By Romeo Alex Penheiro



Introduction


The use of regenerative medicine approaches for the treatment of disease like Parkinson’s is relatively new and is currently in the experimental stages. The novelty of the technique should not overshadow the possible benefits that can be achieved from it. Parkinson’s disease, a genetic neurodenegerative disorder, is usually categorized by muscular rigidity, 4-6 Hz of resting tremor and at advanced stages of the disease, postural instability[1] . This occurs due to the loss of dopaminergic neurons in the striatum that maintains a balance between the excitation and inhibition of movements by way of D1 and D2 receptors. There are currently several treatments for the disease: pharmacological treatments, namely use of MAO-inhibitors, and other invasive treatments but current research in stem cells therapy looks very promising and may have revolutionary treatment options. Experiments with transplanted medial ganglionic eminence (MGE) cells in animal models are paving the path for the implementation of the technique in human participants to test for its effectiveness and further its use as a treatment option for diseased individuals.[5]

Experimental Approach

The transplanted MGE cells survive for one year in the striatum, migrate from the injection site and spread throughout the striatum[5] . This and other results (MGE cells mature into GABAergic interneurons and display markers common to striatal interneurons) further confirms the results of some previous studies done in the sciences, namely:
1) Some MGE cells survived in the lateral ventricle of embryonic mice but did not migrate[2]
2) Postnatal transplantation of MGE cells in the striatum resulted in migration[3]
3) Postnatal MGE transplanted cells differentiated into GABA+ neurons in the striatum[4]

Figure_1._Transplanted_MGE_Cells_Migrated_Long_Distances_from_the_Site_of_Injection_throughout_the_Striatum

GABAergic cell transplantation in basal ganglia

The striatum of 6-OHDA rats were transplanted with MGE cell[5] . These cells expressed GFP reporter protein to identify the migration of the transplanted cells later on. Around 250,000 cells were introduced into the striatum of the 6-OHDA rats but most had survival rates of only three weeks. At four weeks, only around 1% of the original remained and these survived for one year after transplantation. The results were similar to the ones that have been previously published[6] .The cells underwent no proliferation since labeling the cells with Ki67, a marker expressed by dividing cells, yielded no Ki67+ MGE cells.

MIGRATION OF TRANSPLANTED MGE CELLS THROUGH THE STRIATUM
The cells migrated from the injection site and diffused throughout the host striatum [5] . At week 1, GFP+ cells migrated from injection site and started to gain an oblong soma with axonal and dendritic processes. At week 2, the GFP-MGE cells migrated from 2.0-3.5 mm in all directions (only in the striatum) from the transplanted site, and the cells were more mature with prominent neural processes (**Figure1A**). At week 4, most of the cells matured with huge neuronal projections that dominated the entire striatum. Since these cells did not develop spines, they were not medium spiny projection neurons (**Figure 1B**).

Figure_2._Most_MGE_Cells_Adopted_an_Inter-_neuronal_Fate_when_Transplanted_into_the_Adult_StriatumDevelopment to GABA+ neurons

For determining cell differentiation, cell specific markers were examined.[5] Most of the cells expressed NeuN, a marker for mature neurons. Most cells also expressed markers specific to GABAergic cells: markers of GABA, GABA-synthesizing enzyme, and GABA transporter (**Figure 2**). These, along with early data, indicate that the MGE cells expressed cell-specific markers that were expressed in adult rat striatum This form of marker expression was preserved but with certain minor exceptions. [5] The results also confirmed that 1/4th of the transplanted cells were NeuN negative meaning they did not develop into mature neurons9. However, they stained positive for CNPase, a myelin protein; so, some of the cells differentiated into oligodendrocytes. [5] Since no GFAP was expressed, none of the cells differentiated into astrocytes. It was also observed that cell body size of the transplanted cells was similar to that of the host cells. [5]

Integration of the cells to the basal ganglia circuitryFigure_3._MGE_Cells_Transplanted_into_the_Striatum_Displayed_the_Basic_Membrane_Properties_Characteristic_of_Mature_Forebrain_Interneu-_rons_and_Showed_Evidence_of_Synaptic_Integration

After week 4 of transplantation, more than 50% of the cells underwent synaptic growth [5] (**Figure 3A**). At 5-20 weeks, whole-cell patch-clamp recordings were recorded from the GFP+ MGE cells to study the membrane properties. A dye was used to confirm that the recordings were from the selected cells (**Figure 3B**). Most of the cells exhibited physiological properties of neurons (**Figure 3C**). The rest had properties similar to glial cells (**Figure 3D**).[5]

The cells were also treated with many toxins that have many effects on the cells (**Figure 3F**). The transplanted cells had many processes that stretched away from the cell body (**Figure 3G**) suggesting that the transplanted cells made connections to both inhibitory GABAergic neurons in the striatum and the excitatory glutamatergic neurons in the cortex[5] . It was found that the membrane properties of the transplanted cells were similar to previous findings in the host striatal interneurons and also to MGE cells that were that were transplanted in the cerebral cortex [7] , [8] . Therefore, majority of the MGE cells that became incorporated into the host striatum became inhibitory interneurons.

GABAergic cell transplantation in the STN

Increasing the inhibition in subthalamic nucleus (STN) in Parkinson’s disease is an effective way to reduce parkinsonian symptoms. That is why deep brain stimulation (DBS) is directed to STN. It was investigated whether MGE cells once transplanted in the STN differentiated in 6-OHDA-lesioned rats [5] . Four weeks after the transplantation, it was found that, contrary to cells in the straitum, the cells in the STN did not migrate (**Figure 7A**) and neither did they differentiate into neurons (did not express the neuronal marker, NeuN). However, a small proportion of the cells that were located outside the STN did express NeuN (**Figure 7B**). No cells in the STN expressed calcium-sequestering proteins, CR or CB, or the inhibitory interneuron markers, GABA or GAD (**Figure 7E, G-I**). The majority of the cells expressed the astrocyte marker, GFAP unlike the cells in the striatum. These convincing data suggest that most cells in the STN become glial cells and not neurons. [5]

Factors affecting the impact of transplanted cells on behavior

Although a small number of the transplanted neurons survived, the efficacy of the experiment depends on the number of transplanted neurons compared to the total number of host striatal interneurons which was about 5% [5] . Also, the behavioral studies done on 6-OHDA treated and untreated rats showed there was a significant change in motor behavior. Transplantation ameliorates the deficits associated with 6-OHDA lesions.

Details on whether transplantation of more MGE cells will result in more surviving neurons in the striatum, or larger integration into the striatum or greater improvement in animal models of Parkinson’s disease is still to be understood. Also, implantation of different sorts of cell-types into the striatum can lead to understanding of different aspects of the Parkinson’s disease in animal models [5] . The environment of the transplanted neurons also plays a major role in determining their fate; for instance, MGE cells in the striatum expressed neuronal markers but not astrocyte marker, however, it is the contrary in the STN

Four Stages of Clinical Trials

Before a drug or treatment procedure is clinically practiced, they need to undergo a series of tests to determine their efficacy and safety. Here is a diagrammatic description of the four phases.[9]
Four_Stages_of_Clinical_Trials

Human Trial 1

Two Double-blind placebo-controlled trials [10]

Forty patients within the age range of 35-75 years underwent either a bilateral transplantation of human embryonic dopaminergic neurons from the mesenphalic region in the putamen (four embryos in each side) or a sham surgery[10] . The study was conducted for a year and 13 of the sham operated patients were transplanted after one year. For sham surgery, holes were drilled in the skull but the dura was not pierced. The efficacy of the treatment was measured by a subjective global rating of severity of the disease symptoms where a positive score indicated wearing of symptoms and negative scores indicated increase in severity of symptoms4.

Human_Trials_1.JPGHuman_Trials_1.1.JPGThere was no significant difference in the mean global rating scores between the transplanted and sham group. However, some of the younger patients, who were less than 60 years of age, showed improvement [10] . Also, 15% of the participants, less than 60 years of age, in the transplanted group showed improvement early in the study but later developed dystonia and dyskinesia even after discontinued use of leva dopa [10] . The participants also showed increased uptake of 18F-Flurodopamine after transplantation of embryonic dopamine neurons but not for the sham surgeries. This uptake increased in 16/20 transplant patients and indicates growth of neural processes which was detected by a positron emission tomography (PET) scan [10] . Also, functional test revealed overall transplanted patients did better (lower score in UPDRS and higher score in Schwab and England Scores).

The experiment proved that the transplanted cell mortality and effectiveness of treatment are determinant on the age of the patients [10] . Post-mortem study of a patient who underwent the transplanted surgery showed that the transplanted cells grew, got stained for tyrosine hydroxylase and made neuromelanin. The treatment proved beneficial but whether this would be more effective than a sham surgery is yet to be discovered. [10]

Human Trial 2

Human_Trials_1.2.JPG
Thirty-four patients between the ages of 30-75 were divided into three groups to undergo either of the following transplantation in the putamen:

Bilateral fetal nigral transplant (1 embryo/side)
Bilateral fetal nigral transplant (4 embryos/ side)
Placebo procedure (partial burr openings that did not penetrate the inner table of the skull)

The study was conducted for 2 years. The results showed that there were no significant differences in the overall treatment effect (Unified Parkinson’s Disease Rating Scale was used to ascertain) but the treatment had effect on patients with mild form of the disease[11] . The fluordopa uptake in the striatum significantly increased in the transplanted groups and post mortem studies revealed a strong survival of dopamine neurons10. 56% of the transplanted patients developed dyskinesia that persisted after overnight withdrawal of dopaminergic medication. [11] In conclusion, the therapy was not recommended for clinical trials as of yet because further research was suggested to acquire the required results.

Three Follow-up studies

Post-Mortem_Study_1.JPGPlacebo effects in Parkinson’s disease[12]
On average 16% of the patients with Parkinson’s disease improve with placebo treatments.
Post-Mortem_Study_2.JPG
Post-mortem studies of long-term grafts[13] [14]
Post-mortem study of two subjects, 11 and 16 years after the transplant showed that the grafts survived and stained positive for tyrosine hydroxylase, and neuromelanin. But it was also found that lewy bodies were present in the grafted cells.

Electrophysiological recordings from transplant patients[15]
Recordings obtained from single neurons in patients who underwent bilateral transplantation of dopamine cells in the striatum during rest and movement showed that transplantation decreased the symptoms of dyskinesia in off-medication patients. There was a reduction in activity in globus pallidus interna (GPi) during movement.

Conclusion

The results discussed here are promising but there are many hurdles that need to be dealt with before they can be clinically practiced. Research in potential sources of cells for transplantation, ways to control dopamine release, and methods to reduce dyskinesia side effects are essential for the success of the procedure.

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