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 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 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 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

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 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

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