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

WT4

0 5 10 15 200

255075

100125150 CF1

WT3-

0 5 10 15 20

40

0

20

50100150200

-NCAM-1AFPVEGFR2

Mea

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ld c

hang

e in

targ

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ne e

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n (T

rans

crip

t cop

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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

0

20

40

60

80

100

120

PSA-NCAM Nucleostemin Sox-2 Musashi-1

Neural stem cell marker

Mea

n pe

rcen

tage

of i

mm

uno-

reac

tive

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 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)