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STEM CELL BIOLOGY
Reprogramming, Pluripotency and Differentiation
1
Outline
- Background
- Applications
A li ti
off stem
t
cell
ll research
h
- Methods for generating stem cells
- SBI’s tools for stem cell research
What Are Stem Cells?
Stem cells are unspecialized cells that are thought to be
able to reproduce themselves indefinitely and, under the
right conditions
conditions, to develop into a wide variety of mature
cells with specialized functions.
Two important
T
i
t t characteristics:
h
t i ti
- Self-renewal
- Pluripotency
Human embryonic
stem cell colony
Types of Stem Cells
Embryonic stem cells (also known as ES cells)
come from a 4 to 7-day-old embryo. They have
the ability to form virtually any type of cell found
in the human body.
Adult stem cells are more specialized than
embryonic stem cells. They are found in the
majority of tissues and organs in our body and
generate the mature cell types within that tissue
or organ. They have a restricted ability to
produce different cell types and to self-renew.
Types of Stem Cells
Stem cells that have the potential to develop into
any of the celltypea found in an adult organism
are called pluripotent. Embryonic stem cells are
pluripotent.
Stem cells that only have the potential to make a
few cell types in the body are called multipotent.
Adult stem cells are multipotent.
Applications of Stem Cell Research
Study cell
differentiation
Pluripotent
Stem Cells
Applications of Stem Cell Research
Drug screening
&
Toxicity testing
Study cell
differentiation
Pluripotent
Stem Cells
Applications of Stem Cell Research
Drug screening
&
Toxicity testing
Study cell
differentiation
Pluripotent
Stem Cells
Bone Marrow
for leukemia &
chemotherapty
Nerve cells for
neurodegenerative disorders
Heart muscle
cells for heart
disease
p
py
Cell replacement
therapy
Pancreatic
islet cells for
diabetes
Methods to Generate Stem Cells
Methods to Generate Stem Cells
Methods to Generate Stem Cells
Tools for Stem Cell Research
I. Inducing pluripotency with SBI’s iPSC
factors
Six iPSC factors for stem
cell reprogramming
Oct4
Nanog
Lin28
Sox2
Klf4
c-Myc
Protocol of Inducing Pluripotency
4 ttranscription
i ti factors
f t
Human fforeskin
H
ki
fibroblasts
Yamanaka S., Cell, 2006
Protocol of Inducing Pluripotency
4 ttranscription
i ti factors
f t
Human fforeskin
H
ki
fibroblasts
Replate HFFs on feeder cells
Yamanaka S., Cell, 2006
Protocol of Inducing Pluripotency
4 ttranscription
i ti factors
f t
Human fforeskin
H
ki
fibroblasts
iPS colony formation
Replate HFFs on feeder cells
Yamanaka S., Cell, 2006
Protocol of Inducing Pluripotency
4 ttranscription
i ti factors
f t
Expand best colonies
1
2
3
4
5
6
Pick individual colonies
Human fforeskin
H
ki
fibroblasts
iPS colony formation
Replate HFFs on feeder cells
Yamanaka S., Cell, 2006
Colony Formation
Day 12
Human ES-like Colonies from
Retroviral Based Factors
Retroviral-Based
Teratoma
a
A
AP
SSEA--1/4
Nanog
Characterization of iPSCs
SBI’s Stem Cell Lines
Human iPS cell line(SC101A-1)
Mouse iPS cell line(SC201A-1)
Human or mouse induced pluripotent stem cells were
generated by transducing genetically unmodified human or
mouse dermal fibroblasts with viruses individually encoding
the four transcription factors (Oct4, Sox2, Klf4, and c-Myc)
that have been shown to induce the reprogramming of
somatic cells to a pluripotent state. The cells were derived
using morphological selection criteria and without the use of
fluorescent marker or drug selection.
When cultured under standard ES cell culture conditions,
the morphology of SBI iPSCs are identical to that of ES cells.
The cells also express the pluripotency markers SSEA-4/1
Nanog and demonstrate strong endogenous alkaline
and Nanog,
phosphatase activity.
Tools for Stem Cell Research
II. Stem cell pluripotency reporters
Reporters for human and mouse Oct4 and
Nanog allow monitoring, sorting and selecting
undifferentiated ES or iPS cells
Tools for Stem Cell Research
II Stem cell pluripotency reporters
II.
G
GFP
Phase Co
ontrast
H9 hES cells
pGreenZeo-CMV pGreenZeo-mOct4 pGreenZeo-mNanog
Transduced
Transduced
Transduced
H9 hES cells
H9 hES cells
H9 hES cells
Data courtesy of Drs. Timothy Kamp and Chad H. Koonce
Tools for Stem Cell Research
III Stem
III.
St
cell
ll differentiation
diff
ti ti reporters
t
• Available as
prepackaged virus or
plasmid off the shelf
• Custom construction
for any reporter gene
also offered
Protocol for Neural Differentiation
hMAP2
hDCX
mGAD67
Mouse
iPSCs
Mouse
Neurons
Mouse
NSCs
Mouse
Astrocytes
mGFAP
Tools for Stem Cell Research
Stem Cell Differentiation Reporters
- Astrocyte reporter construct
GFAP: marker for astrocytes
Tools for Stem Cell Research
Stem Cell Differentiation Reporters
- Immature neuron reporter construct
DCX: marker for immature neurons
Tools for Stem Cell Research
Stem Cell Differentiation Reporters
- Mature neuron reporter construct
MAP2: marker for mature neurons
Comparison of SBI Products with
Competitors
Human
iPSC
Mouse
iPSC
iPSC
Factors
Pluripotency
Reporters
Differentiation
Reporters
SBI
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Invitrogen
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Open
Bi
Biosystems
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Stemgent
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Clontech
System Biosciences (SBI)
www.systembio.com
System Biosciences (SBI)
265 North Whisman Rd.
Mountain View, CA 94043
Tel:
650-968-2200
Fax: 650-968-2277