Download Slides - Workforce Development in Stem Cell Research

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Differentiation
of pluripotent cells and transdifferentiation
What is differentiation
of pluripotent cells?
Pluripotent
Multipotent
Ectodermal cell
ES/iPS cell
Mesodermal cell
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Differentiated cells
brain
heart
Endodermal cell
Differentiation of ES/iPS
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 cellular-based
treatment or development
of new therapies.
pancreas
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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 (MEFs)
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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References
(I) Directed differentiation of ES/iPS cells
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Pluripotent
ES/iPS cell
Multipotent
Ectodermal cell
✗
✗
✗
✗
Feeders
Feeders
Differentiated cells
brain
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References
(I) Directed differentiation of ES/iPS cells
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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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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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References
(I) Induced differentiation of ES/iPS cells
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Pluripotent
ES/iPS cell
Multipotent
Ectodermal cell
✗
✗
Differentiated cells
brain
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References
(I) Induced differentiation of ES/iPS cells
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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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ICM 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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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 ALS)
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+)
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
Oct4
Sox2
Klf4
C-Myc
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
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.
Transdifferentiation
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Transdifferentiation, also known as
lineage reprogramming, is a process
where one mature somatic cell
transforms into another mature
somatic cell without undergoing an
intermediate pluripotent state or
progenitor cell type
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Induced Transdifferentiation
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TRANSCRIPTION FACTORS!
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Induced in vivo Transdifferentiation
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• Cardiac fibroblasts represent 50% of the cells in the mammalian heart
• Cardiac fibrobalsts can be directly reprogrammed to adult
cardiomyocyte-like cells in vitro by the addition of Gata4, Mef2c and Tbx5
(GMT)
• Can in vivo over-expression of GMT reprogram in situ the cardiac
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fibroblats?
Induced in vivo Transdifferentiation
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Summary
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• Directed differentiation of ES/iPS cells is the production of various mature cell
types (e.g. motor neurons, dopaminergic neurons) using defined growth factors or
cytokines.
• The defined growth factors are crucial for generating these cells during normal
embryonic development.
• Induced differentiation of ES/iPS cells is the production of various mature cell
types (e.g. motor neurons, dopaminergic neurons) using defined transcription
factors.
• The defined transcription factors are overexpressed and are sufficient to promote
the differentiation of the cells into the appropriate cell type
• Transdifferentiation is the production of various mature cell types (e.g. epithelial
cells) from another mature cell type (e.g. blood cell).
• Induced Transdifferentiation is the production of various mature cell types (e.g.
motor neurons, dopaminergic neurons) from another mature cell type (e.g.
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fibroblasts, epatocytes) using defined transcription factors.