Establishing an in-vitro model of Parkinson's disease.
Parkinson's disease (PD) is a major neurodegenerative disorder that manifests primarily after the age of 60 and whose main typical features include slowed physical movement (bradykinesia), rigidity, tremor and weakness (Reviewed by Lees, et al., 2009 & Berdarelli, et al., 2001). The precise mechanisms that trigger the onset of this disorder are not yet fully understood and even though various studies have been carried out in order to shed some light on this problem, the question remains as to what exactly promotes the development of this disease. At the cellular and molecular level, the most important characteristics of PD are a reduction of dopamine (DA) release from the substantia nigra mesenchephalic neurons due to their gradual degeneration; and the presence of Lewy bodies, which are small cytoplasmic inclusions that are positive for a-synuclein and ubiquitin (Spillantini, 1997 cited by Sherer, et al., 2002).
Current therapeutic approaches for PD are limited to ameliorating some of the symptoms by, either the use of dopamine replacement therapies, such as L-dopa and DA agonists administration or the use of surgical treatments like Deep Brain Stimulation (DBS) (Lindvall & Kokaia, 2009). While the employment of these therapies offers a certain reduction of the symptoms for a limited time only, there is hope that a "disease-modifying" treatment will emerge sooner than later for millions of people around the world who are suffering from this condition.
However, in order to achieve this goal, scientists first need to develop a reliable method of studying the disease by getting to know the precise mechanisms that give rise to it. Several studies have been carried out using animal models of the disease, in which different drugs (Table 1) are used to induce the symptomatology of PD (Reviewed by Shimohama, et al., 2003).
Huge steps have been taken in the context of biomedical research since both mouse and human embryonic stem cells were first derived more than twenty and ten years ago respectively (Martin, 1981; Evans & Kaufman, 1981; Thomson, et al., 1998). Since then, new discoveries in the field of stem cell biology have further enhanced our understanding of the pathogenesis of different diseases and in doing so, have allowed scientists to develop novel therapeutic approaches to treat them. Within this array of therapies lies the challenging but nonetheless promising potential of developing tailor-made cellular or tissue transplants using either human embryonic stem cells (hESCs) or induced pluripotent stem cells (hiPSCs) (Crook & Kobayashi, 2008). Moreover, with the advent of these technologies it is becoming more feasible than ever to try and create in-vitro disease models that in turn offer advantages over their animal-based counterparts, since some of the latter sometimes do not provide a means of rendering human disease with complete accuracy when it comes to resembling the particular traits that define the pathology (Lengerke & Daley, 2009).
The ability of hESCs and hiPSCs to differentiate in-vitro into potentially all of the specialised cell types in the body is clearly the feature that makes them perfect candidates for studying a wide range of biological processes, including pathological states (Lengerke & Daley, 2009). In the context of PD in particular, there have been a number of reports that address the issue of realising methods for modelling this condition in-vitro (Sherer, et al., 2002; Park, et al., 2008) Soo Cho, et. al. 2008). In most cases, and given the pathogenesis of the disease, their main focus is on trying to generate dopaminergic neurons that are subsequently suitable for transplantation into animal models (Friling, et al., 2009) (Arenas, 2002) Cai et al., 2009; Kim et al., 2002)
Embryonic stem cells are a type of pluripotent stem cells that are derived from the Inner Cell Mass (ICM) of the blastocyst (Martin, 1981). Human embryonic stem cells have the capacity to differentiate into all the tissues of the three germ layers (Thomson, et al., 1998) and the germline (Amabile & Meissner, 2009). But, unlike zygotes, they cannot give rise to extra-embryonic tissues (trophoblast and placenta) (Arenas, 2002; Amabile & Meissner, 2009). The basic defining characteristic of hESCs, which is their ability to give rise to every cell in the human body, is also a major point of concern. This is due to the fact that at the time of inducing their differentiation, they can also unexpectedly yield some types of cells that are not desired or even worse, give rise to tumours (teratomas) (Thomson, et al., 1998; Biehl & Russell, 2009). Nevertheless, current methodologies for generating hESCs are now much better than they were ten years ago. Newer and better approaches of achieving more stable cultures of hESCs means that the possibility of attaining the higher goal of regenerative medicine is not too far away. And if all this advances with hESCs were not enough, now there is an even more novel technology that allows scientists to practically turn back time in the cell clock. Just a few years ago, it was discovered that completely differentiated somatic adult cells could be de-differentiated to an state in which they display common features of embryonic stem cells, by just a handful of transcription factors (Takahashi, et al., 2007). Human induced pluripotent stem cells are a perfect example of the versatility of cells. Their generation brings about very important insights into the understanding of how the molecular events that occur inside the cell may determine its fate. One major argument in favour of the use and generation of hiPSCs is that they effectively circumvent the ethical problems regarding the use of hESCs, as the former can be obtained directly from a patient's own tissue and be set back to an embryonic state and then re-programmed into the cell type of interest (Takahashi, et al., 2007). However, it is still unkown how far can this technology reach, given the fact that there is still a lack of knowledge about the precise molecular mechanisms that occur during the re-programming events (Nishikawa, et al., 2008). These human induced pluripotent stem cells, together with their "original" counterparts (hESCs) may account for some of the most important findings that are still to come in the near future, specifically, regarding the issue of modelling human disease in-vitro.
Parkinson's disease (maladie de Parkinson) is a neurodegenerative disorder that was first described by James Parkinson in 1817 as the shaking palsy and was later attributed with its current name (Parkinson's disease) by Jean Martin Charcot (Lees, et al., 2009). To date, PD represents one of the most common neurodegenerative disorders whose incidence increases profoundly with age and that affects 1-2% of people over the age of 65 (Lees, et al., 2009; Arenas, 2002; Schle, et al., 2009). Although the precise mechanisms that lead to the onset of the disease are not fully understood yet, important genetic and pathological evidence has recently come out to light (Lees, Hardy, & Revesz, 2009).
It is believed that environmental factors could play a role in the onset of this disorder, which is commonly regarded as a sporadic disease. However, there are few environmental cues characterised to date that could hint towards what the triggers of this disorder are (Tanner, 2003; Taylor, et al., 2005 & Dick, et al., 2007 cited by Lees, et al., 2009). The pathological cellular features that are the hallmark of this disease are mainly found in nigrostriatal and mesolimbic dopaminergic neurons, which undergo a gradual and progressive loss that ultimately leads to the characteristic motor dysfunctions seen in PD: bradykinesia (slowed physical movement), rigidity and tremor (Reviewed by Arenas, 2002; Berdarelli, et al., 2001; Lees, et al., 2009; Taylor & Minger, 2005). Nevertheless, neuronal loss alone does not account for all of the defects present in PD. The presence of small intracellular fibrillar inclusions, known as Lewy bodies, which display an abnormal accumulation of proteins, such as a-synuclein and ubiquitin, are also part of the pathological features of PD (Spillantini, 1997 cited by Sherer, et al., 2002 & Arenas, 2002).
It is unknown how these selective detrimental changes in the dopaminergic neuron population come about, but there is evidence that a multi-factorial cascade of insults, such as the presence of oxidative stress, mitochondrial dysfunction and excyto-toxic damage are key players in the physiopathology of the disease (Reviewed by Arenas, 2002). Parkinson's disease is more common as a sporadic disorder. Hence, there is sufficient rationale for the implication of different genetic and environmental factors in the appearance of the disease (Arenas, 2002). On the other hand, it is the study of the more infrequent cases of the familial form of Parkinson's disease that has shed some light on the pathogenesis of this condition. There are at least four genes that can undergo loss-of-function mutations, which in turn lead to recessive early onset parkinsonism: parkin, DJ-1, PINK1 and ATP13A2 (Reviewed by Lees, et al., 2009). The E3 ubiquitin ligase parkin protein is in charge of mediating the engulfment of dysfunctional mitochondria by autophagosomes (Narendra, D. et al., 2008 cited by Lees, et al., 2009). Whilst mutations in the DJ-1, PINK1 and ATP13A2 genes are not very common, Parkin mutations account for the second most common genetic cause of parkinsonism that is responsive to L-dopa treatment (Reviewed by Lees, et al., 2009). Thus, abnormal accumulation of unwanted proteins (a-synuclein) and dysfunctional mitochondria are key factors that could lead to the onset of PD (Reviewed by Lees, et al., 2009).
Since the causes of PD are unknown, there is not an exact scientific preventive measure that can be relied on in order to reduce the risk of developing this condition. Age seems to be the most predominant factor that leads to the onset of the disease; and men have a slightly higher risk of developing PD compared to women (Twelves, D., et al., 2003 cited by Lees, et al., 2009). There are however, some studies that highlight some factors that could have an impact on the probablity of acquiring the disease, but nevertheless, much research remains to be done in this regard. Among these possible factors are that smoking and caffeine consumption relates to lower rates of Parkinson's disease. However, these claims are more to do with the role of dopamine in reward pathways, rather than to a some sort of neuroprotective effect on the side of nicotine of caffeine (Evans, et al., 2006; Quik & Jeyarasasingman, 2000 cited by Lees, et al., 2009).
While there is no documented cure for Parkinson's disease, there are drug-based approaches, as well as surgery options, that offer some restoring effects for some patients. The most common approach is the use of dopamine replacement therapies, in which Levodopa (L-dopa) is
As the major characteristic of Parkinson's disease is the loss of functional dopaminergic neurons, therapy approaches are focused on developing cell replacement strategies, which were first explored in the 1970's and 1980's (Taylor & Minger, 2005). The possible and currently studied cell sources for this purpose vary from the use of autologus dopaminergic cell transplants to fetal ventral mesencephalon tissue and different stem cell lines (Ren & Zhang, 2009). Given the pluripotent capacity of stem cells and their different types, namely, human embryonic stem cells (hESCs), neural stem cells (NSCs), nuclear transfer ES cells (ntESCs), human induced pluripotent stem cells (hiPSCs), carotid body stem cells and mesenchymal stem cells (MSCs), they are the most sought after prospects for the successful generation of dopaminergic neurons (Newman & Bakay, 2008; Bjugstad, et. al., 2008; Tabar, et al., 2008; Yamanaka, 2007; Pardal, et al., 2007; Park, et al., 2008 cited by Ren & Zhang, 2009).
Modelling Human Disease (use of hESCs and hiPSCs in this field)
A key goal in the field of biomedical scientific research is the ability to identify with the greatest possible accuracy, the mechanisms that take place during both normal and pathologic states. With the discovery of stem cells and their different subtypes, understanding of the molecular and cellular events that underlie each of these conditions had never been so close to attain. For many years, scientists have relied on the use of animals as a tool for generating human disease models. In the case of diseases of the Central Nervous System (CNS) however, their study has been hindered by the complexity of the circuitry implicated and by the limitations that animal models impose, not to mention the restrictions for studying human subjects (Crook & Kobayashi, 2008).
The use of hESCs or hiPSCs seems to offer a good work-around to these limitations. Nevertheless, there is a caveat when it comes to rendering a model of a genetic disease. These type of conditions present varied degrees of severity in the symptomatology and the level of penetrance from one patient to another (Colman & Dreesen, 2009). This heterogeneity has its roots in the complexity of the interactions between the specific genetic background of one person and their environment. Prior knowledge of these factors enables for a more informed experimental design and interpretation of data; whereas this would not be possible with disease-specific hESCs, since they obviously lack such clinical history due to their origins (Colman & Dreesen, 2009). Animal models of disease are certainly of enormous importance, since they may serve as a validation tool for either an embryonic stem cell or an induced pluripotent stem cell approach of generating specific cell types (Colman & Dreesen, 2009).
Embryonic stem cells have been successfully employed in various murine disease models, which could suggest that translational clinical therapies of hESCs may be possible within a not so long period of time (Koch, 2009). Furthermore, stem cell therapies offer a particularly good outlook on approaching neurodegenerative disorders, such as Parkinson's disease, since the main feature of the pathology is a marked reduction in the population of nigrostratial dopaminergic neurons, which in turn provides a good reason for developing methods that allow generation of dopaminergic neurons suitable for transplantation (Koch, 2009).
In-vitro disease models have some limitations, but these are counter-balanced by the fact that the cells used for this purpose are from human origin, thus allowing for a better characterisation of the pathological properties that make up the disease and also by reducing the time-consuming effort of developing an in-vivo model (Schule, 2009). The use of tissue culture systems for the study of neurodegenerative disorders is particularly useful because gene manipulation is readily achieved, such as over-expression or knockdown of proteins that allow the study of the functional consequences of these changes (Schule, 2009).
Amabile, G., & Meissner, A. (2009). Induced pluripotent stem cells: current progress and potential for regenerative medicine. Trends in Molecular Medicine , 15 (2), 59-68.
Arenas, E. (2002). Stem cells in the treatment of Parkinson's disease. Brain Research Bulletin , 57 (6), 795-808.
Berdarelli, A., Rothwell, J., Thompson, P., & Hallet, M. (2001). Pathophysiology of bradykinesia in Parkinson's disease. Brain , 124, 2131-2146.
Biehl, J. K., & Russell, B. (2009). Introduction to Stem Cell Therapy. Journal of Cardiovascular Nursing , 24 (2), 98-103.
Colman, A., & Dreesen, O. (2009). Pluripotent Stem Cells and Disease Modeling. Cell Stem Cell , 5, 244-247.
Crook, J. M., & Kobayashi, N. R. (2008). Human Stem Cells for Modeling Neurological Disorders: Accelerating the Drug Discovery Pipeline. Journal of Cellular Biochemistry , 105, 1361-66.
Evans, M., & Kaufman, M. (1981). Establishment in culture of pluripotential cells from mouse embryos. Nature , 292 (5819), 154-6.
Friling, S., Andersson, E., Thompson, L. H., Jnsson, M. E., Hebsgaard, J. B., Nanou, E., et al. (2009). Efficient production of mesencephalic dopamine neurons by Lmx1a expression in embryonic stem cells. PNAS , 106 (18), 7613-7618.
Lees, A. J., Hardy, J., & Revesz, T. (2009). Parkinson's disease. Lancet , 373, 2055-66.
Lengerke, C., & Daley, G. Q. (2009). Disease Models from Pluripotent Stem Cells - Turning back time in disease pathogenesis? Hematopoietic Stem Cells VII: Ann. N.Y. Acad. Sci. , 1176, 191-196.
Lindvall, O., & Kokaia, Z. (2009). Prospects of stem cell therapy for replacing dopamine neurons in Parkinson's disease. Cell , 30 (5), 260-267.
Martin, G. R. (1981). Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Developmental Biology , 78 (12), 7634-7638.
Nishikawa, S.-i., Goldstein, R. A., & Nierras, C. R. (2008). The promise of human induced pluripotent stem cells for research and therapy. Nature Reviews Molecular Cell Biology , 9, 725-729.
Park, I.-H., Arora, N., Huo, H., Maherali, N., Ahfeldt, T., Shimamura, A., et al. (2008). Disease-Specific Induced Pluripotent Stem Cells. Cell , 134, 877-886.
Sherer, T. B., Betarbet, R., Stout, A. K., Lund, S., Baptista, M., Panov, A. V., et al. (2002). An In Vitro Model of Parkinson's Disease: Linking Mitochondrial Impairment to Altered alpha-Synuclein Metabolism and Oxidative Damage. The Journal of Neuroscience , 22 (16), 7006-7015.
Shimohama, S., Sawada, H., Kitamura, Y., & Taniguchi, T. (2003). Disease model: Parkinson's disease. Trends in Molecular Medicine , 9 (8), 360-365.
Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K., et al. (2007). Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors. Cell , 131 (5), 861-872.
Thomson, J. A., Itskovitz-Eldor, J., Shapiro, S. S., Waknitz, M. A., Swiergiel, J. J., Marshall, V. S., et al. (1998). Embryonic Stem Cell Lines Derived from Human Blastocysts. Science , 282 (5391), 1145-47.