Neurological disorders are disorders of the body's nervous system, where changes or abnormalities occur in the brain, spinal cord and there is a loss of neurones and glial cells. This results in hindered coordination, loss of sensation, muscle weakness and paralysis among many other consequences. There are several known neurological disorders such as, Parkinson's disease and Alzheimer's disease which are associated with difficulty in movement and dementia respectively. These disorders can be divided into two broad categories, disorders of the central nervous system (CNS) and disorders of the peripheral nervous system (PNS).
To understand what stem cells can do to help treat severe diseases, we must first appreciate the role of axonal regeneration in the central and peripheral nervous system. Axonal regeneration refers to the ability of axonal processes to re-grow and repair. It differs between the CNS and PNS by the functional mechanisms and the extent and speed. For example, injury to the central nervous system is not followed by extensive neuroregeneration, like it is in the PNS because it is limited by the inhibitory pressures of glial and extracellular environment.
1.2 Regeneration in the PNS
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Peripheral nerves contain axons which are associated with Schwann cells (the glial cells of the PNS) and are surrounded by basal lamina sheaths. Crush injuries to peripheral nerves usually leave the basal lamina intact, but cause degeneration of the axons distal to the site of injury. The degenerative changes in the distal nerve stump (termed Wallerian degeneration) are important since they facilitate subsequent axonal regeneration. During Wallerian degeneration, the myelin sheaths of axons break down and are extruded as globules by the Schwaan cells. Macrophages play an active role in phagocytosis of the myelin and axonal debris. The Schwann cells also proliferate, forming columns of cells within the basal laminae tubes (termed Bands of Bünger). The Schwann cells are extremely important for axonal regeneration since they synthesise a number of factors during Wallerian degeneration which may stimulate axonal growth such as NGF and other neurotrophic growth factors including BDNF, NT-4 and GDNF. In addition, they also express on their cell surface cell adhesion molecules including N-CAM and L1 which can also support axonal growth. The basal lamina of the endoneural tubes contains an important extracellular matrix molecule called laminin which is very effective in stimulating axonal growth. Since the basal lamina of the endoneural tubes usually remains intact after crush injuries, the regenerating axons may be guided back to their peripheral targets, resulting in potentially good recovery of movement and sensation.
If a peripheral nerve is transacted, the proximal and distal stumps tend to pull apart, leaving a gap which the axons must cross in order to reach the distal nerve stump. During the first few days of injury, fibroblasts of the nerve sheath proliferate and migrate from both proximal and distal stumps and may spontaneously establish a bridge of tissue through which the axons can regenerate.
There are ten steps involved in axon regeneration:
Cell Body Response - this starts with the decentralisation of the nucleus
Metabolic Reaction - an increased number of ribosomes around the nucleus
Immune Response - macrophages start attacking the Schwann cells of the distal segment
Nervous System Reaction - all adjacent neurones start extending sprouts of their axons to the target of the injured neurone
Enzymatic Action - The axon of the distal segment is broken down by enzymes and the products of this are carried by retrograde transport to the soma
Rapid Cell Division - The Schwann cell at the end of the proximal segment starts a rapid mitotic division in attempt to locate the target tissue for the severed neurone
Formation of Growth Path - the chain of the Schwann cells that reaches the target tissue will serve as a path for the growth of the axon
Axon Growth and death of extra Schwann cells - The remainder of the axon in the proximal tubule starts growing in the tube prepared by the Schwann cells. In the mean time, the Schwann cells which did not make it to the target cells will start dying and phagocytes will engulf the remains
Always on Time
Marked to Standard
Death of Sprouts - the re-innervation of the target tissue by the regenerating neurone leads to an automatic death of the sprouts of the adjacent neurones
Return to normal - after complete innervation, the nucleus returns to the centre of the soma and the number of the ribosomes declines and the neurone returns to its normal appearance
1.3 Regeneration in the CNS
For many years, it was believed that axons of the CNS were incapable of regeneration. The problem that lies with the CNS axons is that regeneration can fail for two main reasons. The first is due to the environment surrounding CNS lesions which is inhibitory to axon growth. Secondly, the axons themselves only mount a feeble regeneration response after they are cut. However, during the last few years, grafting experiments using segments of peripheral nerve or embryonic spinal cord have shown that CNS axons are capable of regeneration, providing that they are given a suitable environment.
Glial cells of the PNS are Schwann cells whilst in the CNS there are oligodendrocytes and astrocytes. Schwann cells support regeneration of both PNS and CNS axons. Astrocytes seem to be less capable of supporting axonal growth, even though the range of growth factors and cell adhesion molecules that they express seem to be similar to that of Schwann cells. Administration of neurotrophic factors can stimulate axons to regenerate in the CNS in some situations where they would not usually do so.
A further problem in the CNS is that lesions usually result in the formation of a glial scar (associated with astrocyte proliferation) which also tends to block axonal regeneration. This scar contains astrocytes, meningeal cells, oligodendrocyte precursors and microglia/macrophages. It seems likely that the major inhibitory factor(s) may be certain glycoproteins present in scar tissue.
In contrast to Schwann cells, oligodendrocytes do not support regeneration of either PNS or CNS axons. At least 2 proteins expressed by oligodendrocytes, myelin-associated glycoproteins (MAG) and NOGO, are strongly inhibitory to axonal growth. The activity of NOGO can be neutralised by a monoclonal antibody (IN-1). Observations from certain experiments involving destruction of oligodendrocytes suggest that inhibitory factors produced by this cell type may be partially responsible for failure of axonal regeneration in the CNS.
Since astrocytes and oligodendrocytes do not support axonal regeneration, grafting experiments involving other cell types have been carried out. Recent experiments involving ensheathing cells of the olfactory pathway have been very encouraging. Axonal regeneration may occur in the CNS during certain periods of embryonic development, possibly due to the absence of inhibitory factors since grafted embryonic spinal cord can support axonal regeneration of adult CNS neurons. Other grafting experiments have shown that embryonic CNS neurons are capable of axonal regeneration when grafted into the CNS of adult animals, suggesting that embryonic neurons are able to 'ignore' the factors which inhibit axonal regeneration of adult neurons. There is now considerable interest in the use of embryonic stem cells as grafts to restore function in the CNS (e.g. Parkinson's disease).
1.4 An Introduction to stem cells and their use in neurological disorders
Stem cells are cells which can divide by mitotic cell division and have an outstanding potential to develop into any cell type during growth. In tissues, they serve as part of a repair system where they continue to divide until the other cells are replenished. When a stem cell divides, it can differentiate to become a specific type of cell such as a blood cell, muscle cell or it can remain a stem cell for future use in repair. Stem cells differ from other cells in that they undifferentiated and unspecialised which can divide, even after long periods of inactivity. Another distinguishable characteristic is that they can be induced to become specific tissue cells under particular physiological or experimental conditions. In some organs or tissue, stem cells are always dividing to renew and repair damaged tissues.
Neural stem cells are believed to be able to renew themselves without limit and can generate neuronal and glial cells. They are found in the ventricular zone of the central nervous system during growth and development. 1,2 (Rietz et al and Temple 2001) In the adult brain, neural stem cells are found in two specific regions, the first being the subventricular zone of the lateral ventricles3-8, and the second being the subgranular zone of the dentate gyrus.9-11 It is thought that stem cells are multipotent meaning they can differentiate into multiple neuroectodermal lineages but no other tissue type. However, it has recently been proven that cells with larger potential for differentiation can be obtained from the brain.1,12-14
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Embryonic stem cells are mainly derived from embryos that develop from eggs which have been fertilized in vitro. They can be stimulated to differentiate by allowing them to clump together forming embryoid bodies, and they can then specialise into muscle, brain cells or any other type of cell. It is hoped that diseases like Parkinson's and spinal cord injury can be treated by transplanting cells derived from embryonic stem cells.
Stem and embryonic stem cells are used in the therapies for several neurological diseases such as Huntington's where striatal neurones should be replaced and protected, stroke where dead neurones must be replaced and axons remyelinated, and Parkinson's where dopaminergic neurones are needed to be produced in large numbers. Other neurological diseases where stem cells could be useful are Alzheimer's, Multiple Sclerosis, and Spinal cord lesions. In this essay, I will describe how stem cells come into play in some of these diseases.
The potential therapeutic benefits of research using stem cells and embryonic stem cells provide a strong favour for the research. However, some people oppose the idea for several reasons. Some people perceive it as an unnecessary destruction of life which can be easily avoided with the current medical technologies available. There is also some concern that embryonic stem cells have flaws and are not as beneficial as scientists like to believe, for example they may cause tumours to develop in the transplanted organism.
However, since it is very rare that the neurones in the central nervous system regenerate and drug treatments for most neurological disorders so far have only been palliative, stem cell therapy seems to hold the most potential in treating these conditions. Stem cells would either be directly transplanted to the brain or spinal cord or after pre-differentiation or genetic modification in culture to form the specific type of neurone and glial cell. The cells may also produce neuro-protective molecules. In particular therapies, it is encouraged that the patient's own repair mechanisms are utilised, where endogenous stem cells are recruited to the certain regions of the brain and spinal cord affected by the disease, and they would produce new neurones and glia. Stem cells could provide clinical benefits by neuronal replacement, remyelination and neuroprotection (Lindvall 2006).
2.1 Alzheimer's Disease
Alzheimer's disease is one of the most common causes of dementia affecting 5.3 million Americans. It is known for its typical features of amyloid-Î² peptide (AÎ²) plaques and neurofibrilary tangles (21, 27-29), resulting in the death of many different types of neuronal lineage cells within multiple regions of the brain (29-31), especially cholinergic neurones (23). Î³- and Î²-secretases cleave APP (amyloid precursor protein) at specific amino acids (33), and neurofibrillary tangles are made of hyper-phosphorylated tau proteins. This leads to neurone impairment. (34) These characteristics result in cognitive impairment and the loss of memory (29), but the direct development of the disease is still under research.
The current drug treatments for Alzheimer's disease are only palliative, for example- cholinesterase inhibitors (33, 37). These delay the degradation of acetylcholine after its release from the synapse, which improves cognition (33). There are other available drugs but these treatments only have a modest effect and also vary greatly in their effectiveness in different patients. The use of stem cells for treatment of this disease could be revolutionary since it is expected that 959,000 new cases of AD will arise by 2050, see Fig. 1. (26).
Figure 1: Alzheimer's Disease. AD occurs due to neuronal loss in various regions of the brain such as the amygdale and hippocampus, gradually spreading to the rest of the brain. In the pathological Alzheimer's Disease brain, there is a large accumulation of neurofibrillary tangles and Î²-amyloid protein accumulation (forming plaques), resulting in continual cell death of cells in the brain. It has been hypothesised that by transplanting stem cells that have been modified to release growth factors, the progression of the disease will lessen. This will also decreases the severity of the symptoms.
In 2009, a study was published by Blurton-Jones et al (29), where they injected NSCs into the hippocampi of the brain of both a transgenic AD mouse model and an age-matched non transgenic mouse model. To test the efficacy of NSC transplantation on AD-related cognitive and neuropathological effects in this study, 3xTg-AD mice were used as a model that combines many of the prominent features of AD. 100,000 murine NSCs were injected to the hippocampi of eighteen 18-month old mice, as well as 10 age-matched nonTg mice. Furthermore, as a control, 9 age-matched 3xTg-AD mice and 10 nonTg mice were injected with an equivalent volume of vehicle. A month after the injections, the mice were familiarized, trained and tested on 2 hippocampal-dependent behavioural tasks - Morris water maze (MWM) and context-dependent novel object recognition. Fortunately, the NSC-injected mice showed learning and memory improvements, indicated by their shorter latencies during both MWM acquisition and probe trial testing. From exposing the mice to 2 identical round cage objects and then 2 identical square cages, their memory was tested on recognising these objects twenty-four hours later. Blurton-Jones and colleagues observed their behaviour and it was noted that transplanted NSCs rescued the learning and memory impairments.
One of the most important points implied by this study is that the mice improved in cognitive function. However, there was no change to the existing AÎ² plaques or neurofibrilary tangles  . What the authors did discover is a neurotrophic factor important for neurone growth, and synapse formation increased , resulting in improved cognition through increased synaptic density . This showed that cognition can be improved without modifying the existing pathological conditions .
The function of APP remains unclear at the current moment, but it is suggested that it plays an important role in regulating stem cell biology or adult neurogenesis . It has been shown higher levels of APP caused glial differentiation of human NSCs in vitro and in vivo. If APP levels are high, it may be a problem when regenerating neurones by increasing NSCs. In addition, high APP levels in patients with Down syndrome who develop AD later in life, may use up NSC populations because of increased premature glial differentiation of the cells . The high APP levels in the brain not only reduce NSCs (increasing risk of AD), but also augment the level of glial differentiation of stem cells upon transplantation, reducing the efficacy of therapy to improve cognitive function [42, 43]. Researchers decided that reducing the levels of APP before transplantation of stem cells. In their study, they showed significant neurogenesis from NSC transplantation in APP transgenic mice; only after APP level was reduced by phenserine treatment .
NSCs may also aid in increasing growth factors. In a transgenic model of AD, an improvement in cognition was observed by the release of brain-derived neurotrophic factor (BDNF) after NSCs . Furthermore, NSCs express neurotrophic factors and promote axonal growth in spinal cord injury .
Granulocyte colony-stimulating factor (G-CSF), a haemopoietic growth factor, has neuro-protective effects, as do erythropoietin (EPO), granulocyte-macrophage colony-stimulating factor (GM-CSF), stem cell factor, vascular endothelial growth factor (VEGF) and a few others in ischemic stroke [45, 46]. NSCs modified by transfection of VEGF provided neuroprotection after transient focal cerebral ischemia . Although these have shown positive results in animal models, there is no evidence of similar results on neurodegenerative diseases in humans. However recently found in a long-term follow up study of intravenous autologous MSC transplantation in patients with ischemic stroke showed very promising results . This may support the use of stem cells to augment growth factor in AD in the future.
2.2 Parkinson's Disease
Parkinson's disease is the second most common neurodegenerative disease (Glass CK et al 2010). It is a movement disorder often characterized by muscle rigidity, tremor, a slowing of physical movement (bradykinesia), and in some extreme cases, a loss of physical movement (akinesia). The primary symptoms are due to excessive muscle contraction, normally caused by the insufficient formation and action of dopamine, which is produced in the dopaminergic neurones of the brain.
Some symptoms characteristic of this disease are Lewy bodies, found in the cytoplasm of neurones, and are composed of densely aggregated filaments. These filaments contain ubiquitin and alpha-synuclein. Lewy Bodies are often associated with Parkinson's Disease, but they are not unique to it since they also occur in several other medical disorders. Neuritis is another hallmark of this disease, which is the change of the state of nerves resulting in weakness, loss of reflexes and changes of sensation. (diseaseatoz site)
The main pathology of this disease is the degeneration of nigrostriatial dopaminergic neurones. Patients with PD have been given human foetal mesencephalic tissue (abundant in postmitotic dopaminergic neurones) as intrastriatal transplantation. This study showed proof that neuronal replacement can work in the human brain. The grafted neurones survive and reinnervate the striatum for as long as ten years despite the constant progression of the disease, which destroys the patient's own dopaminergic neurones (Kordower et al 1995, and Piccini 1999). The grafts regulate striatal dopamine release (Piccini 1999) and reverse the impairment of cortical activation underlying Akinesia (Piccini 2000). They become functionally integrated into neuronal circuitries in the brain (Piccini 2000), to the point where some patients have been able to withdraw from L-dopa treatment for many years and return to their independent life (Piccini 1999).
Park and colleagues  investigated the use of MSCs in a PD mouse model to observe if there was any neuro-protective effect on neuronal loss. The number of dopaminergic neurones and tyrosine hydroxylase-positive cells in vitro and in vivo  were preserved by the MSCs.
In a different study, adult olfactory stem cells were used for the recovery of dopaminergic neurones . These stem cells were differentiated into NPCs and were able to become dopaminergic-like neurones both in vitro and in vivo .
NSCs have also been used as potential treatments for Parkinson's disease, where they have produced behavioural benefits and protective effects in vivo . When NSCs were transplanted after 6-hydroxydopamine lesion formation in mice, tyrosine hydroxylase neurones were protected and symptoms of the disease were reduced .
Midbrain ESCs derived from mice, have demonstrated their ability to generate dopamine neurones, showing electro-physiological and behavioural properties which is what is expected of neurones from the midbrain . Further research is required to test whether these cells show functions that will aid in treating the disease.
It should not be expected that transplantation of human foetal mesencephalic tissue will become routine treatment for PD due to the struggle with tissue availability and too much variation in functional outcome. However, what stem cell technology is able to do is generate large numbers of dopaminergic neurones in standardized preparations. The cells obtained should release dopamine in a synchronized manner and should show the molecular, morphological and electrophysical properties of substantia nigra neurones (Isacson et al 2003). The animal models that these cells are injected to must show a reversal of the motor deficits that are parallel to those seen in PD patients. In addition, the yield of cells should allow for at least 100,000 grafted dopaminergic neurones to survive over the long term in each human putamen (Hagell et al 2001).
The clinical trials using human foetal dopaminergic neurones have produced long-lasting improvement in some patients (Lindvall 2004). It has been made clear that for stem-cell therapy to work for PD, dopaminergic neurones with the characteristics of substantia nigra neurones must be produced in large numbers (Mendez 2005). Survival of these neurones in animal models has been poor and needs to be markedly increased before application (Perrier et al 2004). Treatment of patients is also quite difficult since each individual will require a custom-made grafting procedure based on where exactly the implants are needed in the brain (Piccini 2005). Also, being able to prevent the death of existing neurones is also needed, therefore, stem cells modified to express neuro-protective molecules like glial-cell-line-derived neurotrophic factor (GDNF) is also important (Behrstock et al 2006).
The use of iPS cells has also been investigated, derived from mouse fibroblasts, to form neural progenitor cells, which are multipotent adult stem cells. These have been injected into 6-hydroxydopamine-lesioned rats , and were able to migrate to several regions of the brain, differentiate into glia and neurones and integrate into the host brain . Almost all the rats showed high numbers of tyrosine hydroxylase-positive cells.
In support of these findings, a different study by Iacovitti et al  showed human induced pluripotent stem cells (hiPS) generated midbrain dopaminergic neurones. They showed that the cells produced followed the same lineage pathway as H9 human ESCs and demonstrated the same expression levels of dopamine and DOPAC (dihydroxyphenylacetic acid) . The midbrain dopaminergic hiPS cells were transplanted into 6-OHDA-lesioned PD rats, which survived in the long term and also integrated into the host brain. Unfortunately, it was noted that Nestin+ tumour-like cells remained at the site of graft . Further success of stem-cell based therapy for PD will lie with the ability to select the appropriate midbrain dopaminergic cell lineage.
2.3 Huntington's Disease
Huntington's disease is a disorder characterized by chorea (excessive spontaneous movements) and progressive dementia and is fatal. It differs from many other neurological disorders in that there is a mutation in the huntingtin gene, which leads to the loss of medium spiny projection neurones in the striatum. Stem cell therapy for this disease involves replacing these neurones to restore brain function.
Intra-striatal grafts of foetal striatal tissue containing projection neurones re-establish connections with the globus pallidus and receive inputs from host cerebral cortex in some animal models (Dunnett et al 2000). However, this reconstruction of corticostriatopallidal circuitry is inadequate to reverse motor and cognitive deficits in rats and monkeys (Dunnett et al 2000, Kendall et al 1998, Palfi et al 1998).
In clinical trials where intrastriatal transplantation of human foetal striatal tissue was carried out, cell replacement in Huntington's disease was supported. The grafts survived, contained striatal projection neurones and interneurones, and received afferents from the patient's brain (77), but the level of clinical benefit was not very clear.
Improvement was found linked to a reduction of striatal and cortical hypometabolism, suggesting that the grafts had restored function in striato-cortical neural loops (80). To achieve greater clinical improvement, cell therapy will require many more grafted striatal neurones to survive than the low numbers achieved in the trials with foetal tissue (77).
The reconstruction of striatal neural circuitry has not been shown in animals, and stem cell based approaches are still at the formative stage. However, human neural stem cells implanted into the brains of rats were recently shown to reduce motor impairments in experimental HD through trophic mechanisms (Ryu et al 2004 and McBride et al 2004). It seems more promising and achievable to use stem cells for the delivery of trophic factors and neuro-protection to prevent disease progression in Huntington's disease than neuronal replacement.
2.4 Amyotrophic lateral sclerosis
ALS, also known as Lou Gehrig's disease, is another neuro-degenerative disease which affects the spinal cord and brain stem and is usually adult-onset, with a mean onset age of 55. Progressive paralysis develops due to upper and lower motor neurones, in the cerebral cortex, brainstem and spinal cord dying . Unfortunately prognosis is poor after diagnosis, between 2 to 5 years only . Stem cell-based therapy could be used to induce neuroprotection or reduce detrimental inflammation by implanting stem cells releasing growth factors.
For therapy to be successful, there are several aspects which must be considered. Some of these are that cells must be delivered at more than one site along the spinal cord; the stem-cell derived motor neurones must integrate into existing spinal cord neural circuitries, receive appropriate regulatory input and be able to extend their axons long distances to reinnervate muscles in humans. However, with these in mind and several other considerations as well, scientific progress on stem cell-based therapy for replacing motor neurones seems promising.
For example, it has been shown that transplantation of stem cells acts to offset motor neurone loss by releasing neurotrophic molecules or adjusting the inflammatory environment .
Pluripotent cells derived from primordial germ cells (human embryonic germ cells) have been injected into the cerebrospinal fluid of rats with motor neurone injury. It was found that the germ cells migrated into the parenchyma and induced motor recovery through neuro-protection due to growth factor production .
In another study, mouse models of ALS were given intraspinal injections of mouse NSCs. This gave rise to neurone formation, a delay in disease onset and progression and protection to the motor neurones. It is believed this occurred through VEGF- and IGF-1-dependent mechanisms .
A separate study found that when human foetal NSCs were transplanted into the spinal cord in a rat model of ALS, neuroprotection was offered and there was a delay in the disease onset . It is probable that GABAergic interneurones synapsing on host motor neurones released GDNF and BDNF (brain derived neurotrophic factor), had a role in producing these results .
These studies serve as preclinical experimental data and encouragement for a new clinical trial to be performed by NeuralStem in the US, in which 12 ALS patients will be treated by injection with human foetal-derived NSCs into the lumbar region of the spinal cord. It is hoped that they will exert a neuro-protective effect. It is evident that there is much more work required before stem cell-based therapies can move to the clinic as a treatment for ALS.
Ischaemic stroke is caused by occlusion of a cerebral artery and leads to focal tissue loss and the death of multiple neurone types, astrocytes, oligodendrocytes and endothelial cells in the cortex and subcortical regions, see Fig. 2. (Lindvall & Kokaia, 2010). Acute thrombolysis is a procedure carried out on patients with stroke, to breakdown the blood clot through pharmacological means, usually by stimulating fibrinolysis. However, this only occurs in the 20% of individuals who arrive in time to be treated with thrombolysis( Dirks et al 2007), as for the rest, there is no effective treatment to encourage recovery (Lindvall & Kokaia, 2010).
Figure 2 -Stroke. Due to a blocked artery, respective sites of the brain have been deprived of oxygen, resulting in a stroke. A stroke due to ischemia leads to the death of multiple neuronal types and glial cells which will result in an overall decrease in function of the brain, with symptoms varying with the site of damage. In this situation, stem cell therapy could be utilised in such a manner to restore neuronal activity in the damaged sites after its oxygen supply has been re-established.
Human ES cell- derived NSCs injected into the ischemic boundary zone of rats that were subjected to stroke have been shown to migrate toward the lesion and improve forelimb performance (Daadi et al 2008). It has also been revealed from electrophysiological recordings that the grafted cells showed functional neuronal properties and synaptic input from host neurones (Daadi et al 2009), when mouse ES cell-derived precursors were implanted into stroke-damaged rats (Buhnemann et al 2006).
Transplanted human foetal NSCs have also shown to move toward the ischemic lesion in rodents (Kelly et al 2004). Human NSCs isolated from embryonic striatum (Kallur et al 2006) however, have produced morphologically mature neurones after transplantation into the striatum of stroke-damaged rats (Darsalia et al 2007). These two findings provide evidence that functional neurones can replace the lost neurones in the stroke-damaged brain, and also imply that this process contributes to the behavioural improvements observed (Lindvall & Kokaia, 2010).
When human NSCs are intravenously injected, they have induced improvements after hemorrhagic stroke in rats. It is believed that this is achieved through anti-inflammatory actions (Lee et al 2008), by overexpression of either VEGF or the antiapoptotic factor Akt1 in the human NSCs, which promote angiogenesis and increases neuronal survival respectively. This enhances the functional improvements in stroke-damaged mice (Lee et al 2007, 2009).
Human MSCs, when intravenously administered, have been shown to minimise stroke-induced deficits in rats, again most likely by inducing angiogenesis but also improving cerebral blood flow (Onda et al 2008). Genetically modified MSCs, able to express angiopoietin or growth factors such as GDNF, neuroprotection and function was much enhanced (Onda et al 2008, Horita et al 2006, Liu et al 2006, Nomura et al 2005).
Initial clinical trials of stem cell delivery in stroke have been completed (check details). For example, an immortalized human teratocarcinoma cell line implanted into ischemic infarcts affecting the basal ganglia and in some cases the cerebral cortex (Kondziolka et al 2005, 2000, Nelson et al 2002), produced small improvements in some patients. However, there are no considerable clinical improvements shown after intravenous injection of autologous MSCs in patients with an ischemic lesion in the region supplied by the middle cerebral artery (Bang et al 2005). This requires more clinical studies with infusions of autologous bone marrow-derived stem cells, intravenously or intraarterially.
For stem cell-based therapy to reach full-scale clinical trials for the treatment of stroke, there are a number of issues still to be investigated further. For example, it is important to learn how to control the proliferation, survival, migration and differentiation of endogenous and grafted stem cells in the stroke-damaged brain, and develop procedures for cell delivery, optimum functional recovery and patient and assessment (130).
2.6 Spinal Cord Injury
Spinal Cord Injury (SCI) involves many pathological changes such as, damage to the nerve roots, loss of neurones and glial cells, inflammation, scar formation and demyelination. As a consequence, patients with SCI experience slowed or loss of movement and loss of sensation.
Stem cell-based therapies could be used to treat patients with spinal cord injury by replacing the damaged and lost segments of interneurones and motor neurones, promoting myelination and by injected stem cells which are capable of releasing different factors which counteract disadvantageous inflammation.
Different types of stem cells have been implanted with aims to improve locomotor function in animal models (131-133), and fortunately this has been achieved. It is suggested that the secretion of neurotrophic factors, the remyelination of axons and the modulation of inflammation have had major roles in this improvement. Human NSCs have been transplanted into the injured mice spinal cord and have become integrated into the spinal cord, generated neurones and oligodendrocytes and induced locomotor recovery (134). It is most probable that transplanting neurones derived from the grafted human cells with host circuitry mediated the functional revival (135).
In a separate study, human NSCs were transplanted into the injured rat spinal cord and were found to differentiate into neurones that produced axons and synapses, and formed connections with host motor neurones (136). Human NSCs have produced functional recovery in injured dogs as well so from a clinical point of view, these implants have been important and successful.
Oligodendrocyte progenitor cells (OPCs) generated from human ES cells in vitro have been shown to differentiate into oligodendrocytes and give rise to re-myelination when transplanted into demyelinated injured mouse spinal cord (143). Only OPCs implanted in rats at an early stage (7 days) showed differentiated oligodendrocytes, promoted re-myelination and produced locomotor recovery, whereas rats injected late at 10 months did not display these enhancements (144).
The very first phase 1 clinical trial with human ES cell-derived OPCs implanted into patients with thoracic spinal cord injuries is planned to take place by Geron, a US company (Lindvall 2010). Patients will be immunosuppressed for two months after being transplanted with the OPCs, and then tested for recovery of sensory and lower extremity motor function. There are concerns that during this trial, patients may be at risk of forming tumours and whether rodent models of OPCs can reflect the human condition of SCI and if the data obtained from them can directly be interpreted to human patients. However, the benefits that may be established as a result of the implantation will outweigh the risks, especially when SCI patients lack other effective treatments. Also, there is preclinical evidence of efficacy and safety.
While the outlook for stem cells in clinical treatment looks hopeful, there are several disadvantages which may occur as a result of transplantation which should not be disregarded. For example, a boy with a neurodegenerative disease not mentioned above - ataxia telangiectasia (AT) was treated with intracerebellar and intrathecal injection of human foetal neural stem cells. A few years after transplantation, he was diagnosed with a multifocal brain tumour - a glioneural neoplasm (Amariglio et al 2009). Through molecular and cytogenic studies using fluorescent in situ hybridization, PCR, microsatellite and HLA analysis (human leukocyte antigen), it was shown that the tumour was not of host origin, suggesting that it originated from the neural stem cell transplantation (Amariglio et al 2009).
The blood brain barrier is composed of microvascular endothelial cells that are connected by tight junctions. It acts to prevent the infiltration of most blood-borne cells and molecules into the brain parenchyma (Prat et al 2001). Stem cell transplantation involves entering the blood brain barrier, leaving it susceptible to damage or to other viral infections.
Despite the safety concerns, the application of stem cells, whether ESCs, FSCs, MSCs, or iPS cells, is progressively becoming a reality. Research in this field could revolutionise the development of new therapies for many neurodegenerative diseases that currently lack effective treatments. There has been constant advancement in developing approaches to generate the types of human-derived neurones and glial cells that are needed for therapy on pathology in the respective diseases (refs on paper). Patient-specific cells that may be useful for transplantation can now be produced from iPS cells (refs). NSCs in the adult brain have been able to generate new neurones and glial cells to counteract neurodegeneration. Through research, it has become apparent that certain characteristics in the pathology of the diseases play a crucial role in the survival, differentiation and function of both grafted and endogenous cells; a good example of this is the magnitude of inflammation. Stem cells are now known to allow improvements that may be of clinical value through immunomodulation, trophic actions, neuro-protection and stimulation of angiogenesis (refs).
These findings will give rise to many more studies and clinical trials in the near future, such as the transplantation of human ES cell-derived OPCs for re-myelination in spinal cord injury. Clinical attempts at neuronal replacement for ALS, stroke, spinal cord injury and AD seem more distant than for PD, which are likely to be implanted in PD patients within the next five years.
Although the approaches have found that modulating inflammation and neuroprotection with supplying neurotrophic molecules aids recovery in these neurodegenerative diseases, much more basic research is required to understand the mechanisms regulating proliferation, migration, differentiation, survival, integration and function of stem cells. It is fundamental to have this understanding to better control these processes and therefore develop more efficient treatment. Most patients with neurodegenerative diseases have little or no therapeutic options, since drugs have mainly been palliative, so they are willing to test any new approaches. Scientists and researchers must find ways to clinically translate their stem cell research and successful results into appropriate applications for patients with these disorders.
One of the companies leading in research of degenerative diseases that is attempting to perform this feat is Geron, a biotech firm in California. In 2010, the very first phase I clinical trial was initiated, involving the injection of hESCs into a human subject, with the spinal cord being the target site. In preparation for the human clinical trial, a preclinal animal study was carried out, injecting hESC-derived oligodendrocyte progenitor cells into adult rat spinal cords that have been subjected to injury. The results showed that the cells enhanced re-myelination and a significant improvement in motor function was observed (Kierstead et al 2005). Following this study, Geron designed the first clinal trial where these cells would be injected into the human spinal cord. Patients eligible for this clinical trial must have functionally complete spinal cord injury between T3 to T10 spinal segments, and they must consent to the administration of GRNOPC1 into the lesion sites between seven and fourteen days after the injury. One of the primary aims of this trial is undoubtedly to test its safety, at which point Geron can seek FDA approval of increasing the dose of GRNOPC1 and extend the study. However, essentially their goals are to achieve restoration of spinal cord function after injury, and also potentially spread this type of treatment to other neurodegenerative disorders such as Alzheimer's disease, Multiple Sclerosis and Canavan Disease.
The reason why so much emphasis has been placed in conducting research for the treatment of neurodegenerative diseases like Alzheimer's and Parkinson's is because currently there is no cure, so treatment for these are aimed at symptomatic relief rather than treating the pathologies. If the clinical trial on the human patient is successful, this will grant opportunities for groundbreaking research to occur, and making the cure for such fatal diseases being within their reach more realistic.