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