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Dopamine neurons derived from embryonic stem cells function in an animal model of Parkinson

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Title: Dopamine neurons derived from embryonic stem cells function in an animal model of Parkinson


1
Dopamine neurons derived from embryonic stem
cells function in an animal model of Parkinsons
diseaseJong-Hoon Kim, Jonathan M. Auerbach,
Jose A. Rodriguez-Gomez, Ivan Velasco, Denise
Gavin, Nadya Lumelsky, Sang-Hun Lee,John
Nguyen, Rosario Sa nchez-Pernaute, Krys
Bankiewicz Ron McKay
Melissa Christensen and Jennifer Yao
2
Parkinsons 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 Parkinsons
    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

3
The NeurotransmitterDopamine
  • 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 Parkinsons 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

4
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 Parkinsons 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

5
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

6
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 Parkinsons disease

7
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

8
Transfected cells were processed through the five
stage differentiation method
9
  • 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

10
  • 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

11
  • 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

12
  • 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

13
  • 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

14
  • 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

15
  • 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

16
  • 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

17
  • 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

18
  • 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

19
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

20
Figure 4
  • Comparison of currentvoltage 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.

21
Figure 4
  • Action potential spikes of TH- neurons 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

22
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

23
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

24
Figure 4
  • The dotted line shows the host- graft 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

25
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

26
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

27
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

28
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

29
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

30
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

31
  • 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
    Parkinsons disease, even though cells were not
    seen dividing in these experiments, continued
    data is needed to show that ES cells dont divide
    in vivo
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