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Directed differentiation
of ES cells into ectoderm
What is directed differentiation
of ES cells?
Pluripotent
Multipotent
Ectodermal cell
ES cell
Mesodermal cell
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Differentiated cells
brain
heart
Endodermal cell
pancreas
Directed differentiation of
ES cells creates specialized
cells in vitro such as neurons,
heart muscle cells,
endothelial cells from blood
vessels and insulin-secreting
cells similar to those found
in the pancreas, all of which
can be used for cellularbased treatment or
development of new
therapies.
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Why do we care about directed
differentiation of ES cells?
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Secreted factors keep ES cells
pluripotent when cultured
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Secreted factors (proteins):
Feeders
ES cells
• Cell feeder layer (fibroblasts) secretes
proteins that interact with receptors in the
ES cell membrane to maintain its
pluripotency.
• LIF (Leukemia Inhibitory Factor) provided
in the media binds LIF receptors in the ES
cell membrane to maintain both mouse ES
pluripotency and the rate of cell
proliferation.
• Serum contains BMPs (bone
morphogenetic proteins) that maintain
pluripotency of mouse ES cells
Mouse ES cells colonies in culture
• FGF-2 and TGFs maintain human ES cell
pluripotency
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Directing the differentiation of ES
cells in culture (I)
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Change growth conditions of ES cells:
• Remove secreted factors that maintain ES cell pluripotency from
the media
• Add growth factors to the culture solution that trigger activation
(or inactivation) of specific genes in ES cells, in order to promote
differentiation into a specific lineage.
Change the surface on which ES cells are growing:
• Grow ES cells on non-adherent substrates so that they aggregate
with each other. These aggregates are called “embryoid bodies”.
• ES cells within aggregates will interact with each other. These cellcell interactions mimic some of the interactions of ES cells in vivo
that normally guide their differentiation.
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Directing the differentiation of ES
cells in culture (II)
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Transfect ES cells with foreign genes:
• Adding an active gene or genes to the ES cell genome.
• The gene(s) trigger(s) ES cells to differentiate along a particular pathway.
• This approach is a precise way of regulating ES cell differentiation.
Problems with this technology:
• It works ONLY if we know which gene(s) must be active at a particular stage
of differentiation.
• The gene(s) must be activated at the right time, i.e. during the correct stage of
differentiation
• The foreign gene(s) are often only required temporarily, but it is difficult to
introduce them without permanently changing or “damaging” the genome.
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ES cells form three germ layers
during embryogenesis
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Amnion
Implantation
Uterus
Blastocyst
Ectoderm
Epithelial skin cells, inner ear, eye,
Yolk sac
mammary glands, nails, teeth,
nervous system (spine and brain)
Endoderm
Stomach, gut, liver, pancreas, lungs,
tonsils, pharynx, thyroid glands
Mesoderm
Blood, muscle, bones, heart,
urinary system, spleen, fat
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Distinct signaling pathways specify
discrete cell types during development
Cell signaling pathways
Shh
Activin/TGF-
Patched/
Smoothened
Progenitor
cell
Motor neuron
BMP-RI
Progenitor
cell
Heart muscle cell
(Cardiomyocyte)
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Erythropoietin (EPO)
EPO receptor
Progenitor
cell
Red blood cells
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Motor neurons and their diseases
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Motor neurons
– One motor neuron per 106 cells in the body
– Reside in the ventral horn of the spinal cord
– Control movements of muscles
– Exist in various subtypes that control different muscle
groups (limbs versus thoracic regions)
Motor neuron diseases
– Paralysis from spinal cord trauma
– Spinal Muscular Atrophy (SMA)
– Amyotrophic Lateral Sclerosis (Lou Gehrig’s disease or
Stem cell-based approaches to
motor neuron diseases
Patients
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Pathways of
degeneration
iPS cells
ES cells
Animal models
References
Motor neurons
Astrocytes…
Drug discovery
Cell replacement
therapy
References
Modeling ALS in a dish
Skin cells from
ALS patients
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Dimos, JT et al. (2008). Induced
Pluripotent Stem Cells Generated
from Patients with ALS Can Be
Differentiated into Motor Neurons.
Science 321: 1218-21.
ALS motor neurons
Yamanaka
method
Klf4
Oct4
Sox2
iPS cells
induced pluripotent
stem cells
Motor neuron nuclei
Axons
Using motor neurons to screen
drugs promoting their survival
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Mouse disease models
– creating ES cells from existing mouse model strains
– genetic modification of existing ES cell lines
Human disease models
– genetically tested blastocysts from IVF clinics (SMA)
– not applicable to ALS
How are motor neurons generated
during development?
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Lineage restrictions
Pluripotent
Multipotent
Differentiation
Neural stem
cell
Neurons
Ectodermal cell
ES cell
Mesodermal cell
Endodermal cell
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Specification of motor neuron fate
depends on nearby secreted signals
Hb9
BMPs
Wnts
MNs
Retinoic
acid
Shh
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Hb9::eGFP
Graded Shh signaling specifies
ventral interneurons and motor neurons
within the neural tube
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Shh
Shh
Patched/
Smoothened
Progenitor
Cell
Motor neuron
(HB9+)
Directed differentiation protocol for
mouse ES cells into motor neurons
RA/Shh
RA
Hb9-GFP
mES cells
day 2
neurectoderm
day 4
motor neuron
progenitors
day 6
motor neurons
ES cells
2 days
Neurectoderm
Witcherle et al., Cell (2002)
1 M Shh
agonist
(~3 nM
Shh protein)
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RA/Shh
Olig2
2 days
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GFP Hb9
2 days
Motor neuron
progenitors
Motor neurons
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Dorsoventral patterning of
differentiating ES cells
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p0
p1
Irx3
p0,1,2
p2
Olig2
pMN
p3
pMN
Nkx2.2
p3
Irx3
Olig2
Nkx2.2
P0,1,2
2 days
ES cells
pMN
Neurectoderm
2 days
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Timeline for directed differentiation
protocol of mouse motor neurons
Witcherle and Pelzo (2009)
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Motor neurons from mouse ES cells
assayed by injection into
chicken neural tube
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RA/Shh
Hb9-GFP
mES cells
Day 2
neurectoderm
Day 4
progenitors
Day 6
motor neurons
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Motor neurons from mouse ES cells
innervate muscles when injected into
chicken neural tube
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Injection of mouse motor neurons into
the embryonic neural tube of chicken
Mouse motor axons exit chicken spinal cord
Muscle innervation
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Directed differentiation protocol for
human ES cells into motor neurons
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RA RA/Shh
RA
hES cells
References
Day 10
primary
neurectoderm
(early rosettes)
Day 14
secondary
neurectoderm
(late rosettes)
Day 26
motor neuron
progenitors
Day 33
motor neurons
Tubulin
Hb9
hES cells
10 days
4 days
RA
RA
Early
rosettes
Li et al., Nature Neuroscience (2005)
Late
rosettes
12 days
7 days
1 M
Shh agonist
+ RA
1 M
Shh agonist
+ RA
Motor neuron
progenitors
Motor
neurons
Dopaminergic neurons and their
diseases
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Dopaminergic neurons:
• Neurons located in the midbrain that secrete
dopamine - an important neurotransmitter in
the brain
• These neurons degenerate in Parkinson’s
disease, a movement disorder.
• Loss of these neurons is associated with
muscle rigidity, tremor, posture and gait
abnormalities as well as slowing or loss of
physical movements.
Dopaminergic
neurons
• These neurons arise during development in
response to two signals: Shh and FGF-8.
Directed differentiation of ES cells
into dopaminergic neurons
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Dopaminergic neurons require
Shh and FGF-8
• Mouse EBs are grown in the absence of
serum for 4 days on a non-adherent
substrate.
• EBs are transferred to an adherent
substrate and grown in a serum-free media
that promotes survival of neuronal
progenitors.
• After 6-10 days, neural progenitors are
exposed to Shh and FGF-8 to induce
differentiation into dopaminergic neurons.
• Differentiation of human ES cells into
dopaminergic neurons takes a longer time.
Summary
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• Directed differentiation of ES cells into neurons is the production of
various neuronal cell types (e.g. motor neurons, dopaminergic
neurons) using defined factors.
• The defined factors are crucial for generating these neurons during
normal embryonic development.
• Shh is a key signaling molecule that is required for the generation of
both motor neurons and dopaminergic neurons.
• However, some factors are uniquely required to produce a particular
type of neuron (e.g. RA for motor neurons and FGF8 for
dopaminergic neurons).
• Directed differentiation of human ES cells into neurons uses factors
similar to those employed for mouse cells.
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