CELULAS TRONCALES EN NEUROPATIAS

Review

Gene Therapy (2011) 18, 1–6;

 published online 30 September 2010

Progress and prospects: stem cells and neurological diseases

S Gögel1, M Gubernator1 and S L Minger1

1Wolfson Centre for Age-Related Diseases, Guy's Campus, King's College London, London, UK

Correspondence: Dr S Gögel, Current address: Medical and Molecular Genetics, King’s College London, 8th Floor Tower Wing, Guy’s Hospital, Great Maze Pond, London SE1 9RT, UK. E-mail: stefanie.gogel@kcl.ac.uk

Abstract

The central nervous system has limited capacity of regenerating lost tissue in slowly progressive, degenerative neurological conditions such as Parkinson's disease (PD), Alzheimer's disease or Huntington's disease (HD), or in acute injuries resulting in rapid cell loss for example, in cerebrovascular damage (for example, stroke) or spinal cord injury. Although the adult brain contains small numbers of stem cells in restricted areas, they do not contribute significantly to functional recovery. Transplantation of stem cells or stem cell-derived progenitors has long been seen as a therapeutic solution to repair the damaged brain. With the advent of the induced pluripotent stem cells technique a new and potentially better source for transplantable cells may be available in future. This review aims to highlight current strategies to replace lost cellular populations in neurodegenerative diseases with the focus on HD and PD and traumatic brain injuries such as stroke, discussing many of the technical and biological issues associated with central nervous system cell transplantation.

Keywords: cell replacement therapy; stem cells; Parkinson's disease; ischaemic stroke; Huntington's disease

In brief

Progress

•Different cell types might be useful in transplantation studies for different neurodegenerative diseases.

•Transplantation studies in neurodegenerative diseases provide data on safety and feasibility of this procedure.

•Cell transplantation provides an improvement of symptoms for a limited period of time for Huntington's disease patients.

•Enhancement of endogenous repair mechanisms are being targeted for further amelioration of symptoms in stroke patients.

•Early clinical trials for cell transplantation with different cell types in ischaemic stroke show no adverse effects in the patients and the functional outcome is highly variable.

•Cell transplantation in Parkinson's disease is feasible and leads to amelioration of motor functions with room for further improvement.

•Improvement of differentiation protocols for human embryonic stem cells (hESCs) into transplantable dopaminergic progenitors is one focus of recent research.

Prospects

•The necessity for cell replacement therapy is stimulated by the rising incidence of degenerative diseases based on demographic changes in an ageing society.

•Different neurodegenerative diseases might require different cell sources that have yet to be determined. In addition, the optimal time point of implantation relative to disease onset will have to be a focus of future research.

•Improvement of differentiation protocols will lead to more pure populations of transplantable cells derived from either hESCs or even induced pluripotent stem cells.

•Logistical, ethical, societal and economical hurdles have to be overcome in order to make cell replacement therapy available for the broad public.

Different cell types might be useful in transplantation studies for different neurodegenerative diseases

Cell replacement therapy could in the future aid in alleviating symptoms or even reversing disease progression of neurological disorders where pharmacological interventions and other treatment modalities are no longer sufficient or are not available. Over the last decade, convincing evidence has emerged of the capability of various stem cell populations to induce regeneration in animal models of Parkinson's disease (PD), Huntington's disease and Alzheimer's disease (AD) along with multiple sclerosis and cerebral ischaemia.

Cell populations derived from embryonic stem cells (ESCs) have tremendous potential to fulfil the requirements to be a cell source for neural cell replacement therapy.1, 2 ESCs are highly expandable pluripotent cells capable of differentiating into all cell types of the human body, including nervous system tissues. They are derived from the inner cell mass of preimplantation blastocysts to yield a homogenous, self-renewing cell population. As the transplantation of undifferentiated human ESCs (hESCs) is associated with high risk of unwanted proliferation leading to cancerous structures, it is no longer seen as a potential therapeutic approach. Rather the directed differentiation of hESCs into specific neural progenitor subtypes is therefore required. Other potential cell sources for central nervous system (CNS) transplantation include foetal or adult neural stem cells (NSCs), mesenchymal stem cells (MSCs) or bone marrow-derived cell populations amongst others. MSCs for example, are easy to obtain and their use is not incriminated with contentious ethical issues.

NSCs can be expanded over a long period of time and as they are already neuralized (for example, committed to a CNS cell fate) there is no need for recapitulating early developmental signals that lead to neuroectodermal commitment. However, the transplantation of foetal NSCs into the adult brain is accompanied by numerous ethical, scientific and legislative hurdles.3, 4 In addition, the prolonged culturing of NSCs leads to an ever increasing glial differentiation pattern at the expense of neuronal differentiation, which significantly reduces the therapeutic potential of foetal NSCs.5 MSCs may prove to be superior to NSCs as they can be derived from a variety of adult tissues such as bone marrow and cord blood, as well as foetal liver and lung and the developing tooth bud. Bone marrow MSCs are, to date, the most commonly used stem cell source with regard to the treatment of haematopoietic diseases and thus their isolation and application is well established. As these cells seem to be able to pass the blood–brain barrier, no invasive intracerebral surgery is required and instead peripheral systemic application can be envisaged. However, the extent to which MSCs can be directed to a neural cellular fate either ex vivo or in vivo following implantation remains a point of contention among researchers.

Recently, a new cell type has emerged in the stem cell field that might be useful in cell transplantation. Takahashi and Yamanaka,6 succeeded in reprogramming mouse fibroblast cells into cells that are highly similar to ESCs, commonly referred to as ‘induced pluripotent stem cells’. Only one year later, this new technique that reverses developmental processes of cells was applied to human somatic cells.7, 8 This exciting technique allows the derivation of induced pluripotent stem cells from patient's own skin and their subsequent differentiation into cell types lost by neurodegenerative diseases or sporadic injury. Ultimately it is envisaged that these tailor-made patient stem cells are used for cellular transplantation in a variety of degenerative processes in the ageing human body. All the different cell types discussed here might be useful in transplantation studies for the different neurological diseases (see Figure 1).

Figure 1.

Schematic drawing illustrating the different cell types and their origin that can be used in cell therapy in brain diseases. Human embryonic stem cell lines are derived from the inner cell mass of the blastocyst; foetal brain cells can be obtained from aborted foetuses; induced pluripotent stem cells are derived by reprogramming of differentiated cells such as human fibroblasts; mesenchymal stem cells are harvested from cord blood or bone marrow. These different cell types can be differentiated into neuronal precursors that are transplanted into the diseased brain.

Transplantation studies in neurodegenerative diseases provide data on safety and feasibility of this procedure

For cell replacement therapies to move towards clinical application, lasting functional and clinical improvement and absence of deleterious side effects in experimental animal models need to be established. Proof-of-principle studies are necessary to show posttransplant cellular survival, host brain integration, synaptic innervation of target brain regions and correct morphological differentiation in vivo functionally to replace cells lost as a result of the underlying lesion. Differentiation protocols, which will yield a highly efficient cell fate commitment to produce high quality transplantable cells, are required. Behavioural and underlying functional improvement should clearly be demonstrated. The understanding of functional recovery on a cellular level is crucial to optimise circuit reconstruction and progenitor cell guidance. Lastly, the clinical stratification of patients needs to be implemented as variable clinical efficacy of cell therapy in early versus late disease progression is assumed. As the pathologies of the CNS are characterized by different underlying mechanisms, it is therefore likely that specific stem cells and derivates need to be transplanted for each condition in order to affect disease-specific repair processes. This review focuses on transplantation studies for neurodegenerative diseases such as PD, HD and stroke, which have, to date, provided data on safety and feasibility of the procedure.

Cell transplantation provides an improvement of symptoms for a limited period of time for HD patients

HD is an autosomal dominant genetic disorder caused by a mutation in the huntingtin locus resulting in a progressive degeneration of predominantly medium-spiny GABAergic neurons in the striatum (see Figure 2). Later in the disease progression more widespread neuronal degeneration can be observed, such as in the neocortex and hippocampus. Patients subsequently develop severe movement and cognitive problems. Currently there are no disease-modifying treatments available and death occurs 10–20 years after disease onset.

Figure 2.

Schematic coronal section of the human brain illustrating the different areas that are affected in diseases such as PD, HD or stroke and in which different cell replacement therapies should be directed. HD affects initially the GABAergic neurons in the striatum, but during the course of the disease extends to other brain areas. PD is caused by the degeneration of dopaminergic neurons in the caudate nucleus and putamen of the neostriatum. Neuronal and glial degeneration following cerebrovascular damage (stroke) can occur in any area of the brain.

As the early form of HD is characterized by the relatively specific loss of one particular cell type, the medium spiny neurons, Huntington's would seem to represent a good example of a ‘simple’ cell-replacement strategy to replace lost tissue with transplantation of specific replacement cell types thus, hopefully inducing successful long-lasting recovery of the lost striato-cortical circuitry. Indeed, in animal models of HD, transplantation of non-human foetal striatal tissue has provided proof-of-principle that such transplants elicit anatomical and behavioural improvements.9

Since 1990, several clinical trials have been conducted using transplanted foetal neural tissue into the striatum of patients with HD.9 The number of patients that have received foetal transplants is rather small and the efficacy of the procedure measured in terms of clinical improvement of the patients is variable (see Table 1). In one pilot study, initiated in Créteil, France, by Marc Peschanski and Anne-Catherine Bachoud-Lévi, three out of five patients showed a plateau of motor and cognitive improvements already 2 years after transplantation of foetal neural grafts into the left and right striatum, which faded over the following four years for motor disabilities, whereas cognitive function remained stable. In a phase II trial following this pilot study, the researchers successfully involved additional immunosuppressive therapy as antibodies against foetal donor antigens were detected in patient's blood samples.10

Similar results have been obtained in a separate study initiated by Kopyov and colleagues in Los Angeles. Post-mortem examination, 6 years after transplantation, of foetal lateral ganglionic eminence from 5–8 donors into bilateral caudate nucleus and putamen in two patients showed viability of grafts. However, they exhibited limited graft–host connectivity and did not induce clinical improvement.11 In one patient the graft led to cyst formation and overgrowth 10 years posttransplantation and the patient died because of advanced HD.12

In an exceptional case, Reuter et al.13 reported improvement in motor function and striatal PET scans over a period of 5 years in one of two patients who received bilateral foetal striatal tissue, and Capetian et al.14 reported in a post-mortem analysis of a single patient 6 months after transplantation, differentiation and integration of the transplanted tissue into the host tissue without improvement of movement or psychiatric symptoms.

The most recent study that analysed the brains of three patients who received a graft a decade before their death suggests that although the grafted cells differentiate and integrate into the host tissue, they degenerate even more rapidly than the patient's own tissue.15 Cellular implants given to patients who were at a more advanced stage of disease with significant atrophy of relevant brain tissues might be the reason for their high incidence of surgical side effects such as haematomas and the poor survival of the grafts implanted into the heavily atrophied caudate. Transplantation therefore seems to provide an improvement of symptoms for a limited period of time, but does not represent a significant disease-modifying strategy for HD. The fact that striatal grafts without abnormal gene expression degenerate over time suggests that the local environment including microglial activation against the graft and excitotoxic glutamatergic inputs from the cortex is not supportive for long-term graft survival. These latest results can be interpreted as a huge drawback to clinical application of cell transplantation for HD, particularly as early results were promising.

Enhancement of endogenous repair mechanisms are being targeted for further amelioration of symptoms in stroke patients

Neuronal and glial degeneration can occur following cerebrovascular damage resulting from the blockage of blood flow in selected brain areas, resulting in motor, sensory and cognitive disturbances. Over time, atrophy leads to further long-term damage and therapeutic approaches are currently limited to thrombolytic treatment, which has a narrow time window and cannot be applied to all patients. Despite the predominantly focal nature of ischaemia, many different cell types can be affected, which in theory makes this neurological disorder not an ideal candidate for cell replacement therapy. However, it is hypothesized that the reconstruction of only a small fraction of lost circuitry could be sufficient in promoting functional improvement for patients.

It has been shown that stroke-induced stimulation of neurogenesis and migration of neural stem or progenitor cells into regions of ischaemic damage occurs in humans, but the extent to which neurogenesis is able to replace lost neurons or contributes to functional improvement in stroke patients is still unclear.16, 17 If neurogenic neuronal cell replacement is compromised because of the local ischaemic environment, these cells could be supported by intracerebral infusion of cytokines or transplantation of cells overexpressing neurotrophic factors such as brain-derived neurotrophic factor, nerve growth factor, vascular endothelial growth factor and glial cell-derived neurotrophic factor.18, 19

Early clinical trials for cell transplantation with different cell types in ischaemic stroke show no adverse effects in the patients and the functional outcome is highly variable

The idea of recovering lost function by replacing damaged tissue with transplants in stroke patients remains relatively unexplored. Very few data are available and early clinical trials have provided data on the safety and feasibility of cell transplantation. Only a small number of patients have been transplanted. For example, neural progenitors from a human immortalized teratocarcinoma cell line (NT2/N) were transplanted into 12 patients from 6 months to 4.5 years after basal ganglia strokes. No adverse events were observed up to 5 years after transplantation and patients displayed some improvement as based on the European Stroke Scale. In a randomized phase II follow-up trial, a total of 18 patients received either 5 or 10 million human neuronal cells 1–6 years after stroke. Again no cell-related adverse effects were observed, but side effects of the transplantation procedure including a seizure and a subdural haematoma were noted in two patients. In all, 6 out of 14 patients showed an improvement in European Stroke Scale scores at 6 months, but there was no improvement in European Stroke Scale motor scores; however, the everyday memory test score improved significantly. In a separate study, Savitz et al.20 transplanted porcine cells pretreated with anti-MHC1 antibody to avoid immune reaction into 5 patients 1.5–10 years after experiencing basal ganglia stroke. Two patients showed worsening of motor deficits or developed seizures briefly after transplantation. This study was, therefore, terminated by the Food and Drug Administration.

Bang et al.21 transplanted autologous MSCs from bone marrow (5 patients) in a phase I/II clinical trial including 30 patients with cerebral infarcts within the middle cerebral arterial territory. No adverse cell-related effects were observed, and a 1-year follow-up assessment showed no significant improvement in function.

Altogether these human trials imply that stem cell therapy in ischaemic brain is feasible, the limited clinical trial data obtained thus, far provide little consistent evidence of clinical benefit.22, 23, 24 Further research to determine the optimal cell population and method of administration is needed to improve the outcome of cell therapy for stroke. In addition, the optimal time point of post-ischaemic transplantation has yet to be determined, as activated microglia might limit survival of transplanted cells.

Basic research on the common animal models, mouse and rat, will help further understand the mechanisms underlying the beneficial effects of stem-cell therapy. For example, the systematic administration of bone marrow mononuclear cells in mice after cortical infarction increased cell proliferation of endothelial cells and neural progenitor cells (NPCs) within the infarct region.25 Nakagomi et al.26 could show that co-transplantation of endothelial cells and NPCs increased survival of ischaemia-induced NPCs even more than transplantation of NPCs alone. It is speculated that endothelial precursor cells increase angiogenesis and consequently promote neurovascular repair.27 A possible role for glial cell-derived neurotrophic factor in cell survival has been demonstrated by administration of this cytokine after middle cerebral artery occlusion in rats and by genetically modifying NPCs with glial cell-derived neurotrophic factor before transplantation in rats after middle cerebral artery occlusion.28, 29 These results suggest that trophic factor mechanism rather than cell replacement per se, contribute predominantly to the behavioural improvement in animal models. Therefore, the combination of stimulating endogenous neurogenesis with cytokines and stem cell technology will likely have a huge impact in the future treatment of stroke patients.

Cell transplantation in PD is feasible and leads to amelioration of motor functions with room for further improvement

Foetal tissue transplantation for PD has spearheaded cell replacement therapy for neurodegenerative diseases. PD is mainly caused by the degeneration of mesencephalic substantia nigral dopaminergic (DA) neurons and a progressive loss of DA neurotransmission in the caudate and putamen of the neostriatum (see Figure 2). Patients experience motor dysfunction such as tremor, rigidity and bradykinesia, as well as disturbances in sleep and cognition. To date, L-DOPA and other dopamine agonists provide motor symptoms relief but are only effective early in the course of the disease. Deep brain stimulation is an additional therapeutic option for PD patients. Given the relatively specific loss of a single neuronal cell type, researchers hypothesized that cellular replacement of DA neurons might be feasible. Beginning in the 1970s, transplantation of post-mitotic foetal ventral mesencephalic cell suspension demonstrated significant behavioural improvement when transplanted into animal models of PD with clinical translation of these procedures into human patients in the mid-1980s.

In total, 300–400 patients worldwide have now been treated with foetal cell transplantation.30 Open-label trials showed functional benefit for motor symptoms; however, the double-blind trials failed to show significant benefit compared with placebo.31 Overall, highly variable clinical results and in some cases the development of dyskinesias, low graft survival together with the large number of early human embryos required for optimal clinical benefits (1–8 per putamen) suggest that alternative and more reliable cell sources are required.32 Recently, strict patient selection and surgical parameters, such as implantation sites, number of needle tracks and cellular deposits have also been highlighted as factors that impact on maximum therapeutic benefit.33

Isolation of tissue from the developing brain to produce expandable populations of NSCs was the logical progression from clinical studies. Several groups developed protocols for the derivation of multipotent NSCs from the developing mesencephalon.34 Although human NSCs are highly expandable ex vivo and protocols improved to enrich DA neuron progenitor, the number of DA neurons produced is at most 15% of the total cellular population.35, 36 Thus, this approach appears to be non favourable for the development of PD therapies, instead future therapies might aim towards the use of embryonic stem cell or even induced pluripotent stem cell-derived dopamine neurons.

Improvement of differentiation protocols for hESCs into transplantable DA progenitors is one focus of recent research

The current major strategy now for CNS transplantation for PD is the generation of transplantable DA progenitor cells from ESCs. Key to success of using ES cells in PD cell replacement therapy is the efficient and stable generation of midbrain A9 DA neurons. A large variety of differentiation protocols have been reported using exposure to soluble growth and neurotrophic factors to drive DA neuronal differentiation.

Roy et al.37 demonstrated that formation of embryoid bodies from hESCs followed by exposure to sonic hedgehog and fibroblast growth factor-8 enhanced the generation of mesencephalic neurons (up to 67% of neurons were TH+) and such cells were able significantly to reverse lesion-induced behavioural deficits in a rodent model of PD. However, the transplants also contained large numbers of residual mitotically active neuroepithelial cells that continued to proliferate following implantation. Alternatively, a number of groups have shown that co-culture of hESCs with a second cellular ‘feeder’ layer can improve lineage specification towards a mesencephalic cell fate. The co-culture of ES cells with the DA-inducing PA6 mouse stromal cell line promoted the differentiation of mouse, primate and human ES cells into functional DA neurons.38 PD-lesioned animals transplanted with PA6-cultured cells displayed significant behavioural recovery, although transplantation still led to teratoma formation in some of the grafted animals.39 A further strategy for the generation of A9 DA neurons involves the gene transfer and overexpression of genetic determinants important for early midbrain development. ESCs transfected with cDNAs encoding transcription factors, important in early mesencephalic lineage specification, have shown enhanced DA differentiation. The proportion of cells differentiating into DA neurons has been shown to increase significantly (50–65% of all neurons are TH+) following transduction with either Lmx1a or Nurr1+Pitx3.40, 41 When these cells were transplanted into the striatum of PD-lesioned mice, significant recovery of drug-induced rotational behaviour was visible 6 weeks following implantation. Collectively these findings suggest that it is possible to generate large number of DA neurons from ES cells, but the presence of contaminating non-DA cells and continued proliferation of transplanted cells suggests that further refinement of these differentiation protocols is required before these cells could be used in a clinical setting. Further research is also required to explore the potential safety risks of genetic manipulation of stem cells for clinical application.

Conclusions

Proof-of-principle studies in experimental animals have shown that the cell replacement of degenerated neural tissue using stem cells of various sources is feasible and can lead to improvement of lost function. In humans, the early transplantation trials show a huge variety of outcomes ranging from significant clinical benefit to worsening of symptoms with severe side effects. As the pathophysiology differs in PD, HD and stroke, different cell sources for transplantation might be required for optimal clinical improvement. Continued basic research is therefore necessary before cell replacement therapy can become a realistic clinical strategy.

Prospects

The use of stem cells in the therapeutic field and in drug development has been of considerable interest in contemporary bioscience. In recent years our knowledge and understanding of stem cell biology and regenerative medicine has increased substantially. ESCs not only continuously divide, they are also able to differentiate into approximately 220 different cell types of the human body. The isolation of tissue stem cells could offer a less ethically contentious and more practical source of replacement tissue for organs that are susceptible to age-related diseases or traumatic injury. These diseases include Alzheimer's and Parkinson's disease, but also stroke, myocardial infarction and diabetes to mention a few. They have become a serious health problem in our societies as people now live longer. A large part of stem cell research aims to identify the ideal cell type and time point of cell transplantation. Future research will also lead to improved protocols that generate more pure populations of transplantable cells. With the continuous progress in stem cell research, modern clinical medicine is at the threshold of transformation.

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