMsx1Msx1 dopaminergic dopaminergic Ptx3Ptx3 dopaminergicdopaminergicNgn2Ngn2 dopaminergicdopaminergicNurr1Nurr1 dopaminergicdopaminergic
In progressIn progressHb9Hb9 motor neuronmotor neuronSox 9Sox 9 chondrocyteschondrocytesRunx2Runx2 osteoblastsosteoblastsPaxPax 44 pancreaticpancreaticSox 10 Sox 10 oligodendroglialoligodendroglialSox17Sox17 pancreaticpancreaticFoxJ1FoxJ1 lunglungGATA6GATA6 lunglungTITF1TITF1 lunglungGsh2Gsh2 ventral forebrainventral forebrainNkx2.1Nkx2.1 ventral forebrainventral forebrainChATChAT cholinergiccholinergic
Results for undifferentiated cellsResults for undifferentiated cellsC-peptide Hoechst Merged image
Insulin Hoechst Merged image
Luo, Wu, Jones and Minger, in preparation
Results for differentiated cellsResults for differentiated cellsGlucagon Hoechst
Merged image
Luo, Wu, Jones and Minger, in preparation
Pure Clonal Populations of Highly Expandable Mesoendodermal Progenitor Cells Generated
from GATA-4-transduced mES cells
Shatapathy & Minger, unpublished data
Stem Cell Biology Laboratory Cellular Targets Stem Cell Biology Laboratory Cellular Targets ––Therapy and Drug DiscoveryTherapy and Drug Discovery
CNS DisordersCNS DisordersSpinal Cord Cell types (McMahon, Bradbury) Spinal Cord Cell types (McMahon, Bradbury) -- traumatic injury, Multiple traumatic injury, Multiple SclerosisSclerosisDopaminergic Progenitor Cells Dopaminergic Progenitor Cells -- ParkinsonParkinson’’s disease (Duty, Jenner) s disease (Duty, Jenner)
Cardiac RegenerationCardiac RegenerationCardiac Progenitor Cells/Endothelial Cells for Heart Failure/HeaCardiac Progenitor Cells/Endothelial Cells for Heart Failure/Heart Attack rt Attack (Mathur/Martin & British Collective on Cell Therapy in the Heart(Mathur/Martin & British Collective on Cell Therapy in the Heart))
Retinal RegenerationRetinal RegenerationRetinal Stem Cells for Macular Degeneration, Retinitis Retinal Stem Cells for Macular Degeneration, Retinitis PigmentosaPigmentosa(Ali, Institute of Ophthalmology)(Ali, Institute of Ophthalmology)
Endocrine DisordersEndocrine DisordersInsulinInsulin--Producing Islet Cells for Type I Diabetes (Jones, Producing Islet Cells for Type I Diabetes (Jones, PersaudPersaud & King& King’’s s Islet Transplant Programme)Islet Transplant Programme)
Hepatic RegenerationHepatic RegenerationHepatic Stem Cells for Hepatic Insufficiency (KingHepatic Stem Cells for Hepatic Insufficiency (King’’s Liver Transplant s Liver Transplant Programme)Programme)
Joint and Bone DestructionJoint and Bone DestructionChondrocytes for Cartilage Repair (Hollander, Oreffo)Chondrocytes for Cartilage Repair (Hollander, Oreffo)OsteoblastsOsteoblasts for Bone Replacement (Grigoriadis)for Bone Replacement (Grigoriadis)
Epithelial CellsEpithelial CellsLung Differentiation (Novartis)Lung Differentiation (Novartis)Epithelial Cells for Genetic Skin Disorders (McGrath)Epithelial Cells for Genetic Skin Disorders (McGrath)
Developments Required for Therapeutic Developments Required for Therapeutic Application of Human ES CellsApplication of Human ES Cells
Generation of AnimalGeneration of Animal--Free, Chemically Defined Free, Chemically Defined Culture ConditionsCulture ConditionsIdentification of hES Growth Factors/Small Identification of hES Growth Factors/Small MoleculesMoleculesControlled Differentiation Controlled Differentiation ––> Generation of > Generation of Specific Cell PopulationsSpecific Cell PopulationsTesting of Therapeutically Important Cell Testing of Therapeutically Important Cell Populations in Animal Models Populations in Animal Models –– Relevance to Relevance to Human Clinical Conditions?Human Clinical Conditions?Species Requirements for Safety/Efficacy?Species Requirements for Safety/Efficacy?
Developments Required for Therapeutic Developments Required for Therapeutic Application of Human ES CellsApplication of Human ES Cells
Delivery/Design of Cells for Therapeutic Delivery/Design of Cells for Therapeutic Applications Applications –– suspension, aggregates, scaffoldssuspension, aggregates, scaffoldsImmune Response Immune Response –– Immunosuppression, Immunosuppression, SCNT, hES Cell Banks, Encapsulation, Induction SCNT, hES Cell Banks, Encapsulation, Induction of Tolerance?of Tolerance?Generation of TherapeuticGeneration of Therapeutic--Grade hES Grade hES CelllCelll Lines Lines –– Will Require Specialised GMPWill Require Specialised GMP--Level Facilities & Level Facilities & Expertise (KingExpertise (King’’s s ££4M 4M –– opening now)opening now)
Stimulation of Adult NeurogenesisStimulation of Adult Neurogenesis
Goal Goal Elucidate Mechanisms underlying IschaemiaElucidate Mechanisms underlying Ischaemia--Induced Induced Enhancement of Neurogenesis Enhancement of Neurogenesis
FundingFundingResearch into AgingResearch into AgingAlzheimerAlzheimer’’s Research Trusts Research Trust
PeoplePeopleDr Antigoni EkonomouDr Antigoni Ekonomou Prof Robert Perry (Newcastle)Prof Robert Perry (Newcastle)Prof Clive BallardProf Clive Ballard Prof Raj Prof Raj KalariaKalaria (Newcastle)(Newcastle)Prof Elaine PerryProf Elaine Perry Dr Omar Pathmanaban (Dr Omar Pathmanaban (MancManc))Mr Christos ToliasMr Christos ToliasDr Mike ModoDr Mike ModoDr Manuel MayrDr Manuel Mayr
Adult Neurogenesis
Human adult braincontains proliferativeneural stem cells in dentate gyrus and lateral ventricle wall (SVZ) that generate new neurons throughout life
Eriksson et al, 1998, NatureMed, 4, 1313
Regulators of Adult NeurogenesisRegulators of Adult Neurogenesis
PositivePositive
Enriched EnvironmentEnriched EnvironmentPhysical Activity (e.g. Running)Physical Activity (e.g. Running)AntidepressantsAntidepressantsCaloric RestrictionCaloric RestrictionLearningLearningStroke (experimental)Stroke (experimental)Pregnancy (Pregnancy (ProlactinProlactin))Inflammatory Blockade (NSAIDS)Inflammatory Blockade (NSAIDS)VEGF, GVEGF, G--CSF, NO, SCFCSF, NO, SCFStatins (AntiStatins (Anti--lipids)lipids)LithiumLithium
NegativeNegative
OpiatesOpiatesMethamphetamineMethamphetamineSeizuresSeizuresAgingAgingStress (Stress (GlucocorticoidsGlucocorticoids))Serotonin Depletion Serotonin Depletion Nitric OxideNitric OxideInflammation Inflammation InterleukinInterleukin--6 (from activated 6 (from activated microglia)microglia)IrradiationIrradiationNicotine Nicotine
Minger et al, 2007
Ekonomou et al, submitted
Ischaemia-induced Proliferation and Migration of NeuralStem/Progenitor Cells into Region ofVascular Damage
Adult Human SVZ Neural Progenitor Cells
Generation of DiseaseGeneration of Disease--Specific Specific Human ES Cell Lines Human ES Cell Lines
Use of PGD Embryos with Genetic Lesions (CF, HD)Use of PGD Embryos with Genetic Lesions (CF, HD)Prof Peter BraudeProf Peter BraudeEmma StephensonEmma StephensonSara HallSara HallGlenda CornwallGlenda CornwallFunded by Medical Research CouncilFunded by Medical Research Council
KnockKnock--in of Disease Genes using Homologous in of Disease Genes using Homologous Recombination (HD)Recombination (HD)
Prof Gillian BatesProf Gillian BatesDr Liza SuttonDr Liza SuttonEva SirinathsinghjiEva SirinathsinghjiDr Stefanie Gogel Dr Stefanie Gogel Funded by the High Q Foundation and HuntingtonFunded by the High Q Foundation and Huntington’’s Disease s Disease AssociationAssociation
Eggs from cow ovaries
Nucleus removedHuman skin cell
Enucleated egg
Nucleartransfer
Stimulus foregg to divide
Cell division
Day 3
Day 6
Embryonic stem cell culture
Colony of embryonic stem cells
Schematic representation of procedure for deriving stem cells from human “admixed” embryos
Inner cell mass (source of stem cells)
Induction of inducedPluripotent Stem (iPS) Cellsfrom Adult Human DermalFibroblasts
Requires Retroviral-MediatedGene Transfer of:
n-MycOct-4Sox2Klf4
Takahashi et al., (2007) Cell 131, 861-872
SCNT/iPS hES Cell LinesSCNT/iPS hES Cell Lines
Use fibroblasts from patients with FAD, SMA, Use fibroblasts from patients with FAD, SMA, ApoEApoE εε4+/4+/εε4+ and genetic forms of PD, MND, 4+ and genetic forms of PD, MND, FTD FTD Clone cells into nonClone cells into non--human oocytes & iPShuman oocytes & iPSGenerate cloned hES cell linesGenerate cloned hES cell linesDifferentiate hES cells Differentiate hES cells --> disease> disease--specific specific selectively vulnerable neural populations selectively vulnerable neural populations Establish Establish ““disease in a dishdisease in a dish”” cellular modelscellular models
Strengths of UK Stem Cell ResearchStrengths of UK Stem Cell Research
WorldWorld--class academic research centresclass academic research centresTight regulation on Assisted Reproduction & Human Tight regulation on Assisted Reproduction & Human Embryo Research by HFEAEmbryo Research by HFEAGovernment Commitment to Stem Cell Research (~Government Commitment to Stem Cell Research (~££45 45 million million –– 20032003--2004); 2004); ££350350--800 million 10800 million 10--year strategy year strategy from Chancellorfrom Chancellor’’s Office announced Dec 2005 s Office announced Dec 2005 Government Funded UK Stem Cell Bank for foetal, adult Government Funded UK Stem Cell Bank for foetal, adult & embryonic human stem cell lines& embryonic human stem cell linesLondon Regenerative Medicine Network London Regenerative Medicine Network –– drive towards drive towards clinical applicationsclinical applicationsUK Stem Cell Network UK Stem Cell Network –– integration of all UK regional integration of all UK regional networknetwork
Stem Cell Biology LabStem Cell Biology Lab
Antigoni EkonomouAntigoni EkonomouYue WuYue WuSarah PringleSarah PringleMiriam GubernatorMiriam GubernatorChetan ShatapathyChetan ShatapathyNoor AlmaaniNoor AlmaaniLiza SuttonLiza SuttonSH CedarSH CedarKaterina VaranouKaterina VaranouAlyma SomaniAlyma SomaniSarah HardwickSarah HardwickStephanie GogelStephanie GogelOmar PathmanabanOmar Pathmanaban
Sara HallSara HallHannah TaylorHannah TaylorDaniel WebberDaniel WebberZhenling Zhenling LuoLuoMinalMinal PatelPatelJessica CookeJessica CookeJulie GhoshJulie GhoshNiloufar SafiniaNiloufar Safinia
Stem Cell Biology Laboratory CollaboratorsStem Cell Biology Laboratory CollaboratorsHuman ES CellsHuman ES CellsPeter BraudePeter BraudeSara HallSara HallGlenda CornwallGlenda CornwallEmma StephensonEmma Stephenson
Spinal Cord RepairSpinal Cord RepairSteve McMahonSteve McMahonLiz BradburyLiz BradburyPaul FeltsPaul FeltsMerion Davies Merion Davies
Retinal RegenerationRetinal RegenerationRobin Ali (UCL/Robin Ali (UCL/MoorfieldsMoorfields))
Pancreatic Islet Cells Pancreatic Islet Cells Peter JonesPeter JonesShantaShanta PersaudPersaud
ParkinsonParkinson’’s Diseases DiseasePeter JennerPeter JennerSusan DutySusan Duty
Tooth ReplacementTooth ReplacementPaul SharpePaul Sharpe
Neural DifferentiationNeural DifferentiationJonathan CorcoranJonathan CorcoranBiaBia GonclavesGonclaves
iNANO (Aarhus)iNANO (Aarhus)Morten FossMorten FossMogens DuchMogens Duch
Medical Research Council, Mrs Lily Safra, BBSRC, ESRC, EPSRC, Medical Research Council, Mrs Lily Safra, BBSRC, ESRC, EPSRC, European Commission, Oliver Bird Foundation, UK DTI, European Commission, Oliver Bird Foundation, UK DTI, Francesca Patrizi,Francesca Patrizi,
High Q Foundation, Wellcome Trust, HuntingtonHigh Q Foundation, Wellcome Trust, Huntington’’s Disease Societys Disease Society, John &, John & Helen Robertson, Helen Robertson, AlzheimerAlzheimer’’s Research Trust, Liz & John Hancock, Guys Research Trust, Liz & John Hancock, Guy’’s & St Thomass & St Thomas’’ Charitable TrustCharitable Trust
Adult NeurogenesisAdult NeurogenesisClive BallardClive BallardOmar PathmanabanOmar PathmanabanManuel MayrManuel MayrChristos ToliasChristos ToliasElaine Perry (Newcastle)Elaine Perry (Newcastle)Robert Perry (Newcastle)Robert Perry (Newcastle)
Vascular DevelopmentVascular DevelopmentKaren Hirschi (Baylor)Karen Hirschi (Baylor)Qingbo XuQingbo Xu
Blood Stem CellsBlood Stem CellsPaul Fairchild (Oxford)Paul Fairchild (Oxford)Karl Karlsson (Oxford)Karl Karlsson (Oxford)
Lung DifferentiationLung DifferentiationCarol Jones (Novartis)Carol Jones (Novartis)Phil Kemp (Novartis)Phil Kemp (Novartis)
Bioethics & PoliticsBioethics & PoliticsClaire WilliamsClaire WilliamsSteve Steve WainrightWainrightSarah Franklin (LSE)Sarah Franklin (LSE)Brian Salter (Norwich)Brian Salter (Norwich)Herbert Gottweis (Vienna)Herbert Gottweis (Vienna)
ElectrophysiologyElectrophysiologyReginald DochertyReginald Docherty
Cardiac RepairCardiac RepairAnthony Mathur (Anthony Mathur (BartsBarts))Ken Suzuki (Ken Suzuki (BartsBarts))
ProteomicsProteomicsTony NgTony NgSimon Simon AmeerAmeer--BegBegManuel MayrManuel Mayr
HuntingtonHuntington’’s Diseases DiseaseGillian BatesGillian BatesEva SirinathsinghjiEva SirinathsinghjiLiza SuttonLiza Sutton
Hepatic Stem CellsHepatic Stem CellsAnillAnill DhawanDhawanRobin HughesRobin HughesRagai MitryRagai Mitry
Joint and Bone Joint and Bone DevelopmentDevelopmentAgi GrigoriadisAgi Grigoriadis
Skin DifferentiationSkin DifferentiationJohn McGrathNoor Almaani
LentivirusLentivirus ProductionProductionAdrian Thrasher (GOSH)Adrian Thrasher (GOSH)