Download Dopamine neurons derived from embryonic stem cells

Dopamine neurons derived from embryonic
stem cells function in an animal model of
Parkinson’s disease
Jong-Hoon Kim*, Jonathan M. Auerbach*†, Jose´ A. Rodrı´guez-Go´mez, Iva´n
Velasco, Denise Gavin, Nadya Lumelsky, Sang-Hun Lee†,
John Nguyen†, Rosario Sa´ nchez-Pernaute†, Krys Bankiewicz† & Ron McKay
Melissa Christensen and Jennifer Yao
Parkinson’s Disease
• Caused by the loss of midbrain neurons
that synthesize the neurotransmitter
dopamine (substantia nigra)
• Diagnosed in more that 50,000 Americans
each year. ~1 million Americans have
Parkinson’s disease, including 1 out of
every 100 people over the age of 60
• Symptoms include:
– Muscle rigidity
– Tremors
– Bradykinesia (the slowing down of
movement and the gradual loss of
spontaneous activity)
– Changes in walking pattern and posture
– Changes in speech and handwriting
– Loss of balance and increased falls
• May be the first disease to be amenable to
treatment using stem cell transplantation
The Neurotransmitter
Dopamine
• Dopamine transmits signals between the areas in the
brain that, when working normally, coordinate smooth
and balanced muscle movement
• May also control functions related to mood
• Dopamine precursors (medications the brain converts to
dopamine) and antagonists (directly stimulate nerves in
the brain that are not naturally being stimulated by
dopamine) are prescribed to patients with Parkinson’s
disease and have shown some effect
• However, more research is now being dedicated to the
use of fetal midbrain precursors and embryonic stem
cells in cell regeneration therapy
Neuron Replacement from Fetal
Midbrain Precursors
• Fetal midbrain precursors (mouse or human)
can proliferate and differentiate into dopamine
synthesizing neurons in vitro
• Transplantation of these cells has led to
recovery of a rat model of Parkinson’s disease
But…
• They are an inadequate source of dopamine
synthesizing neurons because
– These precursor cells generate dopamine neurons for
only a short period in culture
– The ability to generate these neurons is unstable
Embryonic Stem (ES) Cells
• human embryonic stem cells are
derived from fertilized embryos less than
a week old and are pluripotent
• undifferentiated embryonic stem cells
can proliferate indefinitely in culture, and
can potentially provide an unlimited
source of specific, clinically important
adult cells
• many uses in genetic engineering,
including the isolation and functional
analysis of specific cell types
• also, human embryonic stem cells offer
insights into developmental events that
cannot be studied directly in humans in
utero or fully understood through the use
of animal models
• use of stem cells in cell therapy can be
successfully applied to animal models of
disease, however, only a few cases have
be shown
Goals
• To develop a method to further increase
the efficiency of midbrain specific
generation of dopamine neurons from ES
cells
• To demonstrate that these cells can
functionally integrate into host tissue as
well as lead to recovery in a rodent model
of Parkinson’s disease
Generation of midbrain CNS precursors
• Nuclear receptor related-1 (Nurr1)
– Transcription factor that is involved in the
differentiation of midbrain precursors into
dopamine neurons
– Modified to express an antigenic site derived
from the haemagglutinin protein (HA) and
inserted into a cytomegalovirus plasmid to
drive expression of Nurr1 ES cell lines
Transfected cells were processed through the five stage
differentiation method
The anti-HA antibody shows
• the introduced gene is expressed at high levels at stage 4, but much lower
in stage 5
• endogenous Nurr1 gene was expressed at low levels in stage 4, but much
higher in stage 5
Conclusion?
• Nurr1 was successfully expressed through the use of a pCMV prior to
differentiation
• Nurr1 is expressed in differentiated cells
• Nurr1 transfected cells
differentiated appropriately into
TH positive neurons at day 10 in
stage 5
• tyrosine hydroxylase (TH) is the
rate limiting enzyme in dopamine
synthesis and is expressed in
naturally occurring dopamine
synthesizing neurons
• In undifferentiated cells, Nurr1 is
expressed in a restricted site in the nucleus
Conclusions?
• TH positive neurons derived from Nurr1
ES cells are generated from precursor cells
that are responsive to the actions of the
Nurr1 protein
• The number of dopamine synthesizing neurons generated from ES
cells can be increased by treatment with FGF8 and Shh
• The generation of serotonin synthesizing neurons is also promoted
by treatment with FGF4
Conclusions?
• With endogenous mid- and hindbrain CNS precursors, the cell
population at stage 4 is responsive to signals generated by the isthmic
organizer
To further define the cells at stage 4, the
expression of transcription factors that
characterize precursors in different regions of
the CNS was evaluated
• Engrailed 1 (En-1) was highly coexpressed
with Pax2 and Otx2, but not with Bf1
• Similar to En-1 expression patterns in seen
in postmitotic differentiated dopamine
neurons, nearly all ES-derived TH positive
neurons expressed En-1 in their nucleus
Conclusion?
• Midbrain precursors and differentiated
neurons can be efficiently generated from ES
cells
• Expression of Nurr1 in ES cells increase the number of TH positive cells
generated by day 10 of stage 5
• In addition, treatment of the Nurr1 cells with FGF8 and Shh increases
the number of TH positive cells generated even more
• Expression of Nurr1 increases the number of serotonin cells only slightly
• Expression of Nurr1 and treatment with Shh and FGF8 also increases
the amount of dopamine released by stage 5 cells
Expression of genes involved in midbrain neuron development and
function in stage 5 cells
• Midbrain specific genes Nurr1, Ptx3, En-1 and the dopamine
transmitter (DAT) are expressed at low levels in the absence of
Nurr1 overexpression and Shh and FGF8 treatment at stage 4
Nurr1 ES cells were integrated into the striatum of hemiparkinson rats
• Many TH positive processes extend away from the graft into the
parenchyma of the host striatum
• Grafts were detected by staining for a mouse-specific antigen (M2)
as well as for TH
• Many of the M2 positive grafted cells also expressed TH
To characterize the phenotype of grafted cells, the number of neurons
positive for TH, serotonin and glutamate decarboxylase (GAD67) in the grafts
were measured at 4 weeks and 8 weeks after implantation
• The majority of neurons were TH positive and neuron number did not
change significantly between 4 and 8 weeks
• This stability is important because undifferentiated cells can cause
teratomas
Immunostaining for Ki-67 in a graft of dopamine synthesizing neurons
• Ki-67 is an antigen characteristically expressed in dividing cells and
was used to detect areas of cell proliferation in the graft
• No Ki-67 expression evident in the grafts, but were abundant in the
human gliomal cells grafted into an adult rat brain
• Consistently, no teratomas were observed in animals that had
received the grafts of the Nurr1 ES cells
Table 1
• Test the electrophysiological properties of grafted neurons in vivo
• Using infrared differential interference contrast microscope
• The grafted Action potential frequency and duration properties of
TH+ neurons are very different from TH- neurons in the graft and
TH- neurons in the host
• TH+ neurons display electrophysiological characteristics similar to
the dopamine neurons
Figure 4
• Comparison of current–voltage relationship between TH- neurons
in the host and TH+ neurons in the graft
• Circle indicates the immediate reduction in the membrane
potential, triangle indicates the sustained membrane potential
• The pattern in the TH+ neuron graph displayed anomalous
rectification, which also occurs in dopamine synthesizing cells
after hyperpolarizing pulse.
Figure 4
• Action potential spikes of THneurons in the host VS. TH+
neurons in the graft
• TH+ neurons have broader action
potentials at a lower frequency
compare to the TH- neurons in
the host
Figure 4
• Dopamine neurons have a unique
inhibitory postsynaptic potential
(IPSP)
• Dependent on the activation of
metabotropic glutamate receptors
(mGluR1)
• The grafted TH+ neurons
displayed IPSP when stimulated
• MCPG inhibits the activation of
metabotropic glutamate receptors
• After wash, TH+ neurons resume
IPSP
• None of the TH- neurons showed
this IPSP
Figure 4
• Extracellular stimulation was applied to cells within the
graft
• Excitatory postsynaptic potentials (EPSP) were recorded in
both the host neurons and grafted TH+ neurons
• Indicates the presence of graft-to-host and graft-to-graft
synapses
Figure 4
• The dotted line shows the hostgraft interface
• Biocytin-filled TH+ neurons
are in green and non-filled
TH+ neurons are in red
• Biocytin is often used to label
neurons for visualization
• The biocytin-filled TH+
neurons extended into the host
striatum
Figure 5
• Test the behaviour of sham-operated animals and animals grafted
with wild type ES cells or Nurr1 ES cells
• Amphetamine induces ipsi-lateral rotational behaviour in the
animals
• The group grafted with wild type ES cells showed a slight
recovery in rotational behaviour
• The group grafted with Nurr1 ES cells changed to consistent
contra-lateral rotational behaviour
Figure 5
• One week after injection of
Amphetamine, spontaneous
turning behaviour was measured
for 5 minutes
• The turning biases were preserved
in sham grafted groups and
groups grafted with Nurr1 ES
cells
Figure 5
• Results in the step test are expressed
as a percentage of the lesioned paw
relative to the number of steps with
the non-lesion paw
• Nurr1 group showed the most
improvement compare to the other
two groups
Figure 5
• In the paw-reaching test, the
number of pellets eaten with the
lesioned paw were normalized by
the total number of pellets eaten
during the 7-day test period.
• Nurr1 group has the most
significant improvement
Figure 5
• The percentage of use of the
lesioned-side limb relative to the
total number of landings after
rearing is measured
• Nurr1 group has the most
improvement
Conclusion
• Anatomical test → Showed that ES-cell-derived TH+ cells release
dopamine
• Neurochemical test → Showed that ES-cell-derived neurons are able to
extend axons into the host striatum
• Electrophysiological test → Showed that ES-cell-derived neurons can
form functional synaptic connections
• Behaviour test → Showed that ES-cell-derived neurons are capable of
modulating spontaneous and pharmacological induced behaviour
• ES cells have been shown to more efficiently generate precursors and
dopamine neurons than cultures of fetal, neonatal and adult stem cells
• However……………
• Further studies are needed to address the long term safety and
efficiency of these cells
• For example, tumour formation is a problem associated with ES cell
grafting in models of Parkinson’s disease, even though cells were
not seen dividing in these experiments, continued data is needed to
show that ES cells don’t divide in vivo