Download • ES cells from the inner cell mass can give rise to all three germ

Work Force Development: Directed Differentiation (Neurons) Primer
Learning Items B, C, D, E, J, K, S, V
Essential questions (B, J and K)
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How can stem cells be induced to adopt various fates in a dish?
What are the potential therapeutic uses for differentiated stem cells?
How are motor neurons specified during normal embryonic development?
What are some common diseases that affect motor neurons?
How does our knowledge of motor neuron development inform experimental
approaches to create these cells in vitro from stem cells?
Key knowledge and skills you will acquire as a result of this lecture
Students will know:
 Key Terms: directed differentiation, embryoid body, pluripotent, multipotent,
unipotent, transfection, motor neuron, Spinal Muscular Atrophy, Amyotrophic
Lateral Sclerosis, Parkinson’s disease, dopaminergic
 The developmental potential of various stem cell populations.
 Advantages and disadvantages of in vitro approaches to understand and treat
diseases of the nervous system.
 Cellular and genetic basis for some well-described neurodegenerative
diseases.
 Crucial developmental signaling molecules that are used to derive neural
progenitors and neurons from ES cells in a dish.
Students will be able to:
 Describe how secreted factors present during normal development are
employed to direct differentiation of stem cells into neurons.
 Compare and contrast the underlying mechanisms responsible for various
neurodegenerative diseases.
 Discuss the potential pitfalls when using laboratory disease models to arrive
at new treatments for Parkinson’s disease and Amyotrophic Lateral Sclerosis.
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Embryonic stem cells from the inner cell mass can give rise to all three germ
layers and are pluripotent. Cells in each germ layer retain the ability to proliferate
and give rise to a more restricted spectrum of cells. Therefore, they are multipotent
cells. During embryonic development, proliferating precursors or progenitors
eventually appear that have very limited fates and are unipotent, producing only a
neuron or a skin cell. Stem cells or progenitors thus undergo successive steps of
lineage restriction that limit the ultimate cell types they can produce. The directed
differentiation of ES cells in culture is the process of intentionally converting ES
cells in vitro into specialized cells such as neurons, heart muscle cells, endothelial
cells of blood vessels or insulin-secreting cells similar to those found in the
pancreas. Such cells can be used for cellular-based treatments or development of
new therapies (Slide 1).
The differentiated cell types that arise from pluripotent human ES cells have
applications in developmental biology, regenerative medicine and drug discovery.
Mitochondrial heteroplasmy (i.e. the process of transferring mitochondria from a
donor oocyte to a recepient oocyte during the process of nuclear transfer),
combined with subsequent generation of ES cells from these oocytes, offers the
possibility of deriving so-called nuclear transfer (NT) stem cell lines containing
mutations in mitochondrial DNA (mtDNA). This enables the study of mitochondrial
dysfunction and development of in vitro models for specific mitochondrial diseases.
Autologous/heterologous differentiated cells derived from a patient’s hESCs
can be used either as an in vitro model for their disease or, after repairing the
abnormality, transferred back to the patient for treatment. In addition, hESCs, pES
cells, NTSCs or iPS-derived cells offer more realistic material for drug discovery and
toxicological screening. Differentiated embryonic stem cells (e.g. neural, cardiac or
hepatic cells) can serve as a model system and a potential high-throughput
approach to test the pharmaceutical efficacy and toxicity of different drugs or
chemicals. In this case, hESCs derived from different individuals or ethnic
populations will make possible the determination of whether genetic variants result
in altered expression of the target gene or altered response of the gene product to
therapeutic compounds (Slide 2).
Several factors maintain pluripotency of ES cells in culture. These factors can be
divided in two groups: a) secreted factors and b) intracellular factors. ES cells are
cultured on a mitotically-inactivated mouse fibroblast “feeder” layer that maintains
their pluripotency. Fibroblasts promote self-renewal and/or suppress ES cell
differentiation. LIF (leukemia inhibiting factor) is another substance necessary to
maintain pluripotency of mouse ES cells in culture.
LIF is a member of the cytokine family of secreted proteins that binds to a receptor
on the ES cell surface and influences gene expression. LIF may influence: a) the rate
of cell proliferation, b) cell cycle progression, or c) activation of a signaling cascade
involved in maintaining pluripotency. Moreover, serum contains Bone
Morphogenetic Proteins (BMPs) that are important for the self-renewing abilities
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of ES cells. Finally, human ES cells are maintained in a pluripotent state in culture by
the presence of Fibroblast Growth Factor 2 (FGF-2) (Slide 3).
The most common approach to direct differentiation of ES cells is to change
their growth conditions in a defined way. One technique is to remove secreted
factors that maintain pluripotency (for example, LIF or serum for mouse ES cells). A
complementary technique is to add growth factors that promote differentiation into
a specific lineage to the culture medium. These factors trigger activation (or
inactivation) of specific genes in ES cells. This initiates a series of molecular events
that cause cells to differentiate along a particular pathway. Thirdly, the composition
of the surface on which ES cells can be changed. For example, the plastic dishes used
to grow both mouse and human ES cells can be treated with substances that either
allow cells to adhere to the surface of the dish or prevent adhesion and cause cells to
float in the culture solution. In general, an adherent substrate helps prevent ES cells
from interacting with each other and differentiating. In contrast, a nonadherent
substrate allows ES cells to aggregate and thereby interact with each other. These
aggregates are called “embryoid bodies” (i.e. they mimic interactions that occur in
vivo in the embryo). Cell-cell interactions are critical to normal embryonic
development, so allowing "natural", in vivo interactions to occur in the culture dish
is a fundamental strategy for inducing mouse or human ES cell differentiation (Slide
4).
One goal of stem cell research is to identify and control the genes that allow a
stem cell to divide and maintain its pluripotency, as well as to turn these genes off
and express different genes when we want cells to differentiate either in a dish or in
a patient to treat disease. One way to direct differentiation of ES cells is to introduce
foreign genes via transfection or other methods. The result of this strategy is to add
(an) active gene(s) to the ES cell genome, which then triggers differentiation along a
desired pathway. This approach is seemingly a precise way of regulating ES cell
differentiation, but it will only work if it is possible to identify which genes must be
active at which particular stage of differentiation. The genes must then be activated
at the right time—meaning during the correct stage of differentiation—and must be
inserted into the genome at the proper location (Slide 5).
An early specialization during development is segregation of the three basic
germ layers that will each give rise to distinct parts of the adult animal:
Ectoderm: nerve cells, skin cells, inner ear, eye, mammary glands, nails, teeth
and the nervous system (spine and brain)
Mesoderm: blood, muscle, bones, heart, skeleton, gonads, urinary system, fat
and spleen
Endoderm: gut, liver, pancreas, lungs, tonsils, pharynx and parathyroid
glands
The future sperm and egg cells do not come from these three layers, but rather are
partitioned early as primordial germ cells (not to be confused with germ layers).
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Gastrulation also establishes the three main axes of the embryo: anterior-posterior
(head to toe), dorsal-ventral (back to front) and left-right (Slide 6).
During development, immature cells or progenitors found at differing
locations in the developing embryo encounter distinct signals:
a) Those that will become motor neurons encounter a secreted molecule
termed Shh, which binds to receptors (Patched and Smoothened) on the cell
surface. This triggers a signaling cascade inside progenitors that promotes their
differentiation into a motor neuron.
b) Progenitors that will become heart muscle cells express a different
receptor called BMPRI. This receptor interacts with the secreted molecules Activin
or TGF- to trigger a signaling pathway that induces differentiation into heart
muscle cells.
c) Progenitors that become red blood cells express a third type of receptor
that binds to a hormone named erythropoietin, inducing differentiation into red
blood cells. The activation of different signaling pathways in distinct immature cells
during development allows them to acquire different fates (Slide 7).
Many researchers have focused their efforts in developing methods to direct
differentiation of ES cells into motor neurons. Motor neurons represent only one
out of every million cells in the body, reside in the ventral horn of the spinal cord
and control muscle movement. Furthermore, there are different types of motor
neurons present in the body (e.g. at limb versus thoracic positions). There are a
number of diseases that affect motor neurons:
One example is paralysis arising from spinal cord trauma. Spinal cord
injuries cause myelopathy (damage to nerve roots or myelinated fiber tracts that
carry signals to and from the brain). This type of injury can also damage the grey
matter in the central part of the spinal cord, causing losses of both interneurons and
motor neurons.
A second example is Spinal Muscular Atrophy (SMA), a neuromuscular
disease characterized by degeneration of motor neurons that results in progressive
muscle wasting and weakness. The clinical spectrum of SMA ranges from early
infant death to a normal adult life with only mild weakness. Patients often require
comprehensive medical care which can be costly for society. The most common
form of SMA is caused by a mutation in the Smn (Survival of Motor Neurons) gene
and manifests itself over a wide range of severity that affects infants through adults.
Amyotrophic Lateral Sclerosis (ALS) or Lou Gehrig’s disease is yet another
motor neuron disease, a progressive and fatal neurodegenerative disorder caused
by degeneration of motor neurons. There is no known cause for ALS in ~95% of
cases. There is a known hereditary factor for familial ALS (FALS), a condition known
to run in families that only accounts for ~5% of all cases. Among this small subset,
an known autosomal dominant genetic defect (in the superoxide dismutase
enzyme) is associated with approximately 20% of FALS. Children of those diagnosed
with familial ALS have a higher risk factor for developing the disease (Slide 8).
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ES cells can be generated for animal models of motor neuron diseases, such
as SMN1/SMN2 mutant mice or SOD1G93A transgenic mice that mimic the
respective human diseases (SMA or ALS). iPS cells can also be produced from
patients that have diseases such as SMA or ALS. Motor neurons or other CNS cells
such as astrocytes can be derived from iPS cells or ES cells, and can be used for a
variety of scientific and clinical purposes:
a) determining which cellular and molecular pathways are responsible for
degeneration of motor neurons
b) discovering drugs by designing assays to identify molecules that improve
the pathology of neurons
c) cell replacement therapy (Slide 9)
ES cells may provide a new tool for studying disease mechanisms and
identifying drugs to slow progression of ALS. Two studies were recently reported by
the Project A.L.S./Jenifer Estess Laboratory for Stem Cell Research, a privately
funded laboratory focused exclusively on stem cells and ALS. These studies,
performed at Harvard and Columbia, compellingly demonstrate that ES cells can be
used to create an in vitro model for ALS. Until then, scientists had not known
whether motor neurons in ALS die because of a problem within neurons or from
their environment. One study describes a novel embryonic stem cell-based model
for ALS that will help scientists to answer this question (Dimos et al., 2008. Induced
Pluripotent Stem Cells Generated from Patients with ALS Can Be Differentiated into
Motor
Neurons.
(Read
the
following
blog:
http://cellnewsblog.blogspot.com/2008/08/als-ips-cells-created-from-skin-cells.html).
Both
research groups observed that non-neuronal cells called astrocytes may have a toxic
effect on motor neurons in ALS (Slide 10). This discovery of astrocyte-derived
toxicity provides not only insight into how damage associated with ALS occurs plus
a potential biomarker for early diagnosis, but also a target for new therapies aimed
at slowing motor neuron degeneration (Slide 11).
The ectodermal lineage normally gives rise to the nervous system and skin
via neural and skin stem cells. Neural stem cells generate all the neurons in the
central nervous system, including motor neurons, through the process of neural
specification from immature progenitors followed by differentiation (Slide 12).
During CNS development motor neurons are born in the ventral region of the
posterior neural tube, which will become the spinal cord of the adult. Progenitor
cells in the ventral neural tube are specified into motor neurons by two signals:
Sonic hedgehog (Shh) and retinoic acid (RA).
Motor neurons are mature differentiated cells and are characterized by the
unique and early expression of a transcription factor called Hb9. They can be
detected by expression of the Hb9 protein while being specified from immature
progenitors and migrating laterally to occupy their final position. The Hb9 gene
promoter is used to express the marker Green Fluorescent Protein in motor
neurons, including their axons as they exit the spinal cord to innervate peripheral
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muscles. This transgenic mouse has become a valuable tool for understanding the
process of ES cell directed differentiation into motor neurons and testing the ability
of motor neurons that are generated in culture to innervate muscles after
transplantation in vivo (Slide 13).
During embryonic development of the neural tube, progenitor cells that will
become motor neurons express two receptors on their surface (Patched and
Smoothened). These receptors are activated by a secreted protein called Sonic
Hedgehog (Shh) that originates from both a tube-like structure called the
notochord and the tissue above the notochord called the floor plate. Shh mRNA is
detected in these structures before motor neurons are generated. Shh protein
diffuses toward the neural tube and forms a gradient that is crucial for specifying
the fates of several populations of neurons in the ventral spinal cord, including
motor neurons. In culture, motor neurons are generated at a very high efficiency
from neural progenitors using a concentration of 3 nM Shh (Slide 14).
This information has been used to direct differentiation of ES cells into motor
neurons in vitro, which requires production of embryoid bodies (EBs) as floating
balls of cells for two days. This is done in both the absence of serum and the
presence of retinoic acid (RA) at 1 M concentration. RA specifies EBs to become
neuroectoderm, one of the three germ layers that gives rise to the central nervous
system, including motor neurons. Neuroectoderm produces motor neuron
progenitors following incubation for two days with RA plus Shh. The amount of Shh
agonist required to specify these progenitors in culture is 1 m, and they can be
identified by expression of a transcription factor called Olig2. Further incubation of
progenitors for two more days in the presence of RA and Shh yields motor neurons
that express the transcription factor Hb9 and eGFP (Slide 15).
Neuroectoderm can give rise not only to motor neuron progenitors but also
to progenitors for other neurons that populate the spinal cord. This tissue produces
specifically motor neuron progenitors after incubation for two days with retinoic
acid and Shh. The amount of Shh agonist required to specify motor neuron
progenitors in culture is 1 m. Motor neuron progenitors can be identified by
expression of a transcription factor called Olig2. However, if the neuroectoderm is
incubated for two days with retinoic acid and less Shh (10 nM) it is specified instead
as progenitors for a class of interneurons (p0-p2). These interneurons are
normally born dorsal to motor neurons and therefore experience a reduced Shh
signal in the animal, and can be identified by expression of a transcription factor
called Irx3. This is an excellent example of how understanding normal development
of motor neurons or interneurons can be used to direct their differentiation in vitro
from ES cells (Slides 16-17).
To test if mouse motor neurons generated from the directed differentiation
protocol function normally, Witcherle et al. injected these cells into the spinal cord
of a chick embryo. Chick and mouse embryos have many similar features in the
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development of their spinal cords. Several days after the injection, mouse motor
neurons that had been generated in vitro are present in the chick neural tube and
send projections into the periphery (Slides 18-19). Finally, directed differentiation
of human ES cells into motor neurons requires the same two secreted factors as
mouse ES cells, however this process takes 5-6 times longer in human cells (Slide
20).
Several investigators have focused their efforts on developing methods to
direct differentiation of ES cells into dopaminergic neurons. These are neurons
found in midbrain that secrete dopamine, an important neurotransmitter in the
brain. Dopaminergic neurons degenerate in Parkinson’s disease, a movement
disorder. Loss of these neurons is associated with muscle rigidity, tremor, postural
and gait abnormalities as well as slowing or loss of physical movements (Slide 21).
These neurons are born during development in response to two signals: Shh
and FGF-8. Briefly, undifferentiated mouse ES cells are dissociated into single cells
and plated at low density. They proliferate in plastic culture dishes coated with
gelatin. Their growth media contains LIF and fetal calf serum supplemented with
amino acids; conditions that promote the proliferation of undifferentiated ES cells.
Next, ES cells are induced to form embryoid bodies (EB) by dissociation with the
enzyme trypsin. They are then replated at a higher density on a nonadherent
surface. These conditions allow the cells to aggregate and begin the process of
differentiation. After 4 days, the EBs are replated onto an adherent substrate in the
original growth medium and the growth medium replaced with serum-free (ITSFn)
medium one day later. This switch to a serum-free medium causes many cells to die
but allows the survival of cells that express nestin, which is a marker to identify
CNS stem cells and progenitors in vivo and in vitro (but is also expressed by other
cell types). This process marks the formation of neuroectoderm. After 6 to 10 days
in this medium that allows only nestin-positive progenitors to survive, the cells are
dissociated and induced to divide in a medium that contains basic FGF, a growth
factor that induces proliferation. The progenitors are then exposed to Shh and FGF8. This series of signals triggers development of progenitors that give rise to
dopaminergic and serotonergic neurons. This complex, multistage differentiation
procedure yields a high percentage (30%) of neurons that express tyrosine
hydroxylase, the rate-limiting enzyme in the synthesis of dopamine. These neurons
secrete dopamine into the culture medium, show the electrical activity typical of
neurons and respond to a high concentration of potassium ions by releasing more
dopamine, much as they would in vivo. A separate population of neurons from these
cultures produces serotonin (Slide 22).
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