Stem Cell Technology and Its Application to Biomedical...

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

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System Biosciences System Biosciences –– Smart Research ToolsSmart Research Tools

www.systembio.com

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





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





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



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


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


ES cellES cell

IsolationIsolation

Generation of Stem CellsGeneration of Stem Cells

44--7 Days7 Days

ES cellES cell

IsolationIsolation

1.1.Ethical issuesEthical issues

2.2. ImmuneImmune--rejection problemsrejection problems


Nuclear Nuclear

TransferTransfer


NuclearNuclear

ReprogramReprogram

iPSiPS

cellscells


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


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


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


iPS cell linesiPS cell lines

Source cells Source cells and and


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



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



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


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


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


HFF/HEKHFF/HEK

28

2Transfer to Transfer to

feeder cellsfeeder cells


High quality feeder cells

StemPure Medium

4 iPSC factors


HFFHFF

HFFs on feeder cellsHFFs on feeder cells

Pluripotency ReportersPluripotency Reporters

29

3

3

Observe & Wait!Observe & Wait!


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


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


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


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


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


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

http://en.wikipedia.org/wiki/File:ChimericMouseWithPups.jpg

RealReal--timetime

MonitoringMonitoring

Pluripotency reportersPluripotency reporters

A. How do they work?A. How do they work?

Pluripotency MonitoringPluripotency Monitoring

RealReal--timetime



B. Your choicesB. Your choices


ColorColor

•• GreenGreen fluorescent fluorescent

•• RedRed fluorescentfluorescent

SelectionSelection

•• Positive: Positive: ZeoZeo--RR

•• Negative: Negative: GCVGCV

RealReal--timetime


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


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



E. ExamplesE. Examples


pGreenZeopGreenZeo--Oct4Oct4

pGreenZeopGreenZeo--NanogNanog


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

Collection of Reporters at SBICollection of Reporters at SBI

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


MonitorsMonitors


ReportersReporters

SBI: OneSBI: One--stop Providerstop Provider



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

MicroRNAsMicroRNAs

April 12 2009

BrightfieldBrightfield

GFPGFP

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

Visit SBI OnlineVisit SBI Online

www.systembio.com

Stem Cell Technology and Its Application to Biomedical...

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