Stem Cells as A Possible Medical Treatment of Parkinson’s Disease

Leonardo Platon Major in Cell and Molecular Biology

Undergraduate Seminar in Biology

Significance
• Parkinson’s disease is one of the leading neurological disorder today. Through the seminar, the audience will be informed on how neural stem cell transplantation can address the cause of this disorder by replacing degraded dopamine neurons and providing neuroprotection to existing ones. This seminar summarizes significant experimental studies in animal models which demonstrate safety and efficacy prior to human clinical trials.

Objectives
• To characterize and identify uses of neural stem cells • To describe how stem cell transplantation can be a potential treatment to Parkinson’s disease • To present studies showing the application of stem cells in treatment of model animals with Parkinson’s disease

Neural Stem Cells
• Stem cells are capable of generating various committed progenitor cells and ultimately differentiate into mature cells • Neural stem cells are not only present in the developing mammalian central nervous system but also in the adult nervous system of all mammalian organisms including humans • Neural stem cells can also be derived from more primitive embryonic stem cells

Fig. 1. An illustration proposing the classes of mammalian stem cells that can give rise to neurons.

Location of Neural Stem Cells
• The location of adult stem cells and the brain regions to which their progeny migrate is still unclear but a number of viable locations have been identified. In the adult mammalian brain, the genesis of new neurons has been consistently documented in the subgranular layer of the dentate gyrus and the subventricular zone of the lateral ventricles The subventricular zone is the adult brain region with the highest neurogenetic rate, from neural stem cells had first been isolated Neural stem cells are also found in the spinal cord

Fig. 2. View of the adult rodent brain that depicts (in gray) the regions where continous neurogenesis occurs throughout adulthood which are the olfactory bulb and the hippocampus. Areas known to contain NSCs (in black) are the subventricular zone and the dentate gyrus of the hippocampus.

Isolation of Neural Stem Cells
1) Cells are harvested from human brain tissue or the spinal cord 2) Cell suspension is stained with monoclonal antibodies (mAbs) 3) Proprietary mAbs are placed in cells sorter which separates non-stem cells from stem cells 4) Cells are cultured and readied for transplantation

Fig. 3. Procedure for isolation of neural stem cells.

Transplantation of Neural Stem Cells
• Brain sites targeted for stem cell transplantation of different studies vary in location. Common sites of transplantation are areas of the brains where dopamine neurons degrade such as the substantia nigra and corpus striatum • Specific dosage of stem cells to be transplanted is still to be determined

A total of 5x105 GDNF-c17.2 NSCs were grafted in four deposits in the striatum of mice models Single intrastriatal injection of 4ug of 6-OHDA to induce Parkinson’s disease

Fig. 4.  Neural stem cell transplation procedure  used by Akerud et al. (2001).

Medical Properties of Neural Stem Cells
• As the central nervous system’s most primordial cells, neural stem cells have attributes that appear to promote anatomical and functional preservation or restoration in neurodegenerative diseases • Multipotent neural stem or progenitor cell lines can differentiate to neuronal and glial phenotypes, both in vitro and after transplantation into the developing or adult brain • Possible medical applications is also based on the fact that neurogenesis still continues during adulthood within restricted areas of the central nervous system

Present Knowledge on Neural Stem Cells
• It is presently known that isolated fetus or adultderived neural stem cells from rat, mouse and human brain tissue survive well in the developing and adult, intact or damaged brain and can migrate over sizable distances • Neural stems may need to be accustomed to neighboring cells of transplantation site to induce their differentiation toward specific lineages and to function properly

Facts About Parkinson’s Disease
• Parkinson’s disease is the second most common neurodegenerative disorder • It affects approximately 2 out of 1000 people. On average, symptoms first appear when a patient is older than 50. • Parkinson’s affects both men and women and is one of the most common neurologic disorders in the elderly.

• About 60,000 Americans are newly diagnosed with Parkinson’s disease each year with more than 1.5 million Americans affected at any one time. • In 2002, there was an estimated 200 total deaths or 0.3 per 100,000 people in the Philippines died due to the disease. • It is estimated that four million people worldwide have Parkinson’s disease.

Parkinson’s Disease
• Parkinson’s disease is a degenerative disorder of the central nervous system. It is characterized by a massive degeneration of dopamine-containing neurons in the substantia nigra which is in the mid-brain It is both a chronic, meaning it persists over a long period of time, and progressive, meaning its symptoms grow worse over time The disease by itself does not cause people to die. However, general physical and emotional condition of people who are severely affected can cause or exacerbate other illnesses and so contribute to the final cause of death Parkinson’s is not contagious. Some cases appear to be hereditary but most cases are sporadic.

What causes Parkinson’s?
• Degeneration of dopamine (DA) neurons in the substantia nigra (SN) and the consequent deficit of DA release appear to be responsible for the characteristic manifestations of Parkinson’s disease. Dopamine is a chemical messenger responsible for transmitting signals between the substantia nigra and the next relay station of the brain, the corpus striatum, to produce smooth and purposeful movement Dopamine work in balance with acetylcholine to transmit messages between nerve cells and muscles. The transmitted messages allow for performance of coordinated movements. In people with Parkinson’s, this balance is upset because of the degeneration of dopamine neurons leading to lack of dopamine release. Loss of dopamine results in abnormal nerve firing patterns within the brain that cause impaired movement. Neurons are not well-regulated and do not behave in a normal manner. The reason why dopamine neurons degenerate in the brain of people with Parkinson’s is still unknown.

Fig. 5. Neuronal pathways that degenerate in Parkinson’s disease.

• Parkinson’s disease does not affect everyone in the same way and the rate of progression differs among patients. 1) Tremor is one of the primary symptoms and is usually the reason that causes people to seek medical help. 2) Rigidity or resistance to movement affects most people with Parkinson’s disease. 3) Postural instability or impaired balance causes patients to fall off easily. A stooped posture in which the head is bowed and the shoulders are drooped may develop in affected persons.

What are the symptoms of the disease?

• Stage 1 – symptoms on one side of the body only • Stage 2 – symptoms on both sides of the body. No impairment of balance • Stage 3 – balance impairment. Still physically independent • Stage 4 – severe disability but still able to walk or stand unassisted • Stage 5 – wheelchair-bound or bedridden unless assisted

Hoehn and Yahr Staging of Parkinson’s Disease

How is Parkinson’s disease diagnosed?
• There is currently no blood or laboratory tests that have been proven to help in diagnosing the disease • The diagnosis is based on medical history and a neurological examination • The disease is difficult to diagnose since its symptoms can also been as signs of normal aging • Doctors may sometimes request brain scans to rule out other diseases. However, CT and MRI brain scans of people with Parkinson’s disease usually appear normal

How is Parkinson’s treated?
• There is no present cure for Parkinson’s but medications can sometimes provide relief from the symptoms. There are three categories of medications used:

1) Drugs that increase brain levels of dopamine such as Levodopa. These drugs are dopamine precursors which can cross the bloodbrain barrier and then change into dopamine. 2) Drugs which ease the symptoms of the disease by influencing other neurotransmitters of the body. An example are anticholinergic drugs that inhibits uptake of acetylcholine. 3) Drugs that help control non-motor symptoms or those that does not affect movement. • Treatment of Parkinson’s disease through surgery is now only being done in advanced cases.

Findings of Recent Studies

Neuroprotection through Delivery of Glial Cell-Line Derived Neurotrophic Factor By Neural Stem Cells in a Mouse Model of Parkinson’s Disease
• By Akerud et al. (2001) • Objective: to demonstrate that neural stem cells engineered to release glial cell line-derived neurotrophic factor (GDNF) can differentiate into multiple, stable neural cells and prevent degeneration of dopaminergic neurons in the substantia nigra

Materials and Methods
A total of 5x105 GDNF-c17.2 NSCs were grafted in four deposits in the striatum of mice models 56 mice models (32 grafted and 24 non-grafted) received a single intrastriatal injection of 4ug of 6-OHDA to induce Parkinson’s disease Neuroprotective effects of GDNFc.17 NSCs on substantia nigra dopaminergic neurons were characterized through immunohistochemistry

Results
• Morphological analysis of brains engrafted with GDNF-c17.2 NSCs after 30 days showed that the NSCs was able to survive throughout the striatum Immunohistochemisty showed that GDNF-c17.2 NSCs differentiated into different types of multipotent neural stem cells Grafting of GDNF-c17.2 NSCs was able to protect the substantia nigra dopaminergic neurons from degradation

Fig. 6. Immunochemistry showed that GDNF-c17.2 NSCs give rise to neurons (D), astrocytes (E) and oligodendrocytes (F).

Fig. 7. Grafts of GDNF-c17.2 NSCs protected SN dopaminergic neurons against degradation done by 6-OHDA.

Conclusions
• Neural stem cells engineered to release GDNF were able to survive and differentiate to different neural cell types • GDNF-c17.2 NSCs was also able to provide neuroprotection to the substantia nigra dopaminergic neurons against degradation

Transplantation of Human Neural Stem Cells Exerts Neuroprotection in a Rat Model of Parkinson’s Disease

• By Yasuhara et al. (2006) • Objective: to demonstrate the neuroprotective effects of transplanted HB1.F3 human neural stem cells lines against dopaminergic degradation in rats induced with Parkinson’s disease

Materials and Methods
72 Sprague Dawley rats received 6hydroxydopamine (6-OHDA) injections in the right medial forebrain to induce Parkinson’s disease 32 of the rats immediately received HB1.F3 NSCs transplantation while 40 of the rats served as control

morphological analysis to show degree of dopaminergic preservation

Results
• Staining of the striatum and analyzed at three different coronal section levels, demonstrated that the dopaminergic nigrostriatal system of the HB1.F3 transplanted rats was significantly preserved in all levels examined Thyroxine hydroxylase fibers in the striatum and neurons in the substantia nigra were preserved by HB1.F3 cell grafts HB1.F3 cells were able to survive in the lesioned striatum

• •

Fig. 8. HB1.F3 cell grafts were able to preserve the dopaminergic nigrostriatal system. Dopamine neurons in the SN of rats that received HB1.F3 cell grafts were markedly preserved (C and F) compared with vehicle-treated rats (B and E).

Conclusion
• Transplanted human neural stem cells in rat models was able to exert significant neuroprotection against dopaminergic degradation

Behavioral Improvement in a Primate Parkinson’s Model is Associated with Multiple Homeostatic Effects of Human Neural Stem Cells
• By Redmond et al. (2007) • Objective: to demonstrate that Parkinson-induced adult monkey brain retains intrinsic microenvironmental signals that may direct differentiation of an uncommitted human neural stem cell (hNSCs) toward a dopamine (DA) phenotype and that hNSCs have the capacity to respond to DA deficiency resulting to significant functional and behavioral recovery

Materials and Methods
25 African green monkeys were injected systematically with 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine (MPTP) to induce Parkinson’s disease transplantation or injection of hNSCs in to the right substantia nigra

behavioral scoring and statistical analysis

histological analysis

Results
• Observations of monkeys with Stage 5 Parkinson’s disease showed greater improvements in activities of daily living (such as ability to sit, walk and self-feed) compared to sham operated monkeys

Fig. 9. Comparison of behavioral recovery in severely Parkinsonian monkeys after hNSC injections and shamoperated monkeys.

Histological sections from the brains of the monkeys were analyzed to further understand the basis of the behavioral and functional recovery Histological analysis showed that hNSCs, which are transplanted in monkeys with severe Parkinson’s disease, could survive, migrate and yield dopamine neurons

Fig. 10. Survival and migration of hNSCs into the nigrostriatal system. hNSCs were injected unilaterally, dorsal to the right substantia niagra (white arrow). Migration of donor-derived cells (green stars).

Figure 11. Some transplanted hNScs showed key markers of DA neurons.

Conclusions
• Uncommitted human neural stem cells (hNSCs) were able to differentiate to dopamine phenotype because of certain microenvironmental signals • Differentiation of hNSCs to dopamine neurons led to capacity to respond to dopamine deficiency which resulted to behavioral and functional recovery

References Akerud, Peter.  2001. Neuroprotection through delivery of glial cell line­
derived  neurotrophic factor by neural stem cells in a mouse  model of Parkinson’s disease.   Journal of Neuroscience.  21(20):  8108­8118. Gage, Fred.  2000.  Mammalian neural stem cells.  Science.  287(5457):1433­ 1438. Galli, Rossella.  2003.  Neural stem cells.  Circulation Research.  92:598. Redmond, Eugene et al.  2007.  Behavioral improvement in a primate  Parkinson’s model is associated with  multiple homeostatic effects of  human neural stem cells. PNAS.  104(29): 12175­12180. Sanberg, Peter.  2007.  Neural stem cells for Parkinson’s disease: to  protect and repair.  PNAS.  104(29):  11869­11870.   Uchida, Nobuko.  2000.  Direct isolation of human central nervous  system stem cells.   PNAS.  97(26): 14720­14725. Yasuhara, Takao et al.  2006.  Transplantation of human neural stem cells  exerts neuroprotection in a rat model of Parkinson’s  disease.   Journal of  Neuroscience.  26(48): 12497­12511.

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