Cell Therapeutics in Parkinson’s Disease

The main pathology underlying motor symptoms in Parkinson’s disease (PD) is a rather selective degeneration of nigrostriatal dopamine (DA) neurons. Intrastriatal transplantation of immature DA neurons, which replace those neurons that have died, leads to functional restoration in animal models of PD. Here we describe how far the clinical translation of the DA neuron replacement strategy has advanced. We briefly summarize the lessons learned from the early clinical trials with grafts of human fetal mesencephalic tissue, and discuss recent findings suggesting susceptibility of these grafts to the disease process long-term after implantation. Mechanisms underlying graft-induced dyskinesias, which constitute the only significant adverse event observed after neural transplantation, and how they should be prevented and treated are described. We summarize the attempts to generate DA neurons from stem cells of various sources and patient-specific DA neurons from fully differentiated somatic cells, with particular emphasis on the requirements of these cells to be useful in the clinical setting. The rationale for the new clinical trial with transplantation of fetal mesencephalic tissue is described. Finally, we discuss the scientific and clinical advancements that will be necessary to develop a competitive cell therapy for PD patients.     

Current advances in the treatment of Parkinson's disease with stem cells

Stem cell replacement has emerged as the novel therapeutic strategy for Parkinson's disease (PD). Control of motor behavior is lost in PD due to the selective degeneration of mesencephalic dopamine neurons (DA) in the substantia nigra. This progressive loss of DA neurons results in devastating symptoms for which there is no cure. Debilitating side effects often result from chronic pharmacological treatment, hence current investigations into cell transplantation therapy as a substitute and/or adjuvant to other therapeutics. Clinical trials with fetal DA tissue have provided evidence that cell transplantation could be a viable alternative. Limited availability of fetal tissue, combined with variable outcome led to emphasis on other sources of cells, such as stem cells. This review focuses on three stem cell sources (embryonic, neural, and adult mesenchymal). Also discussed is the molecular differentiation into mature DA neurons, the various protocols that have been developed to generate DA neurons from various stem cells, and the current state of stem cell therapy for PD.     

Dopaminergic properties and function after grafting of attached neural precursor cultures

Generation of dopaminergic (DA) neurons from multipotent embryonic progenitors represents a promising therapeutical strategy for Parkinson's disease (PD). Aim of the present study was the establishment of enhanced cell culture conditions, which optimize the use of midbrain progenitor cells in animal models of PD. In addition, the progenitor cells were characterized during expansion and differentiation according to morphological and electrophysiological criteria and compared to primary tissue. Here, we report that CNS precursors can be expanded in vitro up to 40-fold and afterwards be efficiently differentiated into DA neurons. After 4-5 days under differentiation conditions, more than 70% of the neurons were TH+, equivalent to 30% of the total cell population. Calcium imaging revealed the presence of calcium-permeable AMPA receptors in the differentiated precursors which are capable to contribute to many developmental processes. The overall survival rate, degree of reinnervation and the behavioral performance after transplantation of 4 days in-vitro-differentiated cells were similar to results after direct grafting of E14 ventral mesencephalic cells, whereas after shorter or longer differentiation periods, respectively, less effects were achieved. Compared to the amount of in-vitro-generated DA neurons, the survival rate was only 0.8%, indicating that these cells are very vulnerable. Our results suggest that expanded and differentiated DA precursors from attached cultures can survive microtransplantation and integrate within the striatum in terms of behavioral recovery. However, there is only a short time window during in vitro differentiation, in which enough cells are already differentiated towards a DA phenotype and simultaneously not too mature for implantation. However, additional factors and/or genetical manipulation of these expanded progenitors will be required to increase their in vivo survival in order to improve both the ethical and the technical outlook for the use of fetal tissue in clinical transplantation.     

A role for tumor necrosis factor alpha in death of dopaminergic neurons following neural transplantation

Poor survival of transplanted dopaminergic (DA) neurons remains a serious obstacle to the success of cell replacement therapy as an alternative to the current treatments for Parkinson's disease (PD). We have examined the temporal release profile of an inflammatory cytokine, tumor necrosis factor-alpha (TNFalpha), following transplantation of fetal mesencephalic tissue into the rat striatum. The amounts of TNFalpha released in vivo when added to cultures of embryonic DA neurons, significantly reduced the survival of DA neurons in vitro, and this cell death could be prevented by the inclusion of an antibody to the TNFalpha receptor type 1. Inclusion of this antibody in cell suspensions during transplantation also increased the survival of transplanted fetal DA neurons by approximately 250%. Use of this therapeutic antibody approach may offer significant improvements to neural transplantation as a treatment for PD.     

Cell Therapy in Parkinson's Disease

Summary: The clinical studies with intrastriatal transplants of fetal mesencephalic tissue in Parkinson''s disease (PD) patients have provided proof-of-principle for the cell replacement strategy in this disorder. The grafted dopaminergic neurons can reinnervate the denervated striatum, restore regulated dopamine (DA) release and movement-related frontal cortical activation, and give rise to significant symptomatic relief. In the most successful cases, patients have been able to withdraw l-dopa treatment after transplantation and resume an independent life. However, there are currently several problems linked to the use of fetal tissue: 1) lack of sufficient amounts of tissue for transplantation in a large number of patients, 2) variability of functional outcome with some patients showing major improvement and others modest if any clinical benefit, and 3) occurrence of troublesome dyskinesias in a significant proportion of patients after transplantation. Thus, neural transplantation is still at an experimental stage in PD. For the development of a clinically useful cell therapy, we need to define better criteria for patient selection and how graft placement should be optimized in each patient. We also need to explore in more detail the importance for functional outcome of the dissection and cellular composition of the graft tissue as well as of immunological mechanisms. Strategies to prevent the development of dyskinesias after grafting have to be developed. Finally, we need to generate large numbers of viable DA neurons in preparations that are standardized and quality controlled. The stem cell technology may provide a virtually unlimited source of DA neurons, but several scientific issues need to be addressed before stem cell-based therapies can be tested in PD patients.     

Embryonic and adult stem cells as a source for cell therapy in Parkinson's disease

The rationale behind the use of cells as therapeutic modalities for neurodegenerative diseases in general, and in Parkinson's disease (PD) in particular, is that they will improve patient's functioning by replacing the damaged cell population. It is reasoned that these cells will survive, grow neurites, establish functional synapses, integrate best and durably with the host tissue mainly in the striatum, renew the impaired wiring, and lead to meaningful clinical improvement. To increase the generation of dopamine, researchers have already transplanted non-neuronal cells, without any genetic manipulation or after introduction of genes such as tyrosine hydroxylase, in animal models of PD. Because these cells were not of neuronal origin, they developed without control, did not integrate well into the brain parenchyma, and their survival rates were low. Clinical experiments using cell transplantation as a therapy for PD have been conducted since the 1980s. Most of these experiments used fetal dopaminergic cells originating in the ventral mesencephalic tissue obtained from fetuses. Although it was shown that the transplanted cells survived and some patients benefited from this treatment, others suffered from severe dyskinesia, probably caused by the graft's excessive and uncontrolled production and release of dopamine. It is now recognized that cell-replacement strategy will be effective in PD only if the transplanted cells have the same abilities, such as dopamine synthesis and control release, reuptake, and metabolizing dopamine, as the original dopaminergic neurons. Recent studies on embryonic and adult stem cells have demonstrated that cells are able to both self-renew and produce differentiated tissues, including dopaminergic neurons. These new methods offer real hope for tissue replacement in a wide range of diseases, especially PD. In this review we summarize the evidence of dopaminergic neuron generation from embryonic and adult stem cells, and discuss their application for cell therapy in PD.     

Emerging restorative treatments for Parkinson's disease: manipulation and inducement of dopaminergic neurons from adult stem cells

Parkinson's disease (PD) is a common neurodegenerative disease, characterized by a selective loss of midbrain Dopaminergic (DA) neurons. To address this problem, various types of stem cells that have potential to differentiate into DA neurons are being investigated as cellular therapies for PD, including cells derived from embryonic or adult donor tissue, and embryonic stem cells. These cell sources, however, have raised certain questions with regard to ethical and rejection issues. Recent progress in adult stems has further proved that the cells derived from adult tissue could be expanded and differentiated into DA precursor cells in vitro, and cell therapy with adult stem cells could produce a clear improvement for PD models. Using adult stem cells for clinic application may not only overcome the ethical problem inherent in using human fetal tissue or embryonic stem cells, but also open the possibility for autologous transplantation. The patient-specific adult stem cell is therefore a potential and prospective candidate for PD treatment.     

A clonal line of mesencephalic progenitor cells converted to dopamine neurons by hematopoietic cytokines: a source of cells for transplantation in Parkinson's disease

Neural progenitor cells potentially provide a limitless, on-demand source of cells for grafting into patients with Parkinson's disease (PD) if the signals needed to control their conversion into dopamine (DA) neurons could be identified. We have recently shown that cytokines which instruct cell division and differentiation within the hematopoeitic system may provide similar functions in the central nervous system. We have shown that mitotic progenitor cells can be isolated from embryonic rat mesencephalon and that these cells respond to a combination of interleukin-1, interleukin-11, leukemia inhibitory factor, and glial cell line-derived neurotrophic factor yielding a tyrosine hydroxylase-immunoreactive (THir) phenotype in 20-25% of total cells. In the present study, 24 clonal cell lines derived from single cells of mesencephalic proliferation spheres were examined for their response to the cytokine mixture. The clone yielding the highest percentage of THir neurons (98%) was selected for further study. This clone expressed several phenotypic characteristics of DA neurons and expression of Nurr1. The response to cytokines was stable for several passages and after cryopreservation for several months. When grafted into the striatum of DA-depleted rats, these cells attenuated rotational asymmetry to the same extent as freshly harvested embryonic DA neurons. These data demonstrate that mesencephalic progenitor cells can be clonally expanded in culture and differentiated in the presence of hematopoietic cytokines to yield enriched populations of DA neurons. When transplanted, these cells provide significant functional benefit in the rat model of PD.     

Embryonic and adult stem cells as a source for cell therapy in Parkinson’s disease

The rationale behind the use of cells as therapeutic modalities for neurodegenerative diseases in general, and in Parkinson’s disease (PD) in particular, is that they will improve patient’s functioning by replacing the damaged cell population. It is reasoned that these cells will survive, grow neurites, establish functional synapses, integrate best and durably with the host tissue mainly in the striatum, renew the impaired wiring, and lead to meaningful clinical improvement. To increase the generation of dopamine, researchers have already transplanted non-neuronal cells, without any genetic manipulation or after introduction of genes such as tyrosine hydroxylase, in animal models of PD. Because these cells were not of neuronal origin, they developed without control, did not integrate well into the brain parenchyma, and their survival rates were low. Clinical experiments using cell transplantation as a therapy for PD have been conducted since the 1980s. Most of these experiments used fetal dopaminergic cells originating in the ventral mesencephalic tissue obtained from fetuses. Although it was shown that the transplanted cells survived and some patients benefited from this treatment, others suffered from severe dyskinesia, probably caused by the graft’s excessive and uncontrolled production and release of dopamine. It is now recognized that cell-replacement strategy will be effective in PD only if the transplanted cells have the same abilities, such as dopamine synthesis and control release, reuptake, and metabolizing dopamine, as the original dopaminergic neurons. Recent studies on embryonic and adult stem cells have demonstrated that cells are able to both self-renew and produce differentiated tissues, including dopaminergic neurons. These new methods offer real hope for tissue replacement in a wide range of diseases, especially PD. In this review we summarize the evidence of dopaminergic neuron generation from embryonic and adult stem cells, and discuss their application for cell therapy in PD.     

Neuromodulation for Parkinson's disease]

Parkinson's disease (PD) is a neurodegenerative disorder characterized by a progressive loss of midbrain dopaminergic (DA) neurons and a subsequent reduction in striatal dopamine. As a treatment for advanced Parkinson's disease, deep brain stimulation (DBS) of the thalamus was introduced in 1987 to treat tremor, and was applied in 1993 to the subthalamic nucleus. Now high-frequency stimulation of the subthalamic nucleus has become a surgical therapy of choice. Another surgical treatment is a cell replacement therapy. Transplantation of fetal dopaminergic (DA) neurons can produce symptomatic relief, however, the technical and ethical difficulties in obtaining sufficient and appropriate donor fetal brain tissue have limited the application of this therapy. Then, neural precursor cells and embryonic stem (ES) cells are expected to be candidates of potential donor cells for transplantation. We induced DA neurons from monkey ES cells, and analyzed the effect of transplantation of the DA neurons into MPTP-treated monkeys as a primate model of Parkinson's disease. Behavioral studies and functional imaging revealed that the transplanted cells functioned as DA neurons, attenuating the MPTP-induced neurological symptoms. DA neurons have also been generated from several human ES cell lines. Furthermore, functional recovery of rat PD models after transplantation was observed. One of the major problems in ES cell transplantation is tumor formation, which is caused by a small fraction of undifferentiated ES cells in the graft. So, it is essential for undifferentiated ES cells to be eliminated from the graft in order for transplantation to be feasible. These efforts will lead to clinical application of ES cell transplantation to the patients with PD.     

Adeno-associated viral vector-mediated gene transduction in mesencephalic slice culture

Adeno-associated viral (AAV) vector is a non-pathogenic vehicle that is suitable for the delivery of foreign genes into non-dividing neuronal cells. This vector has been utilized for in vivo neurological research and in clinical trials of gene therapy for neurodegenerative disorders. Viral vector-mediated gene delivery has the limitation that progressive changes in cellular phenotype cannot be monitored in living animals. To visualize living neurons transduced with foreign genes in vitro, we used cultured mesencephalic tissue harboring living dopaminergic (DA) neurons and examined cellular tropism of serotype-1 and serotype-2 AAV vectors in a culture system. The viability of DA neurons was evaluated using transgenic mice carrying enhanced green fluorescent protein under the control of the rat tyrosine hydroxylase (TH) promoter, which enables the visualization of living DA cells in the substantia nigra. Apoptosis of a subset of neuronal cells was noted within one day of culture. After 7 days, the serotype-1 AAV vector had successfully delivered the foreign gene into neurons and astrocytes, and serotype-2 AAV vector was able to transduce TH-positive DA neurons efficiently. Our method should be useful for in vitro investigations of pathological changes in DA neurons following transduction with foreign genes.     

Toxicity of Dieldrin for Dopaminergic Neurons in Mesencephalic Cultures

Dieldrin can be retained for decades in lipid-rich tissue and has been measured in some postmortem PD brains. Dieldrin has been reported to deplete brain monoamines in several species and has been shown to inhibit mitochondrial respiration. To further investigate the possibility that it may be involved in the pathogenesis of parkinsonism, its toxicity for dopaminergic (DA) neurons was assessed in a mesencephalic cell culture model. Primary neuronal cultures of mesencephalic neurons were prepared from fetal rats or fetal mice, grown for 1 week and incubated with Dieldrin (0.01–100 μM) for 24 or 48 h. Toxicity for DA neurons was determined by measuring density of surviving tyrosine hydroxylase immunoreactive (TH-ir) cells. Toxicity for gamma-aminobutyric acid (GABA)-ergic neurons was determined by measuring survival of glutamate decarboxylase (GAD)-ir neurons. General, nonselective cytotoxicity was determined by counting cells visualized by phase contrast microscopy or by DAPI-stained cells with fluorescence microscopy. Dieldrin exposure for 24 h resulted in a dose-dependent decrease in survival of TH-IR cells (DA neurons) with a 50% decrease (EC₅₀) produced by 12 μM in rat mesencephalic cultures. Dieldrin also produced a dose- and time-dependent decrease in mouse DA-ergic and GABA-ergic neurons in mouse mesencephalic cultures. GABA-ergic neurons were less sensitive to the toxin compared to DA-ergic neurons. Cellular uptake of³H-DA was also affected by lower concentrations of Dieldrin (EC₅₀ = 7.98 μM) than uptake of³H-GABA (EC₅₀ = 43 μM). Thus, Dieldrin appears to be a relatively selective DA-ergic neurotoxin in mesencephalic cultures. Dieldrin, which may be ubiquitous in the environment, is proposed as an agent which can initiate and promote dopaminergic neurodegeneration in susceptible individuals.     

Cell survival and clinical outcome following intrastriatal transplantation in Parkinson disease

Intrastriatal transplantation of embryonic dopaminergic neurons is currently explored as a restorative cell therapy for Parkinson disease (PD). Clinical results have varied, probably due to differences in transplantation methodology and patient selection. In this review, we assess clinical trials and autopsy findings in grafted PD patients and suggest that a minimum number of surviving dopaminergic neurons is required for a favorable outcome. Restoration of [18F]-fluorodopa uptake in the putamen to about 50% of the normal mean seems necessary for moderate to marked clinical benefit to occur. Some studies indicate that this may require mesencephalic tissue from 3-5 human embryos implanted into each hemisphere. The volume, density and pattern of fiber outgrowth and reinnervation, as well as functional integration and dopamine release. are postulated as additional important factors for an optimal clinical outcome. For neural transplantation to become a feasible therapeutic alternative in PD, graft survival must be increased and the need for multiple donors of human embryonic tissue substantially decreased or alternate sources of donor tissue developed. Donor cells derived from alternative sources should demonstrate features comparable to those associated with successful implantation of human embryonic tissue before clinical trials are considered.     

IL-1 beta is released from the host brain following transplantation but does not compromise embryonic dopaminergic neuron survival

Poor survival of transplanted dopaminergic (DA) neurons remains a serious obstacle to the success of cell replacement therapy as an alternative to the current treatments for Parkinson's disease. We have examined the temporal release profile of an inflammatory cytokine, interleukin-1 beta (IL-1 beta) following transplantation of fetal mesencephalic tissue into the rat striatum. The amounts of IL-1 beta released in vivo when added to cultures of embryonic DA neurons, did not significantly reduce the survival of DA neurons in vitro, and inclusion of the naturally-occurring IL-1 receptor antagonist, IL-1ra, did not appear to affect the numbers of surviving DA neurons present after 5 days in vitro. Neither did inclusion of IL-1ra in cell suspensions during transplantation increase the survival of transplanted fetal DA neurons. Thus, although IL-1 beta is released following implantation of a neural transplant, we suggest that this pro-inflammatory cytokine does not play an active role in reducing survival of transplanted DA neurons, unlike other cytokines such as tumor necrosis factor alpha. Modulation of IL-1 beta activity, therefore, will not offer significant improvements to neural transplantation as a treatment for PD.     

IL-1β is released from the host brain following transplantation but does not compromise embryonic dopaminergic neuron survival

Poor survival of transplanted dopaminergic (DA) neurons remains a serious obstacle to the success of cell replacement therapy as an alternative to the current treatments for Parkinson's disease. We have examined the temporal release profile of an inflammatory cytokine, interleukin-1 beta (IL-1 beta) following transplantation of fetal mesencephalic tissue into the rat striatum. The amounts of IL-1 beta released in vivo when added to cultures of embryonic DA neurons, did not significantly reduce the survival of DA neurons in vitro, and inclusion of the naturally-occurring IL-1 receptor antagonist, IL-1ra, did not appear to affect the numbers of surviving DA neurons present after 5 days in vitro. Neither did inclusion of IL-1ra in cell suspensions during transplantation increase the survival of transplanted fetal DA neurons. Thus, although IL-1 beta is released following implantation of a neural transplant, we suggest that this pro-inflammatory cytokine does not play an active role in reducing survival of transplanted DA neurons, unlike other cytokines such as tumor necrosis factor alpha. Modulation of IL-1 beta activity, therefore, will not offer significant improvements to neural transplantation as a treatment for PD.     

Cryopreservation and storage of embryonic rat mesencephalic dopamine neurons for one year: comparison to fresh tissue in culture and neural grafts

Blocks of embryonic rat ventral mesencephalic tissue containing the developing A8–A10 dopamine (DA) cell groups were cryopreserved and stored for approximately 1 year, at which time this tissue was thawed, dissociated into a cell suspension, and compared to a similar preparation of fresh mesencephalic tissue for viability in tissue culture and neural grafts. Estimates of total cell number immediately prior to plating in culture indicated that cryopreserved tissue yields fewer cells, but when this reduced cell number is compensated for, and equal numbers of cells were plated in culture, approximately equal total numbers of neurons, as well as tyrosine hydroxylase (TH)-positive neurons, were present in cultures from cryopreserved and fresh tissue. Grafting of equal numbers of fresh and cryopreserved mesencephalic cells into the striatum of adult rats with large unilateral lesions of the nigrostriatal DA pathway tended to yield smaller grafts with fewer surviving TH-positive cells with less extensive neuronal processes when tissue was previously cryopreserved. However, grafts derived from freeze-stored tissue provided a similar timecourse and extent of behavioral recovery in amphetamine-induced rotational tests to that provided by fresh tissue grafts. Taken together, our findings indicate that while cryopreservation of mesencephalic tissue has its costs — reduced cell yield in cultures and grafts, and compromised morphology in grafts — sufficient numbers of cryopreserved neurons survive the grafting procedure to ameliorate behavioral signs of DA depletion in the lesioned rat model.     

Transplantation of autologous sympathetic neurons as a potential strategy to restore metabolic functions of the damaged nigrostriatal dopamine nerve terminals in Parkinson's disease

Grafting of catecholamine-producing cells can be a possible therapeutic strategy for attenuating motor symptoms in Parkinson's disease (PD). The potential of autologous sympathetic neurons has been investigated as a donor for cell therapy of PD. The clinical trials of autotransplantation of sympathetic ganglion cells in PD have revealed that the grafts increase the duration of L-DOPA (L-dihydroxy phenyl alanine)-induced beneficial effects, and that the graft-mediated effect is detectable during a follow-up period of at least 1 year postgrafting. In an in vitro analysis of the ability of human sympathetic neurons to release catecholamines, although DA was not detectable under basal conditions, DA levels were significantly increased upon exposure to exogenous L-DOPA. Furthermore, animal experiments with xenografting of human sympathetic ganglionic neurons in the DA-denervated striatum of rats demonstrated that a significant increase in striatal DA levels is noted after systemic L-DOPA treatment, and that the DA levels remain high for longer periods of time in the grafted rats than in control animals with sham surgery. The L-DOPA-induced rise of striatal DA levels was significantly attenuated when given reserpine pretreatment. This suggests that DA derived from exogenously administered L-DOPA is subjected to, at least in part, vesicular storage in grafted sympathetic neurons. Histological examinations indeed showed that the grafts express aromatic-L-amino acid decarboxylase and vesicular monoamine transporter-2, both of which are important molecules for the synthesis and the storage of DA, respectively. Taken together, grafted sympathetic neurons can provide a site for both the conversion of exogenous L-DOPA to DA and the storage of the synthesized DA in the DA-denervated striatum. This might be an explanation for a mechanism by which sympathetic neuron autografts can increase the duration of L-DOPA effects in PD patients. This review article summarizes the clinical effect of transplantation of autologous sympathetic neurons in PD and discusses the underlying mechanism for the effect based on experimental evidence previously obtained.