Cell transplantation in Parkinson's disease: problems and perspectives.

PURPOSE OF REVIEW: We review recent experiments conducted using embryonic tissue and stem cell transplants in experimental models of Parkinson's disease. We also highlight the challenges which remain to be met in order for cell therapy to become clinically effective and safe. RECENT FINDINGS: The outcome of previous clinical transplantation trials was variable in terms of motor recovery. We discuss whether transplants can mitigate L-3,4-dihydroxyphenylalanine (L-DOPA)-induced dyskinesias and consider the risk factors which predispose to graft-induced dyskinesias. In addition, we introduce Transeuro, a new European Union-funded multicenter consortium which plans to perform transplantation trials.Stem cells have emerged as an alternative source for the generation of dopaminergic precursors. We briefly outline progress made in the use of human embryonic stem cells and focus predominantly on the emerging field of induced pluripotency. We conclude by introducing the exciting and novel method of direct reprogramming which involves the conversion of fibroblasts to neurons without inducing a pluripotent state. SUMMARY: The area of cell transplantation has been revitalized by the identification of parameters which predispose patients to graft-induced dyskinesias and by the emergence of novel methods of generating dopaminergic neurons. Hopefully, the Transeuro clinical trials will give further impetus and act as a stepping stone to future trials employing stem-cell-derived neurons.

The R6 lines of transgenic mice: a model for screening new therapies for Huntington's disease.

Huntington's disease (HD) is a hereditary neurodegenerative disorder caused by an expanded CAG repeat in the HD gene that results in cortical and striatal degeneration, and mutant huntingtin aggregation. Current treatments are unsatisfactory. R6 transgenic mice replicate many features of the human condition, show early onset of symptoms and fast disease progression, being one of the most used models for therapy screening. Here we review the therapies that have been tested in these mice: environmental enrichment, inhibition of histone deacetylation and methylation, inhibition of misfolding and oligomerization, transglutaminase inhibition, rescue of metabolic impairment, amelioration of the diabetic phenotype, use of antioxidants, inhibition of excitotoxicity, caspase inhibition, transplantation, genetic manipulations, and restoration of neurogenesis. Although many of these treatments were beneficial in R6 mice, they may not be as effective in HD patients, and thus the search for a combination of therapies that will rescue the human condition continues.

Neurotrophic support of midbrain dopaminergic neurons.

In this chapter we review work on neurotrophic factors for midbrain dopaminergic neurons mainly from the past decade, with a focus on neurotrophins and fibroblast growth factors. We summarize data obtained from animal models of Parkinson's disease, review analyses of neurotrophin, neurotrophin receptor and FGF-2 knockout mice and put these into context with data obtained from patients with Parkinson's disease and from postmortem studies. We provide a brief overview on several other factors (EGF, TGF-alpha, IGF, CNTF, PDGF, interleukins) and their capacity to promote survival and protect lesioned DAergic neurons. TGF-betas are reviewed in a separate chapter (Roussa et al, this volume).

Nerve growth factor in treatment and pathogenesis of Alzheimer's disease.

The etiology of Alzheimer's disease (AD) is still unknown. In addition, this terrible neurodegenerative disease will increase exponentially over the next two decades due to longer lifespan and an aging "baby-boomer" generation. All treatments currently approved for AD have moderate efficacy in slowing the rate of cognitive decline in patients, and no efficacy in halting progression of the disease. Hence, there is an urgent need for new drug targets and delivery methods to slow or reverse the progression of AD. One molecule that has received much attention in its potential therapeutic role in AD is nerve growth factor (NGF). This review will demonstrate data from humans and animals which promote NGF as a potential therapeutic target by (1) outlining the hypothesis behind using NGF for the treatment of AD, (2) reviewing both the normal and AD altered signaling pathways and effects of NGF in the central nervous system (CNS), and (3) examining the results of NGF treatment obtained from animal models of AD and AD patients.

Miracles and molecules--progress in spinal cord repair.

Severe spinal cord injury (SCI) leads to devastating loss of neurological function below the level of injury and adversely affects multiple body systems. Most basic research on SCI is designed to find ways to improve the unsatisfactory cellular and molecular responses of spinal cord to injury, which include an array of early processes of autodestruction and a subsequent lack of functional tissue repair. This research has brought us to the threshold of practical application along three lines of approach, derived from animal model studies: acute neuroprotection, enhanced axonal regeneration or plasticity, and treatment of demyelination. There is a growing commercial interest in this previously neglected therapeutic area.

Olfactory ensheathing glia: their contribution to primary olfactory nervous system regeneration and their regenerative potential following transplantation into the injured spinal cord.

Olfactory ensheathing glia (OEG) are a specialized type of glia that guide primary olfactory axons from the neuroepithelium in the nasal cavity to the brain. The primary olfactory system is able to regenerate after a lesion and OEG contribute to this process by providing a growth-supportive environment for newly formed axons. In the spinal cord, axons are not able to restore connections after an injury. The effects of OEG transplants on the regeneration of the injured spinal cord have been studied for over a decade. To date, of all the studies using only OEG as a transplant, 41 showed positive effects, while 13 studies showed limited or no effects. There are several contradictory reports on the migratory and axon growth-supporting properties of transplanted OEG. Hence, the regenerative potential of OEG has become the subject of intense discussion. In this review, we first provide an overview of the molecular and cellular characteristics of OEG in their natural environment, the primary olfactory nervous system. Second, their potential to stimulate regeneration in the injured spinal cord is discussed. OEG influence scar formation by their ability to interact with astrocytes, they are able to remyelinate axons and promote angiogenesis. The ability of OEG to interact with scar tissue cells is an important difference with Schwann cells and may be a unique characteristic of OEG. Because of these effects after transplantation and because of their role in primary olfactory system regeneration, the OEG can be considered as a source of neuroregeneration-promoting molecules. To identify these molecules, more insight into the molecular biology of OEG is required. We believe that genome-wide gene expression studies of OEG in their native environment, in culture and after transplantation will ultimately reveal unique combinations of molecules involved in the regeneration-promoting potential of OEG.

Oligodendroglial progenitor cell therapy limits central neurological deficits in mice with metachromatic leukodystrophy.

This work describes the first successful oligodendrocyte-based cell therapy for presymptomatic arylsulfatase A (ARSA) null neonate mice, a murine model for human metachromatic leukodystrophy (MLD). We found that oligodendrocyte progenitors (OLPs) engrafted and survived into adulthood when transplanted in the neonatal MLD brain. Transplanted cells integrated nondisruptively, did not produce tumors, and survived as proteolipid protein- and MBP-positive postmitotic myelinating oligodendrocytes (OLs) intermingled with endogenous MLD OLs within the adult MLD white matter. Transplanted MLD mice had reduced sulfatide accumulation in the CNS, increased brain ARSA activity, and full prevention of the electrophysiological and motor deficits that characterize untreated MLD mice. Our results provide direct evidence that healthy OLPs can tolerate the neurotoxic accumulation of sulfatides that evolves during the postnatal development of the MLD brain and contribute to OL cell replacement to limit the accumulation of sulfatides and the evolution of CNS defects in this lysosomal storage disease mouse model.

Xenotransplantation for CNS repair: immunological barriers and strategies to overcome them.

Neural transplantation holds promise for focal CNS repair. Owing to the shortage of human donor material, which is derived from aborted embryos, and ethical concerns over its use, animal donor tissue is now considered an appropriate alternative. In the USA, individuals suffering from Parkinson's disease, Huntington's disease, focal epilepsy or stroke have already received neural grafts from pig embryos. However, in animal models, neural tissue transplanted between species is usually promptly rejected, even when implanted in the brain. Some of the immunological mechanisms that underlie neural xenograft rejection have recently been elucidated, but others remain to be determined and controlled before individuals with neurological disorders can benefit from xenotransplantation.

Developing concepts in neural stem cells.

Stem Cells in the Mammalian Brain: the 4th Brain Research Interactive Symposium, at the 2001 Annual Conference of the Society for Neuroscience, San Diego, CA, USA from November 8-10 2001.

Optimising plasticity: environmental and training associated factors in transplant-mediated brain repair.

With progressively ageing populations, degeneration of nerve cells of the brain, due to accident or disease, represents one of the major problems for health and welfare in the developed world. The molecular environment in the adult brain promotes stability limiting its ability to regenerate or to repair itself following injury. Cell transplantation aims to repair the nervous system by introducing new cells that can replace the function of the compromised or lost cells. Alternatives to primary embryonic tissue are actively being sought but this is at present the only source that has been shown reliably to survive grafting into the adult brain and spinal cord, connect with the host nervous system, and influence behaviour. Based on animal studies, several clinical trials have now shown that embryonic tissue grafts can partially alleviate symptoms in Parkinson's disease, and related strategies are under evaluation for Huntington's disease, spinal cord injury, stroke and other CNS disorders. The adult brain is at its most plastic in the period following injury, offering a window of opportunity for therapeutic intervention. Enriched environment, behavioural experience and grafting can each separately influence neuronal plasticity and recovery of function after brain damage, but the extent to which these factors interact is at present unknown. To improve the outcome following brain damage, transplantation must make use of the endogenous potential for plasticity of both the host and the graft and optimise the external circumstances associated with graft-mediated recovery. Our understanding of mechanisms of brain plasticity subsequent to brain damage needs to be associated with what we know about enhancing intrinsic recovery processes in order to improve neurobiological and surgical strategies for repair at the clinical level. With the proof of principle beginning to emerge from clinical trials, a rich area for innovative research with profound therapeutic application, even broader than the specific context of transplantation, is now opening for investigation.

Establishment and properties of neural stem cell clones: plasticity in vitro and in vivo.

The study of the basic physiology of the neural precursors generated during brain development is driven by two inextricably linked goals. First, such knowledge is instrumental to our understanding of how the high degree of cellular complexity of the mature central nervous system (CNS) is generated, and how to dissect the steps of proliferation, fate commitment, and differentiation that lead early pluripotent neural progenitors to give rise to mature CNS cells. Second, it is hoped that the isolation, propagation, and manipulation of brain precursors and, particularly, of multipotent neural stem cells (NSCs), will lead to therapeutic applications in neurological disorders. The debate is still open concerning the most appropriate definition of a stem cell and on how it is best identified, characterized, and manipulated. By adopting an operational definition of NSCs, we review some of the basic findings in this area and elaborate on their potential therapeutic applications. Further, we discuss recent evidence from our two groups that describe, based on that rigorous definition, the isolation and propagation of clones of NSCs from the human fetal brain and illustrate how they have begun to show promise for neural cell replacement and molecular support therapy in models of degenerative CNS diseases. The extensive propagation and engraftment potential of human CNS stem cells may, in the not-too-distant-future, be directed towards genuine clinical therapeutic ends, and may open novel and multifaceted strategies for redressing a variety of heretofore untreatable CNS dysfunctions.

Cellular replacement therapy for Parkinson's disease--where we are today?

The concept of replacing lost dopamine neurons in Parkinson's disease using mesencephalic brain cells from fetal cadavers has been supported by over 20 years of research in animals and over a decade of clinical studies. The ambitious goal of these studies was no less than a molecular and cellular "cure" for Parkinson's disease, other neurodegenerative diseases, and spinal cord injury. Much research has been done in rodents, and a few studies have been done in nonhuman primate models. Early uncontrolled clinical reports were enthusiastic, but the outcome of the first randomized, double blind, controlled study challenged the idea that dopamine replacement cells can cure Parkinson's disease, although there were some significant positive findings. Were the earlier animal studies and clinical reports wrong? Should we give up on the goal? Some aspects of the trial design and implantation methods may have led to lack of effects and to some side effects such as dyskinesias. But a detailed review of clinical neural transplants published to date still suggests that neural transplantation variably reverses some aspects of Parkinson's disease, although differing methods make exact comparisons difficult. While the randomized clinical studies have been in progress, new methods have shown promise for increasing transplant survival and distribution, reconstructing the circuits to provide dopamine to the appropriate targets and with normal regulation. Selected promising new strategies are reviewed that block apoptosis induced by tissue dissection, promote vascularization of grafts, reduce oxidant stress, provide key growth factors, and counteract adverse effects of increased age. New sources of replacement cells and stem cells may provide additional advantages for the future. Full recovery from parkinsonism appears not only to be possible, but a reliable cell replacement treatment may finally be near.

How the brain repairs itself: new therapeutic strategies in inflammatory and degenerative CNS disorders.

In the early 20th century, seminal work by Tello and Cajal showed that the CNS has the ability to regenerate itself after injury. In the most recent years, this pivotal observation has been rejuvenated by detailed in vitro and in vivo evidence supporting the idea of an innate self-maintenance programme to sustain brain homoeostasis and repair. These observations support the idea that chronic inflammatory and degenerative disorders of the brain might result from defective repair mechanisms rather than uncontrollable pathogenetic events. Investigation of the molecular and cellular events sustaining intrinsic brain-repair mechanisms and a better understanding of why they fail over time in chronic disorders might, therefore, provide an attractive conceptual framework within which to develop new and efficacious therapies for neurological diseases.

Strategies for the augmentation of grafted dopamine neuron survival.

The percentage of grafted embryonic DA neurons that survive transplantation is low, estimated at 5-20%. Significant agreement has emerged from the work of research groups worldwide that specific conditions associated with the transplant procedure and post-transplantation interval render grafted mesencephalic cells susceptible to apoptotic death. Detrimental triggers including hypoxia/ischemia, trophic factor withdrawal, and oxidative stress appear to exert the most impact on grafted DA neuron survival. Treatment strategies that aim to reduce or eliminate the triggers of grafted cell death appear to be more successful than approaches that target the intracellular apoptotic cascade. In particular, treatment of mesencephalic cell suspensions with isolated neurotrophic factors (GDNF, BDNF, NT 4/5) as well as glial-derived factors, antioxidant therapies and augmentation of graft vasculature have demonstrated consistent survival promoting effects. Caspase inhibition, although initially quite promising, has not been demonstrated to reliably increase grafted cell survival. Bcl-2 overexpression similarly has yet to prove beneficial, although this may be due to biologically irrelevant levels of bcl-2 present during the critical immediate post-grafting interval. Future strategies will target a "cocktail" approach in which effective treatment agents are combined to maximize grafted DA neuron survival. Refinements in ex vivo transduction parameters will allow for efficient sustained delivery of survival promoting agents to grafted cells. Once identified, the optimal survival-enhancing treatment of grafted primary embryonic DA neurons should also benefit future transplant therapies utilizing alternatively derived DA neurons.

Cell replacement strategies for neurodegenerative disorders.

Cell transplantation has over the last two decades emerged as a promising approach for restoration of function in neurodegenerative diseases, in particular Parkinson's and Huntington's disease. Clinical trials have so far focused on the use of implants of embryonic mesencephalic tissue containing already fate-committed dopaminergic neuroblasts with the capacity to develop into fully mature dopamine neurons in their new location in the host brain. However, the recent demonstration that immature neural progenitor cells with multipotent properties can be isolated from both the developing and adult CNS and that these cells can be maintained and propagated in culture, has provided a new interesting tool for restorative cell replacement and gene transfer therapies. Embryonic stem cells, obtained from the early stages of embryonic development, and neural stem cells, obtained from the developing brain, may provide renewable sources of cells for therapeutic purposes, and could eventually offer a powerful alternative to primary fetal CNS tissue in clinical transplantation protocols. The purpose of this review is to discuss the prospects of the emerging progenitor cell technology for cell replacement and restorative therapies in neurodegenerative diseases, and consider some of the critical issues that must be solved in order to make progenitor cells useful in studies of brain repair.

Striatal tissue transplantation in non-human primates.

The caudate nucleus and putamen form part of a complex but topographically connected circuitry that links the cortex, the basal ganglia and the thalamus. Within this complex system lie a series of functionally and anatomically segregated loops that allow the concurrent processing of a wide range of cognitive and motor information (Alexander et al., 1986; Alexander and Crutcher, 1990). As a constituent of these loops it has been shown that the striatum is involved in movement initiation, response selection and attentional processes (Robbins and Brown, 1990; Alexander, 1994; Lawrence et al., 1998). Although it is the medium spiny GABAergic projection neurones that are primarily lost in HD, it is not sufficient merely to replace the GABA. Instead it is crucial for striatal tissue transplants to integrate with the host tissue in such a way that the cortico-striatal-thalamic circuitry is restored and is functional. Rodent studies have progressed a long way in establishing the principle that striatal grafts can, at least partially, restore function and integrate appropriately with the host (Dunnett and Svendsen, 1993; Björklund et al., 1994; Sanberg et al., 1998) but the limited behavioural repertoire and the undifferentiated striatum meant that it was inevitable that studies should progress into primate models. Anatomical tracing studies have demonstrated that motor, premotor and somatosensory cortical areas send corticostriatal projections primarily to the putamen region in primates, whereas the head and body of the caudate nucleus mostly receive efferent input from associative cortical areas (Kemp and Powell, 1970; Kunzle, 1975, 1977, 1978; Selemon and Goldman-Rakic, 1985). Based on such anatomical, and functional, studies Alexander and colleagues have proposed the existence of at least five cortico-striatal-thalamic loops including a motor, a dorsolateral-prefrontal and an orbito-frontal loop (Alexander et al., 1986). The concentration of motor inputs to the putamen region suggests a particular involvement of this structure in the motor loop. Indeed, unilateral lesions of the putamen disrupt motor performance in the marmoset and generate apomorphine-induced dyskinesias in larger primates (Burns et al., 1995; Kendall et al., 2000). The implantation of striatal grafts into marmosets that had previously received unilateral putamen lesions ameliorated some of the motor impairments, which suggested at least partial restoration of the motor loop. In support of this we found direct evidence of host-graft cortico-striatal connectivity using an anterograde tracer injected in the primary motor cortical region (Kendall et al., 1998a). In larger primates, with lesions of the caudate and putamen, striatal [figure: see text] allografts and xenografts have been shown to reduce apomorphine-induced dyskinesias (Isacson et al., 1989; Hantraye et al., 1992; Palfi et al., 1998). The mechanism by which dyskinesias are elicited is not fully understood but alterations in firing patterns within both segments of the globus pallidus have been identified during dyskinetic movements (Matsumura et al., 1995). It seems likely that it would actually require re-establishment of afferent connections between the implanted putamen and the globus pallidus as well as of functioning dopamine receptors within the graft for the reduction in the dyskinetic profile to be observed. Certainly there is evidence, from rodent studies and the marmoset study described here, that close proximity of the graft to the globus pallidus yields better functional recovery (Isacson et al., 1986). In addition, anatomical tracing studies in rats have demonstrated connections between the implanted tissue and the host globus pallidus (Wictorin et al., 1989b, 1990) However, the relationship between graft placement and functional recovery remains to be fully substantiated.

Restoration of function by neural transplantation in the ischemic brain.

Stroke remains a major brain disorder that often renders patients severely impaired and permanently disabled. There is no available treatment for reversing these deficits. Hippocampal, striatal and cortical grafting studies demonstrate that fetal cells/tissues, immortalized cells, and engineered cell lines can survive grafting into the ischemic adult brain, correct neurotransmitter release, establish both afferent and efferent connections with the host brain, and restore functional and cognitive deficits in specific models of stroke. The success of neural transplantation depends on several factors: the stroke model (location, extent, and degree of infarction), the donor cell viability and survival at pre- and post-transplantation, and the surgical technique, among others. Further exploitation of knowledge of neural transplantation therapy already available from our experience in treating Parkinson's disease needs to be critically considered for stroke therapy. While the consensus is to create a functional neuronal circuitry in the damaged host brain, there is growing evidence that trophic action of the grafts and host, as well as exogenous application of trophic factors may facilitate functional recovery in stroke. Current treatment modules, specifically that of rehabilitative medicine, should also be explored with neural transplantation therapy. However, validation of neural transplantation and any other treatment for stroke should be critically assessed in laboratory experiments and limited clinical trials. No direct treatment is recognized as safe and effective for reversing the stroke-induced brain damage and functional/cognitive deficits. The first clinical trial of neural transplantation in stroke patients is a mile-stone in stroke therapy, but subsequent large-scale trials should be approached with caution.

Neural precursors as a cell source to repair the demyelinated spinal cord.

Schwann cells and neural precursor cells derived from adult human brain (subventricular zone) and from bone marrow were studied anatomically and physiologically after transplantation into the demyelinated rat spinal cord. All cell types formed myelin and restored conduction velocity. Following transection of the dorsal funiculus, Schwann cells and olfactory ensheathing cells facilitated axonal regeneration and restoration of conduction across the lesion site. There is discussion on the challenges of cell type selection and preparation for a potential clinical cell therapy study in human demyelinating diseases.

Towards a definition of recovery of function.

In this review we consider recovery of function after spinal cord injury, and, in particular, recovery improved following intraspinal cellular transplants. Some recovery occurs spontaneously and this can be especially dramatic in neonates, supporting the notion that developing and adult spinal cord respond differently to injury. Recovery can be improved in both neonates and adults by appropriate cellular transplants into the injury site. We describe several functional tests used in animals with spinal lesions and transplants. We compare the effects of transplants of fetal tissue and genetically modified fibroblasts into neonatal and adult injury sites on recovery of motor and sensorimotor function. Fetal tissue transplants support greater recovery and elicit more regeneration in neonates than in adults. Transplants of fibroblasts modified to produce neurotrophic factors however support both recovery and axonal growth even in adults. The contribution of the transplant to recovery is shown by the loss of function that follows a second lesion just rostral to the original lesion/transplant site. The effect of the re-lesion indicates that the recovery is mediated by the presence of the transplant but the way in which transplants act to promote recovery may include a number of mechanisms, including regeneration and sprouting, neuroprotection, and modifications of organization of spared CNS structures.