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Neural transplantation Forum Neural transplantation for the treatment of Parkinson’s disease Anders Björklund, Stephen B Dunnett, Patrik Brundin, A Jon Stoessl, Curt R Freed, Robert E Breeze, Marc Levivier, Marc Peschanski, Lorenz Studer, and Roger Barker Neural transplants in Parkinson’s disease: trials showed unequivocally that human fetal dopaminergic do they work? neurons can survive and function for more than 10 years in Anders Björklund and Stephen B Dunnett the striatum of patients with PD and show no signs of being Cell transplantation to replace lost neurons is a new approach affected by the ongoing disease process. These studies to the treatment of progressive neurodegenerative diseases. have also provided a clear indication that grafted fetal Replacement of dopaminergic neurons in patients with dopaminergic neurons can be therapeutically effective. On the Parkinson’s disease (PD) has spearheaded the development of basis of the limited, but encouraging, observations in these this approach and was the first transplantation therapy to be early open-label trials,1 the National Institutes of Health tested in the clinic. The success of cell replacement for (NIH) decided to support a second phase; two double-blind the treatment of PD is based on two hypotheses: first, the sham-surgery controlled trials were launched in the early predominant symptoms of PD are dependent on 1990s, the results of which have only recently been reported the dysfunction or loss of the dopaminergic neurons in the and turned out to be more disappointing than expected.2,3 nigrostriatal pathway; and second, dopaminergic neurons Meanwhile, the on-going open-label trials have continued grafted into the dopamine-deficient striatum can replace those to generate more data and further questions that designed neurons lost as a result of the disease process and can reverse, trials may be able to address. The most recent data from four at least in part, the major symptoms of the disease. centres (Sweden, France, USA, and Canada), which includes a Extensive animal experiments over the past two decades total of 26 patients with moderately severe PD, shows a have provided the basic proof of principle for cell replacement. 60–70% increase in striatal uptake of fluorine-18-labelled Immature dopaminergic neurons or neuroblasts—taken from dopa (figure 1) and overall improvements in Unified the mesencephalon of 13–15 day old rat fetuses or 6–8 week Parkinson’s Disease Rating Scale (UPDRS) motor scores of old aborted human embryos—survive and re-establish 6–40%.1 These improvements developed gradually during the dopaminergic innervation when transplanted into the first 6–24 months after transplantation. Moreover, clinical denervated striatum, and restore baseline dopamine synthesis improvements have been associated with recovery of and release in the reinnervated area. Striatal dopaminergic movement-related activation of the premotor and innervation can be restored to 10–40% of normal, which is supplementary motor cortex, a deficit of which is thought to similar to the extent of dopamine depletion seen in the early underlie the hypokinetic symptoms of PD.4 stages of PD. Although these results are clear-cut, these studies A major issue, however, is that the results of these trials were done in rodent and non-human-primate models of the have been quite variable, not only between centres but, more disease, in which parkinsonian symptoms are induced by the importantly, among groups of patients at the same centre selective destruction of the nigrostriatal dopaminergic (who received grafts of the same type of cell preparation and pathway by means of a neurotoxin (ie, the lesion is acute underwent the same surgical technique). Some patients rather than progressive, “cleaner”, and more circumscribed showed minor benefit, or no benefit at all, whereas others had than in PD). Moreover, the functional improvements seen substantial clinical improvement with reductions in UPDRS after intrastriatal transplantation of dopaminergic neurons, in motor scores of up to 50–60%.1 Transplant-induced increases both rats and monkeys, are limited to certain basic motor functions whereas impairments in more complex behaviours AB is at the Wallenberg Neuroscience Center, Section of are resolved to a lesser extent, if at all. This may be because the Neurobiology, Lund University, Sweden. SBD is at the Brain Repair transplanted cells are placed in the striatum—the target of Group, School of Biosciences, Cardiff University, Wales, UK. PB is at dopaminergic neurons and where dopamine exerts its the Wallenberg Neuroscience Center, Section for Neuronal Survival, action—rather than into the substantia nigra, from where the Department of Physiological Sciences, Lund University, Sweden. AJS is at the University of British Columbia, Pacific Parkinson’s normal dopaminergic neurons are lost in PD. This ectopic Research Centre, Vancouver, BC, Canada. CRF and REB are both at placement is necessary for extensive reinnervation of the the Departments of Medicine and Neurosurgery, University of striatum by the transplanted neurons, but may limit the Colorado School of Medicine, Denver, CO, USA. ML is at the Service functionality of the graft. de Neurochirurgie, Hôpital Erasme, Brussels, Belgium. MP is at Clinical trials of fetal-cell transplantation in PD have INSERM U 421, Faculté de Médecine, Créteil, France. LS is at the Developmental Biology Program and the Division of Neurosurgery, developed in two phases. In 1987, a series of small open-label Sloan Kettering Institute, New York, NY, USA. RB is at the Cambridge trials, in which only carefully selected patients were included, Centre for Brain Repair, Cambridge, UK. was initiated to obtain evidence to support the viability of Correspondence: Dr Marc Peschanski, INSERM U 421, Faculté de transplantation as a therapy for PD. These early open-label Médecine, F-94010 Créteil, France. Email firstname.lastname@example.org THE LANCET Neurology Vol 2 July 2003 http://neurology.thelancet.com 437 For personal use. Only reproduce with permission from The Lancet Publishing Group. Forum Neural transplantation in putaminal 18F-dopa uptake have also been highly variable, which suggests that differences in survival and growth of the grafted dopaminergic neurons may be an important factor in the recovery of patients. A systematic patient-by-patient analysis in the larger double-blind trials could help to elucidate the factors underlying this variability. Another challenge is that the reported clinical improvements after transplantation may be due to investigator bias, placebo effects, or both. In this regard, the results of the two NIH-sponsored double-blind studies are reassuring in that none of the patients who underwent sham surgery showed any significant, long-term benefit of the Figure 1. F-dopa PET scan in a patient with PD after transplantation of human fetal neurons into the 18 placebo treatment. However, these right putamen. Left: a defined area of increased F-dopa uptake is visible on the PET image (black 18 studies have raised two major concerns: arrow). Right: this area of increased uptake corresponds to the site of graft placement (red cross) in the the PET image with only modest efficacy was observed in the putamen, as confirmed by coregistration of of Marc Levivier. stereotactic MRI used during the neurosurgical grafting procedure. Courtesy transplantation groups, and a significant number of patients developed off-state dyskinesias after the delivery of transplants—before further double-blind trials transplantation. involving larger groups of patients are warranted to determine It is clear from the disappointing outcome of these two the true value and efficacy of neural transplantation in trials that we still do not fully understand how to use fetal-cell comparison with other available treatments. transplantation to achieve consistent, optimum results. They also highlight several serious issues that must be addressed. How can we make transplants of embryonic First, the mechanism underlying the off-state dyskinesias neural tissue more effective? developed by some patients needs to be investigated. Are some Patrik Brundin and A Jon Stoessl patients particularly sensitive5 and how can this side-effect be Two recent double-blind, sham-surgery-controlled trials, avoided? Second, how do we select the patients who are likely supported by funding from the NIH, of embryonic to derive the greatest clinical benefit? Are patients with mesencephalic transplants for the treatment of PD advanced disease unsuitable candidates for fetal-cell produced disappointing results. The first of these studies2 transplantation and is age a negative factor? Third, where is the did not show any overall improvement on a subjective best site for graft placement? Does the transplantation surgery global rating scale. However, patients under 60 years of need to be tailored to each patient’s disease profile? Fourth, do age had reductions in UPDRS scores. This modest we need to standardise preparation of cells for transplantation? improvement was offset by the emergence of severe Is it feasible to store tissue for days or weeks in vitro—as done dyskinesias in 15% of the patients, which persisted despite in the two NIH trials—without losing functional efficacy? substantial reductions in levodopa doses. The second study Fifth, what about immunosuppression? Can we really exclude has yet to be published, but the preliminary findings were or reduce immunosupressive treatment when multiple reported by Warren Olanow at the Movement Disorders allogeneic donors are used and surgery is done in stages? Society meeting (November 10–14, 2002; Miami, FL, USA).3 Finally, we need to consider the risk–benefit analysis. What is The investigators observed no significant improvement in the balance between clinical benefit and side-effects, and how UPDRS motor scores, activities of daily living, or percentage does it compare with the other treatment options available? of off time. More than half the patients developed Now that the NIH trials have been completed and the dyskinesias that persisted for 12 h after discontinuation of results reported, we are entering a new phase in the levodopa, but which disappeared 36 h after the last dose of development of cell-replacement therapy for PD. In our levodopa. However, there was some improvement after opinon, transplantation of fetal dopaminergic neurons is based transplantation surgery in patients with less severe disease on a solid rationale and open-label trials have provided ( 49 UPDRS score at baseline). convincing proof of principle that transplants can work—and Why did these trials not achieve the extent of work well—in some patients. However, in the light of the NIH symptomatic relief reported in previous open-label trials? trials, it is clear that we need carefully to reassess critical aspects Could the positive results in previous trials simply be of the transplantation procedure and further analyse the issues attributable to placebo effects, or were the results open to raised in well-designed open-label studies. Further carefully unintentional observer bias? Alternatively, could differences planned open-label trials are required to answer various major in transplantation methods, patient selection, or assessment outstanding issues—such as those relating to tissue collection protocols account for the differences between the placebo- and preparation, patient selection, and surgical placement and controlled and open-label studies? Finally, why were severe 438 THE LANCET Neurology Vol 2 July 2003 http://neurology.thelancet.com For personal use. Only reproduce with permission from The Lancet Publishing Group. Neural transplantation Forum become increasingly denervated later in the disease process and Rights were not granted to include this may therefore have a more important role in advanced cases; within the putamen, the pattern of denervation also changes as image in electronic media. Please the disease progresses. Perhaps these facts need to be taken into refer to the printed journal. account when targets for implantation are selected. The transplanted tissue is itself crucial to the function of the transplant: previous studies have used different numbers of donor embryos (2–9 per patient) of various ages (5·5–9·0 weeks); the storage or culture of tissue before surgery has varied from a few hours to 4 weeks; and different methods of tissue preparation have been used to produce different types of transplants (tissue strands, chunks, or cell suspensions).2,6,7 The most recent NIH-funded trial reported by Olanow and colleagues3 suggests that the use of tissue chunks from four embryos may be better than transplantation of tissue from only one embryo, but the differences between these two types of transplants were not significant. Age of donor tissue and preparation method are known to influence survival of transplanted dopaminergic neurons. These factors Catherine Pouedras/Eurelios/Science Photo Library. may also affect other cells in the graft that could influence functional outcome. Dopaminergic neurons transplanted as a cell suspension produce a more homogenous reinnervation than transplants of tissue chunks. A homogenous reinnervation may reduce the risk of dyskinesias and increase the chance of clinically beneficial effects. Storage or culture of donor tissue before transplantation increases the probability of dyskinesias.5 The handling of donor tissue may also affect survival or maturation of non-dopaminergic neurons in the graft that either directly influence the host striatum or change Figure 2. Patient with PD undergoing stereotactic surgery to transplant fetal neurons into the striatum. the activity of the dopaminergic neurons in the graft. Consequently, storage and culture of donor tissue should be avoided. Many of these technical issues may require years of off-medication dyskinesias not observed in the studies done further careful experimentation to develop an optimum before 2001? In this article, we discuss factors that might protocol for clinical transplantation. have contributed to the disappointing findings in the NIH- The immune response of the host may have an funded studies, and suggest ways to improve the outcome of important effect on graft function. Although the brain is an future cell-transplantation trials in patients with PD. immunologically privileged site, intracerebral transplants Patient selection is an essential factor. Freed and co- undergo immune rejection in certain circumstances. In the workers2 suggested that younger patients might benefit more first double-blind study, patients were not given any from cell transplantation. Subsequent meeting reports indicate immunosuppressive treatment2 and in the second study only that levodopa responsiveness at baseline (before surgery) is an a low dose of ciclosporin was given for 6 months. In the important predictor of positive response to transplantation. latter study, evidence of functional grafts was found at The unpublished study by Olanow and colleagues3 suggests 6 month follow-up, but then gradually disappeared, which that patients with less severe disease show some clinical benefit. could be consistent with an immune-mediated rejection of In an open-label trial done in Lund, Sweden, the severity of the graft. However, PET scans revealed continued 18F-dopa off-medication dyskinesias after transplantation was inversely uptake at graft sites 24 months after surgery. Earlier open- correlated with striatal 18F-dopa uptake before surgery.5 Thus, label trials used a more extensive immunosuppression more severe pathology at baseline may reduce clinical benefit regimen with three drugs,5,7 which was maintained in some and predispose patients to the development of dyskinesias. patients for several years. Future clinical trials should Graft location may also be crucial. Different approaches reassess immunosuppression protocols and investigate have been used to target transplants to the putamen (figure 2). immune and inflammatory responses around the grafts by Sagittal stereotactic injections along the longitudinal axis of the use of imaging techniques. putamen2 result in a pattern of reinnervation different to that What of the dyskinesias? One might speculate that the obtained with multiple, dorsoventral injections into the grafts produce an excess of dopamine that causes the postcommissural putamen6 or into a greater volume of this involuntary movements. Two patients in the Tampa–Mount structure.5,7 Could imaging studies provide clues as to which Sinai open-label pilot study6 showed a greater and longer patients might respond better to transplantation? Every patient increase in striatal dopamine concentrations in response to with the disease has a unique profile of dopaminergic levodopa, as assessed by PET scans with raclopride, than is denervation in the forebrain. Regions outside the putamen typical for patients with PD.8 In five patients with dyskinesias THE LANCET Neurology Vol 2 July 2003 http://neurology.thelancet.com 439 For personal use. Only reproduce with permission from The Lancet Publishing Group. Forum Neural transplantation from the first NIH-funded study, 18F-dopa uptake was formed our standard transplantation protocol. We have now increased in small focal areas in the grafted striata.9 However, done this operation in 60 patients. To reduce the number of in the Swedish study, there was no association between needle passes into the brain, we developed a method in which dyskinesia and increased 18F-dopa uptake in the whole the needle trajectories pass through the forehead above the putamen.5 This suggests that an uneven, patchy innervation frontal sinus and project through the long axis of the putamen rather than a generalised overproduction of dopamine could (35–40 mm) bilaterally. Four needle passes are made in total, be the source of the dyskinesia problem. However, graft- one dorsal and one ventral on each side of the brain (separated induced dyskinesias may also depend on other mechanisms— by a distance of about 7 mm). We also kept donor tissue in eg, an inflammatory response around the transplants due to a culture for up to 1 month before use. low-grade, chronic immune response. Three patients from the In our study, mesencephalic dopaminergic neurons from Swedish study, who received immunosuppression for a four aborted human embryos were transplanted and strands restricted time, had maximum off-phase dyskinesias 2–4 years of tissue from a single embryo were grafted along each needle after surgery, whereas clinical benefit reached a peak after track. 40 patients were recruited, 20 were under 60 years of age 1 year.5 The concept of late-onset inflammation as a and 20 were over 60 years of age. The aim of the study was to contributing factor in graft-induced dyskinesias can be tested compare the sham-surgery control group with the in animal models of PD and ought to be addressed before transplantation group, in both younger and older patients. No large-scale clinical trials are reinitiated. immunosuppression was given. At the end of the trial, patients To understand which variables are important for clinical in the sham-surgery group were given the option of receiving efficacy of neural transplants in patients with PD, further trials a transplant; 14 of the 20 patients in the control group took in patients are required. However, these ought to be limited to this option so, in total, 34 patients received transplants. small series of patients with well-defined scientific goals, and 18 F-dopa PET scans 1 year after transplant surgery revealed should be based on improvements in graft technology that can survival of grafts in 85% of patients.2 Survival was the same in only be developed by meticulous experiments in animals. the younger and older subjects. The younger group showed improvement in UPDRS motor off scores (34%) and in Transplantation of embryonic dopaminergic Schwab and England off scores (31%) compared with no neurons in PD: the Colorado experience change in the sham-surgery control group or in the older Curt R Freed and Robert E Breeze patients who received transplants. There were no changes in Since transplantation of embryonic dopaminergic cells was best on scores, which indicates that transplants produce first reported in human beings in the early 1990s,10,11 several symptomatic improvements similar to, but not better than, open-label clinical trials have confirmed the benefits of the best effects of levodopa. Interestingly, the longer the tissue transplantation. Outcomes of these trials have been similar, was kept in culture before transplantation, up to a maximum despite the use of various experimental techniques and of 4 weeks, the better the clinical outcome. transplantation protocols. The validity of these studies has Subsequent analysis has shown that preoperative been uncertain because of small patient numbers, variable response to levodopa, not patient age, determines the inclusion criteria, and the absence of control groups. magnitude of clinical benefit after transplantation. As a After the election of Bill Clinton as President of the USA in group, the older patients had more variable responses to 1992, the NIH agreed to fund a controlled clinical trial. In levodopa before transplant surgery. Older patients who had collaboration with Stanley Fahn (Columbia University) and good responses to levodopa (>60% improvement in motor David Eidelberg (North Shore University Hospital), we scores) showed a similar response to transplantation to that designed a double-blind, placebo-controlled clinical trial to of the younger patients. Reduced efficacy of levodopa in some assess the efficacy of neural transplantation in patients with older patients may indicate a more extensive disease process PD. This was the first double-blind study in neurosurgery. in non-dopaminergic systems that the transplants were Although there was opposition at the time,12 we felt that valid unable to affect. conclusions could only be made from a large study in which Patients in the sham-surgery group who subsequently patients undergoing transplant surgery were compared with a opted to receive transplants were compared with patients control group who would undergo sham surgery. given transplants in the initial, blinded protocol. Clinical Before we did our first transplant into a human being in improvement over the first year was identical in these two 1988, studies in rodent and non-human primates had shown groups, which confirms that there was no measurable placebo that only dopaminergic neurons at an early stage of their effect of the sham surgery. development (7 to 8 weeks after conception in human beings) Graft development and maturation in individual could survive and re-innervate the host brain. Experiments in patients is uncertain, mainly because most transplanted animals also showed that immunosuppression was probably dopaminergic cells die from apoptotic cell death in the first unnecessary for these allografts (grafting of tissue between few days after transplantation. Pretreatment of embryonic individuals of the same species). This finding was critically tissue with neurotrophic factors before transplantation can important because the long-term costs and complications of reduce this cell loss and may make the effects of immunosuppression are difficult to justify for a symptomatic transplantation more uniform. The biggest limitation to treatment. Because the putamen is most affected by dopamine extensive testing and application of neural transplantation is depletion in idiopathic PD, and the structure has many links to the limited availability of human embryonic dopaminergic and from the cortex, bilateral implants into the putamen neurons. Laboratory production of large quantities of 440 THE LANCET Neurology Vol 2 July 2003 http://neurology.thelancet.com For personal use. Only reproduce with permission from The Lancet Publishing Group. Neural transplantation Forum dopaminergic neurons from a stem-cell source could solve a viable treatment for PD, and should we be able to offer this this problem. form of treatment to all patients with advanced PD? The importance of this double-blind design cannot be For us, the answer is no. Whatever the outcome of small- overstated. Other double-blind trials done after our initial scale trials, and whatever improvements in techniques are NIH-funded study, which have investigated transplants of made, transplantation of human fetal neural tissue can never human embryonic tissue or bovine embryonic tissue, did not be a large-scale treatment for all patients with advanced PD. find clinical improvement despite promising results in small The main reason for this rather pessimistic view is that the open-label trials. For cell-replacement therapies to progress, procurement of human fetal tissue requires a chain of more double-blind comparisons must be done. expertise that cannot be “industrialised”. The use of large From our experience, most patients whose motor scores amounts of non-expanded neural cells from several human improve in response to transplant surgery develop dyskinesias fetuses requires an enormous investment in time, that resolve after reduction of levodopa or dopamine agonist organisation, and skill for each patient with PD. There is little treatment.2,10 Others have subsequently made similar hope that these logistical problems could be overcome. There observations. In about 10% of cases, deep brain stimulation of are not enough data to suggest that we may eventually be able the subthalamic nucleus or the internal segment of the globus to amplify neural cells from human fetuses in “cell banks”, pallidus may be necessary to control persistent dyskinesias. from which neurosurgeons could simply draw the number of The ideal outcome of transplantation would be for the dopaminergic cells they require. Conversely, in this forum, patient to regain normal neurological function after the Lorenz Studer and Roger Barker discuss the potential of discontinuation of all their medication. Our experience human embryonic stem cells and xenogeneic fetal neural tissue suggests that such an outcome is only possible in patients who as potential sources for transplantation. These alternative cell appeared free of parkinsonian symptoms and signs while types may eventually be able to meet the need for a substitutive receiving levodopa before transplant surgery. In patients with cell therapy for all patients with advanced PD. advanced disease, the reduction of symptom severity in the off Transplantation of human fetal neural tissue is a genuine state, with maximum benefit about 50% of that with levodopa ethical concern because it is not available on request and, treatment, is a realistic outcome; levodopa and other drugs therefore, cannot meet its social goal. By contrast, new would still be required to fine tune the on state. neurosurgical approaches have been introduced in the past An intriguing question at this time is whether transplants decade that are based on ready-to-use and commercially can prevent the progression of PD. At 5 year follow-up, we available equipment—ie, deep brain stimulation of the have observed no progression of symptoms in our patients. It subthalamic nucleus and, to a lesser extent, lesion or is possible that cell transplantation early in the disease course stimulation of the globus pallidus.15 These approaches are might prevent complications caused by drug therapy now standard procedures in patients with advanced PD. (eg, drug-induced dyskinesias) although this hypothesis will Nevertheless, their expansion to treat the entire population of require clinical testing. patients with advanced PD is severely restricted by their high cost and dependence on very skilled stereotactic Human fetal neural transplants in patients with neurosurgeons. Therefore, the search for alternative PD: ethical considerations neurosurgical approaches remains an issue. Marc Levivier and Marc Peschanski The discussion about whether, and how, clinical trials of There will come a time when patients diagnosed with PD will human fetal neural transplants should be continued must be be treated with therapies that target and halt the considered within this framework. First, whether the neurodegenerative processes of the disease. At present, technique should be investigated in further experimental patients can be treated effectively, for a while at least, with clinical trials is dependent on the benefit to risk ratio. At therapies that pharmacologically replace the missing present, this ratio seems sufficiently positive to continue with dopamine. Unfortunately, as the disease progresses, these such trials, as discussed by Curt Freed. Whether scientists medications lose their efficacy and patients develop severe should invest time and money in doing such clinical trials is side-effects. As a consequence, patients with advanced PD are dependent on the scientific questions that they wish to address candidates for alternative therapies, such as neural and the worth of the conclusions they may draw. We believe transplantation. One of the main ethical constraints of human that there are major questions to be answered, and that the use fetal neural transplants is that they can not be regarded as a of other cell types for transplantation therapies for PD will first-line treatment for repairing damaged neuronal circuitry benefit from the advances made with human fetal neural and rather constitute a “second chance” for patients who no transplants. For example, one major issue that has been raised longer derive benefit from pharmacological therapy. by neural transplantation is cell survival, as discussed by Patrik On this basis, the success of transplants of human fetal Brundin and Jon Stoessl. It is likely that this problem is tissue is a major ethical issue. We are not referring to any independent of cell type and there may be ways to overcome therapeutic benefit in patients receiving this treatment: it, which can be investigated in clinical trials of human fetal therapeutic benefit, and how to improve it, is discussed by neural transplants.7 Other major issues that could be other authors in this forum. Some individuals who have addressed in such trials include assessment of the dopa- received transplants of human fetal dopaminergic neurons minergic system reconstruction in vivo, the immune response have shown clinical benefit—that is beyond any doubt.13,14 But to cells transplanted into the brain, and the combination of can transplantation of human fetal neural tissue ever become transplantation therapies with neuroprotection. Small-scale THE LANCET Neurology Vol 2 July 2003 http://neurology.thelancet.com 441 For personal use. Only reproduce with permission from The Lancet Publishing Group. Forum Neural transplantation clinical trials that address any one of these questions would undoubtedly be very useful in the development of future substitutive treatments based on other cell types. There was much hope for transplantation of human fetal neural cells when the results of the first clinical trials were reported 12 years ago. These studies were done by groups of researchers with strong backgrounds in basic research and focused on a small number of carefully studied patients. It is now evident that clinical application of neural transplantation to the entire population of patients with advanced PD is not feasible. There is little to be gained from the repetition of phase III-style studies, such as those done in the USA during the past decade.2 Incidentally, such “group analyses” may even be detrimental to the scientific goal as they mask interesting results of the treatment in individual patients because they preclude careful and thorough analysis. In contrast, it is our firm belief that there is an ethical need for small-scale, scientifically focused clinical trials of human fetal neural transplants. Such trials would pave the way for the development of other substitutive cell-based therapies that Figure 3. Neurospheres generated from rat cortex at embryonic day 14. may eventually prove to be an effective treatment for all These neurospheres are expanded in culture with mitogens such as basic patients with advanced PD. fibrobast growth factor and epidermal growth factor. These spheres can be used for in vitro studies or in grafting experiments in animal models. Hoescht staining (blue) is used to visualise nuclei, beta III tubulin (red) is an Are stem cells a valid alternative to human fetal early marker expressed by immature neurons, and the presence of glial tissue? fibrillary acid protein (green) denotes glia. Courtesy of Laurent Roybon, Lorenz Studer Jia-Yi Li, and Patrik Brundin (Wallenberg Neuroscience Center, Section for There has been a lot of excitement about the potential use of Neuronal Survival, Lund University, Sweden). stem cells in regenerative medicine. Cell transplantation for the treatment of PD has been proposed as one of the major and induced to differentiate into functional dopaminergic applications for stem-cell therapy. Experience with neurons (figure 4).18 Unfortunately, these midbrain stem cells transplantation of human fetal tissue has provided convincing lose their ability to differentiate into dopaminergic neurons evidence that midbrain dopaminergic neurons can survive after long-term culture, although they can still generate other long-term in patients with PD and can produce functionally neuronal subtypes such as GABAergic and glutamatergic relevant changes in dopaminergic function.16 However, neurons.19 The persistence of neural stem cells in certain disappointing clinical outcomes and the occurrence of regions of the adult brain suggests an alternative approach— significant graft-mediated side-effects in two placebo- the direct recruitment and conversion of endogenous neural controlled trials are raising the bar for the development of a stem cells into dopaminergic neurons. There is, however, no clinically successful transplantation therapy in PD.2,3 evidence that adult endogenous midbrain stem cells can The first step in the development of a stem-cell based differentiate into functional dopaminergic neurons in vivo.20 therapy for PD is to find a renewable source of midbrain Stem cells from non-neural tissues are a readily accessible dopaminergic neurons. Such a source would alleviate many source for autologous grafting. Differentiation of stem cells of the ethical and practical concerns associated with the use from various tissue sources, such as bone marrow or skin, of human fetal tissue, as discussed by Marc Peschanski and into cells that express neural markers has been reported.21,22 Marc Levivier. It would also provide a time window for The most encouraging is a report of derivation of cells that cell characterisation, purification, and banking before express tyrosine hydroxylase from bone-marrow derived transplantation. There are at least four types of stem cells multipotent adult precursor cells.21 However, the that have been studied for this purpose: neural stem cells, dopaminergic function of transdifferentiated cells from these non-neural adult or fetal stem cells (eg, bone marrow or skin sources has not been characterised. cells), embryonic stem cells, and nuclear transfer or By contrast, mouse embryonic stem cells readily parthenogenetic stem cells. differentiate into neurons that have stable genetic and Neural stem cells can be isolated from either the functional characteristics of midbrain dopaminergic cells.23–25 developing or the adult brain (figure 3). Methods for their Transplantation of these cells results in a remarkable propagation and differentiation into neurons, astrocytes, and functional benefit in a rat model of PD.25 The efficient oligodendrocytes have been well established for more than derivation of dopaminergic neurons from human embryonic 10 years.17 However, despite considerable efforts, the efficient stem cells has not yet been reported, but there is little doubt derivation of functional midbrain dopaminergic neurons that such protocols will be available within the next few years. from neural stem cells has not been achieved. Stem cells An ideal source of cells for transplantation would provide isolated from the midbrain just before the time of autologous functional neurons in an unlimited supply. The dopaminergic neuron “birth” can be propagated in culture, successful derivation of dopaminergic neurons from mice 442 THE LANCET Neurology Vol 2 July 2003 http://neurology.thelancet.com For personal use. Only reproduce with permission from The Lancet Publishing Group. Neural transplantation Forum dopaminergic neurons. Furthermore, differentiation of embryonic stem cells in vitro enables access to all stages of dopaminergic neuron development—from neural induction to regional specification, dopaminergic neuron “birth”, and maturation. Systematic transplantation of cells at each of these stages should reveal varied potential for phenotypic differentiation and host integration. Once an optimised cell type has been identified, we need to focus on controlling the interactions of these cells with the host environment. The adult brain provides limited support for neuronal differentiation, migration, and synaptic integration, and the process of transplantation itself might reduce survival by the induction of an inflammatory response. An additional limiting factor in transplantation therapy for PD is the ectopic placement of the graft into the striatum. Despite the ability of such grafts to release dopamine locally, transplanted cells lack the appropriate afferent innervation. Fetal dopaminergic neurons transplanted into the neonatal substantia nigra have the ability to regrow axons into the Figure 4. Dopaminergic neurons (derived from embryonic day 12 striatum.30 However, when the same cells are grafted into the midbrain precursor cells) double labelled for tyrosine hydroxylase (green) substantia nigra of a 20 day old or adult animal, they are no and bromodeoxyuridine (red). The presence of double-labelled cells confirms that these cells were generated from dividing precursor cells. longer able to extend axons into the striatum. The molecular basis of this inhibition has not been identified, but and non-human primates by use of nuclear transfer26 and chondroitin sulfate and other myelin-related factors such as parthenogenetic27 stem cells proves that such a source could be Nogo may be involved. Stem-cell technology could support available in the future. However, progress in this area has been strategies to modulate intracellular responses of the grafted delayed by ethical and political concerns, as well as by cells to this inhibition, or directly change the environment. biological challenges.28 Parthenogenetic stem cells, derived For example, by increasing intracellular concentrations of from unfertilised egg cells, are compatible with the host in cyclic AMP, the response of dorsal-root-ganglion cells to a immunological terms but might function abnormally due to wide range of inhibitory factors in vivo can be reversed.31,32 homozygosity of alleles and purely maternal imprinting. Infusion of chondroitinase ABC into the nigrostriatal tract Teratoma formation is one of the defining properties of allows partial regrowth of axotomised nigral dopaminergic embryonic stem cells, prevention of which is a significant neurons in the adult.33 Such knowledge could be used to challenge in transplantation therapy. Although the incidence develop a stem cell-based strategy for restoring the of tumour formation can be reduced by the transplantation of nigrostriatal pathway. Concerns about the potential for graft- fewer undifferentiated embryonic stem cells,29 the only safe induced dyskinesia could be addressed by genetically approach is to eliminate all undifferentiated embryonic stem modifying cells to express an inducible negative regulator of cells before transplantation. Additional safety concerns will dopamine production. need to be addressed before the use of human embryonic cells After the disappointing results of fetal tissue can be tested clinically. Of particular concern is that all human transplantation in placebo-controlled trials, the stakes to make embryonic stem cell lines listed in the NIH registry that are transplantation therapy a clinically viable option are high. eligible for federal funding have been derived in coculture with However, recent progress in stem-cell technology provides the murine cells, not necessarily in accordance with the good necessary muscle and sophistication to take on the challenge. manufacturing practice standards required by the Federal Drug Administration. Porcine neural xenografts for the treatment of The therapeutic effect of dopaminergic neurons derived neurodegenerative diseases from human embryonic stem cells in various animal models Roger Barker of PD is likely to be proved experimentally over the next few The concept of xenografting—transplantation of tissue from years. However, it remains unclear which parameters will help one species into another—is not a new one. Traditionally, the stem cells exceed the therapeutic benefit that can be obtained pig has been the most logical choice of tissue source. There are by transplantation of human fetal tissue. well-established advantages to the use of pigs, such as big One important variable may be graft composition. Human litters and the potential for greater cell migration and axonal fetal grafts typically contain 5–10% dopaminergic neurons (the outgrowth in the adult CNS.34 Nevertheless, there are some rest of the graft constitutes various other neuronal and glial cell major disadvantages that prevent the clinical use of porcine types). Populations of dopaminergic neurons in grafts may tissue in the UK.35,36 Despite these disadvantages, in the USA, harbour precursors that differentiate into neurons with clinical trials of porcine neural xenograft for the treatment of distinct functional characteristics of both the substantia nigra PD and Huntington’s disease (HD) have already begun.37–39 and the ventral tegmental area. Stem-cell technology should The first main disadvantage is the risk of zoonotic provide the means to generate purified populations of nigral infection. Although pigs can be bred in pathogen-free THE LANCET Neurology Vol 2 July 2003 http://neurology.thelancet.com 443 For personal use. Only reproduce with permission from The Lancet Publishing Group. Forum Neural transplantation conditions, transmission of porcine endogenous retroviruses from donor to host cells is still a risk.40,41 It is not known how real this risk is, although it may be minimised by selective breeding of pigs that are unable to transmit porcine endogenous retroviruses to human cells. Indeed, a meta-analysis of all patients exposed to live pig tissue in 1999 reported no adverse effects, despite the presence of porcine cells in the circulation years after exposure to porcine tissue.42 However, there was no evidence of surviving pig tissue in these individuals (except the few cells in the circulation). Transmission of porcine Figure 5. Grafts of embryonic day 27 porcine ventral mesencephalon transplanted into the 6-hydroxydopamine lesioned striatum of immunosuprressed marmosets. The graft has survived endogenous retroviruses, without 6 weeks post-implantation and has been visualised by use of pig specific neurofilament staining. adverse effect to the host, has Courtesy of Lucy Annett. been reported in severely combined immunodeficient (SCID) mice that received grafts of pig other compounds (eg, 15-DSG, FK506),53–55 although most of tissue.43 The xenografts survived in these mice; however, the these studies have only investigated short survival periods. relevance of this experimental result is debatable, because These results should be interpreted with caution because the xenografted patients would not be as severely drugs may only delay or slow the rejection process, rather immunosuppressed as SCID mice. than prevent it. Nonetheless, the most successful strategies to Transgenic pigs that express regulators of the human date have used monoclonal antibodies targeted to complement cascade44 have been developed to reduce the lymphocytes or the cytokines responsible for their likelihood of complement-mediated hyperacute rejection of recruitment (eg, interleukin 2).56,57 Alternatively, less vascularised whole organ xenografts.45 However, this strategy immunogenic tissue, such as porcine neural precursors or may actually promote the transmission of porcine endogenous stem cells, can be used.58,59 retroviruses by inhibition of complement mediated lysis,41 The third disadvantage relates to the functional capacity although these transgenic pig lines have low expression of such and capabilities of xenografts. Data on this issue are limited regulators in the embryonic brain.46 owing to problems with graft rejection. However, in studies The risk of porcine-endogenous-retrovirus transmission, where grafts have survived (figure 5), basic functional deficits and its implications, is unresolved. This issue represents a in lesioned animals (eg, drug-induced rotation) have been major hurdle to the clinical adoption of this approach, reversed.60 Furthermore, several studies have now shown that although it is ultimately more likely to represent a political and xenografts of porcine tissue from various regions of the a theoretical risk than a real risk. embryonic brain can extend axons, form synapses, and exert The second disadvantage concerns rejection of xenografts. functional benefit in the host rat brain.60,61 Studies of glial When grafted into the rat brain, porcine neural tissue is progenitor cells from embryonic pig brain have shown that rejected by a cellular and humoral process over a 5 week these cells have remyelinating qualities; this suggests that period.47 Humoral rejection seems to have a minor role in the embryonic pig neural cells may have a similar capacity to rejection process in the case of pig-to-rat xenografts because repair the CNS as their human counterparts.62 rats do not have high numbers of circulating naturally Although there are issues that need to be resolved, clinical occurring antibodies to porcine tissue.47 However, human trials have already been initiated for xenotransplantation in beings do have high numbers of naturally occurring HD (although without benefit) and in PD.37–39 In a phase I antibodies to porcine tissue, in particular to the glycoprotein study in PD by Diacrin Inc, greater than 30% improvement in -galactosyl epitope, which is expressed on neural cells in the UPDRS scores was reported in 3 patients 1 year after grafting, embryonic pig brain.48,49 Therefore, in human beings, these although these improvements did not correlate with evidence antibodies can bind to porcine tissue and initiate complement of dopaminergic activity in the graft on PET scanning.37 One mediated lysis of the cells, similar to the response seen during patient in the trial—who died from unrelated causes hyperacute rejection of whole-organ xenografts.45 Various 7–8 months after receiving a graft of porcine ventral genetically modified pigs, designed to either express regulators mesencephalon—had a few surviving dopaminergic neurons of the human complement cascade or that lack -galactosyl at the graft site.63 Therefore, the basis of any beneficial effects epitope, have been developed.50,51 These pigs may thus be of of xenografting in this study is unknown. A phase II study by value for clinical xenograft programmes in the future. the same company, which has been reported in abstract Apart from these genetic approaches, the prevention of form,39 enrolled 18 patients with advanced PD who were porcine neural xenograft rejection by drug therapies has been randomly allocated to receive either a xenograft (and attempted. Monotherapy with ciclosporin A resulted in 40% ciclosporin A monotherapy) or sham surgery. Both groups of survival of xenografts.52 Better results have been obtained with patients showed significant improvement in their motor 444 THE LANCET Neurology Vol 2 July 2003 http://neurology.thelancet.com For personal use. Only reproduce with permission from The Lancet Publishing Group. Neural transplantation Forum scores, although there was no significant difference between moratorium on all clinical studies of this treatment the grafted and the sham-operated groups. The reason for such approach in the UK (and in some other countries). As a a large placebo effect is unclear, particularly because this has result, alternative cell sources are being pursued, including not been reported in sham-operated groups in trials of human the possible use of neural stem cells. fetal allografts.2,3 However, a more detailed analysis of the study is required before any further comments can be made. Acknowledgments In summary, xenografts of porcine neural tissue offer LS’s work cited in this article was supported by the MJ Fox Foundation and the National Institute of Neurological Disorders and Stroke (NIH). some hope but there are major problems with this RB’s work cited in this article was supported by the Medical Research approach that—at the present time—have led to a Council, the Parkinson’s Disease Society, and the Wellcome Trust. References Neuron 2000; 28: 31–40. engineering of the donor as an approach to clinical 1 Dunnett SB, Björklund A, Lindvall O. 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