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Biao Lu, MD, PhDBiao Lu, MD, PhDNov. 23Nov. 23rdrd 20092009
Stem Cell Technology and Its Stem Cell Technology and Its
Application to Biomedical ResearchApplication to Biomedical Research
Stem Cell TechnologyStem Cell Technology
1. History of stem cell technology1. History of stem cell technology
2. Main applications of stem cells2. Main applications of stem cells
3. Methods to generate stem cells3. Methods to generate stem cells
4.4. Core technologies and effective tools that Core technologies and effective tools that
are useful to stem cell researchers are useful to stem cell researchers
OutlineOutline
Embryonic germ cellsEmbryonic germ cells
Mouse embryonic stem cellsMouse embryonic stem cells
Induced pluripotent Induced pluripotent
stem cells (iPS cells)stem cells (iPS cells)
19641964 19811981 19981998
Human embryonic stem cellsHuman embryonic stem cells
20062006
1. History of Stem Cell Research1. History of Stem Cell Research
Types of Stem CellsTypes of Stem Cells
1. ES cells1. ES cells
2. 2. iPSiPS cellscells
Human Stem Cell ColonyHuman Stem Cell Colony
What Are Stem Cells?What Are Stem Cells?
Stem cells Stem cells are unspecialized cells that are able are unspecialized cells that are able
to reproduce themselves indefinitely and, under to reproduce themselves indefinitely and, under
the right conditions, to differentiate into the right conditions, to differentiate into
virtually any mature cell types (>200 cell types).virtually any mature cell types (>200 cell types).
Two critically important properties:Two critically important properties:
1.1. SelfSelf--renewalrenewal
2.2. PluripotencyPluripotency
Stem CellsStem Cells1 Molecular MechanismsMolecular Mechanisms
1.1. PluripotencyPluripotency
2.2. Cell differentiationCell differentiation
3.3. Tissue developmentTissue development
4.4. Organ genesisOrgan genesis
2. Applications of Stem Cells2. Applications of Stem Cells
Stem CellsStem Cells1
Applications of Stem Cells in Applications of Stem Cells in
Biomedical ResearchBiomedical Research
Molecular MechanismsMolecular Mechanisms
Human Disease ModelsHuman Disease Models2Examples:Examples:
1.1. FragileFragile--X syndromeX syndrome
2.2. Cystic fibrosisCystic fibrosis
3.3. Sickle cell anemiaSickle cell anemia
Stem CellsStem Cells1
Applications of Stem Cells in Applications of Stem Cells in
Biomedical ResearchBiomedical Research
Molecular MechanismsMolecular Mechanisms
Human Disease ModelsHuman Disease Models2Examples:Examples:
1.1. FragileFragile--X syndromeX syndrome
2.2. Cystic fibrosisCystic fibrosis
3.3. Sickle cell anemiaSickle cell anemia
4.4. Parkinson diseaseParkinson disease
Stem CellsStem Cells1
Applications of Stem Cells in Applications of Stem Cells in
Biomedical ResearchBiomedical Research
Molecular MechanismsMolecular Mechanisms
Human Disease ModelsHuman Disease Models2
Drug DiscoveryDrug Discovery
1.1. Drug screenDrug screen
2.2. Toxicity studyToxicity study
3
Stem CellsStem Cells
NeuronsNeuronsCardiac Cardiac
Muscle CellsMuscle CellsBetaBeta--cellscells
Cell Replacement TherapyCell Replacement Therapy
4
Applications of Stem Cells in Applications of Stem Cells in
Biomedical ResearchBiomedical Research
3. How Do You Isolate Stem Cells?3. How Do You Isolate Stem Cells?
1. A brief history of stem cell technology1. A brief history of stem cell technology
2. Main applications of stem cells2. Main applications of stem cells
3. Methods to generate stem cells3. Methods to generate stem cells
4. Core technologies and effective tools that 4. Core technologies and effective tools that
are useful to stem cell researchers are useful to stem cell researchers
ES cellES cell
IsolationIsolation
1.1.Ethical issuesEthical issues
2.2. ImmuneImmune--rejection problemsrejection problems
Generation of Stem CellsGeneration of Stem Cells
NuclearNuclear
ReprogramReprogram
iPSiPS
cellscells
Generation of Stem CellsGeneration of Stem Cells
Part 4: Tools and Technologies Part 4: Tools and Technologies
1. A brief history of stem cell technology1. A brief history of stem cell technology
2. Main applications of stem cells2. Main applications of stem cells
3. Methods for generating stem cells3. Methods for generating stem cells
4.4. Effective tools and core technologies that Effective tools and core technologies that
are useful to stem cell researchers are useful to stem cell researchers
CharacterizationCharacterization
& Monitoring& Monitoring
DifferentiationDifferentiation
ReportersReporters
Tools for Stem Cell ResearchTools for Stem Cell Research
Source cells and Source cells and
iPSCiPSC factorsfactors
Source CellsSource Cells
iPSCiPSC factorsfactors
iPS cell linesiPS cell lines
Source cells Source cells and and
iPSCiPSC factorsfactors
Considerations for Source CellsConsiderations for Source Cells
1.1. Purpose of your studyPurpose of your study
2.2. AccessibilityAccessibility
3.3. Reprogramming efficiencyReprogramming efficiency
4.4. Cell typeCell type
Human Foreskin Fibroblasts (HFF)Human Foreskin Fibroblasts (HFF)
•• Most studied cell typeMost studied cell type
•• Accessible Accessible
•• ReprogrammingReprogramming
Features of SBI HFFFeatures of SBI HFF
•• High purityHigh purity
•• Neonatal HFFNeonatal HFF
•• Singular genetic backgroundSingular genetic background
High Quality Source CellsHigh Quality Source Cells
HFFHFF
SBI provides HFF cellsSBI provides HFF cells
Human epidermal keratinocyte (HEK)Human epidermal keratinocyte (HEK)
•• Ectoderm originEctoderm origin
•• Accessibility and reprogramming abilityAccessibility and reprogramming ability
•• Special culture conditionSpecial culture condition
Features of SBI HEKFeatures of SBI HEK
•• Neonatal Neonatal
•• High purityHigh purity
•• Singular genetic backgroundSingular genetic background
•• Complete kit provides an easy solutionComplete kit provides an easy solution
High Quality Source CellsHigh Quality Source Cells
HEKHEK
SBI provides HEK cellsSBI provides HEK cells
Source cells and Source cells and
iPSCiPSC factorsfactors
Induced Pluripotency FactorsInduced Pluripotency Factors
James Thomson
1.Oct4
2.Sox2
5. Nanog
6. Lin28
Shinya Yamanaka
1.Oct4
2.Sox2
3.c-Myc
4.Klf4
SBI provides 6 iPSC factorsSBI provides 6 iPSC factors
Source cells and Source cells and
iPSCiPSC factorsfactors
Highly Efficient Highly Efficient LentivirusLentivirus SystemSystem
••Highly efficientHighly efficient
••Broad tropismBroad tropism
••Success in generating iPS cellsSuccess in generating iPS cells
Induced PluripotencyInduced Pluripotency
FactorsFactorsSource cells and Source cells and
iPSCiPSC factorsfactors
Faithful Expression of Faithful Expression of iPSCiPSC FactorsFactors
InIn--house tested for house tested for
generating iPS cell lines!generating iPS cell lines!
26
44--inin--11Source cells and Source cells and
iPSCiPSC factorsfactors
Oct4
Sox2
Klf4c-Myc
44--inin--1 Provides a Better Solution1 Provides a Better Solution
••More efficientMore efficient
SBI ‘s 4SBI ‘s 4--inin--1 is coming1 is coming
4 iPSC factors
Yamanaka S., Cell, 2006
HFF/HEKHFF/HEK
27
Reprogramming ProceduresReprogramming Procedures
1
1Virus Virus
transductiontransduction
4 iPSC factors
Yamanaka S., Cell, 2006
HFF/HEKHFF/HEK
28
2Transfer to Transfer to
feeder cellsfeeder cells
Reprogramming ProceduresReprogramming Procedures
High quality feeder cells
StemPure Medium
4 iPSC factors
Yamanaka S., Cell, 2006
HFFHFF
HFFs on feeder cellsHFFs on feeder cells
Pluripotency ReportersPluripotency Reporters
29
3
3
Observe & Wait!Observe & Wait!
Reprogramming ProceduresReprogramming Procedures
Colony FormationColony Formation
4 iPSC factors
Yamanaka S. Cell, 2006
Human foreskin Human foreskin fibroblastsfibroblasts
HFFs on HFFs on
feeder cellsfeeder cells
1 2 3 4 5 6
iPS colony formationiPS colony formation
30
5 Expand coloniesExpand colonies
Pickup Pickup
coloniescolonies4
Reprogramming ProceduresReprogramming Procedures
PluripotencyPluripotency
MonitorsMonitors
How to validate iPS Cells?How to validate iPS Cells?
Characterizations & MonitoringCharacterizations & Monitoring
Criteria:Criteria:1.1.MorphologyMorphology
2.2.Growth Properties (AP staining)Growth Properties (AP staining)
3.3.Stem cell markers Stem cell markers
4.4.KaryotypeKaryotype
5.5.Pluripotency testPluripotency test
A Powerful Monitoring Tool:A Powerful Monitoring Tool:
Pluripotency reporter,Pluripotency reporter,
RealReal--time monitoringtime monitoring
PluripotencyPluripotency
MonitorsMonitors
Characterization of iPS CellsCharacterization of iPS Cells
1. Morphology1. Morphology
22--dimensional dimensional
Human iPS CellsHuman iPS Cells
33--dimensional dimensional
Mouse iPS CellsMouse iPS Cells
PluripotencyPluripotency
MonitorsMonitors
Characterization of iPS CellsCharacterization of iPS Cells
2. Growth Property2. Growth Property
AP staining AP staining
AP stainingAP staining
•• Quick and easyQuick and easy
•• Not very specific Not very specific
SBI AP Staining Kit is comingSBI AP Staining Kit is coming
Doubling timeDoubling time
Mitotic activityMitotic activity
PluripotencyPluripotency
MonitorsMonitors
Characterization of iPS cellsCharacterization of iPS cells
3. Stem Cell Markers3. Stem Cell Markers-- immunoimmuno--stainingstaining
Pluripotency markersPluripotency markers
•• More specificMore specific
•• Multiple markers:Multiple markers:
SSEASSEA--3, SSEA3, SSEA--4, TRA4, TRA--11--6060
TRATRA--11--81, TRA81, TRA--22--49/6E49/6E
SSEA-1
Nanog
SBI immunostaining kit is comingSBI immunostaining kit is coming
4. Karyotyping4. Karyotyping
Characterization of iPS cells Characterization of iPS cells
Why do karyotyping?Why do karyotyping?
Requirement for publicationRequirement for publication1
2
34
Abnormal Abnormal KaryotypeKaryotypeSBI Karyotyping Kit is comingSBI Karyotyping Kit is coming
N= 69N= 69
SBI DataSBI Data
PluripotencyPluripotency
MonitorsMonitors
Characterization of iPS cellsCharacterization of iPS cells
5. Pluripotency Testing5. Pluripotency Testing
TeratomaTeratoma formationformation
SBI dataSBI data
Chimera generationChimera generation
WebWeb--PicPic
EmbryoidEmbryoid bodybody
SBI dataSBI data
RealReal--timetime
MonitoringMonitoring
Pluripotency reportersPluripotency reporters
A. How do they work?A. How do they work?
Pluripotency MonitoringPluripotency Monitoring
RealReal--timetime
MonitoringMonitoring
Pluripotency reportersPluripotency reporters
B. Your choicesB. Your choices
Pluripotency MonitoringPluripotency Monitoring
ColorColor
•• GreenGreen fluorescent fluorescent
•• RedRed fluorescentfluorescent
SelectionSelection
•• Positive: Positive: ZeoZeo--RR
•• Negative: Negative: GCVGCV
RealReal--timetime
MonitoringMonitoring
Pluripotency MonitoringPluripotency Monitoring5. Pluripotency reporters5. Pluripotency reporters
C. Your choicesC. Your choices
Target Cell Type Species Promoter/Enhancer
ES/iPS Cells Mouse/Human m/hOct4 promoter
ES/iPS Cells Mouse/Human m/hNanog promoter
ES/iPS Cells Mouse & Human SOX2 enhancer + mCMVp
ES/iPS Cells Mouse & Human Oct4 enhancer + mCMVp
SBI provides a collection of reportersSBI provides a collection of reporters
RealReal--timetime
MonitoringMonitoring
Pluripotency MonitoringPluripotency MonitoringPluripotency reportersPluripotency reporters
D. ApplicationsD. Applications
SBI provides a collection of reportersSBI provides a collection of reporters
pGreenZeopGreenZeo
LentiLenti--reporterreporter
pGreenFirepGreenFire
LentiLenti--reporterreporter
LuciferaseLuciferase
Pluripotency monitoring
Enrichment
Sorting & isolation of stem cells
Pluripotency monitoring
Gene regulation
Drug screening
Sorting &Sorting &
IsolationIsolation
RealReal--timetime
MonitoringMonitoring
Pluripotency reportersPluripotency reporters
E. ExamplesE. Examples
Pluripotency MonitoringPluripotency Monitoring
pGreenZeopGreenZeo--Oct4Oct4
pGreenZeopGreenZeo--NanogNanog
DifferentiationDifferentiation
ReportersReportersHematopoiesisHematopoiesis
BB--cellscells
TT--cellscells
Macrophages/Macrophages/
MicrogliaMicroglia
MyogenesisMyogenesisCardiomyocytesCardiomyocytes
Skeletal muslceSkeletal muslce
Smooth muscleSmooth muscle
NeuralNeuralNeuronsNeurons
PhotoreceptorsPhotoreceptors
AstrocyteAstrocyte
OligodendrocyteOligodendrocyte
EndocrineEndocrineBeta cellsBeta cells
StructuralStructuralChondrocyteChondrocyte
OsteoblastOsteoblast
Stem CellsStem Cells
HematopoiesisHematopoiesisRed blood cellsRed blood cells
> 30 Differentiation Reporters> 30 Differentiation Reporters
Efficient Monitoring in RealEfficient Monitoring in Real--time time
• CellCell--specific promoters drive GFP and Zeocin selection in specific promoters drive GFP and Zeocin selection in
differentiated cells differentiated cells – monitor differentiation in real timemonitor differentiation in real time
• Rapidly create transgenic lines and ES reporter cells for Rapidly create transgenic lines and ES reporter cells for
lineage trackinglineage tracking
Application of differentiation reportersApplication of differentiation reporters
ExamplesExamples
DAPI mGFAP_GFP
GFAP_Ab Merge
Data provided courtesy of Dan Data provided courtesy of Dan HoeppnerHoeppner, McKay Lab, National Institute of Neurological Disorder and Stroke., McKay Lab, National Institute of Neurological Disorder and Stroke.
GFAP : GFAP : AstrocyteAstrocyte markermarker
Differentiation ReportersDifferentiation ReportersGFAP : GFAP : AstrocyteAstrocyte marker marker
Data provided courtesy of Dan Data provided courtesy of Dan HoeppnerHoeppner, McKay Lab, NINDS. , McKay Lab, NINDS.
All CellsAll Cells GFP CellsGFP Cells
Differentiation ReportersDifferentiation Reporters
DCX : Immature neuron markerDCX : Immature neuron marker
iPS cells differentiated into immature neuronsiPS cells differentiated into immature neurons
Differentiation ReportersDifferentiation Reporters
MAP2: Mature neuron markerMAP2: Mature neuron marker
iPS cells differentiated into immature neuronsiPS cells differentiated into immature neurons
Monitor DifferentiationMonitor Differentiation
Undifferentiated Undifferentiated cardiomyoblastscardiomyoblasts differentiate into differentiate into
mature mature cardiomyocytescardiomyocytes with retinoic acid treatmentwith retinoic acid treatment
TNNT: Cardiomyocyte MarkerTNNT: Cardiomyocyte Marker
PluripotencyPluripotency
MonitorsMonitors
DifferentiationDifferentiation
ReportersReporters
SBI: OneSBI: One--stop Providerstop Provider
Source cells and Source cells and
iPSCiPSC factorsfactors
Source CellsSource Cells
iPS factorsiPS factors
iPS celliPS cell
Lentiviral Technology
Why use Lentiviruses?Why use Lentiviruses?
Effective and versatile Effective and versatile
Core Technologies Core Technologies
LentivirusesLentiviruses Get InGet In
•• Dividing or NonDividing or Non--Dividing CellsDividing Cells
(Retroviruses only infect dividing cells)(Retroviruses only infect dividing cells)
•• Useful for hardly transfected stem cellsUseful for hardly transfected stem cells
•• Infect ES/iPS cells and Embryoid BodiesInfect ES/iPS cells and Embryoid Bodies
•• Useful for slowly dividing Primary CellsUseful for slowly dividing Primary Cells
•• Broad cellular tropismBroad cellular tropism
Virus Tropism Virus Tropism
–– very broadvery broad
•• Most Cell linesMost Cell lines
•• Primary cellsPrimary cells
•• Stem CellsStem Cells
•• Animal studiesAnimal studies
Human Primary Human Primary
Neurons (GFP)Neurons (GFP)
Human Human AstrocyteAstrocyte
(GFP)(GFP)
Human EmbryonicHuman Embryonic
Kidney Cell (RFP)Kidney Cell (RFP)
FelineFeline
Kidney Cell (RFP)Kidney Cell (RFP)
Rat Cardiac Rat Cardiac
MyoblastsMyoblasts (RFP)(RFP)Human EmbryonicHuman Embryonic
Stem Cell (GFP)Stem Cell (GFP)
Carotid Arterial Carotid Arterial
SBI’s lentiSBI’s lenti--mirmir--145145
LentivirusesLentiviruses Stay InStay In
•• Stable Integration of Constructs into Host Stable Integration of Constructs into Host
ChromosomeChromosome
•• Good for reporters, and sustained overexpression, Good for reporters, and sustained overexpression,
& knockdown& knockdown
•• Easily create Stable Easily create Stable
cell linescell lines
Pioneers in Pioneers in LentivectorsLentivectorsStably express cDNAsStably express cDNAs
Strong and ubiquitous expression of the gene of interest Strong and ubiquitous expression of the gene of interest
•• Single or double expression cassette with choice of reporter gene Single or double expression cassette with choice of reporter gene
•• Target gene expressed from CMV, EF1, PGK, UbC or Target gene expressed from CMV, EF1, PGK, UbC or MSCV promoter MSCV promoter
Pioneers in Pioneers in MicroRNAsMicroRNAs
Stably express microRNAsStably express microRNAs
Permanent, heritable microRNA Permanent, heritable microRNA
overexpressionoverexpression
•• Efficient delivery and robust expressionEfficient delivery and robust expression
•• Analyze the specific effects of MicroRNAs Analyze the specific effects of MicroRNAs
•• Express single microRNA precursors Express single microRNA precursors
or clusters or clusters
Pioneers in Pioneers in MicroRNAsMicroRNAs
Permanently knockdown Permanently knockdown
microRNAsmicroRNAs
Permanent, heritable microRNA Permanent, heritable microRNA
inhibitioninhibition
•• Efficient delivery and robust interferenceEfficient delivery and robust interference
•• Disrupt microRNA signaling to study Disrupt microRNA signaling to study
pathways pathways
b-actin
c-Myc
Pioneers in Pioneers in LentivectorsLentivectors
Stably express shRNAsStably express shRNAs
Permanent heritable gene knockdownPermanent heritable gene knockdown
•• Efficient delivery and permanent Efficient delivery and permanent
Knock downKnock down
•• Analyze the specific effects of Target Analyze the specific effects of Target
genes genes
•• Single or Double promoter formats Single or Double promoter formats
Pioneers in Pioneers in LentivectorsLentivectors
Efficiently create reporter Efficiently create reporter
cell linescell lines
Sort for GFP/RFP or Zeo/Puro SelectionSort for GFP/RFP or Zeo/Puro Selection
•• Monitor transcription network activityMonitor transcription network activity
•• Track cell differentiation Track cell differentiation
•• Quantify transcription response Quantify transcription response
We make it extremely easy
for a seemingly
complicated system!
Effective Tools Developed at SBI Effective Tools Developed at SBI
CloneClone--it it LentivectorLentivector SystemSystemAccurate and Simple lentivector construction
If you can do PCR, you can do the cloning!!!If you can do PCR, you can do the cloning!!!
HighHigh--titer Virus Productiontiter Virus Production
If you can do transfection, you can produce virus!!!If you can do transfection, you can produce virus!!!
Our pPACK packaging mix makes Our pPACK packaging mix makes
virus production never so easyvirus production never so easy
Virus Concentration Virus Concentration –– PEGPEG--itit
Collect cell culture Collect cell culture
medium containing medium containing
viral particlesviral particles
Incubate at 4Incubate at 4ooC,C,
Centrifuge the mixture at Centrifuge the mixture at
low speed for 30 min. low speed for 30 min.
If you have a table centrifuge, you have highIf you have a table centrifuge, you have high--titer virus!!!titer virus!!!
Virus Virus TiteringTitering –– UltraRapidUltraRapid TiterTiter
Our ULtraRapid Our ULtraRapid Titer Titer
Kit makes virus Kit makes virus
titering very easy titering very easy
and accurate!and accurate!
You do not have to have purified DNA to do virus titering!!!You do not have to have purified DNA to do virus titering!!!
SBI Offers Smart Research ToolsSBI Offers Smart Research Tools
LentivectorLentivector
TechnologiesTechnologies
MicroRNAMicroRNA ProfilingProfiling
Functional AnalysisFunctional Analysis
HighHigh--throughput throughput
RNAiRNAi ScreenScreen
Stem Cell ResearchStem Cell Research
ToolsTools