Stem Cell Technology and Its
Application to Biomedical Research
Biao Lu, MD, PhD
Nov. 23rd 2009
System Biosciences – Smart Research Tools
www.systembio.com
Stem Cell Technology
Outline
1.
2.
3.
4.
History of stem cell technology
Main applications of stem cells
Methods to generate stem cells
Core technologies and effective tools that
are useful to stem cell researchers
1. History of Stem Cell Research
1964
1981
1998
2006
Embryonic germ cells
Mouse embryonic stem cells
Human embryonic stem cells
Induced pluripotent
stem cells (iPS cells)
Types of Stem Cells
1. ES cells
2. iPS cells
Human Stem Cell Colony
What Are Stem Cells?
Stem cells are unspecialized cells that are able
to reproduce themselves indefinitely and, under
the right conditions, to differentiate into
virtually any mature cell types (>200 cell types).
Two critically important properties:
1. Self
Self--renewal
2. Pluripotency
2. Applications of Stem Cells
Stem Cells
1 Molecular Mechanisms
1.
2.
3.
4.
Pluripotency
Cell differentiation
Tissue development
Organ genesis
Applications of Stem Cells in
Biomedical Research
Stem Cells
1 Molecular Mechanisms
2 Human Disease Models
Examples:
1. Fragile
Fragile--X syndrome
2. Cystic fibrosis
3. Sickle cell anemia
Applications of Stem Cells in
Biomedical Research
Stem Cells
1 Molecular Mechanisms
2 Human Disease Models
Examples:
1. Fragile
Fragile--X syndrome
2. Cystic fibrosis
3. Sickle cell anemia
4. Parkinson disease
Applications of Stem Cells in
Biomedical Research
Stem Cells
1 Molecular Mechanisms
2 Human Disease Models
3 Drug Discovery
1. Drug screen
2. Toxicity study
Applications of Stem Cells in
Biomedical Research
Stem Cells
4
Cell Replacement Therapy
Beta--cells
Beta
Cardiac
Muscle Cells
Neurons
3. How Do You Isolate Stem Cells?
1.
2.
3.
4.
A brief history of stem cell technology
Main applications of stem cells
Methods to generate stem cells
Core technologies and effective tools that
are useful to stem cell researchers
Generation of Stem Cells
ES cell
Isolation
4-7 Days
Generation of Stem Cells
ES cell
Isolation
1. Ethical issues
2. Immune
Immune--rejection problems
Generation of Stem Cells
Nuclear
Transfer
Generation of Stem Cells
Nuclear
Transfer
Generation of Stem Cells
Nuclear
Reprogram
iPS
cells
Part 4: Tools and Technologies
1.
2.
3.
4.
A brief history of stem cell technology
Main applications of stem cells
Methods for generating stem cells
Effective tools and core technologies that
are useful to stem cell researchers
Tools for Stem Cell Research
Source cells and
iPSC factors
Source Cells
iPSC factors
iPS cell lines
Characterization
& Monitoring
Differentiation
Reporters
Considerations for Source Cells
Source cells and
iPSC factors
1.
2.
3.
4.
Purpose of your study
Accessibility
Reprogramming efficiency
Cell type
High Quality Source Cells
Human Foreskin Fibroblasts (HFF)
• Most studied cell type
• Accessible
• Reprogramming
Features of SBI HFF
HFF
• High purity
• Neonatal HFF
• Singular genetic background
SBI provides HFF cells
High Quality Source Cells
Human epidermal keratinocyte (HEK)
• Ectoderm origin
• Accessibility and reprogramming ability
• Special culture condition
Features of SBI HEK
• Neonatal
• High purity
HEK
• Singular genetic background
• Complete kit provides an easy solution
SBI provides HEK cells
Induced Pluripotency Factors
Source cells and
iPSC factors
Shinya Yamanaka
1.Oct4
2.Sox2
3.c-Myc
4.Klf4
James Thomson
1.Oct4
2.Sox2
5. Nanog
6. Lin28
SBI provides 6 iPSC factors
Highly Efficient Lentivirus System
Source cells and
iPSC factors
•Highly efficient
•Broad tropism
•Success in generating iPS cells
Faithful Expression of iPSC Factors
Induced
Pluripotency
Source cells
and
Factors
iPSC factors
In-house tested for
Ingenerating iPS cell lines!
4-in
in--1 Provides a Better Solution
Source cells and
iPSC factors
4-in
in--1
Oct4
•More efficient
Sox2
c-Myc
Klf4
SBI ‘s 44-in
in--1 is coming
26
Reprogramming Procedures
4 iPSC factors
1
1
Virus
transduction
HFF/HEK
27
Yamanaka S., Cell, 2006
Reprogramming Procedures
4 iPSC factors
HFF/HEK
Transfer to
2 feeder cells
High quality feeder cells
StemPure Medium
28
Yamanaka S., Cell, 2006
Reprogramming Procedures
4 iPSC factors
Pluripotency Reporters
HFF
Observe
3 & Wait!
3
Colony Formation
HFFs on feeder cells
29
Yamanaka S., Cell, 2006
Reprogramming Procedures
5 Expand colonies
4 iPSC factors
1
2
4
Human foreskin
fibroblasts
3
4
5
6
Pickup
colonies
iPS colony formation
HFFs on
feeder cells
30
Yamanaka S. Cell, 2006
How to validate iPS Cells?
Characterizations & Monitoring
Criteria:
Pluripotency
Monitors
1.Morphology
1.Morphology
2.Growth
2.
Growth Properties (AP staining)
3.Stem
3.
Stem cell markers
4.Karyotype
4.
Karyotype
5.Pluripotency
5.
Pluripotency test
A Powerful Monitoring Tool:
Pluripotency reporter,
Real--time monitoring
Real
Characterization of iPS Cells
1. Morphology
Human iPS Cells
Mouse iPS Cells
2-dimensional
3-dimensional
Pluripotency
Monitors
Characterization of iPS Cells
2. Growth Property
AP staining
Pluripotency
Monitors
AP staining
• Quick and easy
• Not very specific
Doubling time
Mitotic activity
SBI AP Staining Kit is coming
Characterization of iPS cells
3. Stem Cell MarkersMarkers- immuno
immuno--staining
Pluripotency markers
Pluripotency
Monitors
• More specific
Nanog
• Multiple markers:
SSEA--3, SSEASSEA
SSEA-4, TRATRA-1-60
TRA--1-81, TRATRA
TRA-2-49/6E
SSEA-1
SBI immunostaining kit is coming
Characterization of iPS cells
4. Karyotyping
SBI Data Why do karyotyping?
1
Requirement for publication
2
4
3
N= 69
Abnormal Karyotype
SBI Karyotyping Kit is coming
Characterization of iPS cells
5. Pluripotency Testing
Embryoid body
SBI data
Pluripotency
Monitors
Teratoma formation
SBI data
Chimera generation
Web--Pic
Web
Pluripotency Monitoring
Real-time
RealMonitoring
Pluripotency reporters
A. How do they work?
Pluripotency Monitoring
Real-time
RealMonitoring
Pluripotency reporters
B. Your choices
Color
• Green fluorescent
• Red fluorescent
Selection
• Positive: Zeo
Zeo--R
• Negative: GCV
Pluripotency Monitoring
Real-time
RealMonitoring
Target Cell Type
5. Pluripotency reporters
C. Your choices
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 reporters
Pluripotency Monitoring
Real-time
RealMonitoring
Pluripotency reporters
D. Applications
pGreenZeo
pGreenFire
Lenti--reporter
Lenti
Lenti--reporter
Lenti
Luciferase
Pluripotency monitoring
Enrichment
Sorting & isolation of stem cells
Pluripotency monitoring
Gene regulation
Drug screening
SBI provides a collection of reporters
Pluripotency Monitoring
Real-time
RealMonitoring
Pluripotency reporters
E. Examples
pGreenZeo--Oct4
pGreenZeo
Sorting &
Isolation
pGreenZeo--Nanog
pGreenZeo
> 30 Differentiation Reporters
Stem Cells
Structural
Hematopoiesis
Chondrocyte
Osteoblast
Red blood cells
Endocrine
Beta cells
Differentiation
Reporters
Neural
Hematopoiesis
B-cells
T-cells
Macrophages/
Microglia
Myogenesis
Cardiomyocytes
Skeletal muslce
Smooth muscle
Neurons
Photoreceptors
Astrocyte
Oligodendrocyte
Efficient Monitoring in RealReal-time
Application of differentiation reporters
• Cell
Cell--specific promoters drive GFP and Zeocin selection in
differentiated cells – monitor differentiation in real time
• Rapidly create transgenic lines and ES reporter cells for
lineage tracking
Collection of Reporters at SBI
Examples
GFAP : Astrocyte marker
DAPI
GFAP_Ab
mGFAP_GFP
Merge
Data provided courtesy of Dan Hoeppner
Hoeppner,, McKay Lab, National Institute of Neurological Disorder and Stroke.
Differentiation Reporters
GFAP : Astrocyte marker
All Cells
GFP Cells
Data provided courtesy of Dan Hoeppner
Hoeppner,, McKay Lab, NINDS.
Differentiation Reporters
DCX : Immature neuron marker
iPS cells differentiated into immature neurons
Differentiation Reporters
MAP2: Mature neuron marker
iPS cells differentiated into immature neurons
Monitor Differentiation
TNNT: Cardiomyocyte Marker
Undifferentiated cardiomyoblasts differentiate into
mature cardiomyocytes with retinoic acid treatment
SBI: OneOne-stop Provider
Source cells and
iPSC factors
Source Cells
iPS factors
iPS cell
Pluripotency
Monitors
Differentiation
Reporters
Core Technologies
Lentiviral Technology
Why use Lentiviruses?
Effective and versatile
Lentiviruses Get In
• Dividing or NonNon-Dividing Cells
(Retroviruses only infect dividing cells)
• Useful for hardly transfected stem cells
• Infect ES/iPS cells and Embryoid Bodies
• Useful for slowly dividing Primary Cells
• Broad cellular tropism
Virus Tropism
– very broad
• Most Cell lines
• Primary cells
• Stem Cells
• Animal studies
Carotid Arterial
SBI’s lentilenti-mir
mir--145
Human Primary
Neurons (GFP)
Human Astrocyte
(GFP)
Human Embryonic
Stem Cell (GFP)
Rat Cardiac
Myoblasts (RFP)
Human Embryonic
Kidney Cell (RFP)
Feline
Kidney Cell (RFP)
Lentiviruses Stay In
• Stable Integration of Constructs into Host
Chromosome
• Good for reporters, and sustained overexpression,
& knockdown
• Easily create Stable
cell lines
Pioneers in Lentivectors
Stably express cDNAs
Strong and ubiquitous expression of the gene of interest
• Single or double expression cassette with choice of reporter gene
• Target gene expressed from CMV, EF1, PGK, UbC or MSCV promoter
MicroRNAs
Brightfield
GFP
April 12 2009
Pioneers in MicroRNAs
Stably express microRNAs
Permanent, heritable microRNA
overexpression
• Efficient delivery and robust expression
• Analyze the specific effects of MicroRNAs
• Express single microRNA precursors
or clusters
Pioneers in MicroRNAs
Permanently knockdown
microRNAs
Permanent, heritable microRNA
inhibition
• Efficient delivery and robust interference
• Disrupt microRNA signaling to study
pathways
c-Myc
b-actin
Pioneers in Lentivectors
Stably express shRNAs
Permanent heritable gene knockdown
• Efficient delivery and permanent
Knock down
• Analyze the specific effects of Target
genes
• Single or Double promoter formats
Pioneers in Lentivectors
Efficiently create reporter
cell lines
Sort for GFP/RFP or Zeo/Puro Selection
• Monitor transcription network activity
• Track cell differentiation
• Quantify transcription response
Effective Tools Developed at SBI
We make it extremely easy
for a seemingly
complicated system!
Clone--it Lentivector System
Clone
Accurate and Simple lentivector construction
If you can do PCR, you can do the cloning!!!
High--titer Virus Production
High
Our pPACK packaging mix makes
virus production never so easy
If you can do transfection, you can produce virus!!!
Virus Concentration – PEG
PEG--it
Collect cell culture
medium containing
viral particles
Incubate at 4oC,
Centrifuge the mixture at
low speed for 30 min.
If you have a table centrifuge, you have highhigh-titer virus!!!
Virus Titering – UltraRapid Titer
Our ULtraRapid Titer
Kit makes virus
titering very easy
and accurate!
You do not have to have purified DNA to do virus titering!!!
SBI Offers Smart Research Tools
Stem Cell Research
Tools
Lentivector
Technologies
MicroRNA Profiling
Functional Analysis
High-throughput
HighRNAi Screen
Visit SBI Online
www.systembio.com